METHOD OF LYOPHILIZATION OF A CRYOGENIZED CELLULAR COMPOSITION CONTAINING DISSOLVED GAS

Abstract

The invention relates to the field of lyophilizates of biological materials. More particularly, the invention relates to a new method for preparing a lyophilizate of cells comprising a step of cryogenics “under pressure”. In a preferred embodiment, this method is applied to lactic acid bacteria.

Claims

1. A method of lyophilization of a cellular composition, characterized in that the freezing step is carried out by cryogenics under pressure, said method comprising the steps of: a) providing a cellular composition comprising cells in an aqueous medium; b) dissolving a gas in said composition by passage through a dense zone of gas molecules, such a density being obtained (i) either owing to the flow of gas generated by the evaporation of a cryogenic fluid, (ii) or by raising the pressure, (iii) or by the combination of the two phenomena; c) cryogenizing said gas-rich composition obtained in step b) at a pressure that allows said gas to be kept dissolved in said composition for obtaining frozen granules, particles or beads; d) lyophilization of said frozen granules, particles or beads to obtain a lyophilized cellular preparation.

2. A method for preparing a composition of cells frozen by cryogenics under pressure comprising the steps of: a) providing a cellular composition comprising cells in an aqueous medium in the form of a matrix; b) dissolving a gas in said matrix by passage through a dense zone of gas molecules, such a density being obtained (i) either owing to the flow of gas generated by the evaporation of a cryogenic fluid, (ii) or by raising the pressure, which may be up to 10 bar, (iii) or by combining a flow of gas as mentioned in (i) with raising the pressure; c) cryogenizing said gas-rich matrix obtained in step b) at a pressure that allows said gas to be kept dissolved in said matrix for obtaining frozen granules, particles or beads.

3. The method of claim 1, in which said gas is nitrogen.

4. The method of claim 1, in which said cellular composition comprises lactic acid bacteria of probiotic interest, bacteria forming the microbiota, yeasts, plant cells, microalgae, reproductive cells, blood cells, stem cells.

5. The method of claim 4, in which said cellular composition comprises bacteria of probiotic interest for humans and animals.

6. The method of claim 1, in which the cellular composition does not contain cryoprotectant.

7. The method of claim 4, in which said cellular composition is a sample of faecal microbiota.

8. A lyophilized cellular composition obtained by the method of claim 1.

9. The composition of claim 8, further comprising dehydrated maltodextrins.

10. The composition of claim 8, in which the cellular composition comprises bacteria of probiotic interest.

11. The method of claim 2, in which said gas is nitrogen.

12. The method of claim 2, in which said cellular composition comprises lactic acid bacteria of probiotic interest, bacteria forming the microbiota, yeasts, plant cells, microalgae, reproductive cells, blood cells, stem cells.

13. The method of claim 12, in which said cellular composition comprises bacteria of probiotic interest for humans and animals.

14. The method of claim 2, in which the cellular composition does not contain cryoprotectant.

15. The method of claim 12, in which said cellular composition is a sample of faecal microbiota.

Description

DESCRIPTION OF THE FIGURES

[0060] FIG. 1: Viability of the Lactobacillus plantarum ATCC SD-5209 strain after freezing by cryogenics “under pressure”.

[0061] FIG. 2: A—Concentrations of CFUs and ICs in the lyophilizates of the Lactobacillus plantarum BL3504 strain. B—Yields in viability and ICs of the lyophilizates of the Lactobacillus plantarum ATCC SD-5209 strain. C—Cytometric profiles of the lyophilizates of the Lactobacillus salivarius BL2201 strain.

[0062] FIG. 3: A—Loss of viability under conditions of accelerated ageing (30° C./65% RH) of the lyophilizates of the Bifidobacterium animalis ssp lactis BL3803 strain over 3 months. B—Loss of viability under conditions of accelerated ageing (30° C./65% RH) of the lyophilizates of the Lactobacillus plantarum BL3504 strain over 12 months.

[0063] FIG. 4: Comparison of the yields of intact cells of the lyophilizates of a Lactobacillus plantarum ATCC SD-5209 strain with or without cryoprotectant.

EXAMPLES

Example 1

Effect of Cryogenics on the Properties of a Frozen Composition of Lactic Acid Bacteria The samples are prepared from the commercial strain Lactobacillus plantarum ATCC SD-5209.

