Microbial compositions

Abstract

The invention provides a microbial composition in the form of a powder for oral administration comprising or consisting of: (i) micro-organism, preferably probiotic bacteria; (ii) sugar alcohol, such as Erythritol and Xylitol; (iii) moisture absorbent fibre, such as inulin; (iv) a flow agent, such a silicon dioxide optionally; (v) a flavorant; and/or optionally (vi) a bulking agent, such as maltodextrin. The compositions display good storage stability, fast-melt and sensory properties. They are preferably packaged in a single dose in a sealed stick pack aluminium container.

Claims

1. A microbial composition in the form of a powder for oral administration, comprising or consisting of: (i) micro-organisms; (ii) sugar alcohol; (iii) a moisture absorbent fibre; (iv) a powder flow agent; optionally (v) a flavorant; optionally (vi) a bulking agent; wherein: a) the moisture absorbent fibre is inulin, and is in an amount from 20-25% w/w, b) the microbial composition has a water activity of less than 0.15, c) the ratio of (ii) sugar alcohol to (iii) moisture absorbent fibre is 3:1, and d) the sugar alcohol is erythritol, or the sugar alcohol is a combination of erythritol and xylitol in a ratio of 2:1.

2. A microbial composition as claimed in claim 1 wherein the powder flow agent is silicon dioxide.

3. A microbial composition as claimed in claim 1 wherein the bulking agent is maltodextrin.

4. A microbial composition as claimed in claim 1 wherein the micro-organisms are probiotic bacteria.

5. A microbial composition as claimed in claim 4, wherein the probiotic bacteria are selected from Lactobacillus spp. and/or Bifidobacterium spp.

6. A microbial composition as claimed in claim 1 wherein the micro-organisms are present in an amount of from 10.sup.3-10.sup.12 colony forming units (CFU)/dose.

7. A microbial composition as claimed in claim 6, wherein the micro-organisms are present in an amount of from 10.sup.8-10.sup.11 colony forming units (CFU)/dose.

8. A sealed container filled with one or more doses of a microbial composition as claimed in claim 1.

9. A sealed container as claimed in claim 8 in the form of a sachet or in tubular form.

10. A sealed container as claimed in claim 9, wherein the tubular form is a stick pack or straw.

11. A method of making a packaged microbial composition as claimed in claim 1 comprising (a) mixing ingredients (i)-(iv) and optionally (v) and/or (vi); and (b) packaging the mixture in a sealed container.

12. A method as claimed in claim 11 wherein one or more of the ingredients is dried to a water activity of less than 0.15 prior to mixing step (a).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) We describe below non-limiting examples which embody one or more aspects of the invention. These examples are described with reference to the following figures in which:

(2) FIG. 1(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of a preferred composition of the invention (“particularly preferred composition 1”) produced on a lab scale with Lactobacillus plantarum 299v.

(3) FIG. 1(b): shows the water activity measurements for the composition of FIG. 1(a) over time (months).

(4) FIG. 1(c): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of the composition of FIG. 1(a) up to 18 months.

(5) FIG. 1(d): shows the water activity measurements for the composition of FIG. 1(a) over time (months) up to 18 months.

(6) FIG. 2(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) for a preferred composition of the invention (“particularly preferred composition 1”) produced on an industrial scale with Lactobacillus plantarum 299v.

(7) FIG. 2(b): shows the water activity measurements over time (months) for the composition of FIG. 2(a).

(8) FIG. 3(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of a preferred composition of the invention described as “particularly preferred composition 1” except using a combination of Lactobacillus paracasei 8700:2 (DSM 13434) and Lactobacillus plantarum HEAL9 (DSM 15312) in place of Lactobacillus plantarum 299v.

(9) FIG. 3(b): shows the water activity measurements for the composition of FIG. 3(a) over time (months).

(10) FIG. 4(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of a preferred composition of the invention described as “particularly preferred composition 1” except using Lactobacillus paracasei 8700:2 (DSM 13434) in place of Lactobacillus plantarum 299v.

(11) FIG. 4(b): shows the water activity measurements for the composition of FIG. 4(a) over time (months).

(12) FIG. 5(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of a preferred composition of the invention described as “particularly preferred composition 1” except using Lactobacillus plantarum 299 (DSM 6595) in place of Lactobacillus plantarum 299v.

(13) FIG. 5(b): shows the water activity measurements for the composition of FIG. 5(a) over time (months).

