Coated dehydrated microorganisms with enhanced stability and viability

Abstract

The present invention relates to coated dehydrated microorganisms comprising a dehydrated microorganism surrounded by at least one coating, said coating comprising by dry weight at least 25% of hygroscopic salt(s) and wherein the pH of the coating is compatible with viability of the coated dehydrated microorganism. The coating can be partially crystalline, the salt(s) in the coating having preferably a crystallinity degree of up to 60% once applied onto the dehydrated microorganism. The present invention also relates to liquid coating compositions, methods for coating and protecting a dehydrated microorganism. Finally, the present invention relates to a method for the preparation of food products, feed products, consumer healthcare products or agri-products as well as to a food product, feed product, a consumer healthcare product or an agri-product containing such coated dehydrated microorganisms.

Claims

1. A coated dehydrated microorganism comprising a dehydrated microorganism surrounded by at least one coating, said coating contacting the microorganism and comprising by dry weight at least 25% of hygroscopic salt(s), wherein the microorganism is a bacteria, wherein the coating has a Moisture Uptake Rate (MUR) of at least 20% w/w at 25 C. at 75% RH after 7 days, and wherein the pH of the coating is from 5.5 to 7.9 and improves the viability or the stability of the bacteria over the bacteria when uncoated.

2. The coated dehydrated microorganism according to claim 1, wherein the hygroscopic salt(s) is selected from at least one hygroscopic salt from the group consisting of dipotassium phosphate (K.sub.2HPO.sub.4), disodium hydrogen phosphate anhydrous (Na.sub.2HPO.sub.4), sodium hexametaphosphate (NaPO.sub.3).sub.6, sodium acetate anhydrous (CH.sub.3COONa), magnesium nitrate (Mg(NO.sub.3).sub.2), calcium bromide (CaBr), lithium bromide (LiBr), calcium chloride (CaCl.sub.2), magnesium chloride (MgCl.sub.2), lithium chloride (LiCl), phosphorus pentoxide (P.sub.4O.sub.10), disodium hydrogen phosphate dihydrate (Na.sub.2HPO.sub.4.2H.sub.2O), disodium hydrogen phosphate heptahydrate (Na.sub.2HPO.sub.4.7H.sub.2O), ammonium acetate (CH.sub.3COONH.sub.3), calcium acetate (CH3COO).sub.2Ca, potassium acetate (CH.sub.3COOK), potassium carbonate (K.sub.2CO.sub.3), sodium carbonate (Na.sub.2CO.sub.3), sodium formate (NaCHO.sub.2), potassium citrate monohydrate (K.sub.3C.sub.6H.sub.5O.sub.7.H.sub.2O), Sodium citrate pentahydrate (C.sub.6H.sub.5Na.sub.3O.sub.7.5H.sub.2O) and mixtures thereof.

3. The coated dehydrated microorganism according to claim 1, wherein said coating comprises by dry weight at least 28% of the hygroscopic salt(s).

4. The coated dehydrated microorganism according to claim 1, wherein the coating consists of 100% by dry weight of the hygroscopic salt(s).

5. The coated dehydrated microorganism according to claim 1, wherein the hygroscopic salt(s) has/have a MUR of at least 20% w/w at 25 C. at 75% RH after 7 days.

6. The coated dehydrated microorganism according to claim 1, wherein the hygroscopic salt(s) has/have a MUR of at least 30% w/w at 25 C. at 75% RH after 7 days.

7. The coated dehydrated microorganism according to claim 1, wherein the coating also comprises by dry weight from 0 to 60% of a non hygroscopic salt(s).

8. The coated dehydrated microorganism according to claim 1, wherein the coating also comprises by dry weight from 0 to 70% other ingredient(s).

9. The coated dehydrated microorganism according to claim 1, wherein the coating comprises dipotassium phosphate K.sub.2HPO.sub.4 as the hygroscopic salt or comprises a mixture of the hygroscopic salts including dipotassium phosphate K.sub.2HPO.sub.4.

10. The coated dehydrated microorganism according to claim 7, wherein the non-hygroscopic salt(s) is selected from the group consisting of monopotassium phosphate (KH.sub.2PO.sub.4), sodium acetate tri hydrate (CH.sub.3COONa.3H.sub.2O), calcium sulphate dihydrate (CaSO.sub.3.2H.sub.2O), sodium sulphate (Na.sub.2SO.sub.4), magnesium sulphate (MgSO.sub.4), potassium sulphate (K.sub.2SO.sub.4), sodium chloride (NaCl), potassium chloride (KCl), calcium carbonate (CaCO.sub.3), calcium lactate ((CH.sub.3CHOHCOO).sub.2Ca), calcium citrate tetrahydrate ((Ca.sub.3C.sub.6H.sub.5O.sub.7).sub.2.4H.sub.2O), sodium citrate dihydrate (HOC(COONa)(CH.sub.2COONa).sub.2.2H.sub.2O), and mixtures thereof.

11. The coated dehydrated microorganism according to claim 8, wherein the other ingredient(s) can be selected from the group of polyhydroxy compounds, anti-sticking agents, compounds having health and/or nutritional benefits, hydrocolloids, fillers, lubricants, binders, acids, alkali, hydrophobic species, polymers, and mixtures thereof.

12. The coated dehydrated microorganism according to claim 1, wherein the coating comprises K.sub.2HPO.sub.4 (83 wt % dry) and talc (17 wt % dry) or K.sub.2HPO.sub.4 (30 wt % dry), sodium acetate trihydrate (30 wt % dry), sucrose (17 wt % dry) and talc (14 wt % dry).

13. The coated dehydrated microorganism according to claim 1, wherein the coating comprises at least K.sub.2HPO.sub.4 and KH.sub.2PO.sub.4.

14. The coated dehydrated microorganism according to claim 12, wherein the coating is selected from the group consisting of: K.sub.2HPO.sub.4 (40 wt % dry), KH.sub.2PO.sub.4 (29 wt % dry), sucrose (17 wt % dry), and talc (14 wt % dry); K.sub.2HPO.sub.4 (48 wt % dry), KH.sub.2PO4 (35 wt % dry), and talc (17 wt % dry); and K.sub.2HPO.sub.4 (63 wt % dry), KH.sub.2PO.sub.4 (20 wt % dry), and talc (17 wt % dry).

15. The coated dehydrated microorganism according to claim 1, which has at least one outercoating.

16. The coated dehydrated microorganism according to claim 15, wherein said outercoating comprises a compound selected from the group consisting of fats, fatty acids, emulsifiers, oils, waxes, resins, low permeability polymers, hydrocolloids, starches, cyclodextrines, polyols, cellulose, cellulose derivatives and mixtures thereof.

17. The coated dehydrated microorganism according to claim 1, wherein the salt(s) in the coating has/have a salt crystallinity degree of up to 60% once applied onto the dehydrated microorganism.

18. The coated dehydrated microorganism according to claim 1, wherein the coated dehydrated microorganism has a Moisture Uptake Rate of at least 8%, at 25 C. and at 75% relative humidity (RH) after 7 days.

19. The coated dehydrated microorganism according to claim 1, wherein the coating is in a quantity of at least 10% by weight of the coated dehydrated microorganism.

20. The coated dehydrated microorganism according to claim 18, wherein the coating is in a quantity of at least 30% by weight of the coated dehydrated microorganism.

21. The coated dehydrated microorganism according to claim 1, which has a stability of minimum 40% survival upon storage from 15 C. to 40 C. for up to 2 years when stored in sealed conditions.

22. The coated dehydrated microorganism according to claim 1, which has a viability loss of <1.5 LOG upon 12 months storage at temperature from 15 C. to 40 C., in a feed product, a food product, a consumer healthcare product or an agri-product having a water activity (aw) greater than 0.10.

23. The coated dehydrated microorganism according to claim 1, which has a viability loss of <3 LOG upon 2 years storage at temperature from 15 C. to 40 C., in a feed product, a food product, a consumer healthcare product or an agri-product having an aw greater than 0.10.

24. The coated dehydrated microorganism according to claim 1, wherein the bacteria are probiotics or direct fed microbials (DFMs).

25. A method for the preparation of a food product, a feed product, a consumer healthcare product or an agri-product, wherein the coated dehydrated microorganism as defined in claim 1 is subsequently added to a food product, feed product, consumer healthcare product or agri-product.

26. A food product, a feed product, a consumer healthcare product or an agri-product comprising the coated dehydrated microorganism as defined in claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A) is a schematic representation of coated dehydrated microorganisms via the liquid route, wherein the starting biological material is a liquid microorganism concentrate.

(2) FIG. 1B) is a schematic representation of coated dehydrated microorganisms via the solid route, wherein the starting biological material is a freeze-dried microorganism powder.

(3) FIG. 2 A is a survival graph of coated (sample 11) L. acidophilus NCFM into chocolate bar stored at 23 C. Composition of sample 11: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), sucrose (17% dry), talc (14% dry).

(4) FIG. 2 B is a survival graph of coated (sample 11) and uncoated (sample 2) L. acidophilus NCFM into chocolate bar stored at 30 C. * Coating of sample 11: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), sucrose (17% dry), talc (14% dry).

