Lactic acid bacteria compositions

Abstract

The invention relates to a dry compositions for lactic acid bacteria and in particular to a dry composition comprising from 10.sup.9 to 10.sup.13 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized by that it also comprises following amounts of protective agents (all amounts of protective agents below are given relative to 1 g of lactic acid bacteria cells in the composition): from 6 to 9 g of trehalose, from 0.1 to 1 g of inulin and from 0.5 to 3 g of hydrolyzed casein, and by that it does not comprise a salt of alginic acid. The composition has an improved storage stability of the cell of interest. Comparison experiments have been made between compositions with and without alginate and it has been found that there is substantially no difference between compositions with or without alginate with regard to stability. Further, the invention relates to a method for preparing a dry lactic acid bacteria composition.

Claims

1. A dry composition comprising lactic acid bacteria cells in an amount from 10.sup.9 to 10.sup.13 cfu/g of the composition, and further comprising the following amounts of protective agents per g of lactic acid bacteria cells in the composition: from 6 to 9 g trehalose, from 0.1 to 1 g inulin and from 0.5 to 3 g hydrolyzed casein, wherein the composition does not comprise a salt of alginic acid, and wherein the log loss of active cells in the composition after 13 weeks is less than 2.5 when stored at 30% relative humidity (RH) and 35 C.

2. A dry composition according to claim 1, which comprises 75-80% (w/w) trehalose, 3-10% (w/w) inulin and 15-20% (w/w) hydrolysed casein.

3. A dry composition according to claim 1, wherein the lactic acid bacteria cells are from at least one lactic acid bacteria species selected from the group consisting of Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species, Bifidobacterium species, Propioni and Pediococcus species.

4. A dry composition according to claim 3, wherein the lactic acid bacteria cells are from at least one lactic acid bacteria species selected from the group consisting of Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Enterococcus, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

5. A dry composition according to claim 4, wherein the lactic acid bacteria cells are from at least one lactic acid bacteria strain selected from the group consisting of Bifidobacterium animalis subsp lactis deposited as DSM 15954, Bifidobacterium animalis subsp lactis deposited as ATCC 27536, Bifidobacterium animalis subsp lactis deposited as DSM 10140, Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus rhamnosus deposited as ATCC 55826, Lactobacillus reuteri deposited as ATCC 55845, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, and Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012.

6. The dry composition according to claim 1, wherein the weight of the dry composition is from 50 g to 1000 kg.

7. The dry composition according to claim 1, wherein the weight of the dry composition is from 100 g to 1000 kg.

8. The dry composition according to claim 1, wherein the weight of the dry composition is from 1 kg to 1000 kg.

9. An infant powder comprising a composition according to claim 1.

10. A food product comprising a composition according to claim 1.

11. A dietary supplement comprising a composition according to claim 1.

12. A method for preparing a dry composition according to claim 1, comprising: (a) fermenting lactic acid bacteria cells and harvesting the cells to obtain a lactic acid bacteria cell concentrate comprising the lactic acid bacteria cells and water, wherein the concentrate comprises from 10.sup.8 to 10.sup.14 cfu lactic acid bacteria cells per g dry matter of the concentrate; (b) forming a slurry by mixing the lactic acid bacteria cell concentrate with the following amounts of protective agents per g of lactic acid bacteria cells in the slurry: from 6 to 9 g trehalose, from 0.1 to 1 g inulin and from 0.5 to 3 g hydrolyzed casein, wherein the amounts of the lactic acid bacteria cells and protective agents are measured as dry matter in the slurry, and wherein the slurry does not comprise a salt of alginic acid; (c) freezing the slurry to form solid frozen particles/pellets; (d) loading a tray with from 2 kg/m.sup.2 to 50 kg/m.sup.2 of the frozen particles/pellets to obtain material comprising lactic acid bacteria cells; (e) primary drying the material under a vacuum pressure of from 0.7 to 2 millibar at a temperature such that the temperature of the material does not get so high that more than 75% of the lactic acid bacteria cells are inactivated and for a period of time until at least 90% of the water from the slurry of step (b) has been removed; and (f) secondary drying the material of step (e) under a vacuum pressure of from 0.01 to 0.6 millibar at a temperature such that the temperature of the material does not get so high that more than 75% of the lactic acid bacteria cells are inactivated and for a period of time sufficient to reduce the water activity (a.sub.w) to less than 0.30, thereby obtaining the dry composition comprising lactic acid bacteria cells in an amount from 10.sup.9 to 10.sup.13 cfu/g of the composition, wherein the composition further comprises the following amounts of protective agents per g of lactic acid bacteria cells in the composition: from 6 to 9 g trehalose, from 0.1 to 1 g inulin and from 0.5 to 3 g hydrolyzed casein, and wherein the composition does not comprise a salt of alginic acid.

