HUT72846A - Viable bacteria - Google Patents

Description

The present invention relates to a process for the preparation of compositions containing dried microbial cells in a stasis state. The present invention further relates to these compositions and to live cultures prepared therefrom.

A well-known problem in the literature is the storage of viable cultures, which has been attempted several times. For example, U.S. Pat. No. 3,897,307. (i) discloses the use of an ascorbate compound and a glutamate or asparaginate for stabilizing lactic acid-producing bacterial cells; .

Mugnier et al., Applied and Environmental Microbiology 108-114. (1985) describe the use of a combination of polysaccharide gels and various nutrients, such as C 1-3 and C 6-12 compounds, as a matrix of freeze-dried bacterial cells. It has been found that the gel-forming polysaccharides do not collapse during freeze-drying.

Redway et al., Cryobiology 11, 73-79. (1974)] investigated the utility of certain monosaccharides (up to 150 mg / 2 mL sample concentration) and similar compounds as agents for long-term survival of freeze-dried bacteria.

It has been found that if the microbial cells are resuspended in a matrix to be described below and the suspension is dried under defined conditions, the short-term viability of the cells will be improved. We have also found that if you know these dried systems later • ·

- rehydrate under 3 conditions, improving long-term viability of microbial cells.

Furthermore, it has surprisingly been found that the collapse of the matrix used to suspend the microbial cells does not result in a deterioration of short-term viability.

Accordingly, the present invention relates to stabilized dried compositions comprising stasis microbial cells suspended in a collapsed matrix.

By stabilized designation is meant to reduce the degradation of microbial cells resulting in a decrease in recoverable viable cell count.

By stasis state is meant that cells are at rest, that is, they do not metabolize, divide or grow, but can be regenerated by proper treatment.

By regenerative label is meant cells that, when placed in appropriate conditions (i.e., rehydrated and supplied with nutrients), are able to grow and divide.

Viable cells are those cells which, when placed in appropriate conditions (i.e., rehydrated and supplied with nutrients), are capable of growing and dividing.

By collapsed designation, it is understood that (i) the matrix shrinks and becomes so porous that it allows only a small amount of low molecular weight diffusible material to penetrate (e.g., · · · · · · · · · · · · · · * · ··································································

- absorbs only a small amount of water vapor when exposed to 4 moist air); and / or (ii) the matrix was subjected to a temperature higher than the liquid / glass transition temperature (Tg) such that it became a viscous liquid which significantly reduced the specific surface area / volume ratio of the matrix and surrounded the cells as a low porosity barrier.

The present invention further relates to a process for the preparation of a stabilized dried composition comprising a stasis state of microbial cells suspended in a matrix by (a) mixing the microbial cells with an aqueous composition containing the matrix forming material, and (b) viscous flow of the material and However, the cells are dried under conditions that are not significantly damaging.

The composition obtained in step (b) is preferably stored at a temperature lower than the Tg of the matrix, i.e. preferably a composition having a Tg greater than the intended storage temperature of the composition.

Thus, preferably, the composition obtained in step (b) is further dried, i.e. subjected to secondary drying, in order to increase the storage conditions of the composition by increasing the Tg value of the matrix, i.e. making the composition stable at higher temperatures.

Preferably, the stabilized dried compositions of the invention comprise bacterial cells as microbial cell barriers. However, in this process, other microbial cells, such as fungal cells, yeast cells, etc., are used. can also be used. THE

bacterial cells are preferably Gram-negative bacterial cells, although the applicability of Gram-positive bacterial cells is not excluded. Examples of gram-negative bacterial cells include Pseudomonas fluorescens and Escherichia coli and rhizosphere-associated bacterial cells.

Mixtures formed step of the process according to the invention (a) the microbial cells from 10 ⁴ / ml and 10 ¹³ / ml, preferably 10 ^lo / ml and 10 ^{i may} contain a concentration between ^L / mL of j.

In step (a) of the process of the invention, the material to be mixed with the microbial cells is a polyhydroxy compound such as a polyol such as mannitol, inositol, sorbitol, galactite, or preferably a carbohydrate, particularly preferably a saccharide.

The saccharides useful in the process of the present invention may be disaccharides, trisaccharides, oligosaccharides, or preferably monosaccharides. Exemplary monosaccharides include hexoses such as rhamnose, xylose, fructose, glucose, mannose and galactose. Examples of disaccharides are maltose, lactose, trehalose and sucrose (sucrose). Exemplary trisaccharides include raffinose, and oligosaccharides include maltodextrins.

