CZ280595A3 - Viable bacterium - Google Patents

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.

Description

The invention provides a process for preparing compositions containing microbial cells in a stabilized state, such compositions and live cultures which are subsequently prepared therefrom.

BACKGROUND OF THE INVENTION.

Storing viable cultures is currently a recognized problem. For example, US 3,897,307 describes (i) the use of a combination of ascorbate and glutamate or aspartate as stabilizers for lactic acid producing bacterial cells and (ii) the use of certain sugars, particularly inositol at 25 mg / ml solution, as cell protection agents the harmful effects of freezing temperatures wherever these bacterial cells are lyophilized.

Mugnier et al., Applied and Environmental Microbiology, 1985, pages 108-114, describe the use of polysaccharide gels in combination with certain nutrients, for example, C 1 -C ₃ and C ₆ -C ₁₂ compounds, as matrices for lyophilized bacterial cells. It has been found that gel-forming polysaccharides do not collapse upon lyophilization.

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

We have found that when microbial cells are suspended in a particular matrix as described herein and dried under certain conditions, their short-term viability is increased and that these dried systems are stored and rehydrated under certain conditions here

The long-term viability of these microbial cells is also increased.

Furthermore, it has surprisingly been found that the collapse of the matrix in which microbial cells are suspended does not result in low short-term viability.

The invention describes the preparation of a stable, dried composition comprising microbial cells in a stabilized state suspended in a collapsed matrix.

Stable is understood to mean that degradation of microbial cells is reduced (degradation would lead to loss of cell viability recovery).

The term stabilized state refers to the fact that cells do not metabolize, divide and grow (but these functions can be restored when exposed to appropriate conditions).

"Cell viability" refers to the ability of cells to grow and divide when exposed to appropriate conditions (i.e., rehydration and a source of nutrients).

By viable cell is meant cells capable of growing and dividing when subjected to suitable conditions (i.e., rehydration and a source of nutrients).

The word 'collapsed' means the fact that:

(i) the matrix has shrunk, has become less porous and allows only low penetration of low molecular weight diffusers, for example, absorbs only a small amount of water vapor when exposed to humid air; and / or ii) the matrix has been exposed to temperatures in excess of its glass transition temperature (Tg), wherein

3plastic flux caused a significant reduction in the surface / volume ratio and the encapsulation of the cells in a poorly porous sheath.

The invention further provides a process for preparing a stable dried composition comprising microbial cells in a stabilized state suspended in a matrix, comprising the following steps:

A: mixing the microbial cells with an aqueous medium containing material from which the matrix will be derived

B: drying the mixture under conditions such that plastic flow of the material occurs and the matrix collapses, but not excessive cell damage.

The composition prepared according to Step B is preferably stored at temperatures lower than the Tg of the matrix, i.e. the composition has a Tg higher than the expected storage temperature.

In the composition prepared according to step B, it is advantageous to carry out a further drying, the so-called secondary drying, in order to increase the Tg matrix so that the composition is stable over a wide range of storage conditions, i.e., to be stored at a higher temperature.

The cells contained in the stable dried composition of the invention are preferably bacterial cells, but the possibility of using other types of microbial cells, such as fungi, yeasts and so on, cannot be excluded. If the microbial cells used are bacterial cells, gram-negative bacterial cells are preferable, although the use of gram-positive bacteria cannot be excluded. Examples of such gram-negative bacteria include Pseudomonas fluorescens, Escherichia coli and tuberous bacteria.

The microbial cell concentration in the composition prepared according to Step A of the invention is in the range of 10 ⁴ / ml to 10 ^{: 3} / ml, preferably in the range of 10 ¹⁰ / ml to 10 ^µ / ml.

The material that is admixed with the microbial cells by the method of step A of the invention is any polyhydroxyl compound, for example a polyalcohol such as mannitol, inositol, sorbitol, galacitol, or more preferably a sugar and in particular a carbohydrate.

Where a carbohydrate is used as the material, it may be a disaccharide, a trisaccharide, an oligosaccharide, or preferably a monosaccharide. As examples of monosaccharides, mention may be made of hexoses, for example rhamose, xvlose, fructose, glucose, mannose and galactose. Examples of disaccharides include maltose, lactose, trehalose and sucrose. As an example of trisaccharides we can mention raffinose. As an example of oligosaccharides we may mention maltodextrins.