[0064] The bacteria are solubilized in a reconstituted vegetable MRS medium without carbohydrate+sucrose 1%+maltodextrins 5% (cryoprotectants), so that the concentration of intact cells is adjusted to 5.10.sup.9 cells/mL. The −80° C. control corresponds to freezing of 40 mL at −80° C. for a minimum of 48 h. The −20° C. control corresponds to freezing of 40 mL at −20° C. for a minimum of 48 h. For the strains frozen by cryogenics, different pressures were tested. The samples are obtained using equipment making it possible to implement the method as described in patent EP07858410, at a relative pressure of 8 bar in the cryogenics chamber. At the end of the freezing step, all the samples are stored in a chamber at −80° C. before being analysed. The samples are then thawed (1 h at 37° C.) and the viability is measured.

[0065] The results obtained are presented in FIG. 1.

[0066] It can be seen that the loss of viability of the control frozen at −20° C. is far greater than for the sample frozen by cryogenics at 8 bar. This result is entirely significant and shows a real benefit from cryogenics.

Example 2

Effect of Cryogenics on the Properties of a Lyophilized Composition of Lactic Acid Bacteria

[0067] A—Investigation of a Freshly Cultured Composition of Lactobacillus plantarum BL3504

[0068] A bacterial sample is cultured for 24 h in 500 mL of culture medium of the vegetable MRS type (Ref BK176HA from Biokar Diagnostics). The culture medium is then centrifuged for 10 min at a speed of 3400 g.

[0069] The pellet, containing the microorganisms under investigation, is taken up in fresh culture medium so that the concentration of intact cells is adjusted to 5.10.sup.9 cells/mL.

[0070] The preparation is then separated into 4 fractions making it possible to test the 4 different methods of freezing: [0071] The sample “control standard method” is prepared by aliquoting the preparation in 30 mL bottles and placing the latter in a chamber at −80° C. for at least 48 h so as to guarantee that all of the product has frozen and has reached a temperature of −80° C.; [0072] The sample “control nitrogen beads” is prepared by manually producing a drip, using a syringe, above an open vessel, such as a basin or a bowl, containing liquid nitrogen, at atmospheric pressure and ambient temperature; [0073] The sample “cryogenics 5 bar” is obtained using equipment making it possible to implement the method as described in patent EP07858410, at a relative pressure of 5 bar in the cryogenics chamber; [0074] The sample “cryozero” is obtained using equipment making it possible to implement a cryogenics step at a relative pressure of 0 bar, in such a way that the saturation of dissolved nitrogen obtained is about 3 times greater than would be obtained at this same pressure with the “control nitrogen beads”.

[0075] At the end of the freezing step, all the samples are stored in a chamber at −80° C. before being analysed.

[0076] The samples frozen by the 4 methods are also lyophilized, in a Drywinner CT60 lyophilizer (Heto Holten) for 48 h to 72 h. The samples are ground carefully using a pestle and mortar to reduce them to powder. They are then stored in sterile pots at −20° C. before being analysed. For the analyses, the powder samples are first dispersed in buffered peptone water with addition of Tween 80 at 1% (w/v) and homogenized using a Stomacher. The quantities of CFUs and ICs are obtained by counting in MRS agar and by cytometric analysis (protocol B of standard ISO 19344 IDF 232 v2015).

[0077] The results in FIG. 2-A were obtained with the Lactobacillus plantarum BL3504 strain.

[0078] This chart shows the results obtained according to 2 parameters: [0079] the concentration of live cells in the lyophilizate, in CFUs (viability test consisting of testing the capacity of the bacteria to form colonies on an agar medium), [0080] the membrane integrity of the cells in the lyophilizate, expressed as intact cells (ICs) measured by flow cytometry.

[0081] The results show that the use of a faster cooling rate (cryogenics: “control nitrogen beads”) makes it possible to double or triple the concentrations of CFUs and ICs in the lyophilizate with respect to the “control standard method (−80° C)”. The “cryozero” condition makes it possible to increase these concentrations further, since a tripling or quadrupling of the concentrations of CFUs and ICs of the “control standard method (−80° C.)” is achieved. Finally, the “cryogenics 5 bar” condition makes it possible to reach the highest concentrations of CFUs and ICs, 4 and 6 times higher than those in the standard method.