The mixture formed in step (a) of the process of the invention may contain the polyhydroxy compound in a concentration of 10 mg / 10 ¹⁰ cells to 1000 mg / 10 ¹⁰ cells, preferably 200 mg / 10 ¹⁰ cells to 400 mg / 10 ¹⁰ cells. One of ordinary skill in the art can easily determine the concentration limits that can be used to form a collapsed matrix of a given polyhydroxy compound. For example, it has been found that the optimum concentration of inositol for inositol is about 45 mg / ml; inositol at concentrations above 60 mg / ml already causes severe cell damage, whereas when inositol is used at concentrations below 25 mg / ml, the resulting matrix does not collapse.

Some representatives of polyhydroxy compounds also exhibit a wide range of protective activities, but other polysaccharides already exhibit adverse effects above a critical concentration range (which appears to be related to the solubility of the particular polyhydroxy compound in aqueous media).

The invention will now be described in more detail with reference to the accompanying drawings. The drawings illustrate various characteristics of some exemplary representatives of the compositions of the invention.

Figure 1 is a graph showing the change in viability of Pseudomonas fluorescens cells in freeze-dried water or 0.04 M MgSO ₄ solution as a function of the concentration of inositol added. On the vertical axis, viability is raised9 in billions of units (i.e. 1E + 9 for 10 ppb,

1E + 8 for 10 ppb etc. ) and the horizontal axis is the amount of additive in mg / sample units. The points marked with dark squares are the values measured using the inositol / magnesium sulfate mixture, while ·· · ·

light circles correspond to the values measured using the inositol / water mixture.

2-7. The graphs shown in Figures 1 to 5 show the viability of freeze-dried Pseudomonas fluorescens cells in the presence of various monosaccharides as a function of the concentration of that monosaccharide. The viability of the vertical axes is expressed in parts per billion (i.e. 1E + 0 1 trillion, 5E-1 0.5 trillion, 2E-1 0.2 trillion, etc.), while the horizontal axes are expressed as mg / mg in sample units. In each figure, the following experiments with the following monosaccharides were performed.

results illustrated: 2. figure: galactose 3. figure: fructose 4. figure: glucose 5. figure: xylose 6th figure: rhamnose 7th figure: mannose

The graph in Figure 8 illustrates the change in cell death rate (k _x ) versus the Tg value of the matrix and the change in Tg value as a function of relative humidity. Ki values were recorded for the left vertical axis, Tg values for the right vertical axis (° C), and relative humidity (%) for the horizontal axis. In the graph, the dashed line connecting the dark squares represents the change in the rate constant (k ^) of cell death, and the dashed line connecting the light squares shows the change in Tg.

In the drawings, viability is expressed as the ratio of viable bacteria to the 10 bacteria in the initial suspension; therefore, viability refers to the number of bacteria that have survived the treatment, out of a million initially viable bacteria. Viability was expressed in parts per billion (ppb), so the 10 ⁹ ppb indicated for viability was 100% survival, the 10 ⁸ ppb value was 10% survival, and so on. means.

As can be seen from Figure 1, cell viability is maintained at low inositol concentrations, but at higher inositol concentrations (e.g., greater than 100 mg / sample), cell viability is rapidly reduced.

2-7. Figures 1 to 4 show that some monosaccharides protect cells from damage at low concentrations (e.g. less than 10 mg / sample (= 5 mg / ml)) and this protective effect is high [e.g. about 400 mg / sample (= 130 mg / ml)] monosaccharide concentrations are essentially maintained.

It can be seen from Figure 8 that when the Tg value falls below the storage temperature (20-21 ° C according to the upper horizontal line), the cell proliferation rate (k _T ) increases significantly. This fact indicates the importance of maintaining the glassy state during storage.

The concentration at which the matrix forming agent is active, i.e., the matrix formed from it, but no significant cell damage has yet occurred, is due to several factors, including the volume ratio of cells in suspension in step (a) · ♦ ·· ··

9, depends on the apparent liquid / glass transition temperature of the polyhydroxy compound, the change in the liquid / glass transition temperature of the matrix as a function of the water content of the matrix, and the temperature to which the matrix is exposed during and after freeze-drying.

It will be appreciated that the polyhydroxy compound (i) may serve as a cryoprotective agent at low temperatures, in particular to prevent damage caused by ice particles formed during freeze drying; and / or (ii) serve as a lio-protective agent to prevent damage due to water loss during drying and / or storage, and / or (iii) serve as a nutrient source for cell regeneration.

Microbial cells useful in the method of the invention may be cultured in conventional culture media, such as medium or tryptone / soy medium. The cells may be isolated at any suitable stage of growth, preferably in the early stationary stage.