(5) FIG. 3 is a moisture uptake graph for coated L. acidophilus NCFM at 75% RH and 37 C. All are dehydrated L. acidophilus NCFM prepared via the liquid route and surrounded by a coating of: Sample 10: K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), sucrose (0% dry), talc (17% dry). Sample 11: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), sucrose (17% dry), talc (14% dry). Sample 12: K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), sucrose (54% dry), talc (22% dry). Sample 13: Na.sub.2SO.sub.4. Sample 14: MgSO.sub.4.

(6) FIG. 4 is an X-ray diffraction pattern of coated dehydrated microorganism obtained by the liquid route, wherein the coating is a mixture of K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), talc (17% dry) and 0% sucrose, co-processed by spray-coating (sample 10).

(7) FIG. 5 is an X-ray diffraction pattern of coated dehydrated microorganism obtained by the liquid route, wherein the coating is a mixture K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17% dry), co-processed by spray-coating (sample 11).

(8) FIG. 6 is an X-ray diffraction pattern of a coated dehydrated microorganism obtained by the liquid route, wherein the coating is a mixture K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), talc (22% dry) and sucrose (54% dry), co-processed by spray-coating (sample 12).

(9) FIG. 7 is an X-ray diffraction pattern of a coated dehydrated microorganism obtained by the liquid route, wherein the coating is one non hygroscopic salt (KH.sub.2PO.sub.4) with talc processed by spray-coating (sample 16).

(10) FIG. 8 is an X-ray diffraction pattern of a coated dehydrated microorganism obtained by the solid route, wherein the coating mixture is K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17% dry), co-processed by spray-coating (sample 20).

(11) FIG. 9 is an X-ray diffraction pattern of a coated dehydrated microorganism obtained by the solid route, wherein the coating mixture is Na.sub.2SO.sub.4, co-processed by spray-coating (sample 21).

(12) FIG. 10 is a DSC thermogram for sample A (KH.sub.2PO.sub.4).

(13) FIG. 11 is a DSC thermogram for sample B (K.sub.2HPO.sub.4).

(14) FIG. 12 is a DSC thermogram for sample C (KH.sub.2PO.sub.4/K.sub.2HPO.sub.4/sucrose).

(15) FIG. 13 is a water vapor sorption isotherm at 25 C. for dehydrated microorganisms surrounded by a coating comprising different composition, exposed to 0-75% RH at 25 C. at 28 days. All are fluid-bed dried NCFM surrounded by a coating of: Sample 10: K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), sucrose (0% dry), talc (17% dry). Sample 11: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), sucrose (17% dry), talc (14% dry). Sample 12: K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), sucrose (54% dry), talc (22% dry). Sample 13: Na.sub.2SO.sub.4. Sample 14: MgSO.sub.4. Sample 15: K.sub.2HPO.sub.4 (83% dry), talc (17% dry). Sample 16: KH.sub.2PO.sub.4 (83% dry), talc (17% dry). Sample 23: K.sub.2HPO.sub.4 (23% dry), KH.sub.2PO.sub.4 (60% dry), talc (17% dry). Sample 24: K.sub.2HPO.sub.4 (19% dry), KH.sub.2PO.sub.4 (15% dry), sucrose (52% dry), talc (14% dry).

(16) FIG. 14 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 11 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 11: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), sucrose (17% dry), talc (14% dry).

(17) FIG. 15 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 10 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 10: K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), sucrose (0% dry), talc (17% dry)

(18) FIG. 16 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 15 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 15: K.sub.2HPO.sub.4 (83% dry), talc (17% dry).

(19) FIG. 17 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 13 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 13: Na.sub.2SO.sub.4

(20) FIG. 18 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 12 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 12: K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), sucrose (54% dry), talc (22% dry).

(21) FIG. 19 is a water vapor sorption isotherm for uncoated particles (cores; core+dehydrated microorganism) and coated particles (core+salt coating; coated deshydrated microorganisms) with salt composition corresponding to sample 16 exposed to 0-75% RH at 25 C. at 28 days. Coating of sample 16: KH.sub.2PO.sub.4 (83% dry), talc (17% dry)

EXAMPLES

(22) Table 1 is a summary of all tested samples. Some refer to the dehydrated microorganisms coated with the coating of the present invention, while others are dehydrated microorganisms which are coated with a different composition and which do not demonstrate an enhanced microorganism stability in stressed conditions.

(23) TABLE-US-00001 TABLE 1 Dehydrated Sample microorganisms 1.sup.st coating Outercoating(s) Comments 1 Fluid-bed dried NCFM None None Control, uncoated microorganism 2 Freeze-dried NCFM None None Control, uncoated microorganism 3 Freeze-dried Lp115 None None Control, uncoated microorganism 4 Fluid-bed dried NCFM None PS101 Conventional Sun flower oil coating 5 Fluid-bed dried NCFM None Stearine Conventional coating 6 Fluid-bed dried NCFM None Sepifilm LP30 Conventional coating 7 Fluid-bed dried NCFM None Shellac Conventional coating 8 Fluid-bed dried NCFM None HPMC Conventional coating 9 Fluid-bed dried NCFM None PS101/Sepifilm Conventional LP30 coating 10 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (48% dry), None An example of KH.sub.2PO.sub.4 (35% dry), coating according sucrose (0% dry), to the invention talc (17% dry) 11 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (40% dry), None An example of KH.sub.2PO.sub.4 (29% dry), coating according sucrose (17% dry), to the invention talc (14% dry) 12 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (14% dry), None An example of a KH.sub.2PO.sub.4 (11% dry), coating outside sucrose (54% dry), the invention talc (22% dry) 13 Fluid-bed dried NCFM Na.sub.2SO.sub.4 None An example of a coating outside the invention 14 Fluid-bed dried NCFM MgSO.sub.4 None An example of a coating outside the invention 15 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (83% dry), None An example of a talc (17% dry) coating outside the invention (pH not suitable) 16 Fluid-bed dried NCFM KH.sub.2PO.sub.4 (83% dry), None An example of a talc (17% dry) coating outside the invention 17 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (40% dry), Sepifilm LP30 An example of KH.sub.2PO.sub.4 (29% dry), coating according sucrose (17% dry), to the invention talc (14% dry) 18 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (40% dry), Shellac An example of KH.sub.2PO.sub.4 (29% dry), coating according sucrose (17% dry), to the invention talc (14% dry) 19 Fluid-bed dried Lp115 K.sub.2HPO.sub.4 (40% dry), Sepifilm Lp30 An example of KH.sub.2PO.sub.4 (29% dry), coating according sucrose (17% dry), to the invention. talc (14% dry) 20 Freeze-dried NCFM K.sub.2HPO.sub.4 (40% dry), None An example of KH.sub.2PO.sub.4 (29% dry), coating according sucrose (17% dry), to the invention. talc (14% dry) 21 Freeze-dried NCFM Na.sub.2SO.sub.4 None An example of a coating outside the invention 22 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (63% dry), None An example of KH.sub.2PO.sub.4 (20% dry), coating according talc (17% dry) to the invention. 23 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (23% dry), None An example of a KH.sub.2PO.sub.4 (60% dry), coating outside talc (17% dry) the invention 24 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (19% dry), None An example of a KH.sub.2PO.sub.4 (15% dry), coating outside Sucrose (52% dry) the invention talc (14% dry) 25 Fluid-bed dried NCFM MgCl.sub.2 (100% dry) none An example of coating according to the invention. 26 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (30% dry), none An example of sodium acetate tri coating according hydrate (30% dry), to the invention. Sucrose (17%) talc (14% dry) 27 Fluid-bed dried NCFM K.sub.2HPO.sub.4 (83% dry), none An example of talc (17% dry), pH coating according adjusted to 6.5 with to the invention. lactic acid 28 Fluid-bed dried NCFM KH.sub.2PO.sub.4 (83% dry), none An example of talc (17% dry), pH coating according adjusted to 6.5 with to the invention NaOH Note: All of the percentages refer to the dry coating (wt % dry), Samples were made with 30 wt % 1.sup.st coating by weight of the coated dehydrated microorganisms

Example 1

Liquid Route-Type Composition

(24) This trial was carried out in an Aeromatic MP-1 fluid bed, run in top-spray mode, using a distributor plate with 8% opening. The spray nozzle was a Schlick 1 mm nozzle, which was placed in the lowest position throughout the process. A Watson-Marlow pump was used to convey the spray material to the nozzle.

(25) Preparation of Primary Particle:

(26) The primary particle was prepared by spray-coating an inert carrier particle (i.e an inert core) with a liquid composition containing L. acidophilus NCFM.

(27) The liquid composition containing the microorganisms (culture mixture) was prepared by mixing the following ingredients:

(28) TABLE-US-00002 Ingredients Amount (%) Liquid culture 64.0 concentrate* Sucrose 6.7 milk protein (Promilk 6.7 852A) Maltitol 12.0 Wheat Starch 10.6 Total 100.0 *Liquid culture concentrate used was L. acidophilus NCFM

(29) The pH of the mixture was adjusted to 7.5 with 10M NH.sub.4OH.