(14) FIG. 6(a): shows the storage stability over time (months) at 25° C. and 60% relative humidity (RH) of a preferred composition of the invention described as “particularly preferred composition 1” except using Lactobacillus rhamnosus 271 (DSM 6594) in place of Lactobacillus plantarum 299v.

(15) FIG. 6(b): shows the water activity measurements for the composition of FIG. 6(a) over time (months).

(16) In each of the figures, “10B limit” and “16 limit” represent a lower limit of 10 billion and 1 billion CFU respectively, being a desired dose for probiotic effectiveness of the particular strain(s) used in the respective figure. It will be appreciated that the storage stability of the fast melt formulations exemplified herein is such that the viable count of probiotic bacteria in each figure does not fall below the desired dose for the particular strain(s) in each of the exemplary products.

EXAMPLES

(17) Materials and Methods

(18) Many different types of ingredients were evaluated in order to produce a fast-melt powder formulation probiotic bacteria with a low water activity (<0.15) and desired sensory properties.

(19) The water activities of the different ingredients were measured at 25° C. with the instrument Aqualab 4TE. When the water activity values of any ingredient were higher than 0.15, drying of the ingredients was performed on a lab scale by adding desiccant bags together with the powder in sealed aluminium bags for 1-3 days. After the drying step the water activity was measured again.

(20) The sensory properties of the ingredients were evaluated regarding to consistency, mouthfeel, particle size, melting properties in mouth, taste and sweetness. The sensory evaluation was performed by a panel of qualified personnel.

(21) Addition of a moisture absorbent fibre (inulin) was able to achieve a lower total water activity and also a preferred mouthfeel property.

(22) Further, a dry bulking agent, such as maltodextrin, could be added to the formulation in order to lower its total water activity.

(23) A probiotic strain or combination of strains was added to give the preferred probiotic health effects. Lactobacillus plantarum 299v were chosen for this formulation, but other probiotic bacteria in the suggested range of 10.sup.3-10.sup.12 CFU/dose and more preferred 10.sup.8-10.sup.11 CFU/dose should be included.

(24) A silicon dioxide flow agent was able to achieve a satisfactory flow of the powder during process and packaging, but other known alternative powder processing aids and powder flow agents would work as well (for example due to regulatory issues on different markets).

(25) Flavorant(s) were added to the formulation in order to give the product a pleasant taste.

(26) Storage stability at 25° C./60% Relative Humidity was evaluated to examine the shelf life of a preferred product over time (months).

(27) Our next step was to industrialize the large scale production of the probiotic composition. The water activity of the ingredients of the composition was measured. If the water activity value of an ingredient was higher than 0.15, drying of the ingredient was conducted by fluidized bed drying.

(28) Blending of the ingredients were performed using a conventional cone- or v-shaped blenders to achieve a homogenous blend with a minimized loss of the probiotic bacteria.

(29) The final homogenous powder blend was filled in in stick packs. Handling, blending and filling of probiotic formulations are performed in a controlled atmosphere, temperature and relative humidity. The stick packs could be of different materials, but to achieve a good storage stability of the probiotic bacteria they preferably include a barrier of aluminium, to protect the powder from moisture.

(30) Results

(31) Evaluation of Ingredients

(32) The evaluation of ingredients showed that the sugar alcohols were the most influential ingredient for desirable sensory properties. Erythritol has functional benefits such as a clean and natural sweet taste and it gives the desired cool mouthfeel and it melts quickly in mouth. Besides that, it has nutritional benefits such as high digestive tolerance (compared to other polyols) and is non-cariogenic. Also, Xylitol has a sugar pleasant sweet taste and a cooling mouthfeel.

(33) Different qualities of the sugar alcohols were evaluated and the most obvious differences for the sensory experience between the qualities related to the particle size. Qualities with smaller particle sizes melted faster in the mouth and gave a more pleasant mouthfeel than the ones with larger particle sizes. It is also of importance to choose ingredients in the similar range of particle sizes, in order to more easily achieve a homogenous blend in the final composition.

(34) Table 1, Appendix 1 shows evaluation of ingredients and the results of lab scale drying tests, particle sizes and comments from the sensory testing panel.

(35) Formulation of Preferred Probiotic Compositions

(36) Different combinations of ingredients were evaluated with respect to sensory properties and water activity, see table 2. After finding the desired properties a second evaluation was made to provide the most preferred formulation with specific amounts of the ingredients, see table 3.

