WO1998013471A1

WO1998013471A1 - Novel composition

Info

Publication number: WO1998013471A1
Application number: PCT/GB1997/002566
Authority: WO
Inventors: Donna Christine Murray; David Kirk Rodham; Bernard D'alwis; John Burnett Cantwell; David John Rhodes; Sandra Samira Bradley
Original assignee: Zeneca Limited
Priority date: 1996-09-24
Filing date: 1997-09-22
Publication date: 1998-04-02
Also published as: EP0929660A1; JP2001501091A; CA2266756A1; GB9619893D0; AU4312797A

Abstract

A composition comprising a stabilised biological material in a stasis state suspended in a collapsed matrix of a non-carbohydrate polymeric material capable of forming a glassy state; characterised in that the matrix also incorporates urea.

Description

NOVEL COMPOSITION The present invention relates to a novel composition for the stabilisation and storage of biological materials. Particularly, but not exclusively, the present invention relates to a novel composition for biological materials which are used in agriculture, pharmaceuticals. environmental control and the food industry..

A problem associated with the industrial applicability of biological materials has been maintaining the materials in a viable condition until they are used: many biological materials cannot be maintained in a viable condition during storage, particularly when they are not kept under refrigerated conditions, The problem is particularly acute with bacteria which do not form spores and so are particularly vulnerable.

A number of formulations/techniques have been proposed to try to overcome this problem of storing biological materials, for example in EP-A-0320483 and EP-A-0520748. Our earlier application O-A-9425564 relates to formulations comprising a biological material in a stasis state suspended in a collapsed matrix. By means of this type of formulation, it was found to be possible to preserve biological materials for considerable periods of time without significant degradation.

In the formulations previously described, the polymers from which the collapsed matrix are formed have been carbohydrates. Although carbohydrate polymers are suitable matrix materials in formulations for maintaining the stability of biological materials, they are not ideal for applications such as agriculture, sewage treatment and bioremedation as they tend to stimulate the growth of pathogens and other organisms. Clearly, stimulation of pathogens is not acceptable in formulations intended for this type of application. Indeed, in agricultural uses, it has been suggested that stimulation of soil pathogens may make a significant contribution to poor performance of formulated cells in field soil. Performance in field soil is often worse than the performance of the same formulation in. for example, a commercial compost or the performance of non-formulated freshly produced cells in either medium.

In order to retain the activity of a biological material such as a protein or a whole cell, dehydration must take place in the presence of a hydrogen bonding material (a cryo- or lyo- protectant) which will ensure that the three dimensional structure of the material is maintained. Carbohydrates have been popular choices as cryo- and lyo-protectants because their multiple hydroxyl groups provide a very similar hydrogen bonding environment to water. Moreover, they are capable of acting as humectants and so retain a level of water in the vicinity of the biological material. They are thus ideal for stabilising biological materials. However, because of the problems of pathogen stimulation, the present inventors set out to provide an alternative system using non-carbohydrate polymers together with added ingredients to try to replicate the cryo- and lyo-protectant properties of carbohydrates without causing increased pathogen stimulation.

In the course of this work, it was found that the inclusion of urea in non-carbohydrate collapsed matrix formulations leads to increased viability of the biological material. This is particularly surprising because urea is known to destabilise proteins and must generally be used in combination with an agent such as trimethvlammonium oxide which has the opposite effect on hydrogen bonding systems to urea. In this case, however, it was found that the viability of biological materials stored for several weeks in a collapsed matrix formulation containing a combination of urea and a non-carbohydrate polymer was improved when compared with biological materials stored in matrices containing the same polymer and no urea.

Therefore, according to a first aspect of the present invention, there is provided a composition comprising a stabilised biological material in a stasis state suspended in a collapsed matrix of a non-carbohydrate polymeric material capable of forming a glassy state: characterised in that the matrix also incorporates urea.

It would certainly not have been expected that formulations containing urea would have storage stability superior to that of formulations containing the non-carbohydrate polymer alone because of the destabilising effect of urea upon proteins. However, surprisingly, formulations containing urea can be stored at higher temperatures and for longer periods of time with less effect on the viability of the biological material than formulations in a non-carbohydrate matrix with no urea. It appears that the improvement in performance may arise through the ability of urea to increase the glass transition temperature (Tg) of the matrix material but the effectiveness of the invention is not dependent upon this theory being correct. A further advantage of using urea in the formulations is that there is evidence for a slight antifungal effect which can be useful in protecting crops, seeds and the like against the - J

damaging effects of fungi. This effect is particularly advantageous when the biological material itself is applied as an antifungal agent.

