CRYOPROTECTIVE COMPOSITIONS AND USES THEREOF

Abstract

The invention pertains to compositions for preserving biological material or active molecules, particularly microorganisms, and their uses.

Claims

1. A composition comprising: (a) trehalose or a derivative thereof, (b) inositol or a derivative thereof, and (c) a microorganism, wherein: the w/w ratio (a)/(b) of said composition is from 1.1 to 1.9, and said composition does not contain charcoal.

2. The composition according to claim 1, wherein said w/w ratio (a)/(b) is from 1.3 to 1.7.

3. (canceled)

4. The composition according to claim 1, wherein a is trehalose.

5. The composition according to claim 1, wherein (a) is trehalose and (b) is inositol.

6. (canceled)

7. The composition according to claim 1, wherein the microorganism is lactic acid bacteria.

8. The composition according to claim 7, wherein said lactic acid bacteria are selected from the group consisting of the strains of the genus Lactococcus, Lactobacillus, Leuconostoc, Bifidobacterium, Carnobacterium, Enterococcus, Propionibacterium, Pediococcus, and Streptococcus.

9. The composition according to claim 8, wherein said lactic acid bacteria are selected from the group consisting of the strains of the species and subspecies Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbruckii bulgaricus, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus pentoceus, Lactobacillus buchneri, Lactobacillus brevis, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus pervulus, Propionibacterium freudenreichi, Propionibacterium jenseni, and Streptococcus salivarius.

10. The composition according to claim 1, wherein said composition comprises the microorganism in a concentration from 1E8 CFU/g of composition to 5E12 CFU/g of composition.

11. The composition according to claim 1, wherein said composition comprises: (a) at least 4% trehalose, and (b) at least 3% inositol.

12-17. (canceled)

18. The composition according to claim 1, wherein (b) is inositol.

19. The composition according to claim 18, wherein said w/w ratio (a)/(b) is from 1.3 to 1.7.

20. The composition according to claim 18, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

21. The composition according to claim 1, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

22. The composition according to claim 4, wherein said w/w ratio (a)/(b) is from 1.3 to 1.7.

23. The composition according to claim 4, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

24. The composition according to claim 5, wherein said w/w ratio (a)/(b) is from 1.3 to 1.7.

25. The composition according to claim 5, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

26. The composition according to claim 1, wherein the composition comprises at least two microorganisms.

27. The composition according to claim 26, wherein the at least two microorganisms are lactic acid bacteria selected from the group consisting of the strains of the genus Lactococcus, Lactobacillus, Leuconostoc, Bifidobacterium, Carnobacterium, Enterococcus, Propionibacterium, Pediococcus, and Streptococcus.

28. A composition comprising: (a) a non-reducing sugar, and (b) inositol or a derivative thereof, and (c) a microorganism, wherein: the w/w ratio (a)/(b) of said composition is from 1.3 to 1.7, and said composition does not contain charcoal.

29. The composition according to claim 28, wherein (b) is inositol.

30. The composition according to claim 29, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

31. The composition according to claim 28, wherein (a) is sucrose.

32. The composition according to claim 31, wherein (b) is inositol.

33. The composition according to claim 32, wherein said w/w ratio (a)/(b) is from 1.4 to 1.6.

34. The composition according to claim 28, wherein (a) is a derivative of sucrose.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0092] FIG. 1: Recovery of cell after accelerated test as a function of Trehalose/Inositol ratio.

EXAMPLES

[0093] Cells Counts Measurement

[0094] The cell count of the freeze dried material has been evaluated through a typical bacteria enumeration method used for lactic acid bacteria. In this method, the freeze dried bacteria were suspended into a MRS solution with a stomacher and revitalized in that solution for 30 minutes. The suspension was then successively diluted in bottles of peptone buffer and finally cultured on a MRS nutritive media for 48 to 72 hours at 38° C. under anaerobic condition. During that period, the bacteria form colonies on the nutritive media. Those colonies were counted and results were expressed as Colony Forming Units (CFU) per gram.

[0095] Accelerated Stability Test

[0096] Long term stability of freeze dried bacteria as a function of time is a critical characteristic for commercial application. The long term stability can be evaluated by accelerated stability test consisting of placing the freeze dried cells into a sealed laminated aluminum foil in a constant temperature chamber maintained at 38° C. for fourteen days. The cell count of the freeze dried material is measured before and after the exposure to elevated temperature. A recovery rate of cells is calculated by subtracting the cells measured after the accelerated stability test from the initial cells measurement and dividing the subtraction results by the initial count. The accelerated stability test recovery rate gives a relative estimate of the long term cell stability.

Example 1

[0097] Various cryoprotectants (see table 1) were mixed in a suspension containing, Lactobacillus acidophilus at an approximate cell count of 1E11 CFU/gr. The mixture, called stabilized concentrate, was kept at 4-8° C. for 1.5 hrs and continuously agitated before being frozen by dispensing droplets of the stabilized concentrate into liquid nitrogen. The resulting frozen droplets are called frozen pellets.