The concentration of polyhydroxyl compound used in the composition in Step A of the invention is between 10 mg / 10 ¹⁰ and 1000 mg / 10 ¹⁰ cells, but preferably between 200 mg / 10 ¹⁰ cells to 400 mg / 10 ¹⁰ cells cannot be excluded. One of ordinary skill in the art will be able to ascertain the optimum concentrations of the individual polyhydroxyl compounds used to prepare the collapsed matrix by a simple experiment. For example, we have found that the optimal concentration of inositol is around 45 mg / ml and that inositol at concentrations above 60 mg / ml causes considerable cell damage and no matrix collapse at concentrations below about 25 mg / ml.

Some polyhydroxyl compounds exhibit protective properties over a wide concentration range, while others have a detrimental effect at concentrations higher than the critical concentration which appears to be related to the solubility of the polyhydroxyl compound in aqueous solution.

The invention is further illustrated by the accompanying drawings which show the compositions of the invention and are non-limiting examples thereof.

Description of the picture

Figure 1 illustrates in a graph the change in viability of Pseudomonas fluorescens as a function of the concentration of (added) inositol when lyophilized from an aqueous solution of inositol or a solution of inositol in 0.04M MgSO 4. The vertical axis represents viability in parts per billion (i.e. 1Ε + 09 = 10 ^θ ppb, 1E + O8 = 10 ⁸ ppb, and so on) and the horizontal axis represents the concentration of the additive in milligrams per sample. The black squares () in the graph correspond to inositol in magnesium sulfate and the circles (O) correspond to inositol in water.

Figures 2 to 7 illustrate, in graphs, the change in viability of lyophilized Pseudomonas fluorescens as a function of monosaccharide concentration for a number of monosaccharides. The vertical axis represents viability in units of number of particles (in billions) per billion (ie 1E + O = 1 billion, 5E-l = 0.5 billion, 2E-l = 0.2 billion, and so on) and the horizontal axis represents concentration sugar in milligrams per sample. The monosacnarides used are:

Figure 2

Figure 3

Figure 4 galactose fructose glucose

Picture

Picture

Image of xylosa rhamosa manosa

Figure 8 illustrates, as a graph, the change in dead cell frequency (kj depending on Tg matrix and change in Tg depending on relative humidity. The left vertical axis represents k _x , the right vertical axis represents Tg ('C) and the horizontal axis represents relative humidity (%) The black squares () connected by a solid line correspond to the frequency of dead cells (k,) and diamonds

-6g glass connected by the dashed line corresponds to the transition temperature (Tg).

In the figures, viability is expressed as the number of viable bacteria per 10 ⁹ viable bacteria in the original suspension, i.e., the number of surviving bacteria from each billion originally viable bacteria. Viability is expressed in parts per billion (ppb), i.e. 10 ⁹ ppb corresponds to 100% survival, 10 ³ corresponds to 10% survival, and so on.

It can be seen from Figure 1 that at low concentrations of inositol, high cell viability is maintained, whereas at higher concentrations, for example greater than 100 mg / sample (corresponding to 50 mg / ml), the number of viable cells decreases rapidly with increasing concentration.

It can be seen from Figures 2 to 7 that some monosaccharides protect cells from damage at low concentrations, for example below 10 mg / sample (corresponding to 5 mg / ml), and that this protective effect is essentially maintained even at higher concentrations, e.g. about 400 mg / sample (corresponding to 130 mg / ml).

From Figure 8, it can be seen that where Tg falls below the storage temperature (represented by the upper horizontal line at 21-22 ° C), there is a significant increase in dead cell count (k1), demonstrating the importance of maintaining the glass state during storage.

The concentration at which the material used is effective, i.e., collapses without unduly damaging the cells, is dependent on a number of factors including, but not limited to: the volume of the cell fraction in suspension in Step A; the intrinsic glass transition temperature of the polyhydroxyl compound; changing the glass transition temperature of the matrix as a function of the water concentration present therein; the temperature to which the matrix is exposed during and after lyophilization.

that the polyhydroxyl compound

It is necessary to realize when

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The microbial cells used in the method of the invention may be grown in conventional growth media, for example, in nutrient broth or tryptic soy broth. These cells may be harvested during any suitable growth phase, such as the cells.