Although polyvinylpyrrolidone (PVP) and urea were both suggested as components of the formulations described in our earlier application WO-A-9425564, it is important to note that, in that case, the matrix material is a carbohydrate which has the disadvantages discussed above.

In the context of the present invention, the term "stabilised" means that the degradation of the biological material is reduced (which degradation would lead to a loss of recoverable viable cells). The term "stasis state" means that the cells are not metabolising, dividing or growing

(but are recoverable if subjected to a suitable treatment).

The term "recoverable" refers to cells which on exposure to suitable conditions (i.e. rehydration and source of nutrient) are capable of growth and division.

The term "viable cells" refers to cells which on exposure to suitable conditions (i.e. rehydration and source of nutrient) are capable of growth and division.

The term "collapsed matrix" means i) that the matrix has shrunk and become less porous allowing little penetration of low molecular weight diffusive species into the matrix, e.g. it absorbs little oxygen on exposure to 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.

In the formulation of the present invention, it is, of course, advantageous to include as much biological material as possible but cell loading is limited by the need to have a collapsed matrix around the cells. It has been found that the cell loading should be from about 10'° to 10¹³ colony forming units (cfu) per gramme of composition, more preferably from 10" to 10'² cfu per gramme of composition. In the remainder of the present specification, the standard cell loading of the composition is taken to be about 10'² cfu and other ingredients are expressed as amounts per 10¹² cfu. We have also found that there is range in which the effects of the urea are particularly beneficial. It is by no means essential that the urea be present in an amount which falls within this range, but we have found that the balance of beneficial effects afforded by urea versus any biocidal effect is especially good in this range.

Thus, the urea may be present in a concentration up to about lg per 10¹² cfu. preferably up to about O.lg per 10^ι; cfu. In one preferred embodiment the lower range is about 0.025g or more, leading to preferred concentrations of about 0.025g to abput lg per 10¹² cfu. In a more preferred embodiment the lower end of the range is about 0.03g per 10^π cfu, and the top end of the range is about 0.09g. 0.08g. 0.07g or 0.06g per 10¹² cfu. Thus, in a particularly preferred embodiment the range is about 0.03g to about 0.06g per 10'² cfu. optimally about 0.05g per 10¹² cfu. Although, as has already been discussed, urea does have the effect of increasing the viability- of a biological material in this type of formulation, it may still be advantageous to include an agent such as trimethylammonium oxide which has the opposite effect to urea on the hydrogen bonding environment. The amount of trimethylammonium oxide (TMAO) used may vary from about 0.025g to l g per 10¹² cfu, with an amount of about 0.025g to 0.5g per 10¹² cfu being preferred.

The matrix material is. as already mentioned, a non-carbohydrate polymer capable of forming a collapsed glassy state. The matrix material may be present in an amount of from about O. lg to lg per l O¹² cfu. In general, the amount of matrix material will be somewhat less than l per 10¹² cfu and will be from about 0.2 to 0.8g per 10^s2 cfu. A typical amount of matrix material included in a formulation according to the invention is about 0.5g per 10¹² cfu. Suitable matrix materials include polyacrylamide, polyethylene glycol (PEG), polyvinyl alcohol (PVA) and many other groups of polymer. However, the composition of the present invention has proved to be particularly successful when the polymer is polyvinylpyrrolidone (PVP). PVP is a suitable matrix material because it is capable of forming the necessary glassy state but, in addition, it is hydrophilic and so readily dispersed and it does not have a high intrinsic viscosity. Both of these factors mean that compositions containing PVP have good handling properties. When the matrix material is PVP. a wide range of molecular weights can be used, for example, it may be in the range of 10000 to 360000. The molecular weight of PVP selected will depend upon the particular handling properties required for the formulation. In addition to the biological material, the matrix polymer and urea, the composition of the present invention may optionally contain further ingredients. The composition typically also contains one or more suitable additives, As examples of suitable additives maybe mentioned inter alia lyo-protectants. for example polymeric species such as polyvinyl alcohol, polyethylene glycol: or anti-oxidants, for example an ascorbate, preferably sodium ascorbate, or sodium glutamate. We do not exclude the possibility that other additives may be present, for example, so-called bulking agents, for example crystallising sugars e.g. mannitol and osmo-regulants. e.g. betaine. proline and sarcosine.

