STABILIZING AGENT FOR PROBIOTIC COMPOSITION

Abstract

The present invention relates to the use of surface-reacted calcium carbonate as stabilizing agent for a probiotic composition, wherein the surface-reacted calcium carbonate is a reaction product of natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H.sub.3O.sup.+ ion donors, wherein the carbon dioxide is formed in situ by the H.sub.3O.sup.+ ion donors treatment and/or is supplied from an external source.

Claims

1. A stabilized probiotic composition, comprising: a stabilizing agent comprising a surface-reacted calcium carbonate; and a probiotic microorganism culture; wherein the surface-reacted calcium carbonate is a reaction product of a natural ground calcium carbonate or a precipitated calcium carbonate with carbon dioxide and one or more H.sub.3O.sup.+ ion donors, wherein the carbon dioxide is formed in situ by the H.sub.3O.sup.+ ion donors treatment and/or is supplied from an external source.

2. The stabilized probiotic composition of claim 1, wherein the probiotic microorganism culture is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecium, Escherichia coli Nissle 1917, Escherichia coli criodesiccata (083:K24:H31), Saccharomyces boulardii, Saccharomyces cerevisiae, and mixtures thereof.

3. The stabilized probiotic composition of claim 1, wherein the probiotic composition comprises a probiotic microorganism culture in an amount of at least 50 wt.-%, based on the total weight of the probiotic composition.

4. The stabilized probiotic composition of claim 1, wherein the stabilized probiotic composition is a dry composition or an aqueous suspension.

5. The stabilized probiotic composition of claim 1, wherein the surface-reacted calcium carbonate has one or more of: i) a volume median particle size d.sub.50 in the range of 0.1 to 75 μm; ii) a volume top cut particle size d.sub.98 in the range of 0.2 to 150 μm; iii) a specific surface area in the range of 15 m.sup.2/g to 200 m.sup.2/g measured using nitrogen and the BET method; and iv) an intra-particle intruded specific pore volume in the range of 0.1 to 2.3 cm.sup.3/g, calculated from mercury porosimetry measurement.

6. The stabilized probiotic composition of claim 1, wherein the natural ground calcium carbonate is selected from the group consisting of marble, chalk, limestone, and mixtures thereof, or the precipitated calcium carbonate is selected from the group consisting of precipitated calcium carbonates having an aragonitic, vateritic or calcitic crystal form, and mixtures thereof, and/or the one or more H.sub.3O.sup.+ ion donor is selected from the group consisting of hydrochloric acid, sulphuric acid, sulphurous acid, phosphoric acid, citric acid, oxalic acid, an acidic salt, acetic acid, formic acid, and mixtures thereof.

7. The stabilized probiotic composition of claim 1, wherein the weight ratio of probiotic microorganism culture:surface-reacted calcium carbonate is in the range of 5:95 to 40:60.

8. The stabilized probiotic composition of claim 1, wherein the stabilizing agent is a drying stabilizer and/or a shelf live preservative.

9. The stabilized probiotic composition of claim 1, wherein the stabilized probiotic composition is a pharmaceutical probiotic composition, a nutritional probiotic composition, or a cosmetic probiotic composition, and/or the stabilized probiotic composition is in the form of a tablet, a capsule, a chewable tablet, a chewable gum, a chewable pastille, a lozenge, a powder, a granulate, a pellet, a paste, a cream, a food, a feed, or a beverage.

10. The stabilized probiotic composition of claim 1, wherein the concentration of viable probiotic microorganism culture in the stabilized probiotic composition is at least 5% greater after drying the stabilized probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

11. A method for stabilizing a probiotic microorganism culture, comprising the step of mixing a probiotic microorganism culture with a surface-reacted calcium carbonate in an aqueous medium, wherein the surface-reacted calcium carbonate is a reaction product of natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H.sub.3O.sup.+ ion donors, wherein the carbon dioxide is formed in situ by the H.sub.3O.sup.+ ion donors treatment and/or is supplied from an external source, and drying the obtained mixture.