13. The method of claim 12 wherein the temperature of the material in step (e) does not get so high that more than 10% of the lactic acid bacteria cells are inactivated; and the temperature of the material in step (f) does not get so high that more than 10% of the lactic acid bacteria cells are inactivated.

14. The method of claim 12, wherein the composition comprises 75-80% (w/w) trehalose, 3-10% (w/w) inulin and 15-20% (w/w) hydrolysed casein.

15. The composition of claim 1, made by a process comprising: (a) forming a slurry of a concentrate comprising the lactic acid bacteria cells and water, wherein the concentrate comprises from 10.sup.8 to 10.sup.14 cfu lactic acid bacteria cells per g dry matter of the concentrate, with from 6 to 9 g trehalose, from 0.1 to 1 g inulin, and from 0.5 to 3 g hydrolyzed casein, wherein the amounts of the lactic acid bacteria cells and protective agents are measured as dry matter in the slurry, and wherein the slurry does not comprise a salt of alginic acid; (b) freezing the slurry to form solid frozen particles/pellets; (c) primary drying the solid frozen particles/pellets material under a vacuum pressure of from 0.7 to 2 millibar at a temperature such that the temperature of the material does not get so high that more than 75% of the lactic acid bacteria cells are inactivated and for a period of time until at least 90% of the water from the slurry has been removed; and (d) secondary drying the material of step (c) under a vacuum pressure of from 0.01 to 0.6 millibar at a temperature such that the temperature of the material does not get so high that more than 75% of the lactic acid bacteria cells are inactivated and for a period of time sufficient to reduce the water activity (a.sub.w) to less than 0.30.

Description

LEGEND TO FIGURES

(1) FIG. 1 shows storage stability for L. casei 431 compositions in Infant powder with a.sub.w: 0.3 and at a storage temperature of 35 C.

(2) FIG. 2 shows storage stability for six different L. casei 431 compositions in Infant powder with a.sub.w: 0.3 and at a storage temperature of 35 C.

(3) FIG. 3 shows storage stability for L. casei 431 compositions in Infant powder with a.sub.w: 0.3 and at a storage temperature of 35 C.

(4) FIG. 4 shows storage stability for LGG compositions in infant powder with a.sub.w: 0.3 and at a storage temperature of 35 C.

(5) FIG. 5 shows storage stability for L. casei 431 compositions in infant powder with a.sub.w: 0.3 and at a storage temperature of 40 C.

(6) FIG. 6 shows storage stability for LGG compositions in Infant powder with a.sub.w: 0.3 and at a storage temperature of 40 C.

(7) FIG. 7 shows storage stability for LGG compositions in infant powder with a.sub.w: 0.25 and at a storage temperature of 35 C.

