Microencapsulated probiotic substance and process of manufacture

Abstract

Dried powder solid particles are disclosed containing a probiotic microorganism and a carrier phase wherein the probiotic microorganism is encapsulated, and wherein the carrier phase further comprises at least a nutritious source. The dried powder solid particles have a particle size distribution between n and (n+400) m, wherein n is comprised between 100 and 10,000 m, preferably between 300 and 5000 m, more preferably between 400 and 1000 m.

Claims

1. A dried powder composition comprising solid particles containing: a) a probiotic microorganism, b) a carrier phase wherein said probiotic microorganism is encapsulated, said carrier phase further comprising at least a nutritious source as well as an enteric composition, wherein said dried powder composition presents a particle size distribution between n and (n+400) m, wherein n is comprised between 100 and 10,000 m, and said solid particles are spherical particles comprising 50 to 80 wt. % of said probiotic microorganism, and wherein said carrier phase comprises a mixture of alginate and pullulan.

2. The dried powder solid particles according to claim 1, wherein said particle size distribution is between n and (n+200) m wherein n is comprised between 100 and 10,000 m.

3. The dried powder solid particles according to claim 1, wherein each of the solid particles presents a homogeneous composition.

4. The dried powder solid particles according to claim 1, wherein said nutritious source comprises at least a compound selected from the group consisting of a monosaccharide, a polysaccharide, an aminoacid, a peptide, a protein, a vitamin, a yeast extract, a halogen salt of an alkali or earthalkali metal, an antioxidant, glycerol, zinc acetate, zinc chloride, zinc lactate, ascorbic acid, citric acids or a vegetable oil and milk fat.

5. The dried powder solid particles according to claim 1, wherein said nutritious source is present in an amount from 1 to 5 wt. % with respect to the total weight of the dried powder solid particles.

6. The dried powder solid particles according to claim 1, further comprising an external coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatine, carageenan, agar, cellulose, hemicellulose, ethylcellulose, carboxymethylcellulose and their mixture.

7. The enteric composition comprising said dried powder solid particles according to claim 1 in a suitable vehicle.

8. The enteric composition according to claim 7, wherein said suitable vehicle is an enteric coating selected from the group consisting of ethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, and methacrylic acid-ethyl acrylate copolymer.

9. The enteric composition according to claim 7, in the form of a soft or hard capsule, tablet, or sachet.

10. A process for the manufacture of dried powder composition comprising solid particles containing a probiotic microorganism in the form of spherical particles comprising the following steps: mixing a preparation of probiotic microorganisms and a carrier phase comprising at least a nutritious source, wherein the carrier phase comprises at least one substance selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatine, carrageenan, agar, and wherein said nutritious source comprises at least a compound selected from the group consisting of a monosaccharide, a polysaccharide, an aminoacid, a peptide, a protein, a vitamin, a yeast extract, a halogen salt of an alkali or earthalkali metal, an antioxidant, glycerol, zinc acetate, zinc chloride, zinc lactate, ascorbic acid, citric acids or a vegetable oil and milk fat, extruding the mixture of said probiotic microorganisms and said carrier phase to produce microspheres, and collecting said microspheres into a bath containing a solidification solution, and wherein said extrusion step is performed at a predetermined speed of liquid flow of 0.2 to 5 m/s through at least one vibrating nozzle in a laminar flow drip casting to obtain said dried powder particles under the form of spherical particles, said vibrating nozzle having a vibration frequency in a range of 1 to 20,000 Hz. and a vibration amplitude of at least 0.5 m.

11. The process according to claim 10, wherein the laminar flow drip casting from at least one vibrating nozzle is obtained with a vibrational support.

12. The process according to claim 10, wherein the vibration of the vibrating nozzle is orientated in an axial or a lateral direction with respect to the flow to generate droplets.

13. The process according to claim 10, wherein the said produced spherical particles have a diameter in the range of 100 to 10,000 m.

14. The process according to claim 10, wherein two liquids are extruded in a laminar flow with one or multiple double nozzle systems comprising an inner nozzle and an outer nozzle.

15. The process according to claim 10, further comprising an additional step of encapsulating the extruded mixture.

16. The process according to claim 10, further comprising an external coating step.

Description

Example 1

Alginate-EC Beads and Probiotics Paste

(1) Microspheres of L. Rhamnosus in a matrix made of alginate and with an ethylcellulose external coating have been made with the following protocole:

(2) 150 g of L. Rhamnosus paste (510.sup.10 cfu) was dispersed in 150 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a L. Rhamnosus suspension.