(30) The fluid bed was charged with 2.8 kg of sucrose cores. Then 0.8 kg of the culture mixture was spray-coated onto the cores, using the following process parameters:

(31) TABLE-US-00003 Parameters Setting Product Air Temperature 40 C. Inlet Air Temperature 55 C. Fluidizing Air Flow Rate 90 m.sup.3/hr Spray Temperature 5 C. Atomizing Air Pressure 2.2 bar Atomizing Air Temperature 40 C. Spray Rate 1.00 kg/hr

(32) Preparation of the Coating Layer

(33) The following liquid coating composition was prepared:

(34) TABLE-US-00004 Amount (wt % Ingredients Amount (%) dry) Sucrose 7.5 17 KH.sub.2PO.sub.4 12.6 29 K.sub.2HPO.sub.4 17.2 40 Talc 6.2 14 Distilled water 56.5 Total 100 100

(35) This liquid coating composition was prepared by dissolving the sucrose and potassium phosphate salts in water and then dispersing the talc (which is added as an anti-agglomeration agent). The mixture was 43.5% dry solids.

(36) The fluid bed was charged with 2.4 kg of the primary particle. Then, 2.8 kg of the liquid coating composition was spray-coated onto the primary particles, using the following process parameters:

(37) TABLE-US-00005 Parameters Setting Product Air Temperature 50 C. Inlet Air Temperature 56 C. Fluidizing Air Flow Rate 150 m.sup.3/hr Spray Temperature 20 C. Atomizing Air Pressure 2.2 bar Atomizing Air Temperature 40 C. Spray Rate 0.50 kg/hr

(38) After the liquid coating composition has been spray coated on the primary particles, a post-drying step is carried out until a stable humidity value is achieved in the exhaust air. The resultant coated dehydrated microorganism corresponds to sample 11. It has a final composition of: 59% inert core; 8% culture mixture; 33% coating of the invention.

(39) The other related coated dehydrated microorganism of table 1 (samples 10, 12-19, 22-28) were prepared in the same way but using different 1.sup.st coating composition formula.

(40) Some of the coated dehydrated microorganisms were further coated with a conventional outercoating (to get samples 17, 18 and 19. One of the following materials was used: SEPIFILM LP30 composed of Hydroxypropyl methyl cellulose, microcrystalline cellulose, and stearic acid from Seppic (a subsidiary of the Air Liquid Group); Shellac (MARCOAT 125, aqueous based shellac solution (25%), from Innovative Material Technologies);

(41) Particles of samples 4 to 9 were only coated with a conventional outercoating (no hygroscopic salt coating of the invention was added). One of the following materials was used: SEPIFILM LP30; Shellac; triglycerides (GRINDSTED PS101 from Danisco), STEARINE TM 50/50 from Exaflor (mixture of stearic acid and palmitic acid 50/50).

(42) Hydroxypropyl methyl cellulose (HPMC, METHOCEL E15 from Dow Chemicals).

(43) It would also have been possible to use these other conventional outercoating ingredients: Modified starch (LYCOAT RS780 from Roquette Frres SA or PURE-COTE from Grain Processing Corporation); monoglycerides, mixture of mono- and diglycerides, fully hydrogenated triglycerides, fatty acids, hydrocolloids

Example 2

Solid Route-Type Composition

(44) The coated dehydrated microorganisms were prepared in a fluid bed process using a GF-3 (made by Glatt Air Techniques, Binzen, Germany) in bottom-spray mode.

(45) The following liquid coating composition was prepared (same as first coating composition as in example 1):

(46) TABLE-US-00006 Ingredients Amount (%) Amount (wt % dry) Sucrose 7.5 17 KH.sub.2PO.sub.4 12.6 29 K.sub.2HPO.sub.4 17.2 40 Talc 6.2 14 Distilled water 56.5 Total 100 100

(47) This liquid coating composition was prepared by dissolving the sucrose and potassium phosphate salts in water and then dispersing the talc (which is added as an anti-agglomeration agent). The mixture was about 43% dry solids.

(48) 1000 g of the freeze-dried powder of L. acidophilus NCFM were charged and fluidized using a heated bed temperature of 55 C. Then, 1180 g of the liquid coating composition according to the invention was spray-coated onto the freeze-dried powder using the following process parameters:

(49) TABLE-US-00007 Parameters Setting Product Air Temperature 50 C. Inlet Air Temperature 60 C. Fluidizing Air Flow Rate 150 m.sup.3/hr Spray Temperature 20 C. Atomizing Air Pressure 2.5 bar Atomizing Air Temperature 40 C. Spray Rate 0.70 kg/hr

(50) After the liquid coating composition has been spray coated onto the freeze-dried powder, a post-drying step is carried out until a constant humidity is achieved in the exhaust air.

(51) The resultant coated dehydrated microorganisms had a final composition of: 66% freeze-dried microorganism; 34% coating of the invention. The resultant particle corresponds to sample 20.

(52) The other related coated dehydrated microorganisms of table 1 (namely sample 21) were prepared in the same way but using a different first coating formula.

(53) It is possible to further coat the coated dehydrated microorganisms with a conventional outercoating (not done for samples 20 and 21). The same materials used in Example 1 for the outercoating could be used herein.

Example 3

Thermostability

(54) Heat resistance of dehydrated L. acidophilus NCFM coated with a first coating prepared according to Example 1 (liquid route) was compared to dehydrated L. acidophilus NCFM coated with conventional coatings: fat (PS101/sunflower oil) or low permeability polymer (Sepifilm LP30). The control consists of uncoated dehydrated L. acidophilus NCFM cells.

(55) 1 g samples of each preparation were introduced into separate sealed hermetic sachets and kept in a water bath at 50 C.

(56) The sealed hermetic sachet from each preparation was then withdrawn from the water bath after 24 hours and the concentration of viable bacteria was immediately determined after appropriate dilution by standard plating method (Table 2).

(57) TABLE-US-00008 TABLE 2 Before heat After heat treatment treatment Viability Sample (RT) (24 hrs/50 C.) loss (Log) 1 2.50.sup.E+10 1.10.sup.E+09 1.36 4 1.70.sup.E+10 1.72.sup.E+05 4.99 6 7.10.sup.E+09 3.48.sup.E+05 4.31 11 1.40.sup.E+10 6.30.sup.E+09 0.35 RT = room temperature

(58) From the results in Table 2, it can be observed that the coating according to the invention (sample 11) significantly improves thermostability of the dehydrated microorganism compared to the uncoated dehydrated microorganism (sample 1), the dehydrated microorganisms coated with fat coating (sample 4) and the dehydrated microorganisms coated with sepifilm LP30 coating (sample 6).

(59) Moreover, as illustrated in Table 3 below, it can be observed that the coatings K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), talc (17% dry) and sucrose (0% dry) (sample 10) and K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17% dry) (sample 11) are more efficient in protecting the bacteria L. acidophilus NCFM from heat. Indeed, the thermostability of these two samples is much greater than:

(60) the thermostability of coated dehydrated microorganism comprising dehydrated microorganism surrounded by a coating comprising a hygroscopic salt in an too low amount, such as sample 12 (K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), talc (22% dry) and sucrose (54% dry)).

(61) or the thermostability of coated dehydrated microorganism comprising dehydrated microorganism surrounded by a coating that does not comprise any hygroscopic salt (samples 13 and 14 comprise only a non hygroscopic salt, sodium sulphate and magnesium sulphate respectively)

(62) TABLE-US-00009 TABLE 3 Before heat After heat treatment treatment Viability Sample (RT) (24 hrs/50 C.) loss (Log) 10 1.00.sup.E+10 2.80.sup.E+09 0.55 11 1.40.sup.E+10 6.30.sup.E+09 0.35 12 5.00.sup.E+09 2.60.sup.E+07 2.28 13 5.50.sup.E+09 1.90.sup.E+06 3.46 14 5.50.sup.E+09 2.80.sup.E+06 3.29 RT = room temperature

(63) The samples were also tested under more stringent conditions, e.g. 64 C. for 18 hours in order to better discriminate between the salt coatings providing high thermoprotection versus those providing lower thermoprotection.

(64) TABLE-US-00010 TABLE 4 Before heat After heat treatment treatment Viability Sample (RT) (18 hrs/64 C./) loss (Log) 26 1.61E+10 4.81E+09 0.5 27 1.20E+10 2.70E+09 0.6 28 1.90E+10 5.00E+09 0.6 11 3.85E+09 1.60E+08 1.4 25 1.49E+09 4.60E+07 1.5 12 2.88E+09 3.30E+06 2.9 13 3.38E+09 1.30E+06 3.4 16 9.30E+08 <10 000 >5.0 14 1.48E+09 <10 000 >5.2 15 2.20E+09 <10 000 >5.3

(65) As can be seen from Table 4, it can be observed that the following coatings

(66) K.sub.2HPO.sub.4 (30% dry), sodium acetate tri hydrate (30% dry), talc (14% dry) and sucrose (17% dry) (sample 26);

(67) K.sub.2HPO.sub.4 (83%) talc (17%) and, pH adjusted to 6.5 (sample 27);

(68) KH.sub.2PO.sub.4 (83%) talc (17%) and, pH adjusted to 6.5 (sample 28); and

(69) K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17% dry) (sample 11),

(70) MgCl.sub.2 (100% dry) (sample 25)

(71) are very efficient in protecting the bacteria L. acidophilus NCFM from heat.