EXAMPLES

Materials and Methods

(8) Washed concentrate of Lactobacillus rhamnosus LGG and Lactobacillus paracasei subsp. paracasei L. casei 431obtainable from Chr. Hansen A/S, Denmark

(9) Trehalose from Cargill name: Treha 16400

(10) Enzymatically Hydrolyzed Casein from DMV International

(11) Na-alginate from FMC BioPolymer: Manugel DMB

(12) Inulin from BENEO-ORAFTI: Orafti GR

(13) Maltodextrin: Glucidex IT 12 from Roquette

(14) Na-ascorbate from Northeast Pharmaceutical group Co.,

(15) Sucrose from Nordic Sugar: Granulated sugar 550

(16) Remy HC-P (pregelatinised rice) starch, baby food grade from Beneo-remy NV

(17) Infant powder was EnfaGrow sourced from Mead Johnson LCC, Evansville, Ind.

Example 1

Drying of LAB Composition

(18) The Lactic acid bacteria (LAB) cell was the commercially available Lactobacillus cell LGGobtainable from Chr. Hansen A/S, Denmark.

(19) The vacuum drier apparatus was an apparatus, wherein the heating in the apparatus was so-called radiation heating. The tray was situated between two heating plates, wherein both heating plates provide radiation heating to the tray.

(20) Step (a):

(21) 1 kg of LAB cell concentrate was obtainedit comprises around 10% dry matter of cellsi.e. a so-called 10% concentrate with around 90% of water.

(22) Step (b):

(23) 1 kg of a mixture of protective agents (the mixture comprised 30 g Sodium Alginate; 50 g Inulin, 753 g Trehalose and 167 g Casein Hydrolysate) was mixed with the LAB cell concentrate.

(24) Accordingly was obtained a slurry that comprised an amount of protective agents of around 10 gwherein the amount of protective agents is given relative to 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and lactic acid bacteria cells are measured as dry matter in a slurry.

(25) Step (c):

(26) The slurry was frozen to form solid frozen particles/pellets.

(27) It was done by use of liquid nitrogen.

(28) Step (d):

(29) Trays were loaded with 10 kg/m.sup.2 of the frozen particles/pellets to get the herein relevant material on the trays.

(30) Step (e):

(31) Primary drying of the material on the tray was performed under different vacuum pressuressome were within the vacuum pressure range of from 0.7 to 2 millibar (mbar) (e.g. was used 1.3 mbar pressure) and some were performed outside this range (e.g. was used 2.5 mbar pressure).

(32) This step was performed at a temperature wherein the temperature of the material did not get higher than 37 C.

(33) At this maximum temperature significantly less than 50% of the LAB cells were inactivated.

(34) This step was performed a period of time until at least 97% of the water of the slurry of step (b) had been removedthis took around 12 hours.

(35) Step (f):

(36) Secondary drying the material of step (e) was performed under a vacuum pressure of 0.2 mbar.

(37) As for step (e)this step was also performed at a temperature wherein the temperature of the material did not get higher than 37 C. This step was performed a period of time of around 12 hours.

(38) Results:

(39) TABLE-US-00001 TABLE 1 Water activity and process survival at two different pressures # 1 # 2 # 3 # 4 Tray load 10 kg/m2 8 kg/m2 10 kg/m2 8 kg/m2 Heating temp. 50 C. 50 C. 50 C. 50 C. Max product temp. 32 C. 32 C. 40 C. 32 C. Pressure 2.5/0.2 mbar 2.5/0.2 mbar 2.5/0.2 mbar 1.3/0.2 mbar Drying time 23 hours 23 hours 23 hours 23 hours Aw 0.29 0.25 0.25 0.13 % active cells 34 26 34 40

(40) From table 1 It is seen that within the products dried at 2.5 mbar the water activity is higher than preferred (<0.15) whereas the sample dried at 1.3 mbar has a water activity as preferred. The process survival is furthermore higher for the sample at 1.3 mbar than for the samples dried at 2.5 mbar.