(3) 150 g of a 5% w/w sterile alginate solution was added to 300 g of the L. Rhamnosus suspension

(4) Drip casting with a laminar flow break-up unit was performed to produce 800 m microspheres by solidification in a 4% w/w CaCl.sub.2 solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(5) 400 g of microspheres were stirred for 1 minute in 400 g of a solution of 1% w/w ethyl cellulose in Ethanol to produce the ethylcellulose coating (EC coating). The separation and washing of the coated microsphere was done in 0.85% w/w NaCl solution. 380 g of microspheres were stored in 380 g of a sterile aqueous solution of 5% w/w glucose before freeze drying in said glucose storage solution. A dry free flowing powder of microspheres of 700-900 m in diameter was obtained.

Example 2

Alginate-Gelatin Beads and Probiotic Paste

(6) Microspheres of L. Rhamnosus in a matrix of alginate and with a gelatine coating has been made following the under mentioned protocol:

(7) 200 g of L. Rhamnosus paste (510.sup.10 cfu) was dispersed in 200 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a L. Rhamnosus suspension.

(8) 200 g of a 5% w/w sterile alginate solution was added to 400 g of the L. Rhamnosus suspension

(9) Drip casting with a laminar flow break-up unit was performed to produce 500 m microspheres by solidification in a 5% w/w calcium lactate solution.

(10) The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(11) 550 g of microspheres were stirred for 1 h in 550 g of a 5% w/w gelatin solution for producing the crosslinked gelatine coating.

(12) The microspheres were then stirred for 2 minutes in 550 g of a 10% w/w glutaraldehyde solution. The separation and washing of the coated microspheres was done in 0.85% w/w NaCl solution.

(13) 550 g of microspheres were stored in 550 g of a sterile aqueous solution of 10% w/w maltodextrin before freeze drying in the maltodextrin storage solution:

(14) A dry free flowing powder of microspheres of 400-600 m in diameter was obtained.

Example 3

Alginate-CMC-Gelatin Beads with Probiotic Paste

(15) Microspheres of L. Rhamnosus in a matrix of alginate and with a carboxy methylcellulose coating and a gelating crosslinked coating have been made as follows:

(16) 300 g of L. Rhamnosus paste (510.sup.10 cfu) was dispersed in 150 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a L. Rhamnosus suspension.

(17) 75 g of a 10% w/w sterile alginate solution was added to 450 g of the L. Rhamnosus suspension

(18) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by solidification in a 3% w/w calcium gluconate solution. The separation and washing of the microsphere was done in 0.85% w/w NaCl solution

(19) 500 g of microspheres were stirred for 10 minutes in 500 g of an aqueous solution of 2% carboxymethylcellulose. The coated microspheres have further been separated and washed in 0.85% w/w NaCl solution.

(20) 500 g of microspheres were further stirred for 1 h in 500 g of a 5% w/w gelatin solution, to produce the crosslinked gelatine coated on the microspheres.

(21) The microspheres were then stirred for 2 minutes in 500 g of a 10% glutaraldehyde solution and separated and washed in 0.85% w/w NaCl solution.

(22) 500 g of microspheres were stored in 500 g of a sterile aqueous solution of 10% w/w glycerol and freeze dryed in the glycerol storage solution:

(23) A dry free flowing powder of microspheres of 800-1200 m in diameter was obtained.

Example 4

Gelatin-Guar Gum-CMC-Beads with Probiotic Paste

(24) Microspheres of Bifidobacterium Lactis in a matrix of gelatine, coated with guar gum and carboxymethylcellulose have been made as follows:

(25) 200 g of Bifidobacterium Lactis paste was dispersed in 100 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a Bifidobacterium Lactis in suspension.

(26) 150 g of a sterile 30% gelatin solution was added to 300 g of the Bifidobacterium Lactis suspension at 37 C.

(27) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by soldification in caprylic/capric triglyceride at 5 C. The separation and washing of the microsphere was done in 0.85% w/w NaCl solution.

(28) 400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 5% w/w guar gum to produce the coated microspheres with guar gum. The coated microsphere were then separed and washed in 0.85% w/w NaCl solution.

(29) 400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 2% carboxymethylcellulose for building the CMC coating. The microspheres were then separated and washed in 0.85% w/w NaCl solution.