(72) On the contrary, coated bacteria L. acidophilus NCFM surrounded by a coating which either does not contain any hygroscopic salt (samples 13 and 14) or which contain hygroscopic salts in an insufficient amount (sample 12) or which does not have the suitable pH (samples 15 and 16) were much less resistant to heat compared to samples 25, 26, 27, 28 and 11.

Example 4

Stability During Recombined Cheese Processing

(73) The recombined cheese trial involved the following steps: i) grinding of the curd, ii) addition of hydrocolloids (no melting salts) to give the right texture to the final product, iii) heating to 60 C., and iv) stirring at 60 C.

(74) Samples of uncoated (control) or coated L. acidophilus NCFM were added to the molten mixture under stirring at 60 C. The stirring was continued for about 1 minute and followed by the moulding phase. Finally the moulded recombined cheese containing L. acidophilus NCFM was rapidly cooled down. The viability of the coated dehydrated L. acidophilus NCFM in the recombined cheese process was then evaluated and compared against uncoated dehydrated L. acidophilus NCFM (control). Results are presented in Table 5.

(75) TABLE-US-00011 TABLE 5 Before heat After heat Survival Viability Protection Sam- treatment treatment to loss in pro- factor over ple (RT) (60 C., 1 min) process cess (Log) control 2 1.00.sup.E+08 1.00.sup.E+04 0.01% 4.0 n.a. 5 1.00.sup.E+07 1.00.sup.E+03 0.01% 4.0 0 8 7.92.sup.E+06 8.00.sup.E+04 1.0% 2.0 10 9 1.00.sup.E+07 2.00.sup.E+05 2.0% 1.7 20 17 4.94.sup.E+06 1.20.sup.E+06 24.1% 0.6 2431 18 1.15.sup.E+07 4.00.sup.E+06 34.9% 0.5 3491 RT = room temperature, n.a. non applicable (control)

(76) From the experiment, it can be observed that the coated dehydrated microorganism comprising dehydrated microorganism surrounded by a first coating composed of the coating of the invention and with an outercoating comprising a polymer (samples 17 and 18) have a significantly enhanced survival rate following the recombined cheese processing. Moreover, they also have an improved stability over the uncoated bacteria (sample 2), the fat-coated bacteria (sample 5), the polymer-coated bacteria (sample 8) and the fat-coated+polymer-subcoated bacteria (sample 9). This shows the efficiency of the coating according to the invention.

Example 5

Feed Pelleting Stability

(77) Dehydrated L. acidophilus NCFM or L. plantarum Lp115 coated with a first coating consisting of a coating according to the invention followed by a polymer outercoating were pelletized using harsh conditions (see below). The stability of these bacteria following pelletization was compared to that of the uncoated dehydrated L. acidophilus NCFM or L. plantarum Lp115 (controls).

(78) Specifically, each test sample (i.e. 240 g of coated dehydrated bacteria) was mixed into 10 kg of premix feed and mixed for 10 min. This 10 kg premix feed (containing the 240 g of coated dehydrated bacteria) was then added to 150 kg of feed in a large horizontal mixer and mixed for 15 min before conditioning. The feed was then treated for 30 seconds at 75 C. by injecting dry steam (i.e. 3.5 to 4.5% wt water) directly into the feed prior to feed pelletization. The feed pellets were obtained using a 3 mm dye. The pellets were then cooled by blowing air around them using a fan. After 5 minutes, the temperature was lowered to 30 C. After 15 additional minutes, the temperature was again lowered to room temperature. At this stage, the pellets had a dry matter content (% DM) of about 80-90% and aw>0.10. The concentration of viable bacteria remaining after pelletization was subsequently determined by standard plating methods.

(79) Composition of the Premix Feed (Corn-Based Feed):

(80) TABLE-US-00012 Corn Diet Ingredients Percent Corn 61.01% Soybean meal 48 31.52% Soy oil 4.00% Sodium bicarbonate 0.40% DL Methionine 0.20% Limestone 1.16% Dicalcium Phos 1.46% VIT/MIN Beta Avitren 90 0.25% TOTAL 100.00%

(81) TABLE-US-00013 TABLE 6 Before heat After heat Viability loss Protection treatment treatment in processing factor over Sample (RT) (60 C., 1 min) (Log) control 2 2.70.sup.E+08 3.79.sup.E+05 3.00 n.a. 17 1.48.sup.E+05 9.90.sup.E+03 1.6 18 3 6.90.sup.E+05 1.56.sup.E+05 3.6 n.a. 19 3.23.sup.E+05 1.04.sup.E+04 0.96 545 RT = room temperature, n.a. non applicable (control)

(82) As can be observed in Table 6 above, the stability of the dehydrated bacteria coated with the coating according to the invention (samples 17 and 19) following pelletization was enhanced compared to the uncoated dehydrated bacteria (samples 2 and 3).

Example 6

Stability in Intermediate Moisture Powder

(83) The viability of dehydrated L. acidophilus NCFM in freeze-dried form (control) and as coated dehydrated L. acidophilus NCFM (made by the liquid route) has been studied in the following conditions:

(84) Maltodextrine powder Glucidex IT6 from Roquette initially having a water activity (aw) equal to 0.2, was exposed to an environment in which the relative humidity was 40% until the aw of the maltodextrine reached and equilibrated at an aw=0.4.

(85) The bacteria preparation was mixed into the maltodextrin powder (aw=0.4) at a ratio of 10% wt of coated bacteria and 90% wt of maltodextrin powder. 10 g of the mixture was then placed into glass vials which were subsequently sealed with a moisture proof cap. The vials were kept at 30 C. in an incubator. Each month, a vial of the mixture was analyzed for its content of viable cells by standard plating methods. The results are listed in Table 7. The concentration of viable cells is expressed as CFU/g and as a percentage of the concentration of each samples at T=0.

(86) TABLE-US-00014 TABLE 7 Protection factor over Sample T = 0 1 month % survival control 2 1.50.sup.E+11 6.80.sup.E+09 5% n.a. 10 8.80.sup.E+09 1.56.sup.E+09 18% 4 11 1.40.sup.E+10 9.00.sup.E+09 64% 13 15 1.90.sup.E+09 1.08.sup.E+08 6% 1 16 5.10.sup.E+09 6.65.sup.E+07 1% 0 n.a. non applicable (control)

(87) The coating comprising a hygroscopic salt such as K.sub.2HPO.sub.4 but with an uncompatible pH (sample 15) or comprising a non hygroscopic salt such as KH.sub.2PO.sub.4 (sample 16) offers no benefits or very limited benefits, whereas the coating according to the present invention (samples 10 and 11) clearly improve the viability of the dehydrated L. acidophilus NCFM at intermediate moisture and provide an increase in stability up to 13 times over uncoated dehydrated L. acidophilus NCFM (see Table 7). See example 13 showing the effect of the pH on K.sub.2HPO.sub.4 coating.

Example 7

Stability in Nutritional Bar

(88) Granola bars were prepared as follows: hydrating pectin (GRINDSTED Pectin CF 140 B) in hot water under high agitation, blending it together with corn syrup (42 DE Corn Syrup) and sugar, heating the pectin slurry-corn syrup+sugar mixture to 106 C. (12.5% water loss, 82% dry matter) to make the binding syrup, cooling the binding syrup to 55 C., pouring 410 g of the cooled binding syrup over 590 g of Cascadian Farms granola cereal, and mixing thoroughly, rolling out the preparation between two sheets of oiled parchment paper and allowing to cool, cutting the rolled out preparation into 34 g bars (each bar having a moisture content=8.1% and an aw=0.5 at the time of manufacture), melting chocolate and adding the test samples containing the bacteria L. acidophilus NCFM in the chocolate at 28 C., depositing 6 g of the chocolate-bacteria mixture onto the bar (i.e. 6 g chocolate-bacteria mixture onto approximately 34 g of granola bar). the bars with the chocolate-bacteria mix were stored at 23 C. (tables 8 and 9) or 30 C. (table 10) and the concentration of viable cells was measured after a storage period of 1, 2, 3, 5, 9 and 12 months at 23 C. or at 30 C., by standard plating methods.

(89) TABLE-US-00015 TABLE 8 Protection Aw at factor Time Time 1 2 3 5 over Sample 0 0 Month Months Months Months control 2 0.51 9.93.sup.E+09 5.10.sup.E+09 3.86.sup.E+09 1.21.sup.E+09 8.02.sup.E+08 n.a. (100%) (51%) (39%) (12%) (8%) 4 n.a. 1.33.sup.E+10 6.54.sup.E+09 3.29.sup.E+09 1.63.sup.E+09 2.14.sup.E+08 0 (100%) (49%) (25%) (12%) (1.6%) 6 n.a. 1.02.sup.E+10 5.98.sup.E+09 2.80.sup.E+09 1.06.sup.E+09 2.32.sup.E+08 0 (100%) (58%) (27%) (10%) (2%) 7 0.54 1.16.sup.E+10 1.05.sup.E+10 5.51.sup.E+09 1.74.sup.E+09 5.72.sup.E+08 0 (100%) (90%) (47%) (15%) (5%) n.a. non applicable (control)

(90) The results in Table 8 shows that when stored at 23 C., the viability of dehydrated L. acidophilus NCFM coated with conventional coatings such as fat (sample 4), low permeability polymer Sepifilm LP30 (sample 6) or shellac (sample 7) as previously described in the prior art is not maintained, the concentration of viable cells decreasing more than 1.5 LOG over a 5 months storage at 25 C. in granola bars and providing no improved stability over the uncoated dehydrated bacteria (sample 2).

