Probiotics for aggregation with disease-associated species in the oral cavity

Abstract

The present invention relates to certain microorganisms or mixtures thereof for use in the treatment and/or prevention of inflammation in the oral cavity, preferably for the treatment and/or prevention of gingivitis and/or peridontitis. In particular, the present invention relates to microorganisms or mixtures thereof for use as probiotic agents for forming co-aggregates with microorganisms associated with gingivitis and/or peridontitis. Furthermore, the present invention provides oral pharmaceutical compositions, oral care products or products for nutrition or pleasure comprising one or more of the probiotic microorganisms as active agents as well as a method of production thereof.

Claims

1. A method for treating gingivitis and/or periodontitis comprising orally administering to a subject a microorganism or a mixture of two or more microorganisms selected from the group consisting of Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus fermentum 35D (DSM 32130), Lactobacillus gasseri NS8 (NCIMB 11718), Lactobacillus salivarius NS 12 (NCIMB 8816), Streptococcus salivarius NS18 (CBS 142430), and Streptococcus salivarius NS19 (CBS 142431).

2. The method of claim 1, wherein the microorganism or the mixture of two or more microorganisms is (an) attenuated or (a) dead microorganism(s).

3. The method of claim 1, wherein the microorganism or the mixture of two or more microorganisms has an inhibitory activity on the growth of one or more disease-associated microorganisms selected from the group consisting of Porphyromonas gingivalis, Porphyromonas endodontalis, Tannerella forsythia, Filifactor alocis, Eubacterium saphenum, Parvimonas micra, and Lachnoanaerobaculum saburreum, and wherein the microorganism is Lactobacillus salivarius NS12 (NCIMB 8816).

4. The method of claim 1, wherein the microorganism or the mixture of two or more microorganisms is additionally capable of treating inflammation in an oral cavity by inhibiting the release of one or more inflammatory factors selected from the group consisting of interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), isoprostanes, matrix metallopeptidase 9 (MMP9) and NF-κB, and wherein the microorganism or the mixture of two or more microorganisms is/are selected from the group consisting of Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus fermentum 35D (DSM 32130), and Lactobacillus salivarius NS1.2 (NCIMB 8816).

5. The method of claim 1, wherein the microorganism or the mixture of two or more microorganisms forms co-aggregates with oral bacterial species selected from the group consisting of Porphyromonas gingivalis, Porphyromonas endodontalis, Tannerella forsythia, Filifactor alocis and Eubacterium saphenum.

6. The method of claim 1, wherein the microorganism or the mixture of two or more microorganisms is orally administered to the subject in an oral composition, wherein the microorganism or the mixture of two or more microorganisms is present in the oral composition in a range from 0.01 to 100%, with respect to the total weight of the oral composition, and/or wherein the total amount of the microorganism or the mixture of two or more microorganisms is in the range from 1×10.sup.3 to 1×10.sup.11 colony forming units (CFU).

7. The method of claim 6, wherein the oral composition is selected from the group consisting of a toothpaste, a tooth gel, a tooth powder, a tooth cleaning liquid, a tooth cleaning foam, a mouth wash, a mouth spray, dental floss, a chewing gum, and a lozenge.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the aggregative power of the indicated probiotics against type strains of disease-associated bacteria in a co-aggregation assay. Results feature the decrease of total optical density upon co-incubation of pathogenic and probiotic bacteria expressed as aggregation index in percent. aggregation index [%]=(OD.sub.total−OD.sub.supernatant/OD.sub.total)×100; OD.sub.total=OD.sub.target strain+OD.sub.probiotic strain. For each strain of disease-associated bacteria shown on the x-axis the aggregation index is represented by the columns for Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus fermentum 35D (DSM 32130), Lactobacillus gasseri NS8 (NCIMB 11718), Lactobacillus salivarius NS13 (NCIMB 8816), Streptococcus salivarius NS18 (CBS 142430) and Streptococcus salivarius NS19 (CBS 142431) from left to right.

(2) FIG. 2 shows the aggregative power of indicated probiotics against wildtype (wt) strains of disease-associated Filifactor alocis and type strain Filifactor alocis (ATCC 35896.sup.T) in a co-aggregation assay. Results feature the decrease of total optical density upon co-incubation of pathogenic and probiotic bacteria expressed as aggregation index in percent. Aggregation index [%]=(OD.sub.total−OD.sub.supernatant/OD.sub.total))×100; OD.sub.total=OD.sub.target strain+OD.sub.probiotic strain. For each wildtype (wt) strain of disease-associated Filifactor alocis and type strain Filifactor alocis (ATCC 35896.sup.T) shown on the x-axis the aggregation index is represented for Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652) (left columns) and for Lactobacillus fermentum 35D (DSM 32130) (right columns).