(41) TABLE-US-00002 TABLE 2 Water activity and process survival at different temperatures # 5 # 6 # 7 Tray load 10 kg/m2 10 kg/m2 10 kg/m2 Heating temp. 60 C. 70 C. 60 C. Max product temp. 32 C. 32 C. 37 C. Pressure 1.3/0.2 mbar 1.3/0.2 mbar 1.3/0.2 mbar Drying time 31 hours 24 hours 24 hours Aw 0.11 0.33 0.12 % active ceils 63 26 65

(42) From table 2 it is seen that the water activity as well as the process survival is in an unacceptable range when the drying temperature is too high (70 C.) whereas there is no differences on neither the water activity nor process survival whether the drying temperature is 50 or 60 C. In both cases the values are in an acceptable range.

(43) For this exampleone may say that the herein essential parameter that was varied was in primary drying step (e)wherein different vacuum pressures were usedsome were within the vacuum pressure range of from 0.7 to 2 millibar (mbar) (e.g. was used 1.3 mbar pressure) and some were performed outside this range (e.g. was used 2.5 mbar pressure).

(44) The experimental results essentially demonstrated that when there was used a vacuum pressure outside the range of 0.7 to 2 mbar there was not obtained a herein satisfactory drying of the LAB composition. The pressure is to be selected to be slightly above the transition temperature of the formulation for the reasons previously explained. The transition temperature of the formulation used in example 1 is about33 C. At 2.5 mbar the temperature is about 10.5 C. which is much higher than the transition temperature. The results in table 1 thus demonstrate that 1.3 mbar is more suitable.

(45) When the vacuum pressure was within the range of from 0.7 to 2 millibar (mbar) (e.g. 1.3 mbar pressure) then it was possible to make a proper and efficient drying to get the dry formula composition with a water activity (a.sub.w) of less than 0.15.

(46) Conclusions:

(47) The results of this Example 1 essentially demonstrated that it is only by working within the vacuum pressure range of from 0.7 to 2 mbar in step (e) that one gets a herein satisfactory method for drying a herein relevant LAB composition comprising relatively high amount of protective agents.

Example 2

Particle Size of Frozen Particles/Pellets of Step (c)

(48) An experiment was made essentially as described for Example 1but wherein the vacuum pressure in step (c) was kept constant at around 1.3 mbar pressure.

(49) In this experiment the essential variable was the particle size of frozen particles/pellets of step (c).

(50) Experiments were made, wherein at least 97% of the frozen particles/pellets in step (c) were particles/pellets that were capable of passing through a mesh with maximum size of the opening/holes of different sizes.

(51) Results:

(52) The experimental results essentially demonstrated that when the sizes of the particles/pellets were above 10 mm then there was not obtained an optimal drying result.

(53) But when the sizes of the particles/pellets were below 5 mm then there was obtained very good and efficient drying.

(54) Conclusions:

(55) The results of this Example 2 essentially demonstrated that it is herein preferred that at least 95% (more preferably at least 97%) of the frozen particles/pellets in step (c) are particles/pellets that are capable of passing through a mesh with maximum size of the opening/holes of 10 mm (preferably with maximum size of the opening/holes of 5 mm).

Example 3

Drying of Other LAB Compositions

(56) Example 1 was essentially repeated but with use of other LAB cells and other protective agents. Drying at pressures of 0.9/0.2 mbar and heating temperatures of 32 C. and 37 C. has been evaluated as well (for formulations with and without alginate) and for both temperatures the water activity of the dry products were <0.15 after 24 hours of drying.

(57) The formulations without alginate and the formulations with sucrose were run as #7 and resulted in dry products with water activity <0.15 and process survival of 50%. The sucrose formulation resulted in dry products with water activity <0.3 but by increasing the drying time the water activity might be much lower.

(58) For step (b) were in all experiments obtained a slurry that comprised an amount of protective agents of around 6 g to 15 gwherein the amount of protective agents is given relative to 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and lactic add bacteria cells are measured as dry matter in a slurry.

(59) For all the experimentsat least 50% of the used protective agents were saccharides.