12. A process for preparing a dry stabilized probiotic composition, comprising the steps of: a) providing an aqueous probiotic composition comprising at least 75 wt.-% of a probiotic microorganism culture, based on the total weight of the probiotic composition, b) providing an aqueous suspension comprising 10 to 30 wt.-%, based on the total weight of the aqueous suspension, of surface-reacted calcium carbonate, wherein the surface-reacted calcium carbonate is a reaction product of natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H.sub.3O.sup.+ ion donors selected from the group consisting of hydrochloric acid, sulphuric acid, sulphurous acid, phosphoric acid, oxalic acid, or mixtures thereof, wherein the carbon dioxide is formed in situ by the H.sub.3O.sup.+ ion donors treatment and/or is supplied from an external source, wherein the surface-reacted calcium carbonate has a volume median particle size d.sub.50 from 0.1 to 75 μm, and a volume top cut particle size d.sub.98 from 0.2 to 150 μm, a specific surface area of from 15 m.sup.2/g to 200 m.sup.2/g, measured using nitrogen and the BET method, and wherein the weight ratio of probiotic microorganism culture:surface-reacted calcium carbonate is from 5:95 to 40:60, c) mixing the probiotic composition of step a) and the surface-reacted calcium carbonate of step b), and d) spray drying the mixture obtained in step c), at an inlet temperature from 130 to 210° C. and an outlet temperature from 50 to 130° C.

13. A dry stabilized probiotic composition obtainable by the process according to claim 12.

14. A product comprising the dry stabilized probiotic composition according to claim 13, wherein the product is a tablet, a capsule, a chewable tablet, a chewable gum, a chewable pastille, a lozenge, a powder, a granulate, a pellet, a paste, a cream, a food, a feed, or a beverage.

15. The product according to claim 14, wherein the product is a pharmaceutical, nutritional or cosmetic.

16. The stabilized probiotic composition of claim 1, wherein the one or more H.sub.3O.sup.+ ion donor is selected from the group consisting of hydrochloric acid, sulphuric acid, sulphurous acid, phosphoric acid, oxalic acid, H.sub.2PO.sub.4.sup.−, HPO.sub.4.sup.2−, and mixtures thereof, wherein wherein H.sub.2PO.sub.4− is at least partially neutralised by a cation selected from Li.sup.+, Na.sup.+ and/or K.sup.+; and wherein HPO.sub.4.sup.2− is at least partially neutralised by a cation selected from Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, and/or Ca.sup.2+.

Description

EXAMPLES

1. Methods

[0168] In the following, measurement methods implemented in the examples are described.

Particle Size Distribution

[0169] Volume determined median particle size d.sub.50 (vol) and the volume determined top cut particle size d.sub.98(vol) was evaluated using a Malvern Mastersizer 3000 Laser Diffraction System (Malvern Instruments Plc., Great Britain). The d.sub.50 (vol) or d.sub.98(vol) value indicates a diameter value such that 50% or 98% by volume, respectively, of the particles have a diameter of less than this value. The raw data obtained by the measurement was analyzed using the Mie theory, with a particle refractive index of 1.57 and an absorption index of 0.005. The methods and instruments are known to the skilled person and are commonly used to determine particle size distributions of fillers and pigments. The measurement was carried out in an aqueous solution of 0.1 wt.-% Na.sub.4P.sub.2O.sub.7. The samples were dispersed using a high-speed stirrer and supersonicated.

[0170] The weight median particle size d.sub.50 (wt) is determined by the sedimentation method, which is an analysis of sedimentation behaviour in a gravimetric field. The measurement is made with a Sedigraph™ 5120, Micromeritics Instrument Corporation. The method and the instrument are known to the skilled person and are commonly used to determine grain size of fillers and pigments. The measurement is carried out in an aqueous solution of 0.1 wt % Na.sub.4P.sub.2O.sub.7. The samples were dispersed using a high speed stirrer and supersonicated.