When used, the anti-oxidant may be present in an amount of from about 0.01 to 0.25g per 10'² cfu. It has been found that particularly successful formulations contain about 0.05e anti-oxidant per IO¹² cfu.

It will be appreciated that the composition of the present invention can comprise more than one type of biological material, for example, a combination of two different bacteria. The biological material may be any of a wide range of materials, and the present invention is particularly applicable to materials which are unstable at normal storage conditions, i.e. ambient temperature. Non-exhaustive examples of biological materials to which the present invention is applicable are given below, and others will be apparent to a skilled worker in the relevant art. The present invention is particularly applicable to microbes such as bacteria, fungi and yeasts. Where the microbial cells are bacterial cells they can be Gram-negative or Gram- positive bacterial cells.

One group of biological materials to which the present invention is applicable comprises biological materials which are useful as biological agricultural agents for both commercial and garden usage, for example herbicides, fungicides and insecticides and the like. Again such agents are generally in the foπn of microbes such as bacteria, fungi and yeasts. As examples of such cells may be mentioned inter alia Pseudomonas βuorescens, Escherichia coli, Bacillus thuringiensis, a Rhizobium species, a Bradyrhizobium species and rhizosphere-associated bacteria. In a particularly preferred embodiment of the present invention the biological material is Pseudomonas βuorescens identified by deposit numbers NCIMB 40186, 40187, 40188, 40189 or 40190. Among other agricultural agents for both commercial and garden usage there may be mentioned: compost inoculants. agents for nitrogen fixation, silage inoculants. agents, paπicularly bacteria, associated with frost protection and agents, such as the fungi mycorhizi, associated with the Christmas tree growing industry. Other areas of application include agents for use in the food industry, such as food inoculants, for example for yoghurt and the like, and wine inoculants. Another area is environmental applications such as bioremediation. sewage treatment (both industrial and home use) biocontrol and microbial biosensors.

The formulation of the present invention is particularly well adapted to use with Gram negative bacteria such as Pseudomonas species because these bacteria do not form resting spores. Whilst this may mean that no energy is used to form spores during the growth phase of the bacteria, it also means that they are unable to survive desiccation and so formulation presents a technical problem. For this reason, the collapsed matrix formulation of the present invention is paπicularly suited to this type of bacteria. A further advantge of the present invention is that there is evidence for a good speed of pseudomonad reactivation [data?].

The composition of the present invention is also especially useful for biological materials which are intended to combat organisms which utilise available sugar based substrates in the environment. An example of such an organism is the fungus Pythium ultimum. A preferential food source for such organisms are carbohydrates and so conventional glassy matrix formulations which are based on carbohydrates are of little value since the fungus grows at a rate which is faster than the rate at which the biological material destroys or inhibits it. Pseudomonas species such as Pseudomonas βuorescens, for example those strains identified by deposit numbers NCIMB 40186, 40187, 40188, 40189 and 40190 are examples of biological materials used to combat Pythium species.

Furthermore, as already discussed above, there is some evidence that urea has a slight anti-fungal effect. This effect has been noted on the fungus Pythium where it appears that the urea is able to inhibit the growth of hyphae.

Thus, the formulation of the present invention, which is a collapsed matrix formulation in which the matrix is formed of a non-carbohydrate material and which also contains urea, is particularly well suited to use with biological materials such as various species of Pseudomonas. especially the strains of P. βuorescens mentioned above, which are intended to combat organisms such as Pythium uhimum which grow rapidly on available sugars.

Thus, according to a second aspect of the invention, there is provided a composition comprising stabilised celis of Pseudomonas βuorescens in a stasis state suspended in a collapsed matrix of polyvinylpyrrolidone: characterised in that the matrix also incorporates urea.

For compositions containing Pseudomonas βuorescens, it is preferred that the PVP has a molecular weight of from about 25000 to about 60000 and compositions with especially favourable properties have been prepared using PVP with a molecular weight of about 40000. Advantageously sodium ascorbate is included in the composition which may also contain TMAO.

The compositions of the present invention may be prepared by mixing the ingredients but in order for successful results to be obtained it is helpful to add the ingredients in a specified order.

According to a third aspect of the invention there is provided a process for the preparation of a composition comprising a stabilised biological material in a stasis state suspended in a collapsed matrix comprising a polymeric material capable of forming a glassy state and urea, the process comprising mixing the biological material with an aqueous composition comprising the polymeric material and urea and drying the resultant mixture.