(60) Conclusions:

(61) The results of this Example 3 essentially demonstrated the same as in Example 1i.e. that it is only by working within the vacuum pressure range of from 0.7 to 2 mbar in step (e) that one gets a herein satisfactory method for drying a herein relevant LAB composition comprising relatively high amount of protective agents. The exact vacuum pressure range suitable for the individual composition is selected by determining the transition temperature of the composition and correlating it with a water vapour pressure table as explained above.

(62) One may say that this Example 3 confirmed this conclusion for different LAB compositions that may be characterized as comprising a relatively high amount of saccharides as protective agents.

Example 4

Preparation of Formulations without Alginate

(63) To one part LGG concentrate was added two parts demineralized water and the concentrate was centrifuged back to the original volume (1.2710.sup.11 active cells/g). The cell concentrate used below had around 10% dry matter of cellsi.e. a so-called 10% concentrate.

(64) Formulation 1: To 100 g washed concentrate was added 30 g sucrose+17.5 g maltodextrin (Glucidex IT 12)+13 g Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was vacuum dried.

(65) Formulation 2: To 100 g washed concentrate was added 70 g sucrose+17.5 g maltodextrin (Glucidex IT 12)+13 g Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was vacuum dried.

(66) Formulation 3: To 100 g washed concentrate was added 37.4 g sucrose+60 g Trehalose+2.6 g Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was vacuum dried.

(67) Reference 1 (reference formulation): To 100 g washed concentrate was added 6 g sucrose+3.5 g maltodextrin (Glucidex IT 12)+2.6 g Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was vacuum dried.

(68) If the mixtures are dried in a vacuum belt dryer it might be necessary to add a small amount of gelatinizing agent e.g. pectin to get an appropriate viscosity

Example 5

Test of LGG Formulations without Alginate in Open Bags and in Infant Powder at 30 C.

(69) The stability of the products has been tested in open bags stored at 30 C. and 30% RH and when mixed into infant powder with a water activity of 0.3. See stability data in Table 3 and Table 4. As reference is used a LGG containing 20% of the amount of additives in Formulation 1 as outlined in Example 4 above. The powder was moisturized to obtain a water activity of 0.27-0.30.

(70) TABLE-US-00003 TABLE 3 Storage in open bags stored at 30 C./30% RH. Start 1 week 10 days 2 weeks 3 weeks Log Log Log Log Log Log active active active active active loss 3 Formulation cells/g cells/g cells/g cells/g cells/g weeks Formulation 1 11.3 11.3 11.3 11.3 11.3 0 Formulation 2 11.0 11.0 11.0 11.0 11.0 0 Formulation 3 11.0 11.0 11.1 11.1 11.0 0 Reference 1 11.5 11.4 11.2 11.0 0.5

(71) The samples are measured by Flow Cytometry (active cells/g).

(72) Conclusion:

(73) From Table 3 it is seen that the highest loss of % active cells is found in Reference 1. By increasing the amount of protective agents by a factor 5 the stability is significantly increased (comparison of Formulation 1 and Reference 1). Also formulations 2 and 3 demonstrate an increased stability.

(74) TABLE-US-00004 TABLE 4 Storage stability at 30 C. in Enfagrow with a.sub.w: 0.3. Start 9 weeks 15 weeks Log Log Log Log Log loss 9 loss 15 Formulation CFU/g CFU/g CFU/g weeks weeks Formulation 1 11.4 10.9 11.1 0.5 0.3 Formulation 2 11.1 11.0 10.9 0.1 0.2 Formulation 3 11.2 10.8 10.7 0.4 0.5 Reference 1 11.5 8.0 5.7 3.5 5.8

(75) The bags were flushed with N.sub.2 and sealed before storage.

(76) Conclusion:

(77) The Infant powders made with formulations 1-3 have a better stability than Reference 1.