[0171] The processes and instruments are known to the skilled person and are commonly used to determine grain size of fillers and pigments.

Specific Surface Area (SSA)

[0172] The specific surface area was measured via the BET method according to ISO 9277:2010 using nitrogen, following conditioning of the sample by heating at 250° C. for a period of 30 minutes. Prior to such measurements, the sample was filtered within a Büchner funnel, rinsed with deionised water and dried at 110° C. in an oven for at least 12 hours.

Intra-Particle Intruded Specific Pore Volume (in cm.sup.3/q)

[0173] The specific pore volume was measured using a mercury intrusion porosimetry measurement using a Micromeritics Autopore V 9620 mercury porosimeter having a maximum applied pressure of mercury 414 MPa (60 000 psi), equivalent to a Laplace throat diameter of 0.004 μm (— nm). The equilibration time used at each pressure step was 20 seconds. The sample material was sealed in a 5 cm.sup.3 chamber powder penetrometer for analysis. The data were corrected for mercury compression, penetrometer expansion and sample material compression using the software Pore-Comp (Gane, P. A. C., Kettle, J. P., Matthews, G. P. and Ridgway, C. J., “Void Space Structure of Compressible Polymer Spheres and Consolidated Calcium Carbonate Paper-Coating Formulations”, Industrial and Engineering Chemistry Research, 35(5), 1996, p 1753-1764.).

[0174] The total pore volume seen in the cumulative intrusion data can be separated into two regions with the intrusion data from 214 μm down to about 1-4 μm showing the coarse packing of the sample between any agglomerate structures contributing strongly. Below these diameters lies the fine inter-particle packing of the particles themselves. If they also have intra-particle pores, then this region appears bi-modal, and by taking the specific pore volume intruded by mercury into pores finer than the modal turning point, i.e. finer than the bi-modal point of inflection, the specific intra-particle pore volume is defined. The sum of these three regions gives the total overall pore volume of the powder, but depends strongly on the original sample compaction/settling of the powder at the coarse pore end of the distribution.

[0175] By taking the first derivative of the cumulative intrusion curve the pore size distributions based on equivalent Laplace diameter, inevitably including pore-shielding, are revealed. The differential curves clearly show the coarse agglomerate pore structure region, the inter-particle pore region and the intra-particle pore region, if present. Knowing the intra-particle pore diameter range it is possible to subtract the remainder inter-particle and inter-agglomerate pore volume from the total pore volume to deliver the desired pore volume of the internal pores alone in terms of the pore volume per unit mass (specific pore volume). The same principle of subtraction, of course, applies for isolating any of the other pore size regions of interest.

2. Materials

[0176] Stabilizer

[0177] SRCC: Surface-reacted calcium carbonate (d.sub.50 (vol)=6.6 μm, d.sub.98 (vol)=13.7 μm, SSA=59.9 m.sup.2/g). The intra-particle intruded specific pore volume is 0.939 cm.sup.3/g (for the pore diameter range of 0.004 to 0.51 μm).

[0178] SRCC was obtained by preparing 350 litres of an aqueous suspension of ground calcium carbonate in a mixing vessel by adjusting the solids content of a ground limestone calcium carbonate from Omya SAS, Orgon having a weight based median particle size d.sub.50 (wt) of 1.3 μm, as determined by sedimentation, such that a solids content of 10 wt.-%, based on the total weight of the aqueous suspension, is obtained.

[0179] Whilst mixing the slurry at a speed of 6.2 m/s, 11.2 kg phosphoric acid was added in form of an aqueous solution containing 30 wt.-% phosphoric acid to said suspension over a period of 20 minutes at a temperature of 70° C. After the addition of the acid, the slurry was stirred for additional 5 minutes, before removing it from the vessel and drying using a jet-dryer.

[0180] Maltodextrin (comparative stabilizer).

Probiotic Microorganism

[0181] Lactobacillus plantarum WCFS1.