(3) FIG. 3 shows the aggregative power of indicated probiotics against wildtype (wt) strains of disease-associated Porphyromonas gingivalis and type strain Porphyromonas gingivalis (ATCC 33277.sup.T) in a co-aggregation assay. Results feature the decrease of total optical density upon co-incubation of pathogenic and probiotic bacteria expressed as aggregation index in percent. Aggregation index [%]=(OD.sub.total−OD.sub.supernatant/OD.sub.total)×100;

(4) OD.sub.total=OD.sub.target strain+OD.sub.probiotic strain. Aggregation indices are represented by grouped columns for each of the probiotics Lactobacillus fermentum 35D (DSM 32130), Lactobacillus gasseri NS8 (NCIMB 11718), Lactobacillus salivarius NS13 (NCIMB 8816), Streptococcus salivarius NS18 (CBS 142430) and Streptococcus salivarius NS19 (CBS 142431) from left to right and within each group the columns for the Porphyromonas gingivalis strains represent ATCC 33277T, 1807 (wt), 2836 (wt), 7681 (wt), 2103 (wt) and 9066 (wt) from left to right.

(5) FIG. 4 shows the aggregative power of indicated viable or attenuated probiotics against wildtype (wt) strains of disease-associated Porphyromonas gingivalis and type strain Porphyromonas gingivalis (ATCC 33277.sup.T) in a co-aggregation assay. Results feature the decrease of total optical density upon co-incubation of pathogenic and probiotic bacteria expressed as aggregation index in percent. Aggregation index [%]=(OD.sub.total−OD.sub.supernatant/OD.sub.total)×100; OD.sub.total=OD.sub.target strain+OD.sub.probiotic strain. For each strain of Porphyromonas gingivalis, shown on the y-axis, ATCC 33277T, 1807 (wt), 2836 (wt), 7681 (wt), 2103 (wt) and 9066 (wt) from left to right, the aggregation index for viable Lactobacillus fermentum 35D (DSM 32130) (left columns) and attenuated Lactobacillus fermentum 35D (DSM 32130) (right columns) as well as viable Lactobacillus salivarius NS13 (NCIMB 8816) (left columns) and attenuated Lactobacillus salivarius NS13 (NCIMB 8816) (right columns) is shown alternately.

EXAMPLE 1: ESTABLISHING THE CULTIVATION AND HANDLING OF PROBIOTIC STRAINS

(6) In order to identify the optimal growth conditions and points of harvest and to determine the colony forming units (CFU) for the probiotic bacteria to be screened, first the log phase and the end of the growth phase were determined.

(7) Bacterial Growth

(8) The frozen (−80° C.) pro-biotic stocks were thawed over night at 4° C. and 6 ml of sterile 9% NaCl solution added to the 1.2 ml of bacteria at the next morning. The samples were centrifuged (5 min, 5000 rpm), the supernatant discarded, the pellet washed with 8 ml 9% NaCl and again centrifuged for 5 min at 5000 rpm. The pellet was then resuspended in 1.2 ml 9% NaCl and 1 ml of the sample added to 50 ml 37° C. warm media (MRS Bouillon, Carl Roth KG, Karlsruhe) and incubated at 37° C. The incubations were performed in a 50 ml sterile polypropylen tube (Greiner) and probes were harvested at different time points to evaluate the growth curve.

(9) OD-Determination

(10) For determination of OD, 500 μl of the bacterial suspension were removed and diluted in 1 ml MRS Bouillon in a 1.5 ml-PS-cuvette (Brand). OD-determination was performed at 600 nm (ThermoScientific, Helios Epsilon) 1.5 ml MRS Bouillon were used as blanc.

(11) Determination of CFU

(12) For determination of CFU, bacteria were diluted (1:10.000.000, 1:50.000.000 and 1:100.000.000), plated on MRS-agar-plates (MRS Agar, X924, Carl Roth) and incubated for 2 days at 37° C. The grown colonies were then counted and the CFU was calculated.

(13) The bacteria approached the log phase right from the beginning until 7 to 8 hours when they start to reach the plateau phase. The amount of bacteria to be seeded does not change the shape of the curve. 5 hours were chosen as the point at the steepest growing phase to harvest the bacteria in the log phase and 7 hours to harvest them at the end of the log phase.