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For example, the culture may be grown in or on a suitable medium, for example, kennel medium or non-culture medium with nutrient medium until the desired cell concentration is reached. The cells are usually isolated by centrifugation. Further, they are suspended in an aqueous medium which contains, on the one hand, the material from which the matrix is formed, and, on the other hand, optionally other additives as mentioned herein.

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The drying of step B in the methods of the invention may

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plastic flow as said

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The invention is further illustrated by the following examples.

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These examples illustrate compositions of the invention wherein the matrix comprises rhamose.

Pseudomonas fluoroscens were grown in standard medium (twice-concentrated nutrient broth) and isolated by centrifugation in the early stationary phase of growth. Cellular

The concentrate was suspended in sterile water and a concentrated autoclave sterilized rhamose solution was added in a volume sufficient to prepare approximately 200 aliquots to a final concentration of 200 mg sugar per 2 x 10 6 cells in a total volume of 4 mL water. Aliquots were placed in 5 ml lyophilization vials.

The vials were placed in the freeze-dryer chambers and the temperature in the chambers was set to -30 ° C, first freezing the contents of the vials and then dropping the temperature of the contents to -28 ° C to -30 ° C over two hours. High vacuum was then applied and the primary drying was carried out for a total of 48 hours. The temperature in the chambers was raised to 0 ° C and the secondary drying was performed for a total of 24 hours. Finally, the vials were brought to room temperature and sealed under vacuum before being removed from the lyophilizer.

The vials were stored at 4 ° C under vacuum (Example 1) or in humidity controlled air at 21 ° C (Examples 2 to 6).

The samples were rehydrated in sterile water and the number of viable bacterial cells was determined by a stepwise dilution method followed by sowing on King with B growth medium. The number of cells capable of forming colony forming units (cfu) on the largest dilution plates was used to determine the number of bacterial cells that survived lyophilization and storage conditions per unit volume.

Immediately after lyophilization, cell viability was 3x10 ^and ppb.

The results obtained using bacteria stored at 4 ° C under vacuum are shown in Table 1.

-10Table 1

Example number Viability (ppb) storage for (weeks) 30 50 1 ND 5x10 ⁸ CTI 0 -

ND: Not determined

CTI is a comparative test in the absence of rhamosis.

It can be seen from Table 1 that the presence of the rhamose matrix significantly improved the viability of the bacteria.

The results obtained using bacteria stored at 21 ° C in controlled humidity chambers are shown in Table 2.

Table 2

Example number: Relative humidity (RH%) Viability (ppb) storage for (days) 13 27 Mar: 3 2 1 8 x 10 ⁷ 3 x 10 ⁷ 4 x 10 ⁷ 3 4 3 x 10 ⁷ 7 x 10 ⁷ 2 x 10 ⁷ 4 9 9 x 10 ⁷ 5 x 10 ⁷ 4 x 10 ⁷ 5 23 4 x 10 ⁷ 4 x 10 ⁷ 8 x 10 ⁷ 6 44 3 x 10 ⁷ 1 x 10 ⁷ 3 x 10 ⁷ CT2 1 10 ³

CT2 is a rhamose-free comparative test.

It can be seen from Table 2 that the presence of sugar significantly improved the viability of the bacteria even at low relative humidity, as can be seen from the comparison of Example 2 with CT2.

-11Examples 7 to 10

These examples illustrate compositions of the invention in which the matrix comprises rhamose and magnesium sulfate.

The procedure was the same as in Examples 1 to 6, except that the cell concentrate was suspended in 0.04 M magnesium sulfate instead of sterile water before the rhamose solution was added.

Immediately after lyophilization, cell viability was 5x10 ³ ppb.

Lyophilized cells were stored at different temperatures and for different periods of time as shown in Table 3.