(91) As some final products have a water activity aw which could be lower, a similar experiment was conducted, but with a reduced aw, giving somewhat dryer conditions.

(92) TABLE-US-00016 TABLE 9 aw *Protection at 1 2 3 5 9 12 factor over Sample T = 0 T = 0 Month Months Months Months Months Months the control 2 0.35 5.91.sup.E+09 4.43.sup.E+09 3.48.sup.E+09 7.76.sup.E+09 9.36.sup.E+08 1.29.sup.E+08 1.34.sup.E+08 n.a. (100%) (75%) (59%) (30%) (16%) (2%) (2%) 11 0.36 5.56.sup.E+09 3.41.sup.E+09 4.25.sup.E+09 3.83.sup.E+09 3.36.sup.E+09 3.88.sup.E+09 3.36.sup.E+09 35 (100%) (61%) (76%) (69%) (60%) (70%) (60%) 17 0.31 8.46.sup.E+09 6.93.sup.E+09 6.69.sup.E+09 6.75.sup.E+09 3.56.sup.E+09 2.96.sup.E+09 not 27 (100%) (82%) (79%) (80%) (42%) (35%) measured 18 0.32 8.58.sup.E+09 5.65.sup.E+09 5.02.sup.E+09 5.36.sup.E+09 4.00.sup.E+09 3.53.sup.E+09 3.42.sup.E+09 20 (100%) (66%) (59%) (62%) (47%) (41%) (40%) n.a. non applicable (control) *Protection factor calculated at 9 month- storage, 25 C.

(93) Results from Table 9 show that when stored at 23 C., presence of the coating according to the invention (samples 11, 17 and 18) results in improved bacteria stability over uncoated dehydrated bacteria (sample 2). Sample 11 offers the greatest protection over a long-time period (12 months).

(94) After 12 months storage at 23 C. in the chocolate coating of the Granola bar, the viability of L. acidophilus NCFM coated with the coating according to the invention remains high, for example, a viability of 60% is obtained after 12 months of storage with sample 11 (see FIG. 2 A).

(95) TABLE-US-00017 TABLE 10 aw *Protection at 1 2 3 5 7 9 factor over Sample T = 0 T = 0 Month Months Months Months Months Months the control 2 0.35 5.91.sup.E+09 3.15.sup.E+09 1.51.sup.E+09 9.00.sup.E+08 1.66.sup.E+06 9.50.sup.E+06 1.52.sup.E+07 n.a. (100%) (53%) (26%) (15%) (0.03%) (0.2%) (0.3%) 11 0.36 5.56.sup.E+09 4.23.sup.E+09 2.92.sup.E+09 2.05.sup.E+09 1.81.sup.E+09 1.35.sup.E+09 4.36.sup.E+08 24 (100%) (76%) (53%) (37%) (33%) (24%) (7.3%) 17 0.31 8.46.sup.E+09 5.6.sup.E+09 1.60.sup.E+09 2.58.sup.E+09 5.82.sup.E+08 Not 2.99.sup.E+09 117 (100%) (66%) (19%) (31%) (7%) measured (35.3%) 18 0.32 8.58.sup.E+09 4.905.sup.E+09 2.14.sup.E+09 2.49.sup.E+09 4.36.sup.E+08 2.68.sup.E+09 2.07.sup.E+09 80 (100%) (57%) (25%) (29%) (5%) (20%) (24%) n.a. non applicable (control) *Protection factor calculated at 9 month-storage, 30 C.

(96) When stored at 30 C., the protection offered by the coating of the present invention (samples 11, 17 and 18) was significantly enhanced compared to the uncoated dehydrated bacteria (sample 2).

(97) After 9 months storage at 30 C. in the chocolate coating of the Granola bar, the viability of L. acidophilus NCFM coated with the coating(s) according to the invention is significantly higher than the one obtained with uncoated microorganisms, for example, it is 1.5 LOG or 24 times higher after 9 months of storage with sample 11 (see FIG. 2 B). It is more than 2 LOG or more than 80 times higher for samples 17 and 18.

(98) In Table 11, the tested samples containing the microorganisms L. acidophilus were added directly into the cooled binding syrup and Cascadian Farms granola cereal, and mixed thoroughly, before making the bars and storing them at ambiant temperature. After a six-month storage, the stability of the L. acidophilus NCFM coated with the coating according to the invention (sample 11) was improved by a factor of 300 over uncoated bacteria.

(99) TABLE-US-00018 TABLE 11 aw *Protection at 1 3 6 factor over Sample T = 0 T = 0 Month Months Months the control 2 0.32 3.15.sup.E+08 2.23.sup.E+08 2.77.sup.E+07 8.83.sup.E+05 n.a. (100%) (71%) (9%) (0.3%) 11 0.32 1.78.sup.+08 2.14.sup.+08 2.51.sup.E+08 1.5.sup.E+08 290 (100%) (100%) (100%) (87%) n.a. non applicable (control) *Protection factor calculated at 6 month- storage, 25 C.

Example 8

Stability in SlimFast Optima French Vanilla Shake Mix

(100) The viability of L. acidophilus NCFM in freeze-dried form (sample 2) and in the coated particles (samples 11, 17 and 18) has been studied in the following conditions:

(101) The test samples were blended into SlimFast powder (commercial SlimFast Optima French Vanilla shake mix with aw=0.35) at a ratio of test sample to Slimfast powder of 1:10.

(102) The mixtures were then divided into 10 g samples in separate sealed hermetic sachets.

(103) The sachets were kept in an incubator at 30 C.

(104) At time 0 and after 3, 6, 9 and 12 months storage at 30 C., a sachet of each blend was analysed for the content of viable cells, by standard plating methods.

(105) TABLE-US-00019 TABLE 12 *Protection 3 6 9 12 factor over Sample Aw T = 0 T = 0 Months Months Months Months the control 2 0.35 2.5.sup.E+10 1.22.sup.E+10 2.2.sup.E+09 2.5.sup.E+08 1.1.sup.E+07 n.a. (100%) (49%) (9%) (1%) (0.04%) 11 0.36 1.37.sup.E+09 8.63.sup.E+08 5.03.sup.E+08 2.3.sup.E+08 7.0.sup.E+07 128 (100%) (63%) (36%) (17%) (5.1%) 17 0.31 8.46.sup.E+09 6.93.sup.E+09 2.23.sup.E+08 1.28.sup.E+08 4.2.sup.E+07 132 (100%) (82%) (29%) (17%) (5.3%) 18 0.32 8.58.sup.E+09 5.65.sup.E+09 4.23.sup.E+07 8.03.sup.E+06 1.8.sup.E+06 6 (100%) (66%) (6%) (1%) (0.25%) n.a. non applicable (control) *Protection factor calculated at 12 month-storage, 30 C.

(106) The results from Table 12 show that the presence of the coating according to the invention (samples 11, 17 and 18) results in improved stability over uncoated dehydrated bacteria (sample 2).

Example 9

Moisture Uptake Rate (MUR) Determination

(107) Glass desiccators containing saturated salt solutions of NaCl (in order to adjust the enclosed salt to 75% RH) were stored at 25 C. Various samples in open trays were loaded in the desiccators and incubated over time.

(108) The weight of the test material used is adjusted in order to ensure accurate weighing (e.g sample mass>2 grams) as well as a low sample thickness (e.g. less than 1 cm) to avoid moisture content heterogeneity within the test sample.