TABLE-US-00001 TABLE 1 Formulation of Trehalose based cryoprotectant tested on a suspension of Lactobacillus acidophilus. Trehalose concentration Additional cryoprotective (% W/W of component stabilized (% W/W of stabilized Cryoprotective solution concentrate) concentrate) Trehalose alone experiment 1 8 0 Trehalose alone experiment 2 14 0 Trehalose with phosphate 13 1 as KHP04 Trehalose with EDTA 14 0.0021 as EDTA Inositol alone 0 16.7 as Inositol Trehalose with Inositol 14 3.4 as Inositol experiment 1 Trehalose with Inositol 13 6.7 as Inositol experiment 2 Trehalose with Inosine Mono 14 3.4 as IMP Phosphate (IMP)

[0098] The frozen pellets were freeze dried in a Virtis® freeze drier at 100 mT and a cell count measurement was performed just after freeze drying by performing an accelerated stability test.

[0099] Table 2 shows the initial cell counts, the cell counts after the accelerated stability test and the recovery of freeze dried cells after the accelerated testing.

TABLE-US-00002 TABLE 2 Cell counts and accelerated stability results for Trehalose based cryoprotectant tested on a suspension of L. acidophilus. Recovery of Viable cell viable cell Viable cell counts after after counts after accelerated accelerated freeze drying stability test stability test Cryoprotection solution (CFU/gr) (CFU/gr) (%) Trehalose alone experiment 1 7.4E+11 2.2E+11 29.0 Trehalose alone experiment 2 6.6E+11 2.4E+11 38.0 Trehalose with phosphate 6.4E+11 1.3E+11 19.9 Trehalose with EDTA 7.0E+11 1.9E+11 27.1 Inositol alone 3.2E+11 1.3E+11 41.0 Trehalose with Inositol 5.8E+11 3.2E+11 54.8 experiment 1 Trehalose with Inositol 5.0E+11 4.4E+11 89.0 experiment 2 Trehalose with Inosine Mono 5.2E+11 1.2E+11 23.3 Phosphate (IMP)

[0100] Table 2 shows that the best recovery after the accelerated testing is achieved when the trehalose solution is at 13% and the Inositol is at 6.7%, with a ratio Trehalose/Inositol=1.9.

Example 2

[0101] Various ratio of Inositol over Trehalose have been tested. The cryoprotectants were mixed for 1 to 3 hrs at 10-30° C. to a suspension containing Lactobacillus acidophilus at an approximate cell count of 1E11 CFU/gr. The mixture was frozen and freeze dried, and a cell count was performed just after freeze drying by performing an accelerated stability test, as disclosed in Example 1. Table 3 gives the results of this testing.

TABLE-US-00003 TABLE 3 Cell counts and accelerated stability results for Trehalose based cryoprotectant tested on a suspension of L. acidophilus including Trehalose to Inositol ratio and percent recovery of cells after accelerated stability testing. Trehalose Inositol Recovery of Viable cell Recovery of concentration concentration Viable cell viable cell from counts after viable cells after (% W/W of (% W/W of Ratio counts after frozen pellets to accelerated accelerated stabilized stabilized Trehalose/ freeze drying freeze dried pellets stability test stability test concentrate) concentrate) Inositol (CFU/gr) (%) (CFU/gr) (%) 6.7 4.4 1.5 4.03E+11 104.4 3.70E+11 91.81 6.7 4.4 1.5 4.21E+11 75.3 3.71E+11 88.12 6.7 4.4 1.5 4.14E+11 79.3 4.01E+11 96.86 6.7 4.4 1.5 4.15E+11 90.7 3.51E+11 84.58 8.2 2.9 2.8 6.26E+11 83.0 3.05E+11 48.72 8.2 2.9 2.8 5.95E+11 75.2 3.50E+11 58.82 8.2 2.9 2.8 6.20E+11 87.0 3.25E+11 52.42 8.2 2.9 2.8 5.70E+11 67.3 3.77E+11 66.14 0.0 9.1 0.0 5.14E+11 81.2 3.72E+11 72.37 12.0 8.0 1.5 4.53E+11 82.5 3.73E+11 82.34 8.3 8.3 1.0 4.49E+11 81.1 3.99E+11 88.86 4.3 8.7 0.5 5.33E+11 89.0 3.80E+11 71.29 12.1 7.3 1.7 4.41E+11 87.5 4.16E+11 94.33 12.3 5.7 2.1 4.30E+11 84.6 3.54E+11 82.33 12.4 5.0 2.5 4.86E+11 81.9 3.48E+11 71.60 12.5 4.2 3.0 4.58E+11 81.7 3.23E+11 70.52 0.0 20.0 0.0 3.70E+11 94.7 2.42E+11 65.41

[0102] The recovery of cells after the accelerated stability test were graphed as a function of the Trehalose/Inositol and shown in FIG. 1. The curve clearly shows a synergistic effect between Trehalose and Inositol. Initially, the recovery increases when the ratio increases. An optimum is achieved around 1.5, after which the recovery decreases. The best recovery values are obtained when the ratio Trehalose/Inositol is comprised between 1.1 and 1.9.