3. Example 1

3.1. Fermentation and Harvesting of Probiotic

[0182] Firstly, a pre-inoculum was made from Lactobacillus plantarum strain in 5 mL of MRS-B culture medium. This was incubated for approximately 10 hours at 37° C. Afterwards, 1% of the pre-inoculum was used to inoculate the actual fermentation. The fermentation medium used was again MRS-B, which was then incubated over night (˜16-17 hours) at a temperature of 37° C. After the fermentation, a sample was taken for CFU determination.

[0183] For harvesting, the finished fermentation was centrifuged for 15 minutes at 5000 rpm (4° C.). The resulting pellet was resuspended in 100 mL of PBS (phosphate-buffered saline) buffer and subdivided in 2 equally sized portions. The concentration of probiotic microorganisms in said suspensions was 4.2×10.sup.10 CFU/g. Each of these portions was mixed with 450 g of stabilizer solution containing 20% (w/w) of stabilizer, resulting in 2 solutions of −500 g. One of the stabilizer solutions contained maltodextrin while the other contained the inventive stabilizing agent. Note that both stabilizer solutions were sterilized (15 minutes at 121° C.) before mixing with the resuspended pellet portions.

3.2 Spray Drying and Shelf Life Testing

[0184] Before spray drying, a sample of each stabilizer solution was taken for CFU determination before drying. The CFU analysis was carried out according to ISO 11133-1:2009.

[0185] For the spray drying trial, a Büchi benchtop spray drying system was used. An inlet temperature of 170° C. and an outlet temperature of 80° C. as used to spray dry all material. Again, the resulting powder was sampled for CFU analysis for yield determination after drying.

[0186] The remaining powder was used for shelf life analysis. For each stabilizer powder, 0.5-1 g was dosed in reactor tubes with filter caps. These tubes were consequently incubated horizontally (for a larger air contact area) at 30° C. with a humidity of 35% for 2 weeks. After this incubation period, samples were again taken for CFU analysis.

3.3 Results

[0187] The results for the CFU measurements at different steps in the benchmark study are shown in Table 1. It can be seen that the concentration of viable probiotic microorganisms (CFU/g) of spray dried powder decreases from before spray drying all the way to the end of the shelf life study. The amount after fermentation appears to be slightly lower than the amount before spray drying, but this is simply caused by the approximation of the dry matter used for the back calculation of this value. Additionally, the standard deviation for each of the measurements is relatively high (as is usually for CFU determinations). These numbers for the standard deviations are shown in Table 2.

[0188] With regards to product properties, the maltodextrin solution before spray drying was clear, while the suspension of the inventive stabilizing agent was turbid (milky). The spray dried maltodextrin powder was more difficult to resuspend then the mineral stabilizer powder, though the latter was prone settling. The maltodextrin powder was also much denser with a bulk density of around 470 g/L compared to the approximate 200 g/L for the mineral stabilizer powder.

TABLE-US-00001 TABLE 1 CFU/grams of powder for each step in the benchmark study. The concentration after fermentation was back calculated based on approximate dry matter. Maltodextrin (comparative) SRCC (CFU/g) (CFU/g) After fermentation (back calculated)  4.2E+10 Before spray drying 6.19E+10 5.91E+10 After spray drying 1.09E+10 1.45E+10 After shelf life testing 9.16E+07 1.95E+08

TABLE-US-00002 TABLE 2 Standard deviation for CFU determination for each step in the benchmark study. Maltodextrin (comparative) SRCC (CFU/g) (CFU/g) After fermentation (back calculated)  1.2E+10 Before spray drying 2.64E+10 2.51E+10 After spray drying 2.73E+9  6.42E+9  After shelf life testing 4.35E+07 1.42E+08

[0189] The results compiled in Tables 1 and 2 show that the concentration of viable probiotic microorganisms is significantly higher in the inventive sample after spray drying and the inventive sample exhibits a significantly increased shelf life.