(109) For example, the MUR test on K.sub.2HPO.sub.4 salt was done by adding 4.7 grams of K.sub.2HPO.sub.4 salt in a plastic tray. The plastic tray had the following characteristics:

(110) Mass of the tray: 3.4 grams

(111) Area of the tray: 10.2 cm2 (circular tray with a diameter of 3.6 cm)

(112) Height of the tray: 1.5 cm

(113) For MUR experiments, the circular plastic tray is filled to half of its height with the sample to be tested. Subsequently, the moisture absorption of the sample was determined by weighing the samples before and during the incubation period at various time intervals. The MUR was determined for each measurement according to the equation 1.
MUR(t) in %=(mtmt0)/mt0Equation 1:

(114) MUR (t): percentage of the water uptake

(115) mt0: initial mass of sample

(116) mt: mass of sample at the measurement time

(117) Table 13 gives the moisture uptake rate (MUR) of various salts after 2, 5 and 7 days exposure at 25 C. and 75% RH:

(118) TABLE-US-00020 TABLE 13 Moisture uptake rate (MUR) of various salts measured at 75% RH, 25 C. at 2, 5 and 7 days (% moisture of the salt weight) Salts 2 Days 5 Days 7 Days Calcium chloride (CaCl2) 36.2% 63.4% 88.3% Sodium acetate anhydrous (CH3COONa) 36% 54% n.m. Magnesium chloride (MgCl.sub.2) 18.7% 41.8% 53.7% Potassium carbonate (K.sub.2CO.sub.3) 21.1% 37.9% 50.7% Dipotassium phosphate (K.sub.2HPO.sub.4) 21.0% 36.5% 45.6% Magnesium sulfate (MgSO.sub.4) 0.1% 0.2% 0.2% Calcium lactate (CH.sub.3CHOHCOOCa) 0.4% 0.7% 0.7% Sodium sulfate (Na.sub.2SO.sub.4) 0.0% 0.0% 0.1% Calcium carbonate (CaCO.sub.3) 0.0% 0.1% 0.1% Monopotassium phosphate (KH.sub.2PO.sub.4) 0.0% 0.0% 0.0% Sodium acetate trihydrate 0.0% 0.0% 0.0% ((CH.sub.3COO).sub.2Na3H2O) Tri-sodium citrate dihydrate 0.1% 0.2% 0.2% (HOC(COONa)(CH.sub.2COONa).sub.2)

(119) It can be concluded from this table that calcium chloride (CaCl.sub.2), sodium acetate anhydrous (CH3COONa), magnesium chloride (MgCl.sub.2) dipotassium phosphate (K.sub.2HPO.sub.4), potassium carbonate (K.sub.2CO.sub.3), and are considered as hygroscopic salts according to this invention as they have MUR values (25 C., 75% RH) at 7 days significantly above to 20% w/w.

(120) It can be concluded also from the same table that magnesium sulfate (MgSO.sub.4), calcium lactate ((CH3CHOHCOOCa), sodium sulfate (Na.sub.2SO.sub.4), calcium carbonate (CaCO.sub.3), monopotassium phosphate (KH.sub.2PO.sub.4), sodium acetate tri hydrate ((CH.sub.3COO).sub.2Na.3H.sub.2O) and tri-sodium citrate dihydrate (HOC(COONa)(CH.sub.2COONa).sub.2) are considered as non hygroscopic salts according to this invention as they have MUR values (25 C., 75% RH) at 7 days significantly less than 20% w/w.

(121) FIG. 3 shows the moisture uptake rate of coated dehydrated microorganisms samples. Glass desiccators containing saturated salt solutions of NaCl (in order to adjust the coated dehydrated microorganisms samples to 75% RH) were stored at 37 C. Various samples in open trays were loaded in the desiccators and incubated for 3 days. Subsequently, the moisture absorption of the samples was determined by weighing the samples before and during the incubation period at various time intervals. FIG. 3 shows the % weight gain of the coated dehydrated microorganisms as a function of time at 75% RH and 37 C. for 3 days.

(122) It can be concluded from FIG. 3, that a coating which contains either 40% or 48% hygroscopic salt (K.sub.2HPO.sub.4) (samples 10 and 11) is better suited to absorb moisture, than a coating comprising only 14% hygroscopic salt (K.sub.2HPO.sub.4) and 76% optional other ingredients (talc and sucrose) (sample 12). This difference in moisture absorption is related to amount of hygroscopic salts and/or to the structure of the coating containing the hygroscopic salts, e.g. to the partially crystalline degree of the coating once processed. Coatings with a too low amount of hygroscopic salt (i.e. under 25%) at the expense of a too high optional other ingredients content (i.e. above 70%, sample 12) or coatings using only non hygroscopic salts (i.e. Na.sub.2SO.sub.4 in sample 13, MgSO.sub.4 in sample 14), are inefficient to absorb adequate amounts of moisture.

Example 10

X-Ray Powder Diffractometry

(123) Investigation into the nature of salt coatings as it relates to the stabilization effects on dehydrated microorganisms. The crystalline vs amorphous nature of the different salt coating has been determined using X-ray diffraction analysis.

(124) Description of Method:

(125) An XRD-powder analysis is made by placing the material in the sample holder and the upper surface is leveled. The sample is placed in the X-ray diffractometer, where X-rays are focussed upon it. The X-rays are scattered depending on the arrangement of atoms in the sample. If the atoms are arranged in an ordered, repeating structure, as in crystals, the XRD pattern will show a series of sharp peaks. As every crystalline material produces a unique X-ray diffraction pattern, it is possible to determine the composition of a sample by subtracting known components from the sample pattern and then comparing the residual pattern with those in a library (e.g. The International Centre for Diffraction Data (ICDD) reference collection) or known reference patterns.

(126) Coated Dehydrated Microorganism: Coated dehydrated microorganism obtained by the liquid route, wherein the coating is K.sub.2HPO.sub.4 (48% dry), KH.sub.2PO.sub.4 (35% dry), talc (17% dry) and 0% sucrose, co-processed by spray-coating (sample 10) (FIG. 4weakly crystalline). Coated microorganism obtained by the liquid route, wherein the coating is K.sub.2HPO.sub.4 (40% dry), KH2PO4 (29% dry), talc (14% dry) and sucrose (17% dry), co-processed by spray-coating (sample 11) (FIG. 5weakly crystalline, salts not crystalline). Coated microorganism obtained by the liquid route, wherein the coating is K.sub.2HPO.sub.4 (14% dry), KH.sub.2PO.sub.4 (11% dry), talc (22% dry) and sucrose (54% dry) co-processed by spray-coating (sample 12) (FIG. 6weakly crystalline, small amount of crystalline sucrose as anhydrate, salts not crystalline) Coated micro-organism prepared by the liquid route but with a coating of one non hygroscopic salt (KH.sub.2PO.sub.4) with talc co-processed by spray-coating (sample 16) (FIG. 7Predominantly crystalline) Coated microorganism obtained by the solid route, wherein the coating is K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17%), co-processed by spray-coating (sample 20) (FIG. 8weakly crystalline, thus largely amorphous) Coated microorganism obtained by the solid route, wherein the coating is Na.sub.2SO.sub.4, co-processed by spray-coating (sample 21) (FIG. 9largely crystalline, thus weakly amorphous).

(127) The physical state of the coating produced according to the invention is shown to be partially crystalline. At high sucrose content (sample 12), the salts and sucrose mixture show the onset of sucrose crystallization, but there is no salt crystallization. However, as shown from FIG. 3, high sucrose content coatings (e.g. sample 12) reduced the MUR of the coated layer and therefore the benefits conferred by the low crystallinity solid state is significantly reduced.

Example 11

Degree of Crystallinity and Glass Transition Temperature by Differential Scanning Calorimetry (DSC)

(128) Method:

(129) The glass transition temperature of the coating and degree of crystallinity of the salt(s) in the coating of the present invention were obtained using DSC. A Netzch DSC 204 (Netzsch, Germany) equipped with an intracooler was used for all measurements with N.sub.2 as flushing agent. Samples of 10 mg were weighed into standard aluminium pans as quickly as possible to avoid water sorption. The pans were sealed and each sample was cooled and held at an initial temperature of 50 C. for 2 minutes. The samples were then heated from 50 C. to 330 C. at 12 C. min.sub.1 and held at 330 C. for 3 minutes.

(130) Preparation of Samples:

(131) Tests were performed on the dried coating produced under the same process conditions as those used to apply the coating onto the dehydrated microorganisms of the present invention. The coating analyzed in this test was not applied onto the primary particle to avoid interference from the core. Indeed, peaks from the core material were found to occur at the same temperature as some of the peaks of the coating materials

(132) 30 wt % aqueous solutions of the mono-potassium phosphate (KH.sub.2PO.sub.4) dipotassium phosphate (K.sub.2HPO.sub.4) and mixture of 45% K.sub.2HPO.sub.4, 35% KH.sub.2PO.sub.4, and 20% sucrose were prepared and each solution was atomized and dried in the fluid-bed, resulting respectively in sample A, B and C

(133) Table 14 described the composition of the dried salt coatings (samples A, B and C) analysed by DSC and Table 15 summarizes the process parameters used to these samples. The samples were post-dried for 15 min at product temperature of 55 C.

(134) TABLE-US-00021 TABLE 14 Dried salt coating composition KH.sub.2PO.sub.4 K.sub.2HPO.sub.4 Sucrose Sample (dry wt %) (dry wt %) (dry wt %) A 100% B 100% C 35% 45% 20%

(135) TABLE-US-00022 TABLE 15 Process parameters Values Step-up Bottom spray Nozzle 1.2 mm Inlet temperature 70 C. Product temperature 55 C. Atomization pressure 3.5 bars Air Flow 120 m3/h
Interpretation of DSC Data (Part 1):

(136) The degree of crystallinity of the salt(s) in the coating of the present invention was determined based on the heat of crystallization of peaks measured for samples A, B and C. The heat of crystallization will depend on the amount of crystalline material present in the sample. Interpretation of thermogram of these three samples are shown in FIGS. 10, 11 and 12. The crystalline melting point (onset temperature) and the heat of crystallization (area in J per g) were determined for each crystallization peak observed and are reported in Table 16.