Example 3

[0103] Cryoprotectants made of Inositol with non reducing sugar such as Trehalose or Sucrose were mixed with a suspension of Bifidobacterium lactis at an approximate cell count of 1E11 CFU/gr. The mixture called stabilized concentrate was kept at 4° C. and continuously agitated before being frozen by dispensing droplets of the stabilized concentrate into liquid nitrogen. The resulting frozen droplets are called pellets. The frozen pellets have then been freeze dried in a Virtis® freeze drier under a vacuum at 100 mT. The freeze dried pellets were evaluated by measuring the cells counts just after freeze drying and by performing an accelerated stability test. In addition, the recovery of cells from the frozen pellets to freeze dried pellets was calculated. Tables 4 and 5 give the results of the test.

TABLE-US-00004 TABLE 4 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Trehalose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Trehalose centration Trehalose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 8.80 6.7 1.3 91.3 84.9 13.33 6.7 2.0 94.4 81.8

TABLE-US-00005 TABLE 5 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Sucrose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Sucrose centration Sucrose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 8.80 6.7 1.3 102.0 82.5 13.33 6.7 2.0 91.1 84.9

[0104] The recovery of biomass stabilized with Trehalose or with Sucrose have similar cell recovery after freeze drying or after the accelerated test. Non reducing sugar can be used in combination with Inositol to successfully dry and preserve Bifidobacterium lactis.

Example 4

[0105] Cryoprotectants made of Inositol with non reducing sugar such as Trehalose or Sucrose were mixed with a suspension of Bifidobacterium animalis subspecies lactis at an approximate cell count of 1E11 CFU/gr. The mixture called stabilized concentrate was kept at 4° C. and continuously agitated before being frozen by dispensing droplets of the stabilized concentrate into liquid nitrogen. The resulting frozen droplets are called pellets. The frozen pellets have then been freeze dried in a Virtis® freeze drier under a vacuum at 100 mT. The freeze dried pellets were evaluated by measuring the cells counts just after freeze drying and by performing an accelerated stability test. In addition, the recovery of cells from the frozen pellets to freeze dried pellets was calculated. Tables 6 and 7 give the results of the test.

TABLE-US-00006 TABLE 6 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Trehalose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Trehalose centration Trehalose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 8.0 4 2.0 94.1 97.6 13.33 6.7 2.0 80.3 92.2

TABLE-US-00007 TABLE 7 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Sucrose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Sucrose centration Sucrose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 8.0 4.0 2.0 90.4 94.4 13.33 6.7 2.0 91.1 84.9

[0106] The recovery of biomass stabilized with Trehalose or with Sucrose have similar cell recovery after freeze drying or after the accelerated test. Non reducing sugar can be used in combination with Inositol to successfully dry and preserve Bifidobacterium animalis subspecies lactis.

Example 5

[0107] Cryoprotectants made of Inositol with non reducing sugar such as Trehalose were mixed with a suspension of Bieliobacterium bifidum at an approximate cell count of 1E11 CFU/gr. The mixture called stabilized concentrate was kept at 4° C. and continuously agitated before being frozen by dispensing droplets of the stabilized concentrate into liquid nitrogen. The resulting frozen droplets are called pellets. The frozen pellets have then been freeze dried in a Virtis® freeze drier under a vacuum at 100 mT. The freeze dried pellets were evaluated by measuring the cells counts just after freeze drying and by performing an accelerated stability test. In addition, the recovery of cells from the frozen pellets to freeze dried pellets was calculated. Table 8 gives the results of the test.

TABLE-US-00008 TABLE 8 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Trehalose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Trehalose centration Trehalose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 6.7 4.5 1.5 106 68.4 13.33 6.7 2.0 106.6 66.9

[0108] Non reducing sugar can be used in combination with Inositol to successfully dry and preserve Bidiobacterium bifidum.

Example 6

[0109] Cryoprotectants made of Inositol with non reducing sugar such as Trehalose were mixed with a suspension of Lactobacillus salivarius at an approximate cell count of 1E11 CFU/gr. The mixture called stabilized concentrate was kept at 4° C. and continuously agitated before being frozen by dispensing droplets of the stabilized concentrate into liquid nitrogen. The resulting frozen droplets are called pellets. The frozen pellets have then been freeze dried in a Virtis® freeze drier under a vacuum at 100 mT. The freeze dried pellets were evaluated by measuring the cells counts just after freeze drying and by performing an accelerated stability test. In addition, the recovery of cells from the frozen pellets to freeze dried pellets was calculated. Table 9 gives the results of the test.

TABLE-US-00009 TABLE 9 Comparison of recovery after freeze drying and recovery after an accelerated stability test for a cryoprotectant containing Trehalose and Inositol. Non- Recovery of Recovery of reducing Inositol viable cell from viable cell after sugar = con- Ratio frozen pellets to accelerated Trehalose centration Trehalose/ freeze dried stability test (% W/W) (% W/W) Inositol pellets (%) (%) 6.7 4.5 1.5 90.4 84.9 13.33 6.7 2.0 89.1 78.8

[0110] Non reducing sugar can be used in combination with Inositol to successfully dry and preserve Lactobacillus salivarius.

[0111] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.