Example 6

Test of L. paracasei Subsp. Paracasei L. casei 431 Formulations without Alginate in Infant Powder at 35 C.

(78) To one part L. paracasei subsp. paracasde L. casei 431 concentrate (LC 431) was added two parts demineralized water and the concentrate was centrifuged back to the original volume (1.2710.sup.11 active cells/g). The cell concentrate used below had around 10% dry matter of cellsi.e. a so-called 10% concentrate.

(79) Formulation 1.sub.LC 431: To 100 g washed concentrate was added 30 g sucrose+17.5 g maltodextrin (Glucidex IT 12)+13 g Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was pelletised in liquid nitrogen before it was vacuum dried.

(80) The mixture was added to infant powder and the stability of the product tested in an Infant powder with a water activity of 0.3 stored at 30% RH at 35 C. See stability data in Table 3. As reference is used infant powder containing LC 431 in Reference 1 as outlined in Example 4 above.

(81) TABLE-US-00005 TABLE 5 Storage stability at 35 C. in infant powder with a.sub.w: 0.3. Start 3 weeks 6 weeks 9 weeks Log Formulation Log CFU/g Log CFU/g Log CFU/g Log CFU/g loss Formulation 1.sub.LC 431 10.0 9.3 9.4 9.1 0.9 Reference 1.sub.LC 431 11.9 9.9 9.1 7.7 4.2

(82) The bags were flushed with N.sub.2 and sealed before storage.

(83) Conclusion:

(84) From Table 5 and FIG. 1 it is seen that also for L. casei 4310 and at a storage temperature of 35 C. the highest loss of % active cells is found in Reference 1. By increasing the amount of protective agents by a factor 5 the stability is significantly increased also for L. casei 4310.

Example 7

Test of Six Different L. paracasei Subsp. Paracasei L. Casei 431 Formulations without Alginate in Infant Powder at 35 C.

(85) To one part L. casei 431 concentrate was added two parts demineralized water and the concentrate centrifuged back to the original volume (1.2710.sup.11 active cells/g). The cell concentrate used below had around 10% dry matter of cellsi.e. a so-called 10% concentrate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was pelletised in liquid nitrogen before it was vacuum dried.

(86) Composition 1: To 1000 g washed concentrate was added 753 g trehalose+191 g maltodextrin+26 g Na-ascorbate+30 g Remy HC-P.

(87) Composition 2: To 1000 g washed concentrate was added 753 g trehalose+50 g inulin+167 g hydrolysed casein.

(88) Composition 3: To 1000 g washed concentrate was added 366 g trehalose+213 g maltodextrin+26 g Na-ascorbate.

(89) Composition 6: To 1000 g washed concentrate was added 615 g trehalose+358 g maltodextrin+26 g Na-ascorbate.

(90) Composition 7: To 1000 g washed concentrate was added 459 g trehalose+267 g maltodextrin+26 g Na-ascorbate.

(91) Composition 8: To 1000 g washed concentrate was added 213 g trehalose+124 g maltodextrin+26 g Na-ascorbate.

(92) The various compositions were added to infant powder and the stability of the products tested in infant powder with a water activity of 0.3 stored at 30% RH at 35 C. in sealed bags flushed with N.sub.2. See stability data in Table 4 and FIG. 2. As reference is used infant powder containing L. casei 431 in Reference 1 described in Example 4 above.

(93) TABLE-US-00006 TABLE 6 Storage stability (log active cells/g) at 35 C. in infant powder with a.sub.w: 0.3. Compo- 6 9 13 17 sition 0 3 weeks 5 weeks weeks weeks weeks weeks 1 10.5 9.4 9.7 8.7 8.4 2 10.7 10.4 10 9.5 9.6 9.4 3 10.8 9.4 9 8.8 8.6 6 10.4 9.3 8.8 8.8 8 7 10.3 9.3 8.6 8.6 8 8 10.7 9.6 8.9 8.5 Refence 12.0 9.0 7.8 1
Conclusion:

(94) From Table 6 and FIG. 2 it is seen that for L. casei 431 and at a storage temperature of 35 C. the highest loss of % active cells is found in Reference 1. All the tested compositions and in particular composition 2 demonstrate a substantially improved stability.