Any further ingredients of the composition, such as an antioxidant. will preferably be incorporated into the aqueous composition either before or after the addition of the biological material.

Preferably, the process for the preparation of the composition comprises the steps of : A. mixing the biological material with an aqueous composition comprising at least one material from which the matrix will be derived;

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.

The other components may also be present in the aqueous composition, or at least one component may be added dry, provided that a solubilised or hydrated composition is obtained before the drying process of Step B is carried out. Preferably the composition prepared in Step B is stored at a temperature below the Tg of the matrix, i.e. the composition has a Tg above its anticipated storage temperature. Thus for a composition which is intended to be stored at room temperature, it is preferred that the Tg of the matrix material is from about 40°C to 50°C. Accordingly, the composition prepared in Step B is preferably dried further to increase the Tg of the matrix such that the composition is stabilised to a broader range of storage conditions, i.e. it can be stored at a higher temperature. As discussed above, it appears that the inclusion of urea in the composition of the present invention may lead to an increase in the Tg of the matrix material and so to an increase in the stability of the composition.

The concentration of the biological material in the mixture prepared in Step A of the process according to the present invention is between 10 cfu/ml and lθ'^Jcfu'ml and preferably is between 10"cfu/ml and 10'²cfu/ml.

The biological material for use in the process of the present invention may be grown in conventional growth media, e.g. 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, e.g. liquid or solid plates, to give a desired cell concentration. The cells are isolated, typically by centrirugation. They are resuspended in an aqueous composition comprising the components which will form the matrix and optionally certain other additives as mentioned hereinafter.

Preferably, the biological material used in the process of the present invention is isolated from the growth medium, resuspended in a solution comprising the components of the composition and suitable additives, etc. and dried. However, we do not exclude the possibility that the components of the present invention and suitable additives, etc. are added to the cells in the growth medium and the resulting mixture dried. It will also be appreciated that the polymer and urea can be added together or separately and in any order to the aqueous composition/growth medium.

Where the biological material is resuspended, it is resuspended in a suitable aqueous medium, e.g. aqueous MgS04 solution, or preferably water, containing the components. The drying in Step B of the process according to the present invention may be carried out by, for example, evaporation, freeze drying, spray-drying, air-drying or vacuum-drying. Evaporation, freeze-drying and vacuum drying are preferred as they keep the sample temperature down during drying. Vacuum drying is especially preferred on economic grounds.

It is preferable to achieve \ iscous flow during at least the drying step. Step B. or any subsequent step

The optimum water content of the compositions of the present invention will depend largely on the particular polymer from which the matrix is formed and the storage conditions under which it will be kept In general, the higher the storage temperature required, the lower the water content of the formulation should be. For example for a formulation in which the matrix polymer is PVP which is intended to be stored at room temperature, the water content is preferably below about 15% and more preferably less than about 10%

The ph sical form of the composition according to the present in ention is generally flakes Flakes are easier to handle than conventional freeze dried formulations The formulation of the present invention may also be prepared in other forms such as prills, pellets and cakes

Various further preferred features and embodiments of the present invention will now be described by way of non-limiting example, in which Pseudomonas βuorescens is the biological material, and with reference to the drawings in which

Figure 1 shows the effect of urea on cell iability during a sealed storage study at 40°C.

Figure 2 shows the effect of urea on cell v iability during a sealed storage study at 25°C,

Figure 3 shows the effect of urea on cell viability during a sealed storage study at 5°C;

Figure 4 shows the effect of urea on cell viability during a relative humidity study at 5% relative humidity;

Figure 5 shows the effect of urea on cell iability during a relative humidity study at 15% relative humidity;

Figure 6 shows the effect of urea on cell viability during a relative humidity study at 37% relative humidity; Figure 7 illustrates the effect of altering the urea content of the composition on the immediate viability of material rehydrated immediately following the drying process;

Figure 8 illustrates the effect of altering the sodium ascorbate content of the composition on the immediate viability of the biological material; Figure 9 illustrates the effect of altering the PVP content of the composition on the immediate viability of the biological material;

Figure 10 illustrates the effect of altering the cell loading of the composition on the immediate viability of the biological material;

Figure 11 shows the effect of various formulation ingredients on the growth of hyphae in Pythium ultimum: and

Figure 12 shows the effect of various formulation ingredients on the growth of hyphae in Pythium ultimum over a shorter timescale than in Figure 1 1.