In an exemplary embodiment, the cell culture is cultured in a suitable medium / medium (e.g., liquid or solid plates) until the desired cell concentration is reached. The cells are harvested, typically by centrifugation, and resuspended in aqueous media containing the matrix material and optionally other additives (s) to be listed below.

In a preferred embodiment, the cells to be used in the method of the invention are separated from the culture medium and the cells are again reconstituted in a solution containing the polyhydroxy compound and optionally suitable additives.

slurry and dry the slurry. However, it is also possible to add the polyhydroxy compound and any additives to the cells in the culture medium and freeze-dry the resulting mixture.

If the microbial cells are resuspended, a suitable aqueous medium containing the polyhydroxy compound, such as an aqueous magnesium sulfate solution or, preferably, water, is used as the resuspending medium.

The drying according to step (b) can be carried out, for example, by evaporation, vacuum drying, spray drying, air drying or, preferably, freeze drying.

As stated above, it is an essential feature of the present invention to achieve a viscous liquid state, at least during the drying step (b) or any subsequent step.

The dried compositions of step (b) typically have a water content of less than 15% by weight.

When freeze-drying is used for the drying in step (b), one or more suitable additives are usually added to the composition. Suitable additives include cryoprotective agents such as sugars or polymers (e.g. polyvinyl alcohol, polyvinylpyrrolidone); lyoprotective agents such as sugars or polymers (such as polyvinyl alcohol, poly / ethylene glycol); or preferably, antioxidants or so-called potentiators such as ascorbates or glutamates. Other additives may also be present in the compositions, of which ···· ··· ··· «··

Examples include so-called bulking agents such as crystallising sugars (e.g., mannitol) and osmosis regulating agents (e.g., betaine, urea / trimethylamine N-oxide, proline and sarcosine).

Further details of the invention are illustrated by the following examples.

1-6. example

Preparation of a composition comprising a Ramnose based matrix

Pseudomonas fluorescens cells were grown on standard medium (double concentration medium) and the cells were separated by centrifugation in the early stationary phase. The cell concentrate was resuspended in sterile water and concentrated aliquots of concentrated rhamnose solution pre-sterilized by autoclaving were added. The final suspension contained 200 mg of sugar per 2x10 ¹⁰ cells. The suspension was filled into 5 ml vials for freeze-drying containing 4 to 4 ml.

The vials were placed in freeze-drying adjustable tray trays, and the trays were frozen to a final temperature of -30 ° C and frozen within 2 hours to -28 ° C to -30 ° C. The vacuum extraction was then turned on and the primary drying continued for 48 hours. Subsequently, the plates were raised to 0 ° C for 24 hours and secondary dried. The vials were left at room temperature

and the vials were sealed under reduced pressure before being removed from the freeze dryer.

The vials in Example 1 were pressurized under reduced pressure at 4 ° C. and, in the case of Example 1, it was stored in a controlled humidity atmosphere at 21 ° C.

Samples were rehydrated with sterile water and, after serial serial dilution in water, plated on King's B medium to determine the number of viable bacterial cells. The number of bacterial cells that survived the freeze-drying and storage conditions was calculated from the number of colony forming units (tU) obtained on the highest dilution culture plate.

Cell viability immediately after freeze-drying was 3x10 ppb.

The results of testing the samples stored under reduced pressure at 4 ° C are shown in Table 1.

Table 1 The example Viability, ppb number After 3 weeks of storage After 50 weeks of storage 1. nv 5x10 ⁸ KV1 0 -

nv = not tested

KV1 = Comparative study without rhamnose

Table 1 shows that the presence of rhamnose matrix greatly increases bacterial viability.

·· *; ···; ·. ..

• · _Λ · · ·· · ·· -. * ·· ·· .. · · ·· ···. .,

The results of the analysis of samples stored at -13A in a controlled humidity atmosphere at 21 ° C are shown in Table 2.

Table 2

The number of the example Relative Humidity,% Viability, ppb 13 days 27 days storage u 34 hrs sunny n 2. 1 8x10 ⁷ 3x10 ⁷ 4x10 ⁷ Third 4 3x10 ⁷ 7x10 ⁷ 2x10 ⁷ 4th 9 9x10 ⁷ 5x10 ⁷ 4x10 ⁷ 5th 23 4x10 ⁷ 4x10 ⁷ 8x10® 6th 44 3x10 ⁷ lx10 ⁷ 3x10 ⁵ KV2 1 10 ³

KV2 = comparative study without rhamnose

Table 2 shows that the presence of sugar significantly increases cell viability even at low relative humidity (cf. Example 2 and KV2 experiment data).