Example 8

Comparison of Compositions with and without Alginate for L. paracasei Subsp. Paracasei (LC 431) and for LGG

(95) 10% concentrates of LGG and L. paracasei subsp. paracasei (LC 431) were prepared as described above and added to composition 2 or to composition 2.sub.alginate (see below). Two different LGG concentrates and two different L. casei 431 concentrates were used in order to test reproducibility. Further, two different formulations were prepared, the difference between the two formulations being that composition 2 does not contain alginate whereas composition 2.sub.alginate contains 30 g Na-alginate per kg washed concentrate. The mixture was stirred until the additives were dissolved and frozen in liquid nitrogen. Afterwards the mixture was vacuum dried.

(96) Composition 2.sub.alginate: To 1000 g washed concentrate was added 753 g trehalose+50 g inulin+167 g hydrolysed casein+30 g Na-alginate.

(97) TABLE-US-00007 TABLE 7a Storage stability data (log (CFU/g) after storage at 35 C. in infant powder with a.sub.w: 0.3 in sealed bags 0 1 2 3 5 6 weeks Week Weeks Weeks Weeks Weeks Composition 2.sub.LGG+Na-alginate 11.2 11 10.7 Composition 2.sub.LGG 11.0 10.7 10.3 Composition 2.sub.LGG 11.0 10.6 10.2 Composition 2.sub.LC 431+Na-alginate 10.3 10 9.6 Composition 2.sub.LC 431 10.9 10.2 9.8 Composition 2.sub.LC 431 10.7 10.4 10.0

(98) TABLE-US-00008 TABLE 7b Storage stability data (log (CFU/g) after storage at 40 C. in infant powder with a.sub.w: 0.3 in sealed bags. 0 1 2 3 Weeks Week Weeks Weeks Composition 2.sub.LGG+Na-alginate 11.2 11 10.2 9.1 Composition 2.sub.LGG 11.0 10.8 10.0 8.9 Composition 2.sub.LGG 11.0 10.7 9.7 8.7 Composition 2.sub.LC 431+Na-alginate 10.3 10.1 9.2 8.0 Composition 2.sub.LC 431 10.7 9.8 Composition 2.sub.LC 431 10.9 10.4 9.4 8.6
Conclusion:

(99) From Tables 7a and 7b and FIGS. 3-6 it is demonstrated that there is substantially no difference between compositions with or without Na-alginate with regard to stability.

Example 9

Heat Treatment of Compositions of LGG with and without Alginate

(100) 10% concentrates of LGG were prepared and added to composition 2 or to composition 2.sub.alginate as described in Example 8 above. As heat treatment is relevant for production scale products two of the compositions were subjected to heat treatment in order to compare the stability of the compositions with or without alginate. As reference is used infant powder containing LGG in Reference 1 described in Example 4 above.

(101) TABLE-US-00009 TABLE 8 Storage stability data (log (CFU/g) after storage at 35 C. in infant powder with a.sub.w: 0.25 in sealed bags 0 3 6 9 13 17 weeks Week Weeks Weeks Weeks Weeks Composition 11.1 11 10.9 10.9 10.7 10.6 2.sub.alginate Composition 2 10.9 10.8 10.7 10.6 10.5 10.5 Composition 11.0 10.9 10.8 10.5 10.3 10.2 2.sub.alginate+heat Composition 10.8 10.8 10.6 10.5 10.4 2.sub.heat Reference 1 11.9 11.5 11.2 11.0 10.7 9.8
Conclusion:

(102) From Table 8 and FIG. 7 it is seen that for the composition with sodium alginate the heat treatment has a negative impact on stability.