(30) 400 g of microspheres were stored in 400 g of a sterile aqueous solution of 4% w/w glycerol before freeze drying in this glycerol storage solution:

(31) A dry free flowing powder of microspheres of 800-1200 m in diameter was obtained.

Example 5

Alginate-Chitosan-Gelatin Beads with Probiotic Paste

(32) Microspheres of L. Rhamnosus in a matrix of alginate and wjtb a chitosan coating with further gelatine coating was made as follows:

(33) 400 g of L. Rhamnosus paste (510.sup.10 cfu) was dispersed in 200 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a L. Rhamnosus suspension.

(34) 100 g of a 10% sterile alginate solution was added to 600 g of the L. Rhamnosus suspension.

(35) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by soldification in a 2% w/w CaCl.sub.2 solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(36) 600 g of microspheres were stirred for 10 minutes in 1200 g of an aqueous solution of 1% w/w chitosan for manufacturing chitosan coated microspheres. The separation and washing of the coated microspheres was done in 0.85% w/w NaCl solution.

(37) 600 g of microspheres were stirred for 1 h, in 1200 g of a 5% w/w gelatin solution to further coat the microsphere with gelatine. The separation and washing of the coated microspheres was done in 0.85% w/w NaCl solution.

(38) 600 g of microspheres were stored in 600 g of a sterile aqueous solution of 4% w/w glycerol before freeze drying in the glycerol storage solution:

(39) A dry free flowing powder of microspheres of 800-1200 m in diameter was obtained.

Example 6

Alginate Beads with Probiotic Paste

(40) Microspheres of L. Rhamnosus in a matrix of alginate has been made as follows:

(41) 200 g of L. Rhamnosus paste (510.sup.10 cfu) was dispersed in 150 g of a sterile solution of 6.7% w/w polysaccharide and 0.85% w/w NaCl to form a L. Rhamnosus suspension.

(42) 230 g of a 3% w/w sterile alginate solution was added to 350 g of the L. Rhamnosus suspension.

(43) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by solidification in a 2% w/w CaCl.sub.2 solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(44) 550 g of microspheres were stored in 550 g of a sterile aqueous solution of 5% w/w glucose and 3% w/w glycerol before freeze drying in the glycerol storage solution:

(45) A dry free flowing powder of microspheres of 400-900 m in diameter was obtained.

(46) The enumeration of the viable bacteria in microspheres is made as follows:

(47) Two samples were prepared with the alginate L. Rhamnosus microsphere, one from the dried powder and one in a wet state: Sample 1: Lactobacillus rhamnosus microspheres, diameter ca 400-900 microns, dried with glucose/glycerol. Sample 2: Lactobacillus rhamnosus microspheres, diameter ca 800-1200 microns, wet in glucose/glycerol solution.

(48) The Microspheres have to be dissolved before enumeration of viable bacteria. Dissolution procedures were adapted to differences during the drying stage of the microspheres:

(49) Sample 1 has been prepared by aseptically weighing 100 mg of dried microspheres to a 15 ml conical sterile tube and adding 2.9 ml Na citrate 0.1M. The mixture is vortexed for 15 minutes (dilution 30).

(50) Sample 2 has been prepared by first separating the microspheres from the storage solution (glucose/glycerol solution) with a sterile sieve (whatman filter paper). 100 mg of wet microspheres were added to a 15 ml conical sterile tube with 1.9 ml Na citrate 0.1M. The mixture is vortexed for 3 minutes (dilution 20)

(51) Sample dissolution was made in duplicate for both samples.

(52) 15 ml of the MRS agar have been poured approximately into each plate and allowed for solidification at room temperature on a cool level surface.

(53) In sterile tubes filled with sterile 9 ml 0.1% peptone dilution blanks 1 ml of the primary dilution (from the conical tube) is added to the 9 ml of diluent with a 1 ml pipette so as to obtain a 10.sup.1 dilution. This operation is repeated until the desired dilution series is obtained. Dilution tubes shaked as stated in standard Methods for the examination of dairy products The experiments are made in triplicate. 0.1 ml of each appropriate dilution is transferred on the surface of labelled, sterile Petri plates poured with circa 15 ml MRS agar nutrient medium. The plates were incubated at 35 C. for minimum 72 hours till 144 hours.

(54) Count colonies on the MRS agar plates and record as viable Lactobacillus rhamnosus cell count per gram, taking into account the dilution factor of the counted plates. Only plates having between 25 and 250 colonies should be counted. (See Standard methods for the examination of dairy products, 16.sup.th edition, pages 213-246).

