SK134695A3 - Viable stabilized microbial cells - 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

Storing viable cultures is currently a recognized problem. For example, U.S. Pat. No. 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. Enviromental Microbiology, 1985, pages 108 to 114, describe the use of polysaccharide gels in combination with certain nutrients, for example up to C ₃ and

C _p and C compounds, such as matrices for the freeze-dried bacterial cells. It has been found that gel-forming polysaccharides do not collapse upon lyophilization.

Redway et al., Cryobiology, 1974, 11, 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.

It has been found that if microbial cells are suspended in a particular matrix as described herein and dried under certain conditions, their short-term viability is increased, and if these dried systems are stored and rehydrated under certain conditions defined herein, 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 discloses the preparation of a stable dried composition comprising microbial cells in a stabilized state suspended in a collapsed matrix.

Stable means that the degradation of microbial cells is reduced (degradation would lead to a loss of cell viability recovery).

By the term stabilized state is meant the fact that cells do not metabolize, divide and grow (but these functions can be restored if exposed to appropriate conditions).

By cell viability is meant the ability of cells to grow and divide when exposed to suitable conditions (i.e., rehydration and a source of nutrients).

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

The word collapsed means the fact that

(i) the matrix has shrunk, become less porous and allows only a low penetration of low molecular weight diffusive substances, 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), the plastic flow resulting from a substantial reduction in the surface / volume ratio and encapsulation of the cells in a poorly porous sheath.

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

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

B: drying the mixture under conditions such that there is a flat material flow and a matrix collapse, but not major 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 further drying, so-called secondary drying, in order to increase the Tg of the 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 microbial cells, bacterial cells are used, then gram-negative bacterial cells are preferable, but the use of gram-positive bacteria cannot be excluded. Examples of such gram-negative bacteria include Pseudomonas fluorescens, Escherichia coli and tuberous bacteria.

The concentration of microbial cells in the mixture prepared according to Step A of the invention is in the range of 10 ⁴ / ml to

10 ^{X 3} / ml, preferably in the range of 10 ^x 10 / ml to 10 ^x 10 / ml.

The material that is admixed with the microbial cells according to the process 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 saccharide has been used as the material, it may be a disaccharide, a trisaccharide, an oligosaccharide, or preferably a monosaccharide. As examples of mayaccharides, mention may be made of hexoses, for example ramose, xylose, fructose, glucose, mannose and galactose. Examples of disaccharides are maltose, lactose, trehalose and sucrose. As an example of trisaccharides, mention may be made of raffinose. As examples of oligosaccharides, mention may be made of maltodextrins.

The concentration of the polyhydroxy compound used in the composition of Step A of the invention is between 10 mg / ^10x0 and 1000 mg / ^10x0 cells, preferably between 200 mg / ^10x0 cells and 400 mg / ^10x0 cells. One of ordinary skill in the art will be able to ascertain the optimum concentration of the individual polyhydroxyl compounds used to prepare the collapsed matrix by simple experiment. For example, we have found that the optimal concentration of inositol is about 45 mg / ml, and that inositol at concentrations above 60 mg / ml causes significant cell damage and no matrix collapse at concentrations below about 25 mg / ml.

Some polyhydroxyl compounds exhibit protective properties over a wide concentration range, but 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 according to the invention and are non-limiting examples thereof.

The concentration at which the material used is effective, i.e., collapsing, without damaging the cells too much, is dependent on a number of factors including, but not limited to: the volume fraction of cells 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.

It will be appreciated that the polyhydroxyl compound may function (i) at low temperatures as a cell protection agent against the deleterious effects of freezing temperatures, particularly against damage caused by ice particles during lyophilization; and / or (ii) as a cell protection agent against damage caused by water loss during drying and / or storage; and / or (iii) as a source of nutrition during cell regeneration.

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 phase of their growth, but preferably in an early stationary phase.

For example, the culture may be grown in or on a suitable medium, for example in a liquid medium or on culture medium plates, until the desired cell concentration is reached. 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 then formed, and, on the other hand, optionally other additives as mentioned herein.

Preferably, the microbial cells used in the method of the invention are isolated from the growth medium, suspended in a solution containing the polyhydroxyl compound, suitable additives and so on and dried. However, we do not exclude the possibility that the polyhydroxyl compound and suitable additives, and so on, are added to the cells in the growth medium and the resulting mixture is dried. Where the cells are suspended, they are suspended in a suitable aqueous medium, for example an aqueous solution of MgSO 4 or, more preferably, in water containing the polyhydroxyl compound.

The drying according to step B in the process according to the invention can be carried out, for example, by evaporation, vacuum drying, spray drying, air drying or preferably lyophilization.

During the drying (step B) or in the next step, a plastic flow is to be achieved as stated above.

The water content of the composition dried according to Step B is typically less than 15 weight percent (w / w).

Where the drying according to Step B is carried out as a lyophilization, the composition typically comprises one or more suitable ingredients. Examples of suitable additives include, but are not limited to, cell protection agents from the harmful effects of freezing temperatures, for example sugars or polymeric compounds such as polyvinyl alcohol, polyvinylpyrrolidone; cell protection agents against damage caused by water loss, for example sugars or polymeric compounds such as polyvinyl alcohol, polyethylene glycol; or more preferably, antioxidants or so-called potentiating agents such as ascorbate or glutamate. Nor does it exclude the possibility that other additives may be present, for example so-called magnifying substances, for example crystallizing sugars such as mannitol, and osmotic pressure regulators such as betaine, urea / trimethylamine-N-oxide, proline, sarcosine.