(37) In earlier studies using formulations with inulin and probiotic bacteria, we found that storage stability was improved considerably by the inclusion of inulin fibres. We reasoned that the fibres were binding free water in the compositions and at higher temperatures the fibres released the water. This led to decreased storage stability, that is, the count of viable bacteria fell at higher temperatures.

(38) With reference to Table 1 it can be seen that, even after drying, the water activity of the preferred sugar alcohol erythritol was higher (0.24) than would normally be acceptable (less than 0.15). However, in light of our earlier studies we reasoned that inclusion of a moisture absorbent fibre such as inulin, could compensate for the higher than desired water activity of the sugar alcohol.

(39) FIGS. 1(a) and 2(a) confirm that the probiotic fast melt compositions of the invention have unexpectedly good storage stability as well as desirable sensory properties.

(40) By combining sugar alcohols, which have too high water activity on their own and which were difficult to dry to the full extent, with a moisture absorbent fibre and drying the blend, a fast-melt probiotic powder a formulation with unexpected good stability and improvement of mouthfeel was obtained.

(41) The moisture absorbent fibre functions in two ways. It helps to bind up free water in the formulation so that the probiotic bacteria are protected from moisture even though the water activity is somewhat higher than one would normally desire due to the inclusion of sugar alcohols. This is shown with good trends of the storage stability testing of the formulation.

(42) The other benefit of including the moisture absorbent fibre together with the sugar alcohol is that it gives the formulation the right consistency and mouthfeel, which makes the flavorant stay in mouth the desired time for the best taste experience.

(43) A particularly preferred composition 1 of the invention with excellent storage stability and sensory properties consists of:

(44) TABLE-US-00001 Weight percent Content Ingredient % w/w (mg/stick pack) Erythritol (sugar alcohol) 45 450 Inulin (moisture absorbent fibre) 22.75 227.5 Xylitol (sugar alcohol) 22.75 227.5 Lactobacillus plantarum 299v 8 80 (probiotic bacteria) Lemon flavour 1 10 Silicon dioxide 0.5 5 (powder flow agent) Total weight 100 1000

(45) Another particularly preferred composition 2 of the invention with excellent storage stability and sensory properties consists of:

(46) TABLE-US-00002 Weight percent Content Ingredient % w/w (mg/stick pack) Erythritol 42.5 425 Inulin 21.5 215 Xylitol 21.5 215 Maltodextrin 5 50 (dry bulking agent) L. plantarum 299v 8 80 Lemon flavour 1 10 Silicon dioxide 0.5 5 Total weight 100 1000

(47) In FIGS. 3-6, when changing the identity of the probiotic strain in the recipe of “particularly preferred composition 1”, the combined mass of erythritol, inulin and xylitol was adjusted according to the mass of powder of the replacement probiotic strain(s) without altering the internal proportion between the erythritol, inulin and xylitol.

(48) FIGS. 3-6 confirm that the probiotic fast melt compositions of the invention have unexpectedly good storage stability as well as desirable sensory properties with several different probiotic strains. Hence, the fast melt properties of the probiotic fast melt compositions of the invention are not expected to differ according to the identity of the probiotic strain therein.

(49) In accordance with the most preferred embodiment of the invention, in the probiotic fast melt compositions of the invention, including “particularly preferred composition 1” and “particularly preferred composition 2”, the particle size profile of the sugar alcohol(s) (e.g. erythritol and/or xylitol) and moisture absorbent fibre(s) (e.g. inulin) is as described above in the section entitled “Powdered form”.

(50) TABLE-US-00003 TABLE 1 Evaluation of ingredients Water activity Water activity (Aw) after (Aw) before drying 1-3 Ingredient drying days Particle size Sensory evaluation Erythritol 1 0.25 0.24 <250 μm Max 20% Mouthfeel like granulated sugar. Too large particles. Erythritol 2 0.37 0.24 >150 μm Max 5% Nice mouthfeel and >250 μm Max 0.5% sweet taste. Melts quickly in mouth. Cooling effect. Xylitol 1 0.29 0.11 <100 μm Max 6% Nice taste, a bit >500 μm Max 5% sugary. Melts quickly in mouth. Some cooling effect. Xylitol 2 >800 μm Max 5% Too large particles. <200 μm Max 10% Sorbitol 1 0.27 0.07 <100 μm Max 6% Nice taste, a bit >200 μm Min 50% sugary. Melts quickly Max 75% in mouth. No cooling >500 μm Max 2% effect. Isomalt 1 0.29 0.04 >500 μm Max 5% Nice taste. >250 μm 20-70% Disappears very <63 μm Max 15% quickly in mouth. Not so sweet. No cooling effect. Isomalt 2 0.27 0.12 >500 μm Max 5% Nice neutral taste. >250 μm 20-70% Disappears very <63 μm Max 15% quickly in mouth. Not so sweet. No cooling effect. Inulin 1 0.16 0.04 <25 μm Max 10% No fast melt mouth >100 μm Min 20% feeling. Becomes like a sugary lump directly into mouth. Inulin 2 0.17 0.08 <60 μm Max 20% No fast melt mouth >150 μm Min 20% feeling. Becomes like a sugary lump directly into mouth. Oligofructose 1 0.20 0.07 <50 μm Max 10% No fast melt mouth >165 μm Min 20% feeling. Becomes like a sugary lump directly into mouth. Sweeter taste than inulin.