7-10. example

Preparation of compositions containing rhamnose and magnesium sulfate in matrix

1-6 were repeated. except that the cell concentrate was resuspended in 0.04 M aqueous magnesium sulfate instead of sterile water and the rhamnose solution was added.

Cell viability was 5xl0 ⁸ immediately after freeze-drying.

Freeze-dried cells were stored at the temperatures indicated in Table 3 for the time indicated in Table 3. Measured viability data are reported in Table 3.

Table 3

For example - Storage - Viability, ppb da temperature - 50 days 100 days 175 days 365 days after storage, ° C

7th -20 5x10 ⁸ 5x10 ⁸ 5x10 ⁸ 5x10 8th 4 2x10 ⁸ 2x10 ⁸ lx10 ⁸ 9th 15 2x10 ⁸ lx10 ⁸ 5x10 ⁷ 10th 20 2x10® 2x10 ⁷

It can be seen from the data in Table 3 that the invention provides significant protection for bacteria over a wide temperature range over a long period of time.

11-15. example

Preparation of Compositions Containing Sodium Ascorbate and Sodium Glutamate in a Matrix

1-6. except that a concentrated aqueous solution of sodium ascorbate and sodium glutamate was added to the mixture of cells suspended in water and rhamnose.

Cell viability is directly determined by frozen θ

after drying it was 2x10 ppb.

Samples were stored in a humidified atmosphere at 21 ° C and periodically tested for bacterial viability. The results are shown in Table 4.

* ··· ·· *

Table 4

The mouse Relative Numbers Viability, ppb da vességtar- 34 days 128 days Tg, C number % of volume after storage

11th 0 lx10 ⁸ lx10 ⁸ 25 12th 14 IxlO ⁸ lx10 ⁸ 24 13th 30 lx10 ⁸ 4x10 ⁷ 14 14th 40 9x10 ⁷ 5x10 ⁶ 12 15th 53 3x10 ⁷ 3x10 ³ 8

From the data described above in Examples 11 and 12, it can be seen that when the dried cells are stored below the Tg of the matrix, the cells are stabilized for a long time.

The results also confirm that by using a glassy, collapsed matrix and incorporating antioxidant, we obtain a matrix that is capable of stabilizing viable cells over a long period of time, even in relatively aggressive environments.

Example 16

Pseudomonas fluorescens cell pellets (4 g), containing ⁵ x ^{10 11} viable cells, separated by centrifugation, were mixed with 14 g of commercial grade maltodextrin (Glucidex IT19) and 1.6 g of sodium ascorbate, and initially dried at room temperature. The evaporated water was condensed in a cooling trap at -50 ° C.

Drying was completed after about 18 hours. At this time, the pressure was about 1 mbar.

Samples of the resulting material were rehydrated with clear water and distributed on nutrient plates. Typically, about 50-60% of the cells remain viable.

The matrix of materials prepared by the above method is a collapsed amorphous material having a glass / liquid transition temperature (when exposed to room air at normal humidity) of more than 20 °.

Claims (11)

What is claimed is: 1. A stabilized, dried composition containing stasis microbial cells suspended in a collapsed matrix, characterized in that it contains Gram-negative bacterial cells as microbial cell barriers. 2. The composition of claim 1, wherein the gram-negative cells are Pseudomonas fluorescens cells, Escherichia coli cells, or rhizosphere associated bacterial cells. 3. A process for the preparation of a stabilized dried composition comprising a stasis-state Gram-negative bacterial cell in a matrix, characterized by (a) mixing the microbial cells with an aqueous composition containing the matrix-forming material, and (b) mixing the mixture with a however, the cells are dried under conditions that do not significantly damage the matrix. The process of claim 3, wherein the composition obtained in step (b) is further dried to increase the glass / liquid transition temperature of the matrix and to widen the storage conditions of the composition. 5. The method of claim 3, wherein said step (a) comprises a cell concentration of 10 ⁴ cells / ml to 10 ¹³ cells / ml. * ··· ·· 6. The method of claim 5, wherein a mixture of 10 to ¹⁰ cells / ml to 10 ¹¹ cells / ml is formed. The process according to claim 3, wherein the matrix forming agent in step (a) is a polyhydroxy compound. 8. A process according to claim 7 wherein the polyhydroxy compound is a monosaccharide. 9. The method of claim 7, wherein the mixture of step (a) comprises 10-1000 mg of polyhydroxy compound per 10 ¹⁰ cells. The method of claim 9, wherein the amount of polyhydroxy compound is 200-400 mg per 10 ¹⁰ cells. 11. The composition of the process of claim 3, wherein the glass / liquid conversion temperature is higher than the expected storage temperature of the composition.