(137) TABLE-US-00023 TABLE 16 Endothermic peaks (T C. and area in J/g) K.sub.2HPO.sub.4 KH.sub.2PO.sub.4 *Area in J/g *Area in J/g sample T C. (K2HPO4) T C. (KH.sub.2PO.sub.4) A 255.1 244.2 B 45.5 38.7 C 46.8 9.2 256.3 13.1 * area corresponds to heat of crystallization

(138) For KH.sub.2PO.sub.4, a crystalline melting point with onset T C.=255 C. and heat of crystallization 244.2 J/g was measured.

(139) For K.sub.2HPO.sub.4, a crystalline melting point with onset T C.=45 C. and heat of crystallization 38 J/g was measured.

(140) For the KH.sub.2PO.sub.4/K.sub.2HPO.sub.4/sucrose mixture, a peak was measured at 256.3 C. i.e. in the same T C. range as that of KH.sub.2PO.sub.4 but its heat of crystallization 13.1 J/g was lower than that measured for KH.sub.2PO.sub.4 (sample A) and a peak was measured at 46.8 C., i.e. in the same T C. range as that of K.sub.2HPO.sub.4 but its heat of crystallization 9.2 J/g was lower than that measured for K.sub.2HPO.sub.4 (sample B)

(141) Taking into account the quantity of each phosphate salt present in the mixture, e.g. 35 wt % KH.sub.2PO.sub.4 and 45 wt % K.sub.2HPO.sub.4, and if both salts were 100% crystalline in the mixture, each salt should result in a heat of crystallization of

(142) 85.5 J/g for KH.sub.2PO.sub.4 (85.5 J/g=244.2 J/g35 wt % of KH.sub.2PO.sub.4) and

(143) 17.4 J/g for K.sub.2HPO.sub.4 (17.4 J/g=38.745 wt % of K.sub.2HPO.sub.4)

(144) However, the measured values for the coating (sample C) were much lower than estimated if both salts were 100% crystalline in the mixture

(145) 13.1 J/g for the peak with onset 256.3 C. corresponding to KH.sub.2PO.sub.4 and

(146) 9.2 J/g for the peak with onset 46.8 C. corresponding to K.sub.2HPO.sub.4.

(147) The degree of crystallinity of each salt in the coating can hence be determined as following:

(148) 13.1/85.5=15.3%, e.g. KH.sub.2PO.sub.4 is 15.3% crystalline and 84.7% amorphous

(149) 9.2/17.4=52.9%, e.g. K.sub.2HPO.sub.4 is 52.9% crystalline and 47.1% amorphous

(150) In 1 g of the coating, there is 0.35 g of KH.sub.2PO.sub.4 and 0.45 g of K.sub.2HPO.sub.4

(151) 0.35 g15.3% crystallinity=0.053 g of KH.sub.2PO.sub.4 in a crystalline form

(152) 0.45 g52.9% crystallinity=0.238 g of K.sub.2HPO.sub.4 in a crystalline form

(153) Hence, the total amount of salts (KH.sub.2PO.sub.4+K.sub.2HPO.sub.4) in a crystalline form is 0.2916 g (0.053 g+0.238 g) per g of coating, so the crystallinity degree of the salts in the coating, once processed, is of 29.1%.

(154) Interpretation of DSC Data (Part 2):

(155) In addition, a glass transition temperature was measured for the sample C (35% KH2PO4, 45% K2HPO4 and 20% sucrose), as shown from FIG. 4. The presence of a Tg implies the presence of amorphous structure for the coating, validating the above approach using the heat of crystallization.

(156) Conclusion from DSC data interpretation: a semi-crystalline structure was identified for fluid-bed dried sample C composition with a glass transition temperature. The degree of crystallinity of the salt(s) in the coating has been quantified to around 29%.

Example 12

Water Vapor Sorption Isotherm

(157) Preparation of Samples:

(158) Coated dehydrated microorganisms were produced with various coatings following the process according to the invention. These coatings were the equivalent to the ones described in table 1, 1st coating, of sample references n.sup.o 10, 11, 13, 14, 15, 16, 23, 24, 25, 26, 27 and 28.

(159) The coating was applied either on the dehydrated microorganism (labelled + cells) or directly on the core in the absence of the dehydrated microorganism (labelled cells). The amount of coating applied was 30 wt %. The process parameters were:

(160) TABLE-US-00024 Process parameters Values Step-up Bottom spray Nozzle 1.2 mm Inlet temperature 65-70 C. Product temperature 55 C. Atomization pressure 2-2.5 bars Air Flow 120-200 m3/h
Method:

(161) In order to generate the sorption isotherms, each of the different samples were placed in a series of glass dessicators, each containing saturated salt solutions of certain salts, in order to adjust the samples to relative humidities (% RH) of 11.3%, 22.5%, 32.8%, 43% and 75%. Table 17 summarizes the salts used to achieve the targeted relative humidity at 25 C.

(162) TABLE-US-00025 TABLE 17 Salt LiCl KCH.sub.2CO.sub.2 MgCl.sub.2 K.sub.2CO.sub.3 NaCl % RH (25 C.) 11.3% 22.5% 32.8% 43% 75%

(163) The glass dessicator containing the samples were kept at constant temperature, e.g. 25 C. Each sample was weighed at regular interval (t). The Moisture Uptake Rate MUR(.sub.t) was determinate for each measurement according to the equation 1 (previously described in example 9) and an water vapour sorption isotherm was drawn (MUR(.sub.t)=f (t), were MUR (t) represent the percentage of the water uptake as measured by % weight gain of the sample at the time of measurement.

Brief Explanation of the Figures

(164) FIG. 13 is a water vapor sorption isotherm at 25 C. for microorganisms coated with different salt coating compositions, exposed to relative humidity conditions ranging from 11.3%, 22.5%, 32.8%, 43% and 75% RH at 25 C. for 28 days.

(165) FIGS. 14 to 19 concern water vapor sorption isotherms at 25 C. for particles including coated dehydrated microorganism exposed to various relative humidity conditions ranging from 11.3%, 22.5%, 32.8%, 43% and 75% RH for 28 days.

(166) The particles are composed of:

(167) the core only (.circle-solid.)

(168) the core+the cell layer (*)referred as reference sample

(169) the core+the salt coating ( )

(170) the core+cell layer+salt coating ()referred as product or coated dehydrated microorganism

(171) The salt coatings were the ones described as 1st coating in table 1, i.e. sample 11 (FIG. 14), sample 10 (FIG. 15), sample 15 (FIG. 16), sample 13 (FIG. 17), sample 12 (FIG. 18), sample 16 (FIG. 19).

(172) Data Interpretation (FIG. 13, Table 18 and Table 19):

(173) The water sorption curves of the water vapor sorption isotherms allow a quantification of the effect of humidity on the moisture content of the coated dehydrated microorganisms. The Moisture Uptake Rate of the coated dehydrated microorganism or product was measured at 25 C., 75% RH at 2, 7 and 28 days (Table 18). The MUR values presented in Table 19 were calculated for the coating according to equation 2.
MUR dehydrated coated microorganism(t) in %=(mtmt0)/mt0Equation 2:

(174) MUR (t): percentage of water uptake by dehydrated coated microorganisms at time T

(175) mt0: initial mass of dehydrated coated microorganism sample

(176) mt: mass of dehydrated coated microorganism at the time of measurement

(177) TABLE-US-00026 TABLE 18 Moisture uptake rate (MUR) of the coated dehydrated microorganism at 75% RH, 25 C. at 2, 7 and 28 days (% moisture of the coated dehydrated microorganism weight) 2 days 7 days 28 days Sample 25 10% 19% 39% Sample 15 8% 17% 26% Sample 10 6% 11% 19% Sample 11 5% 9% 15% Sample 14 5% 7% 9% Sample 23 3% 5% 10% Sample 24 3% 5% 8% Sample 13 0.6%.sup. 2% 6% Sample 16 0.5%.sup. 2% 5%

(178) The results from Table 18 show a big difference in how much water is absorbed by the coated dehydrated microorganism, depending on the salt coating used. Samples 25, 15, 10, 11 are highly hygroscopic, absorbing between 9 wt % and 19 wt % of moisture per weight of the coated dehydrated microorganism. Sample 25 (MgCl.sub.2) and sample 15 (K.sub.2HPO.sub.4) are the most hygroscopic. In addition, the water sorption profile in FIG. 13 shows a gradual increase in water content, which is characteristic of an amorphous or partially amorphous structure. In contrast, samples previously shown to offer little protection to the microorganism, e.g. sample 13 (Na.sub.2SO4), sample 14 (MgSO4), sample 16 (KH.sub.2PO.sub.4) absorbed negligeable amount of water.