(55) Results

(56) Initial Weigh:

(57) Sample 1: duplicate 1: 100 mg duplicate 2: 103 mg Average: 101.5 mg. Sample 2: duplicate 1: 102 mg duplicate2: 99 mg Average: 100.5 mg
Initial Dilution Rate: Sample 1: 101.5 mg in 2.9 ml=dilution 29.6. Sample 2: 100.5 mg in 1.9 ml=dilution 19.9
Count cfu (Colony-Forming-Unit):

(58) TABLE-US-00001 Dilution Dilution Dilution Dilution 10.sup.5 10.sup.4 10.sup.3 10.sup.2 Replication 1 2 3 1 2 3 1 2 3 1 2 Sample 1 Duplicate 1: 3 6 1 18 6 19 299 192 279 > > Duplicate 2: 1 1 1 17 12 16 151 114 160 > > Sample 2 Duplicate 1: 8 6 8 32 88 52 171 203 > > Duplicate 2: 8 6 35 85 39 ? 123 > >
The Results of the Enumeration is as Follows: Sample 1: 192.10.sup.329.6=5.68 10.sup.6 cfu/g microspheres (dry weight) Sample 2: 635.10.sup.319.9=1.26 10.sup.7 cfu/g microspheres (wet weight)

(59) As it can be seen, with a correct choice of diameter and nutrious agent, a survival rate of 1:1000 can be achieved during all processing. While the larger diameter preserves a higher number of living cells through the process, the yield of living microorganisms of more than 1.10.sup.7 cfus is sufficient for a probiotic effect.

Example 7

Alginate-CMC-Gelatin Beads with Lyophilised Probiotics

(60) Microspheres of L. rhamnosus in an alginate matrix coated with carboxymethylcellulose and gelatine has been manufactured as follows:

(61) 150 g of lyophilized L. rhamnosus powder (8.810.sup.11 cfu/g) was dispersed in 300 g of sterile NaCl solution (0.85% w/w NaCl) to form a L. rhamnosus suspension. The L. rhamnosus provided are therefore 1.32.10.sup.14 cfu in 150 g.

(62) 75 g of a 10% w/w sterile alginate solution was added to 450 g of the L. rhamnosus suspension

(63) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by solidification in a 3% w/w calcium gluconate solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(64) 500 g of microspheres were stirred for 10 minutes in 500 g of an aqueous solution of 2% w/w carboxymethylcellulose to obtain the CMC coating. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(65) Further, 500 g of microspheres were stirred for 1 h, in 500 g of a 5% w/w gelatin solution, and the microspheres were then stirred for 2 minutes in 500 g of a 10% w/w glutaraldehyde solution for reaching a crosslinked gelatine coating. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(66) 500 g of microspheres were stored in 500 g of a sterile aqueous solution of 10% w/w glycerol before freeze drying in storage solution. After double coating and crosslinking which do not absorb all coating material but only a small quantity of 0.1-1%, the spheres have been dried and 50 g of glycerol was added (500 g of 10% w/w glycerol), yielding to a dry matter of 203.93 g, under the form of a dry free flowing powder of microspheres of 800-1200 m in diameter with a cell count of 2.910.sup.11 cfu/g was obtained. This means that from the 1.32.10.sup.14 cfu engaged from the lyophilized L. rhamnosus, it still remains 0.61.10.sup.14 cfu (203.93 g. 2.9.10.sup.11). Consequently, the yield of the living probiotics is about 50% being drastically higher than with the process of the prior art.

Example 8

Alginate-EC Beads with Lyophilised Probiotics

(67) Microspheres of L. rhamnosus in an alginate matrix coated with ethylcellulose has been manufactured as follows:

(68) 67.5 g of lyophilized L. rhamnosus powder (8.810.sup.11 cfu) were dispersed in 217.5 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a suspension of L. rhamnosus.

(69) 150 g of a 5% sterile alginate solution was added to 300 g of the L. rhamnosus suspension;

(70) Drip casting with a laminar flow breakup unit was performed to produce 800 m microspheres by solidification in a 4% w/w CaCl.sub.2 solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(71) Further, 400 g of microspheres were stirred for 1 minute in 400 g of a solution of 1% w/w ethyl cellulose in ethanol to prepare EC coated microspheres separation and washing of the microspheres are made in 0.85% w/w NaCl solution.