[0082] B—Investigation of a Commercial Composition of Lactobacillus plantarum ATCC SD-5209

[0083] The samples were prepared from the commercial strain Lactobacillus plantarum ATCC SD-5209.

[0084] The bacteria are solubilized at a level of 10% dry matter in buffered peptone water (tryptone 1.0g/L+NaCl 8.5 g/L+K.sub.2HPO.sub.4 2.5 g/L+KH.sub.2PO.sub.4 2.5 g/L) with addition of 5% (w/w) maltodextrins (cryoprotectant). The −80° C. control corresponds to freezing of 10 mL at −80° C. for a minimum of 48h. The −40° C. control corresponds to freezing of 10 mL at −40° C. for a minimum of 48 h. For the cryogenically frozen samples, various pressures were tested. The samples are obtained using equipment making it possible to implement the method as described in patent EP07858410, at a relative pressure of 0 and 5 bar in the cryogenics chamber (“cryozero” and “cryogenics 5 bar” samples). The cryogenically frozen samples are stored in a chamber at −80° C. before being analysed. Some of the samples are then thawed (30 min at ambient temperature) and the viability is measured.

[0085] The samples frozen according to the 4 methods are in addition lyophilized, in a Drywinner CT60 lyophilizer (Heto Holten) for 72 h. The samples are ground carefully using a pestle and mortar to reduce them to powder. They are then stored in an aluminium sachet at −20° C. before being analysed. For the analyses, the powder samples are first dispersed in buffered peptone water with addition of Tween 80 at 1% (w/v) and homogenized using a Stomacher. The quantities of CFUs and ICs are obtained by counting in MRS agar and by cytometric analysis (protocol B of standard ISO 19344 IDF 232 v2015).

[0086] The results obtained are presented in FIG. 2-B.

[0087] This chart presents 3 yields, calculated either from the concentration of live cells measured by viability (CFUs), or from the concentration of intact cells measured by flow cytometry (ICs): [0088] “Freezing” yield: concentration in the frozen samples relative to the concentration before freezing, [0089] “Lyophilization” yield: concentration in the lyophilizates relative to the concentration in the frozen samples, [0090] “Global” yield: concentration in the lyophilizates relative to the concentration before freezing.

[0091] The results show that the use of cryogenics as a method for freezing samples of lactic acid bacteria makes it possible to increase the yields in viability and in intact cells at the level of the lyophilization step as well as in the overall method. In fact, the yields after the freezing step are not affected by the method used: 100% yield of CFUs and ICs is obtained whatever the sample. The positive effect of freezing by cryogenics clearly occurs during lyophilization with yields from 70 to 80% against 30 to 50% for the −40° C. and −80° C. controls. This effect is also observed for the method as a whole (global yield). However, these results do not make it possible to discriminate the two conditions of cryogenics: the same yields are obtained for the “cryozero” and “cryogenics 5 bar” samples. It may therefore be deduced from this that cryogenics under pressure under the conditions of molecular density described above, is one means for better preserving the intact cells and therefore improving the viability of samples of lactic acid bacteria produced by lyophilization.

[0092] C—Investigation of a Composition of Lactobacillus salivarius BL2201

[0093] The samples are prepared from the Lactobacillus salivarius BL2201 strain, according to the method described in Example 2-B.

[0094] The results obtained are presented in FIG. 2-C.

[0095] This chart presents the composition of the samples as intact cells, damaged cells and dead cells, measured by flow cytometry, at each step of the method (sample before freezing, frozen sample and lyophilizate).

[0096] The aim of these tests is to demonstrate the advantage obtained by freezing by cryogenics under pressure, even for a strain regarded as fragile, since the initial preparation used contains 45% damaged and dead cells. The results obtained also show that the effect from cryogenics occurs at the level of the lyophilization step. In fact, the cytometric profile of the different frozen samples is the same as that of the preparation before freezing, with about 55% intact cells. However, differences are observed in the cytometric profiles of the lyophilized samples: the lyophilizates of the −80° C. and −40° C. controls contain 4 and 5% ICs respectively, whereas the lyophilizates of the samples cryogenized under cryozero and 5 bar conditions contain 13% ICs. It can therefore be concluded that carrying out the freezing step by cryogenics “under pressure” is one means for preserving the level of intact cells in a lyophilized preparation of a fragile strain that does not easily withstand the standard method of lyophilization.