4. Example 2

4.1. Fermentation and Harvesting of Probiotic

[0190] Firstly, a pre-inoculum was made from Lactobacillus plantarum strain in 5 mL of MRS-B culture medium. This was incubated for approximately 10 hours at 37° C. Afterwards, 1% of the pre-inoculum was used to inoculate the actual fermentation. The fermentation medium used was again MRS-B, which was then incubated over night (˜16-17 hours) at a temperature of 37° C. After the fermentation, a sample was taken for CFU determination.

[0191] For harvesting, the finished fermentation was centrifuged for 15 minutes at 5000 rpm (4° C.). The resulting pellet was resuspended in 250 mL of PBS (phosphate-buffered saline) buffer and subdivided in five equally sized portions. Three of these portions was mixed with 450 g of a stabilizer solution containing (based on the total weight of the final mixture): [0192] Stabilizer solution 1: only PBS (control buffer solution) [0193] Stabilizer solution 2: 5 wt.-% maltodextrin [0194] Stabilizer solution 3: 5 wt.-% SRCC

[0195] This resulted in three sample solutions of 500 g. The stabilizer solutions were sterilized (15 minutes at 121° C.) before mixing with the resuspended pellet portions. The composition of the sample solutions is indicated in Table 3 below.

TABLE-US-00003 TABLE 3 Composition of sample solutions produced according to Example 2. Sample Amount of PBS Stabilizing Biomass Total solids solution [g] agent [g] [wt.-%] 1 4.95 — ~5 1.99 2 0.494 5 g Maltodextrin ~5 2.10 3 0.494 5 g SRCC ~5 2.10

4.2. Spray Drying

[0196] Before spray drying, a sample of each sample solution was taken for CFU determination as reference. A Büchi benchtop dryer was used for spray drying.

[0197] For phase 1, an inlet temperature of 200° C. and an outlet temperature of 100° C. was used to spray dry all sample solutions. Since the main aim of phase 1 was to obtain observable yield differences between the different sample solutions, the inlet and outlet temperature were set relatively high to ensure more inactivation and consequently more contrast between the samples.

[0198] During phase 2 the drying conditions were, 180 and 80° C. for inlet and outlet temperature respectively. The main aim of phase 2 was to define effect of stabilizers and formulations on gut and shelf-life survival. Consequently, relatively mild spray drying conditions were chosen to maximize starting CFU's for consequent testing.

4.3. Formulations (Phase 2)

[0199] Three different probiotic formulations were prepared to further asses possible applications of the inventive stabilizing agents, using the spray dried sample solutions 2 and 3 containing the stabilizing agent maltodextrin and SRCC, respectively, and subjected to digestion and/or shelf life experiments. For the different formulations, the powders, which were obtained by spray drying the corresponding sample solutions according to the phase 2 conditions, were mixed with specific bulk components indicated below in a weight ratio of 1:1.

Milk Powder for Liquid Ingestion

[0200] Spray dried powders were mixed in a 1:1 weight ratio with standard skim milk powder. Part of this dry blend was used for shelf life analysis (dry) while the other part was solubilized for the digestion study.

Tablet for Ingestion

[0201] Spray dried powders were mixed in a 1:1 weight ratio with lactose powder. This powder blend was then manually pressed into tablets of −1 gram. These tablets were subjected to the digestion study and shelf life experiments.

Cream for Skin Application

[0202] A base cream was prepared by mixing oleylalcohol and hexadecanol in a 1:1 weight ratio, creating a Vaseline-like cream. Cream and spray dried powder was then mixed in a 1:1 weight ratio, after which it was subjected to shelf life analysis.

4.4. Digestion Study and Shelf Life Experiments

In Vitro-Digestion Testing

[0203] An in-vitro digestion model was used to evaluate the gut survival of L. Plantarum WCFS1 using different stabilizers and formulations. This model has been validated to show comparable strain-specific GI persistence to in-vivo methods (Van Bokhorst-van de Veen et al., 2012).