(179) The MUR of the salt coating was determined based on equation 3 and is reported in Table 19.
MUR dehydrated coated microorganism(t) in %=(mtmt0)/mt0Equation 2:

(180) MUR (t): percentage of the water uptake by dehydrated coated microorganism at time T

(181) mt0: initial mass of dehydrated coated microorganism sample

(182) mt: mass of dehydrated coated microorganism at the time of measurement
MUR reference(t) in %=(mtmt0)/mt0Equation 3:

(183) MUR (t): percentage of the water uptake by the reference sample (the core+the cell layer) at time T

(184) mt0: initial mass of the reference sample

(185) mt: mass of the reference sample at the time of measurement
MUR salt coating(t) in %=[MUR product(t)(MUR reference(t)*wt % reference)]/wt % salt coatingEquation 4:
where wt % reference sample=70%
where wt % salt coating=30%

(186) TABLE-US-00027 TABLE 19 Moisture uptake rate (MUR) of the salt coating at 75% RH, 25 C. at 2, 7 and 28 days (% moisture of the coated salt layer weight) 2 days 7 days 28 days Sample 25 28% 54% 114% Sample 15 22% 46% 70% Sample 10 14% 27% 47% Sample 11 12% 20% 34% Sample 14 12% 14% 14% Sample 23 5% 9% 18% Sample 24 4% 9% 13% Sample 13 4% 2% 5% Sample 16 4% 3% 1%

(187) The results from Table 19 show a big difference in how much water is absorbed in the different coating layer depending on the salt used to coat the dehydrated microorganisms. Samples 25, 15, 10 and 11 absorb a high amount of moisture and exhibit a strong change in the water content of the coating. Sample 25 (MgCl.sub.2) and sample 15 (K.sub.2HPO.sub.4) are the most hygroscopic. In contrast, Na.sub.2SO.sub.4 (in sample 13), (MgSO.sub.4 (in sample 14) and KH.sub.2PO.sub.4 (in sample 16) absorbed negligeable amount of water and resulted in a very small change in its water content of the coating.

(188) The graphs from FIGS. 14 to 17 show significant differences in how much water is absorbed in particles with different cores materials (core only, core+cell layer) but identical salt coating.

(189) As shown in FIG. 14, FIG. 15 and FIG. 16, salt formulations containing hygroscopic salts (samples 10, 11 and 15) exhibit no difference in the quantity of moisture absorbed by particles with and without cells.

(190) As shown in FIG. 17, FIG. 18, FIG. 19, salt formulations with less than 25% of hygroscopic salts (sample 12) or composed of only non hygroscopic salts (samples 13 and 16), exhibit a significant difference in the quantity of moisture absorbed with and without cells. The particle with the cell layer (e.g. the coated dehydrated microorganisms) absorbs a greater amount of moisture compared to particles without the cell layer. This results shows that if the coating allows moisture to pass, the underlaying material will absorb greater amount of moisture. This is seen for samples containing the cell layer when the coating contains less than 25% of hygroscopic salts (sample 12) or do not contain any hygroscopic salts but non-hygroscopic salt instead (samples 13 and 16). These samples do not maintain cell viability under stressful conditions.

Example 13

The Effect of pH of the Salt Coating on the Survival of the Coated Dehydrated L acidophilus NCFM

(191) Results from the effect of pH of the salt coating on the survival of the coated dehydrated L acidophilus NCFM are presented in Table 20.

(192) K.sub.2HPO.sub.4 is an alkaline salt and the pH of the K.sub.2HPO.sub.4 solution prior to coating was adjusted to a pH=6.5 using lactic acid. This resulted in a reduction of pH of the coated dehydrated microorganisms from pH 8 to pH 6.5 when measured as 10 wt % solution of the coated dehydrated microorganism in water. A significant improvement in the resistance of L. acidophilus NCFM was observed: i) during fluid-bed coating with process survival for L. acidophilus NCFM raised from 65% to 90%, ii) after 14 days of storage at 37 C. in dry conditions with recovery of viable L. acidophilus NCFM increased from 7% to 83%, iii) after heat treatment (64 C./18 hrs) with LOG loss for L. acidophilus NCFM reduced from more than 5.3 to 0.6.

(193) K.sub.2HPO.sub.4 coating with pH adjusted to 6.5 remained hygroscopic with a moisture uptake for the coated dehydrated microorganism of 12.8 wt % moisture absorbed prior to pH adjustment and 11.6 wt % moisture absorbed after pH adjustment. (Conditions: 75% RH, 25 C., 6 days).

(194) These results demonstrate that it is needed to have the pH of the coating compatible with the viability of the microorganism.

(195) KH.sub.2PO.sub.4 is an acid salt and the pH of the KH.sub.2PO.sub.4 solution prior to coating was adjusted to pH 6.5 using Sodium Hydroxide (NaOH). This resulted in an increase of pH from pH 5.0 to pH 6.5 when measured after suspended the coated dehydrated microorganism into 10 wt % of water. A significant reduction in the viability loss of L. acidophilus NCFM was observed:

(196) i) during fluid-bed coating with process survival of L. acidophilus NCFM was raised from 47% to 100%,

(197) ii) after 14 days of storage at 30 C. in dry conditions with an increase in the recovery of viable L. acidophilus NCFM from 2% to 95%.

(198) iii) after heat treatment (64 C./18 hrs) with LOG loss for viable L. acidophilus NCFM reduced from >5.0 to 0.6

(199) iv) storage in maltodextrine Aw=0.4 (37 C./9 days) with an increase in the recovery of viable L. acidophilus NCFM from 1% to 44%.

(200) The hygroscopicity of KH.sub.2PO.sub.4 coating with pH adjusted to 6.5 could be dramatically increased with moisture uptake of the coated dehydrated microorganism going from 1.3% prior to pH adjustment to 12.8 wt % moisture absorbed after pH adjustment with NaOH (conditions: 75% RH, 25 C., 6 days). These results showed that by pH adjustment it was possible to obtain a coating suitable by transformation of the non-hygroscopic nature into a hygroscopic one, hereby enabling stabilization of L. acidophilus NCFM in various stress conditions.

(201) TABLE-US-00028 TABLE 20 Viability % MUR coated Stability loss (log), recovery dehydrated Process (14 days heat (9 days, micoroorganisms 1st pH survival >30 C., (64 C./18 37 C., aw (75% RH, 25 C., sample coating * % dry) hrs) 0.4) 6 days) 15 K.sub.2HPO.sub.4 8.0 65% 7% >5.3 2% 12.8% 27 K.sub.2HPO.sub.4 6.5 90% 83% 0.6 Not 11.6% and pH measured adjusted 16 KH.sub.2PO.sub.4 5.0 47% 2% >5.0 1% 1.3% 28 KH.sub.2PO.sub.4 6.5 100% 95% 0.6 44% 12.8% and pH adjusted * pH of 10% solution of coated dehydrated microorganisms in water

Example 14

Solid Route

(202) The viability of dehydrated L. acidophilus NCFM in freeze-dried form and in the coated particles (solid route) has been studied in the conditions described in example 6.

(203) The bacteria preparation was mixed into the maltodextrin powder (aw=0.4) at a ratio of 10% wt of coated bacteria and 90% wt of maltodextrin powder. The vials were kept at 30 C. in an incubator. After 2 months storage at 30 C., a vial of the mixture was analyzed for its content of viable cells by standard plating methods. The results are listed in Table 21. The concentration of viable cells is expressed as CFU/g and as a percentage of the concentration of each samples at T=0.

(204) TABLE-US-00029 TABLE 21 Protection factor % over Sample T = 0 2 months survival control 2 2.52E + 11 9.78E + 08 0.4% n.a. 20 1.84E + 11 5.30E + 10 29% 74 n.a. non applicable (control) * Protection factor calculated at 2 month- storage, 30 C.

(205) The coating of the invention (sample 20: K.sub.2HPO.sub.4 (40% dry), KH.sub.2PO.sub.4 (29% dry), talc (14% dry) and sucrose (17% dry)) applied onto freeze-dried L. acidophilus NCFM powder is very effective in maintaining the viability of L. acidophilus NCFM at intermediate moisture. The effectiveness of the coating of the invention in stabilizing dehydrated microorganisms was previously demonstrated for the coated dehydrated microorganism prepared according to the liquid route. It is now demonstrated in this example that the coating of the invention is also effective for coated dehydrated microorganism prepared according to the solid route.

Example 15

Stability in Infant Formula

(206) The viability of L. acidophilus NCFM in freeze-dried form (sample 2) and in the coated dehydrated microorganisms according to the present invention (sample 11) has been studied in the following conditions:

(207) The test samples were blended into commercial Good Sense Gentle Plus Instant Baby Formula from Nestle (Aw=0.157) at a ratio of test sample to baby formula powder of 1:10. The mixtures were then divided into 10 g samples in separate sealed hermetic sachets. The sachets were kept in an incubator at 30 C. At time 0 and after 1, 3, 6 and 9 months of storage at 30 C., a sachet of each blend was analysed for the content of viable cells, by standard plating methods.

(208) TABLE-US-00030 TABLE 22 *Protection 1 3 6 9 factor over Sample Aw T = 0 T = 0 Months Months Months Months control (2) 2 0.17 2.7.sup.E+10 1.2.sup.E+10 1.1.sup.E+10 1.0.sup.E+10 1.7.sup.E+09 n.a. (100%) (43.6%) (39.6%) (36.6%) (6%) 11 0.15 1.4.sup.E+09 1.45.sup.E+09 1.47.sup.E+09 l.2.sup.E+09 1.2.sup.E+08 14 (100%) (104%) (105%) (85%) (86%) n.a. non applicable (control) *Protection factor calculated at 9 month- storage, 30 C.

(209) The results from Table 22 show that the presence of the coating according to the invention (samples 11) improves stability over uncoated dehydrated bacteria (sample 2).