Table 3

Example number: Temperature storage Viability (ppb) by days 50 100 ALIGN! 175 365 7 -20 5 χ 10 ^a 5 χ 10 ³ 5 x 10® 5 x 10® 8 4 2 χ 10 ⁸ 2 χ 10 ⁸ 1 χ 10® 9 15 Dec 2 χ 10 ⁸ 1 x 10® 5 x 10® 10 20 May 2 χ 10 ³ 2 x 10®

It can be seen from Table 3 that these mixtures provide substantial protection over the entire temperature range.

Examples 11 to 15

These examples illustrate compositions of the invention wherein the matrix comprises sodium ascorbate and sodium glutamate.

The procedure was the same as in Examples 1 to 6 except that aqueous solutions of sodium ascorbate and sodium glutamate were added to the cell concentrate and rhamose.

-12 Immediately after lyorrlisation, cell viability was 2x10 ® ppb.

The samples were stored in humid air at temperature

Deň: 18 ° C.

Table 4

Example Relative Viability (ppb) Tg number: humidity storage for (days) (RH%) ° c 34 128 11 0 1 x 10 ³ 1 x 10 ⁸ 25 12 14 1 x 10 ³ 1 x 10 ⁸ 24 13 30 1 x 10 ³ 4 x 10 ⁷ 14 14 40 9 x 10 ' 5 x 10 ^sec 12 15 Dec 53 3 x 10 ⁷ 3 x 10 ³ 8

It can be seen from Examples 11 and 12 and Table 4 that storage of dried cells at temperatures below the Tg matrix stabilizes the cells over a long period.

The results indicate that the combination of the collapsed matrix in glassy state and the presence of antioxidants provide a matrix that can maintain viable cells for a long time, even under relatively unfavorable conditions.

Example 16 grams of a Pseudomonas fluorescens pellet containing 5 x 10 ¹² viable cells were centrifuged to separate from the growth medium and mixed with 14 grams of commercial grade maltodextrin (Glucidex IT19) and 1.6 grams of sodium ascorbate and dried under reduced pressure at room temperature first the evaporated water was condensed in a freezer pocket where the temperature was maintained at -50 ° C.

The drying was completed after approximately 18 hours when the vacuum value was of the order of 1 mbar.

The samples were rehydrated in pure water and plated on nutrient agar, which usually showed 50-90% survival of the originally viable cells.

The materials prepared in this way appear to be collapsed amorphous matrices with a glass transition above 20 ° C (if the samples were exposed to standard laboratory relative humidity).

Claims (11)

What is claimed is: 1. A stabilized, dried composition comprising microbial cells in a stabilized state suspended in a collapsed matrix, wherein the microbial cells are gram-negative bacteria. Composition according to claim 1, characterized in that the gram-negative cells are Pseudomonas fluorescens, Escherichia coli or tuberous bacteria. A method for preparing a stabilized, dried composition comprising microbial cells, characterized in that the microbial cells are gram-negative bacteria in a stabilized state suspended in a matrix, the method comprising the steps of: A: mixing the microbial cells with an aqueous solution containing the matrix from which the matrix is derived B: drying the mixture under conditions whereby the viscous flow of the matrix mass occurs such that the matrix collapses and the cells are not too damaged. The method of claim 3, wherein the composition prepared in step B is further dried to achieve an increase in the glass transition temperature of the matrix mass, thereby stabilizing the composition over a wider range of storage conditions. The method of claim 3, wherein the concentration of microbial cells in the composition prepared in Step A is between 10 ⁴ / ml and 10 ¹³ / ml. -15 £ ^UBS rmjTE ~ sm ^ The method of claim 5, wherein the concentration of microbial cells A is in the range between 10 ¹⁰ / ml and 10 ^µ / ml. The method of claim 3, wherein the mass that is mixed with the bacterial cells in step A is a polyhydroxyl compound. The method of claim 7, wherein the polyhydroxyl compound is a monosaccharide. The method of claim 7, wherein the concentrations used in the mixture of 10 mg / 10 ¹⁰ cells and 1000 mg / 10 cells. wherein the polynydroxyl compound of Step A is in the range of between The method of claim 9, wherein the concentration of the polyhydroxy compound is between 200 mg / 10 ¹⁰ cells and 400 mg / 10 ¹⁰ cells. ¹¹ Composition according to Claim 3, characterized in that its glass transition temperature is higher than the expected storage temperature.