[0204] The digestion study was done in an in-vitro setup, using a glass sample holder inside a shaken water bath to simulate intestinal mixing. Initially, 3 grams of formulation (milk powder dry blend or tablets) were added to 27 g of sterile water in a sample holder (resulting in a 10 wt.-% solution). This was done to simulate practical intake of a typical milk powder solution and tablets with water respectively. The four resulting formulations were subjected to the in-vitro digestion scheme, as presented in FIG. 1, in which the time of addition of different digestive juices and setting of pH is indicated in minutes. The in-vitro digestion scheme included the following steps: [0205] 0 minutes: Addition of 2 mL saliva buffer, containing α-amylase [0206] 5 minutes: Adjust pH to 2 using 0.5 M HCl solution over 15 minutes. [0207] 20 minutes: Addition of 8 mL gastric buffer, containing lipase and pepsin. [0208] 80 minutes: Take 0.3 mL sample (after gastric phase) and start adjusting pH to 6 using 1 M NaOH solution over 5 minutes. [0209] 85 minutes: Addition of 10 mL pancreatic buffer, containing pancreatin and bile. [0210] 175 minutes: Take 0.3 mL sample (after pancreatic phase) and finalize experiment.
The samples taken were then used for CFU analysis.

Shelf Life Testing

[0211] For shelf life experiments, the same formulations as used for the digestion study were evaluated, as well as the cream for skin application. For each formulation, 0.5-1 g was dosed in reactor tubes with filter caps. These tubes were incubated horizontally (for a larger air contact area) at 30° C. with a humidity of 35% for 2 weeks. After this incubation period, samples were taken for CFU analysis.

4.5. Results

Spray Drying

[0212] After spray drying a CFU analysis of the buffer solution (control sample) and the stabilized samples was carried out according to ISO 1133-1:2009. The results are compiled in FIG. 2. Comparison in terms of recovery % is generally applied in the literature (Perdana et al. 2013). As can be seen from FIG. 2, the CFU values for the inventive sample comprising SRCC are consistently higher than for the comparative sample comprising maltodextrin. The yield for the maltodextrin sample was in line with literature values of 5-10% (Siemons et al. 2021), while the yield for the control sample with buffer was relatively high compared to maltodextrin.

[0213] Furthermore, there was less material on the wall of the Büchi for the inventive samples comprising SRCC compared to the sample containing maltodextrin. The spray dried maltodextrin sample also had more powder lumps which were less easily dispersible into lose powder.

In-Vitro Digestion Testing

[0214] A CFU analysis of the stabilized samples was carried out according to ISO 1133-1:2009, before and after the in-vitro digestion test. The results are shown in FIG. 3. In this case, the difference in performance for the samples containing the inventive stabilizing agent SRCC and the comparative samples containing maltodextrin is striking. The inventive samples with SRCC maintained 10% viability, i.e., a log 1 reduction after digestion. In contrast, the comparative samples with maltodextrin were more sensitive, with only 0.01% viability remaining, i.e., a log 4 reduction after digestion. The inventive stabilizing agent SRCC outperformed maltodextrin in both formulations, i.e., milk powder as well as lactose tablets.

[0215] Moreover, it was observed that the milk powder formulation performed slightly better than the lactose tablets when comparing final CFU values. The reason for that might be the fact the milk is a rich medium with both buffer capacity and nutritional content.

Shelf Life Testing

[0216] A CFU analysis of the stabilized samples was carried out according to ISO 1133-1:2009, before and after the shelf-life test, FIG. 4 shows the results of the accelerated shelf-life experiments. In line with the results of the in-vitro digestion testing, the inventive samples with SRCC outperformed the comparative samples with maltodextrin for the milk powder formulation and lactose tablets. Moreover, the cream formulation containing the inventive stabilizing agent SRCC performed slightly better than the cream formulation containing the comparative stabilizing agent maltodextrin.

[0217] In summary, it has been shown that the inventive stabilizing agent is an effective stabilizing agent in production (spray drying), delivery (shelf-life), and application (in-vitro digestion) of probiotic compositions. Moreover, the inventive stabilizing agent outperformed the comparative stabilizing agent maltodextrin.