EXAMPLE 2: CO-AGGREGATION OF PROBIOTIC STRAINS WITH ORAL DISEASE-ASSOCIATED BACTERIA

(14) The screening was based on the proprietary OASSYS® co-aggregation. The target strain and probiotic strains were cultivated in specific growth conditions, harvested, washed and co-incubated. The behavior of the strains was monitored:

(15) a) macroscopically, for an appearance of flocculation that was graded 0-3 (with “0” for no flocculation/cloudy supernatant and “3” for a considerable pellet/clear supernatant)

(16) b) by estimation of the aggregation index (% aggregation), which expresses the percentage of applied bacterial cells that have aggregated after co-incubation. If aggregation has taken place, the aggregates will sink to the well bottom when slightly centrifuged, resulting in a supernatant of decreased optical density (measured photometrically at 600 nm) than in case of no interaction between the strains, where optical density of the supernatant will remain unchanged. This approach allows accurate quantification of an aggregation event.

(17) The aggregation index was calculated according to the formula:

(18) $\mathrm{Aggregation}\mathrm{index}\left(\%\right)=\frac{{\mathrm{OD}}_{\left(\mathrm{total}\right)}-{\mathrm{OD}}_{\left(\mathrm{supernatant}\right)}}{{\mathrm{OD}}_{\left(\mathrm{total}\right)}}\times 100$${\mathrm{OD}}_{\left(\mathrm{total}\right)}={\mathrm{OD}}_{\left(\mathrm{target}\mathrm{strain}\right)}+{\mathrm{OD}}_{\left(\mathrm{priobiotic}\mathrm{strain}\right)}$

(19) The tested strains were then classified into 5 categories according to their aggregation index (Table 1). A low aggregation index indicates low grade of stable interaction and aggregation between cells, while a high aggregation index indicates high aggregation capacity and little or no planktonic cells left after co-incubation. In the context of the present invention, it is preferred that the microorganisms used according to the present invention show an aggregation index of at least 60%, preferably at least 80%, against a disease-associated microorganism (as described herein).

(20) TABLE-US-00001 TABLE 1 Aggregation Index [%] 0-20% 20-40% 40-60% 60-80% 80-100% Aggregation category 1 2 3 4 5

(21) c) (optional) light microscopy; selected strains for that a visible flocculation has been observed, were analysed by light microscopy and photographed.

(22) Cultivation and Characterization of Target Strains

(23) Microbiological methods for cultivation and handling of the target bacteria Eubacterium saphenum, Tanarella forsythia, Filifactor alocis, Porphyromonas endodontails and Porphyromonas gingivalis were established and adapted.

(24) The target strains were delivered as bacterial suspension in broth, flushed from an agar plate and collected in a cryotube. To establish the most suitable growth medium for cultivation of the target strains as suspension culture, a variety of media, such as BHI (Brain Heart Infusion), TYP (Tryptone-Yeast ExtractPeptone), FAB (Fastidious Anaerobe Broth) in some cases supplemented with arginine, cysteine, vitamine K and haemin, were prepared and tested. Optimal growth conditions were established for P. gingivalis, P. endodontalis and F. alocis. However, no appropriate liquid medium could be found for the cultivation of T. forsythia and E. saphenum and thus the strains were adapted to growth on solid medium (Fastidious Anaerobe Agar supplemented with horse blood and Nacetylmuramic acid). For the subsequent assay the cell material was harvested from the plates. This alternative method of cultivation resulted in lower yield in cell material and allowed conduction of only limited number of screening experiments. Therefore, the screening of T. forsythia was reduced to probiotic strains that have already shown positive co-aggregation capacity in screening against other disease-associated species (F. alocis, P. gingivalis and P. endodontalis).

(25) Due to very slow and poor growth E. saphenum was completely excluded from the screening.

(26) Screening

(27) Co-aggregation assay was performed in 24-well plates. The target and probiotic strains were cultured, washed several times and co-incubated. Additionally, self-aggregation capacity of both analyzed probiotic and target strains was tested. For this purpose the target and probiotic strains were incubated alone and the same parameters—macroscopic observation and measurement of optical density in the supernatant—were recorded, as in case of co-incubation. To create a better overview of the co-aggregation capacity of particular strains and to facilitate hit selection, the probiotic strains were classified into categories according to their aggregation indices (see also Table 1). The presence of aggregates in several samples with a high aggregation index was confirmed by microscopical analysis.