EXAMPLE 1 Preparation of a Composition in accordance with the present invention Pseudomonas βuorescens cells were grown in half strength tryptone soya broth to stationary phase, harvested by centrifugation and the cells resuspended in a solution of urea and sodium ascorbate. The polyvinylpyrrolidone was added as a dry powder and the mixture shaken to hydrate the polymer and mix the suspension. The composition of this mixture was 10¹² cfu. 0.25 ml of water 0.05g of sodium ascorbate. 0.05g of urea and 0.5g of polyvinylpyrrolidone (Molecular weight 40.000 : ex Sigma).

The resultant slurry was vacuum dried to approximately 8% residual moisture, holding drier at ambient temperature, over a 24 hour period.

The resultant viability of the material, on re-hydration, was 60%.

EXAMPLE 2 Effect of Urea Content on Tg

Formulations were prepared as in Example 1 but with varying urea content. The Tg of the formulations was measured and is shown in Table I below.

Tg was measured by differential scanning colorimetry (DSC). Samples were weighed out into aluminium pans and sealed under controlled atmosphere to avoid water adsorption. Thermograms were run at scanning rates of between 5 C/min and 10 C/minute, and the Tg taken as the mid-point of a second order transition in the power/time curve in the first heating ramp.

Table I

Urea PVP Sodium Cfu A ° Drvine Is Ascorbate Moisture Method

0 0.5 0.05 IO'² 4.8 vacuum 14

0 0.5 0.05 IO'² 6 freeze 19

0 0.05 0.05 IO'² 6.6 vacuum 15

0 0.05 0.05 I O¹² 9.5 freeze 21

0.025 0.5 0.05 10'- vacuum 48

0.05 0.5 0.05 10'- vacuum 54

0.1 0.5 0.05 10^1: vacuum 53

0.15 0.5 0.05 I O'² vacuum 54

The results show that the urea has a significant effect on the Tg of the formulation since when even a small amount of urea, for example 0.025g is added to the formulation, the Tg increases from a maximum of 21°C when no urea is present to 48°C.

Example 3 Effect of Urea on Cell Viability during Storage

Formulations were prepared as described in Example 1 with following compositions as shown in Table II:

Table II

This table lists the wet weight of cells in a centrifuge pellet plus dry weight of added ingredients, and estimates % dry weight composition. For example, in Formulation 4. there is 7.2g dry weight urea to 36g wet weight of cells, or l Owt % (based on wet weight total formulation prior to drying). On a dry weight basis theire is therefore 11.4wt % urea. These formulations give about IO¹² cfu per g dry weight, (i.e. based on a g dry weight total formulation which is subsequently re-hydrated in order to measure the viability of the cells before any storage period). This value is shown on the y axis of the graph of Figure 3. This therefore corresponds to 0.1 14g urea per IO¹² cfu. Similarly the amounts of urea in Formulations 2 and 3 are 0.03g and 0.06g of urea per 10¹² cfu.

A. Temperature storage.

The samples were stored in the storage ovens at 40°C and 25°C, and also in a 5°C refrigerator.

B. Controlled Relative Humidity storage.

A device for mixing dry and water saturated air was used to create controllable environments of known relative humidity (RH). Adjustable needle valves control the flow rate of wet and dry air and by measuring the RH in the chambers attached to each separate mixed flow air streams, it is possible to establish a known RH for storage. The formulated samples were placed in open, screw top sample vials in the chambers, and were left exposed to the desired RH for a period of 3 months. Samples were removed from storage, immediately stoppered and viability determined after storage periods of 0, 1 and 3 months. The values of RH used for storage were as follows :

1 5.0 ± 2.3%

2 15.4 ± 1.6%

3 37.3 ± 1.0%

Cell viability measurement

Samples were re-suspended in 10 mis sterile distilled water, shaken on an orbital shaker (230rpm) for 30 minutes. Serial 1 :10 dilutions of these suspensions were plated onto tryptone soya agar. plates incubated at 28°C for 24 hours and colony counts were performed. In Figures 1 -6. the number of viable cells is reported a "viable count" or cfu/g, ie the number of colony forming units per g dry weight of formulated material.

A. Storage as a function of temperature, in Vacuum-packed bags

Samples were prepared as described above split into multiple samples to give discrete samples for each viability count, vacuum-sealed in foil bags and stored at fixed temperature of 5°C, 25°C and 40°C. Bags were opened at specified time intervals, samples re-hydrated and viability measured as described above. The results shown in Figures 1 to 3.