Example 3

Effect of the Method of Lyophilization on the Viability of a Composition of Lyophilized Bacteria (Dry Powder) Over Time

[0097] A—Investigation of the Stability of a Freshly Cultured Composition of Bifidobacterium animalis spp lactis BL3803

[0098] The samples are prepared by the method described in Example 2-A. The lyophilizates in powder form are stored for 3 months, after dilution in dehydrated maize maltodextrins, in paper/aluminium/PE three-layer sachets and kept at 30° C. and 65% RH before being analysed. For the analyses, the samples in powder form are first dispersed as stated in Example 2-A. The quantities of CFUs are obtained by counting in MRS agar, at t=0 just after production of the samples, then after storage for 1.5 months and 3 months, respectively. The loss of viability is calculated by difference between the quantity of CFUs at time t and that corresponding to t=0.

[0099] The experiments were carried out using the Bifidobacterium animalis spp lactis BL3803 strain.

[0100] The results obtained are presented in FIG. 3-A.

[0101] It can be seen that the loss of viability is considerable under the standard conditions (“−80° C. control”) since this loss is nearly half of the CFUs present initially. Conversely, the loss observed for the sample obtained from cryogenics at 5 bar is very low and close to 0. The results obtained for the “control nitrogen beads” and for the “cryozero” condition are comparable and intermediate, which also shows the positive effect of cryogenics “under pressure” on the method proposed for preservation of strains in lyophilized form.

[0102] B—Investigation of the stability of a freshly cultured composition of Lactobacillus plantarum BL3504

[0103] The samples are prepared by the method described in Example 2-A. The lyophilizates in powder form are stored for 12 months, after dilution in dehydrated maize maltodextrins, in paper/aluminium/PE three-layer sachets and kept at 30° C. and 65% RH before being analysed. For the analyses, the samples in powder form are first dispersed as stated in Example 2-A. The quantities of CFUs are obtained by counting in MRS agar, at t=0 just after production of the samples, and then after 1.5 months, 3 months, 4.5 months, 6 months, 9 months and 12 months of storage. The loss of viability is calculated by difference between the quantity of CFUs at a time t and that corresponding to t=0.

[0104] The experiments were carried out using the Lactobacillus plantarum BL3504 strain.

[0105] The results obtained are presented in FIG. 3-B.

[0106] It can be seen that the loss of viability is considerable under the standard conditions (“−80° C. control”) since this loss is already above 50% after 1.5 months of storage and the percentage of viable cells is less than 20% starting from 4.5 months. Conversely, the loss observed for the samples obtained from cryogenics is much slower. It is only significant (>20%) starting from the sixth month and about 60% of the cells are still viable after 12 months of storage. For comparison, barely more than 10% of the cells are still viable under the conditions of freezing at −80° C. The results obtained for the “control nitrogen beads” and “cryogenics under pressure” are comparable.

[0107] The loss of viability is slow enough under the three conditions for the difference between them not to be significant.

Example 4

Effect of the Presence of Cryoprotectant on the Membrane Integrity of a Lyophilized Composition of Lactic Acid Bacteria

[0108] The samples are prepared from the commercial strain Lactobacillus plantarum ATCC SD-5209, by the method described in Example 2-B, with or without cryoprotectants (maltodextrins 5% (w/w)).

[0109] The results obtained are presented in FIG. 4.

[0110] This chart presents the yields of the overall method, calculated from the concentration of intact cells (ICs) in the lyophilizates (measured by flow cytometry) relative to the concentration before freezing. The tests were done in triplicate, and error bars corresponding to the standard deviation of each condition are shown.

[0111] These results show that the presence of cryoprotectants in the samples allows better preservation of the intact cells during the freezing and lyophilization process. In fact, higher yields of ICs are observed for the samples containing maltodextrins, whatever the freezing conditions. It can therefore be deduced from this that the combined use of cryoprotectants and cryogenics gives an improvement in the quality of the lyophilizates of lactic acid bacteria.

[0112] Conclusion: The method of lyophilization of a cellular composition comprising a freezing step carried out by cryogenics “under pressure” gives a both qualitative and quantitative improvement of the production of cell lyophilizates.