(28) Among the over 60 strains screened, the strains Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus fermentum 35D (DSM 32130), Lactobacillus gasseri NS8 (NCIMB 11718), Lactobacillus salivarius NS13 (NCIMB 8816), Streptococcus salivarius NS18 (CBS 142430) and Streptococcus salivarius NS19 (CBS 142431) showed aggregation indices of over 60% with at least one target strain.

(29) The aggregative power of the probiotic strains with respect to tested disease-associated strains is shown in FIGS. 1 to 4. The grading for macroscopic co-aggregation and the aggregation category for the probiotic strains is shown in tables 2 and 3.

(30) Table 2 shows the aggregative power of indicated probiotics against wildtype (wt) strains of disease associated Filifactor alocis and type strain Filifactor alocis (ATCC 35896.sup.T) in a co-aggregation assay being evaluated macroscopically. Flocculation is graded as “0” for no flocculation and “3” for a considerable pellet or clear supernatant. In addition, the table shows grading of aggregation indices within aggregation categories for easier interpretation of assay results.

(31) TABLE-US-00002 TABLE 2 Macroscopic co-aggregation 0-1 2 3 ATCC 35896.sup.T 1550 (wt) 2282 (wt) 7684 (wt) 9351 (wt) 9077 (wt) L. fermentum (DSM 32130) 3 2 3 2 2 0 L. delbrueckii (CCTCC M 2016652 3 3 3 1 3 0 Aggregation category 1 2 3 4 5 Aggregation index <20% 20-40% 41-60% 61-80% >80% ATCC 35896.sup.T 1550 (wt) 2282 (wt) 7684 (wt) 9351 (wt) 9077 (wt) L. fermentum (DSM 32130) 4 4 3 2 2 1 L. delbrueckii (CCTCC M 2016652 3 4 2 1 1 1

(32) Table 3 shows the aggregative power of indicated probiotics against wildtype (wt) strains of disease-associated Porphyromonas gingivalis and type strain Porphyromonas gingivalis (ATCC 33277.sup.T) in a co-aggregation assay being evaluated macroscopically. Flocculation is graded as “0” for no flocculation and “3” for a considerable pellet or clear supernatant. In addition, the table shows grading of aggregation indices within aggregation categories for easier interpretation of assay results.

(33) TABLE-US-00003 TABLE 3 Macroscopic co-aggregation 0-1 2 3 1 ATCC 33277.sup.T 1807 (wt) 2836 (wt) 7681 (wt) 2108 (wt) 9066 (wt) L. fermentum (DSM 32130) 3 3 3 0 0 1 L. gasseri (NCIMB 11718) 3 2 3 1 0 1 L. salivarius (NCIMB 8816) 3 3 3 3 3 3 Aggregation category 1 2 3 4 5 Aggregation index <20% 20-40% 41-60% 61-80% >80% ATCC 33277.sup.T 1807 (wt) 2836 (wt) 7681 (wt) 2108 (wt) 9066 (wt) L. fermentum (DSM 32130) 5 4 4 2 1 2 L. gasseri (NCIMB 11718) 5 4 4 3 1 2 L. salivarius (NCIMB 8816) 5 5 5 5 4 5

EXAMPLE 3: INHIBITORY ACTIVITY OF PROBIOTIC STRAINS AGAINST DISEASE-ASSOCIATED BACTERIA

(34) The inhibitory activity of selected probiotic strains, inter alia L. salivarius NS 13, against peridontitis-associated oral bacterial species (indicator strains) was determined. The indicator strains were: Porphyromonas gingivalis ATCC 33277 Porphyromonas endodontalis ATCC 35406 Tannerella forsythia ATCC 43037 Filifactor alocis ATCC 35896 Eubacterium saphenum ATCC 49989 Parvimonas micra ATCC 33270 Lachnoanaerobaculum saburreum ATCC 33271

(35) A delayed antagonism method was performed in triplicate. The test strains were grown on blood agar as streak for 24 or 48 h and then killed by exposure to chloroform. Indicator strains were cross-streaked and incubated for up to 72 h.