After samples had been stored for 3 months, the samples which had been held at 25 and 40 °C were stored for 19 days at 5°C before being returned to their previous storage temperature, to mimic the effect of samples being used on-farm. and un- opened samples being returned to a refrigerated store before being re-used.

(Therefore, the time intervals marked -*-. ie 4. 5 and 6 months, had been stored for an additional 19 days).

It is clear that without urea (formulation 1) and with larger amounts of urea (formulation 4), the samples degrade when stored at 40°C, losing up to 99% of viable cells over three months.

However, no detectable sample degradation occurs at this temperature over at least a three month period with intermediate urea content samples (formulations 2 and 3).

When samples are cooled (for 19 days) and re-warmed, formulations 1 and 4 continue to degrade over time.

However, the two intermediate urea-content samples, (Formulations 2 and 3) lose some viable cells during the cooling and re-warming stage, but thereafter, do not lose further activity on further storage.

B. Storage in Relative Humidity

Samples were prepared as described above, and subjected to a much harsher storage condition, where samples were removed from the foil bags and left exposed to a continuous flow of humid air. Samples were re-hydrated and viability assessed, as described above. Figures 4, 5 and 6 show the viability as a function of storage time under these conditions at 5%, 15% and 37% relative humidity, at 21°C. It is clear that at all of these humidities the samples with intermediate concentrations of urea (formulation 2 and 3), perform significantly better than samples with no urea (Formulation 1 ) or high levels of urea (Formulation 4).

EXAMPLE 4 Effect of Urea on Cell Viability on Storage

Further formulations were prepared as described in Example 1 except that the urea content was varied. The test formulations contained, respectively, 0.05g urea (as for Example 1), 0.025g urea and no urea. Other ingredients were present in the amounts set out in Example 1. The test formulations were dried either by vacuum or freeze drying as indicated in Table III. The samples were removed from the drier individually and the remaining samples put back under vacuum to prevent moisture uptake. Each sample was divided up and vacuum sealed into bags to give a discrete sample for each viability count. The samples were stored at 25° and 37°C with counts being performed at 37 weeks. In order to test for cell viability, samples were dried for 36 hours in a vacuum drier. After this drying period, the samples were re-suspended in 10ml sterile distilled water by shaking on an orbital shaker, set to 230rpm. for 30 minutes. The suspensions were used in 1 :10 dilution series and plated out onto tryptone soya a ar. The plates were incubated at 28^CC for 24 hours before colony counts were performed. The samples were tested both for immediate cell viability and cell viability after 37 weeks at storage temperatures of 25°C and 40°C. The results are set out in Table II in which E means "exponential". Thus, for example. 7E+1 1 represents 7x10" etc.

Table III

Urea Content (g Initial Viability Viability after 37 Viability after 37 weeks at 25°C weeks at 37°C

0.05 7.20E+1 1 5.2E-+-U 7.8E+9

0.025 6.9E+1 1 3.3E+11 7.2E+9

0 6.3E+1 1 1.8E+1 1 6.9E+8

Table III shows that, although the urea does not have a significant effect on the initial viability of the cells, the effect is considerable after a storage time of 37 weeks. It is particularly significant that the viability at a storage temperature of 37°C increases by a factor of ten when urea is present in the formulation. It may be that this improvement in viability after storage at 37°C arises because, as shown in Example 2. urea increases the Tg of the matrix.

EXAMPLE 5 Effect of Varying the Urea Content on Initial Cell Viability

Formulations were prepared as described in Example 1 except that the amount of urea was varied. The formulations prepared contained 0.025g, O.lg, 0.15g, 0.2g, 0.25g. 0.3g, 0.35g, 0.4g, 0.45g and 0.5g of urea respectively. Amounts of all other ingredients remained as for Example 1 , These formulations were compared with the formulation of Example 1. The samples were vacuum dried for 24 hours, resuspended in sterile distilled water and the cell viability measured using the method set out in Example 4. The effect of altering the urea content is shown in Figure 7 from which it can be seen that all of the compositions exhibited cryo- and lyo-protectant effects.

EXAMPLE 6 Effect of Ascorbate Content on Cell Viability

Formulations were prepared as described in Example 1 except that the amount of sodium ascorbate added to the formulation was varied. The formulations prepared contained 0.01 g. 0.02g, 0.03g, 0.04g, 0.06g, 0.07g. 0.08g. 0.09g and 0.1 g of sodium ascorbate respectively and the viability of the cells after storage was tested as set out in Example 4 and compared with the formulation of Example 1 which contained 0.05 g of sodium ascorbate. The results are shown in Figure 8 from which it can be seen that the effect of ascorbate reaches a plateau at about 0.06g.