(72) 380 g of microspheres were stored in 380 g of a sterile aqueous solution of 5% w/w glucose before freeze drying in the glucose storage solution:

(73) 96.32 g of a dry free flowing powder of microspheres of 700-900 m in diameter with a cell count of 1.910.sup.11 cfu was obtained. That means that from the 5.94.10.sup.13 cfu engaged at the first step, there remains 1.8310.sup.13 cfu of (8.8.10.sup.1167.5) living probiotics (96.321.910.sup.11) corresponding to about 31% of probiotics kept alive.

Example 9

Alginate-Gelatin Beads with Lyophilised Probiotics

(74) Microspheres of L. rhamnosus in an alginate matrix coated with gelatine has been manufactured as follows:

(75) 100 g of lyophilized L. rhamnosus powder (8.810.sup.11 cfu) was dispersed in 300 g of sterile NaCl solution (0.85% w/w NaCl) to form a L. rhamnosus suspension. The probiotics engaged therefore of 8.8.10.sup.13 cfu for preparing the alginate gelatine beads.

(76) 200 g of a 5 w/w sterile alginate solution was added to 400 g of the L. rhamnosus suspension.

(77) Drip casting with a laminar flow breakup unit was performed to produce 500 m microspheres by solidification in a 5% w/w calcium lactate solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(78) 550 g of microspheres were stirred for 1 h, in 550 g of a 5% w/w gelatin solution for obtaining a crosslinked gelatine coating.

(79) The microspheres were then stirred for 2 minutes in 550 g of a 10% w/w glutaraldehyde solution. The separation and washing of the microspheres was done in 0.85% w/w NaCl solution.

(80) 550 g of the microspheres were stored in 550 g of a sterile aqueous solution of 10% maltodextrin before freeze drying in the maltodextrin storage solution:

(81) 168.25 g of a dry free flowing powder of microspheres of 400-600 m in diameter with a cell count of 8.510.sup.10 cfu was obtained, corresponding to 1.4310.sup.13 cfu in the 168.25 g.

(82) As conclusion, an alginate-gelatine microcapsule with a crosslinked coating has a substantially high number of surviving microorganisms, essentially for a commercial viable process since the ration of probiotics kept alive is 16.25 yielding to a powder containing 8.910.sup.10 cfu (largely greater than 10.sup.7 cfu required).

Example 10

Gelating-Guar Gum-CMC BeadsLyophilised Probiotic

(83) Microspheres of Bifidobacterium lactis in a matrix of gelatine coated with guar gum and carboxymethylcellulose was prepared as follows:

(84) 100 g of lyophilized Bifidobacterium lactis powder was dispersed in 200 g of sterile NaCl solution (0.85% w/w NaCl) to prepare a Bifidobacterium lactis suspension.

(85) 150 g of a sterile 30% w/w gelatin solution was added to 300 g of Bifidobacterium lactis suspension at 37 C.

(86) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by solidification in caprylic/capric triglyceride at 5 C. The separation and washing of the microspheres was done in 0.85% NaCl solution.

(87) 400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 5% w/w guar gum to coat the microspheres with guar gum and the separation and washing of the microspheres is carried out in 0.85% w/w NaCl solution.

(88) 400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 2% w/w carboxymethylcellulose to coat the microspheres with CMC and the separation and washing of the microspheres is carried out in 0.85% w/w NaCl solution.

(89) 400 g of microspheres were stored in 400 g of a sterile aqueous solution of 4% w/w glycerol before freeze drying in the glycerol storage solution:

(90) A dry free flowing powder of microspheres of 800-1200 m in diameter with a cell count of 2.910.sup.11 cfu was obtained being higher than the 10.sup.7 value required for such application.

(91) A conclusion it was shown that the carboxy methyl cellulose coating with glycerol as nutritious source during freeze drying yields very high survival rates in an enteric microsphere.

Example 11

Alginate-Chitosan-Gelatin Beads with Lyophilised Probiotics

(92) Microspheres of L. rhamnosus in an alginate matrix coated with chitosan and gelatine has been manufactured as follows:

(93) 200 g of lyophilized L. rhamnosus powder (8.810.sup.11 cfu) was dispersed in 400 g of sterile NaCl solution (0.85% w/w NaCl) to form a L. rhamnosus suspension (1.76.10.sup.14 cfu of L. rhamnosus engaged).