(36) L. Salivarius NS 13 had activity against multiple indicator strains as shown in table 4:

(37) TABLE-US-00004 TABLE 4 Lactobacillus salivarius NS 13 Porphyromonas gingivalis ATCC 33277 — Porphyromonas endodontalis ATCC 35406 — Tannerella forsythia ATCC 43037 — Fillfactor alocis ATCC 35896 + Eubacterium saphenum ATCC 49989 + Parvimonas micra ATCC 33270 + Lachnoanaerobaculum saburreum ATCC — 33271

EXAMPLE 4: PROBIOTIC LOZENGE OR COMPRIMATE

(38) TABLE-US-00005 Isomalt Comprimates Probiotic No Block Ingredients Placebo only +Flavor 1 A Magnesium Stearate 1.800% 1.800% 1.800% 2 Acesulfam 0.050% 0.050% 0.050% 3 Sucralose 0.025% 0.025% 0.025% 4 Probiotic Material 1.000% 1.000% 5 Flavor (e.g. 134229 0.500% Optamint Peppermint s/d) 6 B Isomalt 98.125%  97.125%  96.625%  Sum total 100.00%  100.00%  100.00% 

(39) Production Method: Components 1 and 6 are dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly components 1, 2, 3, 4 and 5 combined and thoroughly mixed (block A). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Block A is subsequently added to component 6 and mixed thoroughly for 5 minutes. The powder mixture is pressed into tablets in a tablet press EKO (Korsch AG, Berlin) at an adjusted pressure of 15-20 kN target parameters: tablet diameter: 20 mm tablet weight: 2.0 g. Storage at RT in sealed aluminum sachets. Per 5 lozenges 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

EXAMPLE 5: POWDER DENTIFRICE

(40) TABLE-US-00006 Toothpowder Probiotic No Block Ingredients Placebo only +Flavor 1 A Magnesium Carbonate 3.00% 3.00% 3.00% 2 Sodium Bicarbonate 2.00% 2.00% 2.00% 3 Sodium Fluoride 0.25% 0.25% 0.25% 4 Sodium Saccharin 0.60% 0.60% 0.60% 5 B Probiotic Material 4.00% 4.00% 6 Flavor (e.g. 134229 2.00% Optamint Peppermint s/d) 7 C Calcium carbonate 94.15% 90.15% 88.15% Sum total 100.00% 100.00% 100.00%

(41) Production Method: Component 7 is dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Components 1, 2, 3 and 4 are combined and thoroughly mixed together (block A). Components 5 and 6 are, if necessary, combined and thoroughly mixed (block B). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Blocks A and B are subsequently combined and thoroughly mixed together. The mixture is added to component 7 and mixed thoroughly for 5 minutes. The powder mixture is made up into portions of 0.5 g each storage at RT together with 1 g of desiccant per portion (activated by 3 h storage at 105° C. in a vacuum compartment drier) in sealed aluminum sachets.

EXAMPLE 6: POWDER DENTIFRICE

(42) TABLE-US-00007 Toothpaste No Block Ingredients tablets 1 A Magnesium Carbonate 3.00% 2 Sodium Bicarbonate 2.00% 3 Sodium Fluoride 0.25% 4 Sodium Saccharin 0.60% 5 Sodium Laurylsulphate 0.50% 6 Magnesium Stearate 1.00% 7 B Flavor (e.g. 134229 Optamint Peppermint s/d) 2.00% 8 Probiotic Material 6.67% 9 C Calcium Carbonate 17.00% 10 Microcristalline Cellulose 66.98% Sum total 100.00%

(43) Production Method: Components 6, 9 and 10 are dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Components 1, 2, 3, 4, 5 and 6 are combined and thoroughly mixed together (block A). Components 7 and 8 are combined and thoroughly mixed together (block B). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Blocks A and B are subsequently combined and thoroughly mixed together. Components 9 and 10 are combined and thoroughly mixed together (block C). The two mixtures (Block NB and Block C) are combined and mixed thoroughly for 5 minutes. The powder mixture is pressed into tablets in a tablet press EKO (Korsch AG, Berlin) at an adjusted pressure of 15-20 kN target parameters tablet diameter: 9 mm tablet weight: 0.3 g Storage at RT in sealed aluminum sachets. Per 3 tablets 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

EXAMPLE 7: CHEWING GUM

(44) TABLE-US-00008 Chewing gum Chewing with Vegetable gum with Oil, Probiotics Vegetable Oil, No Ingredients in Flavor Probiotics in Oil 1 Gum Base (e.g. Geminis T) 30.00% Block A 30.00% Block A 2 Isomalt (here: Isomalt ST-PF) 65.00% Block B 65.00% Block B 3 Sucralose coated (10% in 1.00% 1.00% wax) 4 Deoiled Soy Lecithin 0.30% 0.30% (here: Emulbur IP) 5 Vegetable Oil - Triglyceride 1.60% Block C 1.60% Block C 6 Probiotic Material 0.80% Block D 0.80% 7 Flavor (e.g. 203191 1.30% 1.30% Block D Optamint Peppermint)