EXAMPLE 7 Effect of PVP Content on Cell Viability

Formulations were prepared as described in Example 1 except that the amount of PVP was varied. The formulations prepared contained O.lg, 0.2g, 0.4g, 0.6g, 0.8g and lg and the cell viability (measured by the method set out in Example 3) was compared with the formulation of Example 1 which contained 0.5g PVP. The results are shown in Figure 9 which shows that the cell viability reaches a maximum at a PVP content of about 0,8g. EXAMPLE 8 Effect of Cell Content on Cell Viability

Clearly, it is an advantage to be able to achieve as high a cell loading as possible in the formulations of the present invention. However, cell loading will be limited by the need to have a collapsed matrix around the cells. Formulations were prepared as described in Example 1 except that the cell loading was varied and the immediate cell viability was measured as described in Example 4. The results are shown in Figure 10 which demonstrates that there is a relatively narrow range of cell content over which optimum viability can be achieved

EXAMPLE 9 Effect of Formulation Additives on Pythium ultimum

Treatments were applied at the rates given below to loam compost infested with oospores of P ultimum mixed with 0 3g wheatgerm /I

Treatments mg/trav

Urea 10, 50

PVP 40,000 MW 100, 500

PVP 360,000 MW 100, 500

These treatments were compared with an unprotected control and a healthy control with no pathogen or wheatgerm Pea seeds were planted. The results are shown in Table IV

Table IV

Treatments me/trav Mean Emergence

Urea 10 14.500

Urea 50 14.500

PVP 40,000 MW 100 14.000

PVP 40,000 MW 500 16.500

PVP 360,000 MW 100 15.000

PVP 360,000 MW 500 16.750

Unprotected 13.250

Healthy 23.250 At both the lower and higher concentrations neither the urea nor the PVP had any effect on the pathogen.

EXAMPLE 10 Effect of Formulation Additives on Hyphae of P. ultimum

A hyphal suspension was prepared and sonicated for 45 seconds. The hyphal suspension was prepared by culturing the pathogen Pythium ultimum on 9cm plates of potato dextrose agar at 20°C for 5 days. After this period the plates were covered by a layer of mycelial growth. 10ml of sterile de-ionised water (SDW) was added to each plate in a sterile air flow. The mycelia were then scraped into suspension using a sterile plastic loop. This suspension was then sonicated. Sonicating the hyphae breaks the mycelium into small fragments which do not have hyphal tips, therefore the pathogen must re-form hyphal tips in order to grow. The suspension was added to wells of cell culture dishes at a rate of 0.5ml/well. 0.5ml of the solutions of the formulation additives were also added. There were 8 replica wells for each ingredient with a SDW control, the treatments were:

Formulation additives (mg/2ml SDW):

GABA (γ-amino-butyric acid) 85mg

Urea 40mg

Urea Omg Glucose 200mg

Maltodextrin 30mg

PVP 40,000 MW lOOmg

PVP 360,000 MW lOOmg

The dishes were incubated at 20°C and examined at intervals for growth of the hyphae. Samples were removed from each replicate, stained in lacto phenol cotton blue, mounted in well slides and examined under a microscope at x400 magnification. Samples from the PVP treatments were not stained as they bound the phenol in the stain to form a cloudy suspension. The results for the experiment are set out in Figure 5 from which it can be seen that, initially, the water control, both concentrations of urea, PVP 40,000 MW, and glucose all appeared to be less favourable to pathogen growth than the maltodextrin control which is known to encourage growth in this assay. However, after 9 hours incubation all treatments. except the urea and the PVP 40,000 MW treatments, had encouraged growth of P. ultimum and actively growing hyphae could clearly be seen. This was also true of the water control. Hyphae in the urea and PVP 40.000 MW treatment had not produced many new hyphal tips and were not actively growing, even after 144 hours of incubation.