(51) TABLE-US-00004 TABLE 2 Formulation Evaluation 1 Formu- lation Bulk Water number formulation % Sensory evaluation activity 1 Erythritol 2 70 Like Erythritol in both N/A Isomalt 1 30 appearance and taste. With lumps. Cannot be poured. 2 Erythritol 2 99 No lumps and easier to pour. N/A Silicon dioxide 1 Same taste as only Erythritol. 3 Erythritol 2 69 Free flowing. Melts quickly in 0.13 Silicon dioxide 1 mouth. Cooling effect. More Isomalt 1 30 balanced sweetness than for only Erythritol 4 Erythritol 2 69 Relatively free flowing 0.11 Silicon dioxide 1 powder. First a cooling effect Oligofructose 1 30 and it disappears quickly. Then a few particles left in mouth, that disappears quickly. 5 Erythritol 2 29 Free flowing powder. Melts a 0.06 Silicon dioxide 1 bit slower than no 3 and 4, Isomalt 1 70 but still rather quickly. Not so cooling effect. 6 Erythritol 2 29 Relatively free flowing 0.04 Silicon dioxide 1 powder. Clear inulin flavor. It Inulin 1 70 forms clumps in the mouth, but not that much as for only inulin. No cooling effect. 7 Erythritol 2 49 Relatively free flowing 0.08 Silicon dioxide 1 powder. The particle size of Inulin 2 25 xylitol is perceived a bit Xylitol 1 25 larger than for the other ingredients. Very nice mouthfeel. Is perceived very sweet. Melts nice in mouth, but remains for a short while. The best formulation. 8 Erythritol 2 49 Stays in mouth a bit longer 0.06 Silicon dioxide 1 than formulation no 7. Inulin 2 35 Clearer sweet aftertaste of Xylitol 1 15 the inulin. A bit less cooling effect than no. 7. Certain graininess. 9 Erythritol 2 49 Free flowing powder. More 0.08 Silicon dioxide 1 cooling effect and melts Inulin 2 15 faster in mouth than no. 8. Xylitol 1 35 Certain graininess.

(52) TABLE-US-00005 TABLE 3 Formulation Evaluation 2 Formu- lation Bulk Water numb formulation % Sensory evaluation activity 10 Erythritol 2 59 Disappears quickly in 0.18 Silicon Dioxide 1 mouth, the mouthfeel gets a Xylitol 1 40 bit flat. 11 Erythritol 2 69 Nice mouthfeel and taste. Silicon Dioxide 1 Inulin 2 30 12 Erythritol 2 49 Nice cooling effect. Melts Silicon Dioxide 1 quick in mouth but still with Inulin 2 25 a certain body that gives a Xylitol 1 25 nice overall mouthfeel. The best formulation. 13 Erythritol 2 49 Too much taste of inulin. Silicon Dioxide 1 Inulin 2 35 Xylitol 1 15 14 Erythritol 2 49 Too sweet taste. Silicon Dioxide 1 Disappears quickly in Inulin 2 15 mouth. Xylitol 1 35 15 Erythritol 2 39 Not that cooling effect. Silicon Dioxide 1 Clear inulin taste Inulin 2 30 Xylitol 1 30

(53) TABLE-US-00006 TABLE 4 Weight % and particle sizes for preferred composition Weight Max Ingredient percentage 8.3 <60 63 >90 <100 >125 >150 >250 >500 >710 μm Erythritol 2   45% max 5% max 0.5% Xylitol 1 22.75% max 6% max 5% Inulin 2 22.75% max 20% Min 20% Silicon dioxide  0.50% 100% Lp299v    8% 10% 5% 30% 40% 15% 3%