(45) Production Method: Component 2 is dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Component 1 is tempered to 45-59° C. in a chewing gum lab-kneader with the heating kneaded until a homogenous mass is obtained. The heating is on during the whole mixing process. Components 2, 3 and 4 are added subsequently and kneaded until the mixture is homogenous and no powder is visible anymore. According to the formula component 6 is either worked into component 5 (block C) or component 7 (block D). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. The components are mixed until an even suspension is obtained. First, block C is added to the chewing gum mass and kneaded again until a homogenous mass is obtained. Last, block D is processed accordingly. After addition the composition has to be kneaded until an even chewing gum mass is obtained. The mass is taken out of the mixer and is formed into mini-sticks by an embossing roller using the embossing set “slabs”. Storage at RT in sealed aluminum sachets. Per 7 chewing gums 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

EXAMPLE 8: PROBIOTIC BEADLETS

(46) TABLE-US-00009 probiotic beadlets probiotic beadlets probiotic beadlets probiotic beadlets with high load, with high load, diets with low with low without aroma, without aroma, load, without load, with without dye, without dye, aroma, with aroma, with without gellan without gellan dye, with dye, with gellan gum, high water gum, low water gellan gum gum content content components wt. % wt. % wt. % wt. % Alginate 1.75 1.65 1.44 1.57 Gum arabic 1.25 1.18 0.60 0.65 Wheat fiber 1.125 1.06 0.52 0.57 Dye 0.0125 0.018 — — Aroma — 1.41 — — Glycerol 0.1875 — — — probiotic 1.125 1.35 7.20 7.83 Gallon Gum 0.0625 0.059 — — Water Add to 100 Add to 100 Add to 100 Add to 100 load approx. 20% approx. 20% approx. 74% approx. 74%

(47) Production Method: Production of the calcium chloride bath for precipitation of the alginate beadlets: A 2% calcium chloride solution is produced from distilled water and calcium chloride. Care has to be taken that the CaCl.sub.2 is completely dissolved. Production of the alginate solution (instead of alginate also pectin or gellan gum may be used): In a reaction vessel with a stirrer and which is suitable to the batch size, water is provided. The stirrer is turned on and, while stirring at a high level, the respective amounts of alginate, gum arabicum, wheat fiber and probiotic, as well as the optionally required gellan gum are added. The mixture is heated to 80° C. while stirring and kept at this temperature for 5 minutes—during this step the gel forming components are dissolved. Afterwards, the heating is turned off and the hot gel solution is further stirred for at least 30 minutes until it is free of lumps. Subsequently, the solution is cooled by refrigeration to 39-43° C. while stirring. In a further vessel, the aroma and the dye are provided if required and thoroughly mixed In case no aroma is used, the dye is mixed with glycerol. When the dye dispersion is mixed homogenously, it is added to the batch vessel with the alginate solution. The mixing vessel is washed several times with approx. 10% of the amount of alginate solution used of water and added to the dispersion. The alginate dispersion is stirred further for at least 5 minutes. Subsequently, the batch is stirred for further at least 15 minutes at a low speed to remove potentially present air.

(48) Dripping of the Alginate Solution into the Calcium Chloride Solution for Precipitation of the Beadlets: The alginate dispersion is moved to a tightly sealable pressure stable reaction vessel having two outlets. At one outlet pressurized air is applied. The second outlet leads to the nozzles of the dripping unit via a tube. The reaction vessel is tempered over a heating plate so that the alginate solution reaches a temperature of approx. 45° C. The solution is slightly stirred with a magnet stirrer. After application of pressure to the reaction vessel, alginate solution is pressed towards the nozzles, which are set to oscillation by an oscillator. By adaption of pressure and the frequency of the oscillator, the size of the resulting drops at the tips of nozzles may be adjusted. The drops of alginate solution forming at the tips of the nozzles fall into a collection vessel in the form of a funnel in which the calcium chloride solution prepared at the beginning circulates. The cured alginate beadlets pass with the calcium chloride solution through the funnel and are collected in a sieve, the collected calcium chloride solution is pumped back into the funnel below the dripping unit and thus recycled. The beadlets are dried in an Aeromatic flowbed-drier at an supply air temperature of 80° C. until an exhaust air temperature of 45° C. is reached.