(94) 100 g of a 10% sterile alginate solution was added to 600 g of the L. rhamnosus suspension.

(95) Drip casting with a laminar flow breakup unit was performed to produce 1000 m microspheres by solidification in a 2% w/w CaCl.sub.2 solution. The separation and washing of the microspheres was carried out in 0.85% w/w NaCl solution.

(96) 600 g of microspheres were stirred for 10 minutes in 1200 g of an aqueous solution of 1% w/w chitosan and the separation and washing of the microspheres was carried out in 0.85% w/w NaCl solution.

(97) 600 g of microspheres were further stirred for 1 h, in 1200 g of a 5% w/w gelatin solution and the separation and washing of the microspheres was carried out in 0.85% w/w NaCl solution.

(98) 600 g of microspheres were stored in 600 g of a sterile aqueous solution of 4% w/w glycerol before freeze drying in the glycerol storage solution:

(99) 238.8 g of a dry free flowing powder of microspheres of 800-1200 m in diameter with a cell count of 2.910.sup.11 cfu was obtained, corresponding to a total of 0.6910.sup.14 cfu (yield of living probiotics=39.3%).

(100) As conclusion, it was shown that the chitosan coating with glycerol as nutritious source during freeze drying yields very high survival rates in an enteric microsphere.

Example 12

Alginate Beads with Lyophilized Probiotics

(101) 75 g of lyophilized L. rhamnosus powder was dispersed in 250 g of a sterile solution of 3.6% w/w polysaccharide and 0.85% w/w NaCl to form a L. rhamnosus suspension.

(102) 175 g of a 5% w/w sterile alginate solution was added to 325 g of the L. rhamnosus suspension

(103) Drip casting with a laminar flow breakup unit was performed to produce 1100 m microspheres by solidification in a 2% w/w CaCl.sub.2 solution. The separation and washing of the microspheres is carried out in 0.85% w/w NaCl solution.

(104) 450 g of microspheres were stored in 450 g of a sterile aqueous solution of 5% w/w glucose.

(105) The enumeration made as previously described reveals 8.1 10.sup.9 cfu/g microspheres wet weight. A content of 1.87.10.sup.11 cfu/g was present in the lyophilised powder, instead of the 45.10.sup.11 declared. As the starting lyophilized powder represents 15% of the total weight of the wet microspheres, this content corresponds to (8.1.10.sup.9100) % 15=5.410.sup.10 cfu/g equivalent powder.

Example 13

Alginate Beads with Freeze Dried Bifidobacterium lactis

(106) The following mixtures were prepared: 7.5% of B. lactis (Bifido 300 Blyophilized probiotic powder Bif. Lactis) 1.5% alginate at a concentration of 5% w/w 89.5% NaCl solution at a concentration of 0.85% w/w 1.5% of one of the following additive used as carriers Brace Mix=pullulan Starch 1 Starch 2 Dextrin Na CML Cellulose (CMC) Hydroxypropyl methylcellulose (HPMC) Microcrystalline cellulose (MC)

(107) Drip casting with laminar flow breakup unit was performed to produce 1100 m microspheres by solidification in a 4% w/w CaCl.sub.2 solution. The separation and washing of the microspheres is carried out in 0.9% w/w NaCl solution.

(108) Microspheres were then directly plated (fresh beads) for CFU count or were frozen in nitrogen at 196 C. and then freeze dried at 50 C. before being plated (dried beads) for CFU count. After incubation at 37 C. during 72 h, CFU was determined for fresh beads and dried beads, as previously described.

(109) The results obtained are presented in the Table below.

(110) TABLE-US-00002 Cell count Sample Form (average CFU/gram) Brace Mix Fresh 3.43 10.sup.14 Dried 3.3 10.sup.11 HPMC Fresh 1.9 10.sup.12 Dried 8.2 10.sup.11 CMC Fresh 1.5 10.sup.10 MC Fresh 3.7 10.sup.09 Starch 1 Fresh 4.1 10.sup.09 Starch 2 Fresh 3.8 10.sup.09 Dextrin Fresh 2.9 10.sup.06 The CFU count made as previously described reveals that Brace Mix (fresh and dried forms), HPMC (fresh and dried forms), CMC (fresh form), MC (fresh form), Starch 1 (fresh form) and Starch 2 (fresh form) used as carriers (1.5% of the mixture) ensure a viability of B. lactis (7.5% of the mixture) of at least 10.sup.09 CFU/gram.