Description of pictures

Figure 1 illustrates as 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 (ie 1E + O9 = 1O ⁹ ppb, 1Ε + 08 = 10 ^θ ppb, and so on) and the horizontal axis represents the concentration of the additive in milligrams per sample, black squares ( ^w ) in the graph correspond to inositol in magnesium sulphate and the rings (o) correspond to inositol in water.

Figures 2 to 7 illustrate, in graphs, the change in viability of lyophilized Pseudomonos fluorescens as a function of monosaccharide concentration for a number of monosaccharides. The vertical axis represents viability in units of number of particles (in millimeters) per billion (that is, 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 monosaccharides used are:

Figure 2

Figure 3

Figure 4 galactose fructose glucose

A picture

A picture

Image xylose ramosis mannose

Figure 8 illustrates as a graph the change in the number of dead cells (kj depending on the Tg matrix and the change in Tg depending on the relative humidity. The left vertical axis represents k _i , 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 number of dead cells (k ^) and the diamonds connected by a dashed line correspond to the glass transition temperature (Tg).

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

It can be seen from Figure 1 that at low concentrations of inositol high cell viability is maintained, but 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 less than 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 substantial increase in the number of dead cells (k 2), demonstrating the importance of maintaining the glass state during storage.

Examples ugWočneriiá. invention

Examples 1 Q

These examples illustrate compositions of the invention in which matrices contain ramose.

Pseudomonas fluoroscens were grown in standard medium (twice concentrated nutrient broth) and isolated by centrifugation in the early stationary growth phase. The cell concentrate was suspended in sterile water and a concentrated autoclave-sterilized solution of ramose was added to a volume sufficient to prepare approximately 200 aliquots, resulting in a concentration of 200 mg of sugar per 2x10 10 cells in a total volume of 4 mL of 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, initially 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 carried out 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 viable bacterial cell count was determined by sequential dilution followed by sowing on King B growth medium. The number of colony forming units (cfu) on the largest dilution plates was used to determine the number of bacterial cells survived lyophilization and storage conditions per unit volume.

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

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

Table 1

Example number Viability (ppb) storage during (weeks) 30 50 1 CTI ND 5X10® 0

ND: not specified

CTI is a comparative test in the absence of ramose.

It can be seen from Table 1 that the presence of the ramose 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 during (days) 13 27 3 2 1 8 x 10 ⁷ 3 X 10 ⁷ 4 X 10 ⁶ 3 4 3 x 10 ⁷ 7 x 10 ⁷ 2 X 10 ⁶ 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 comparative test in the absence of ramose.

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

Examples 7 to 10

These examples illustrate compositions of the invention wherein the matrix comprises ramose and magnesium sulfate.

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

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

The 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 50 100 175 365 7 -20 5 x 10® 5 X 10® 5 X 10® 5 x 10® 8 4 2 x 10® 2 x 10® 1 x 10® 9 15 2 x 10® 1 x 10® 5 x 10® 10 20 2 x 10® 2 x 10®

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

Examples 11-15

These examples illustrate compositions of the invention wherein the matrices comprise sodium ascorbate and sodium glutamate.

The procedure is 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 ramose.

Immediately after lyophilization, cell viability was 2x10 ® ppb.

The samples were stored in humid air at 21 ° C.

Table 4

Example number: relative humidity (% RH) Viability (ppb) Tg storage during (days) ° C 13 27 11 0 1 x 10® 1 X 10® 25 12 14 1 X 10® 1 X 10® 24 13 30 1 X 10® 4 x 10 ⁷ 14 14 40 9 X 10 ⁶ 5 X 10® 12 15 53 3 X 10 ⁷ 3 X 10 ³ 8

From Examples 11 and 12 and Table 4, it can be seen that storage of dried cells at temperatures below the Tg matrix stabilizes the cells for a long period of time.

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

Example 16 grams of Pseudomonas fluorescens pellet containing 5.10 ¹² viable cells were separated by centrifugation from the growth medium and mixed with 14 grams of commercial grade maltodextrin (Glucidex IT19) and 1.6 grams of sodium ascorbate, and this material was dried under reduced pressure at room temperature first. while the evaporated water was condensed in a freezer pocket where the temperature was maintained at -50 ° C.

Drying was terminated 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 original viable cells.

The materials prepared in this way appear to be collapsed amorphous matrices with a glass transition greater than 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 process for the preparation of 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 where the matrix mass viscous flow occurs so as to cause the matrix to collapse without causing too much damage to the cells. The method of claim 3, wherein the composition prepared in step B is further dried to achieve an elevated glass transition temperature of the matrix mass, thereby stabilizing the composition over a wider range of storage conditions. Method according to claim 3, characterized in that the concentration of microbial cells in the mixture prepared in step A is between 10 ⁴ / ml and 10 ^{X 3} / ml. Method according to claim 5, characterized in that the concentration of microbial cells A is in the range between ^10x0 / ml and ^10xx / ml. Method according to claim 3, characterized in that the mass which 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 concentration of the polyhydroxyl compound used in the composition of Step A is between 10 mg / 10 ^xo cells and 1000 mg / 10 ^xo cells. The method of claim 9, wherein the concentration of the polyhydroxyl compound is in the range between 200 mg / 10 ^x0 cells and 400 mg / 10 ^x0 cells. Composition according to claim 3, characterized in that its glass transition temperature is higher than the expected storage temperature.