EXAMPLE 1 1 Effect of Formulation Additives on Short-Term Growth of P. ultimum

The aim of this experiment was to repeat Example 10 over a shorter time period to determine the short term effects on growth of hyphae of P. ultimum. The materials and method were as given above in E.xample 10. Samples were examined at 0 hours. 24 hours, 48 hours and 72 hours of incubation. The results are set out in Figure 12 from which it can be seen that by 24 hours of incubation all compounds except urea and PVP 40.000 MW had increased growth of the hyphae. Actively growing hyphae were visible in all treatments including the water control. By 48 hours all compounds except urea and PVP 40,000 MW treatments had induced active growth of the hyphae. Hyphae in the urea and PVP 40.000 MW treatments had not produced many new tips and were not actively growing, even after 72 hours incubation. At 72 hours the samples were examined for spore morphology.

Ingredient Spore Tvpe y-aminobutyric acid Few spores, some spores are distorted in that they are very (GABA) large and have very few contents

Urea 40mg No spores

Urea lOmg No spores

PVP 40,000 MW No spores

PVP 360,000 MW Some sporangia (formed on the end of a hyphum, a septate cross-wall has formed to separate the hyphum from the spore some have re-germinated to form new hyphae) some oospores (antheridia can be seen below the oogonia), few spores in general

Maltodextrin Some spores with similar distortion to the proline samples, however, some sporangia and possibly some oospores. relatively few spores (compared to the TMAO treatment) Glucose Not many spores, appear distorted as in the GABA treatment

Sterile De-ionisedWater Many spores, all sporangia

Claims

1. A composition comprising a stabilised biological material in a stasis state suspended in a collapsed matrix of a non-carbohydrate polymeric material capable of forming a glassy state: characterised in that the matrix also incorporates urea.

2. A composition as claimed in claim 1 , wherein the cell loading is from about I O¹⁰ to I O¹³ cfu per gramme of composition.

3. A composition as claimed in claim 1 or claim 2. wherein the urea is present in a concentration of from about 0.025g to lg per 10¹² cfu.

4. A composition as claimed in any one of claims 1 to 3, which further includes from about 0.025g to l g per I O¹² cfu of trimethylammonium oxide.

5. A composition as claimed in any one of claims 1 to 4, wherein the matrix material is present in an amount of from about O.lg to l g per 10¹² cfu.

6. A composition as claimed in any one of claims 1 to 5. wherein the matrix material is polyvinylpyrrolidone (PVP).

7. A composition as claimed in any one of claims 1 to 6, further containing one or more additives selected from lyo-protectants, for example polymeric species such as polyvinyl alcohol, polyethylene glycol; anti-oxidants, for example an ascorbate or sodium glutamate; bulking agents, for example crystallising sugars, e.g. mannitol; and osmo-regulants, e.g. betaine, proline, sarcosine.

8. A composition as claimed in claim 7 containing an anti-oxidant in an amount of from about 0.01 to 0.25g per I O¹² cfu. - - ->

9. A composition as claimed in any preceding claim, wherein the biological material is one or more bacteria, fungus or yeast.

10. A composition as claimed in claim 9, wherein the biological material is a Pseudomonas species (such as Pseudomonas fluorescens), Escherichia coli. Bacillus thuringiensis. a Rhizobium species, a Bradyrhizobium species or a rhizosphere- associated bacterium.

11. A composition as claimed in claim 10. wherein the biological material is Pseudomonas fluorescens identified by deposit number NCIMB 40186. 40187,

40188. 40189 or 40190.

12. A composition comprising stabilised cells of Pseudomonas fluorescens in a stasis state suspended in a collapsed matrix of polyvinylpyrrolidone: characterised in that the matrix also incorporates urea.

1 . A composition as claimed in claim 12, wherein the PVP has a molecular weight of from about 25000 to about 60000.

14. A composition as claimed in claim 12 or claim 13 which further contains sodium ascorbate.

15. A composition as claimed in any one of claims 12 to 14 which further includes TMAO.

16. An agricultural, pharmaceutical composition, or a composition for use in environmental control or the food industry comprising the composition of any preceding claim.

17. A process for the preparation of a composition as claimed in any one of claims 1 to 16, the process comprising mixing the biological material with an aqueous -.J

composition comprising the polymeric material and urea and drying the resultant mixture.

18. A process as claimed in claim 17 comprising the steps of : A. mixing the biological material with an aqueous composition comprising at least one material from which the matrix will be derived.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.

19. A process as claimed in claim 18 wherein the composition prepared in Step B is preferably dried further to increase the Tg of the matrix.

20. A process as claimed in claim 18 or claim 19, wherein the concentration of the biological material in the mixture prepared in Step A of the process according to the present invention is between 10 cfu/ml and 10'³cfu/ml.