HK1153775B - Bacterial composition - Google Patents

Description

Bacterial compositions

Technical Field

The present invention relates to bacterial compositions having long shelf life, and methods of producing such compositions. The invention also relates to food products comprising the bacterial composition.

Background

Several patent documents disclose bacterial formulations formulated with specific excipients or carriers that claim to increase the storability of the bacterial formulation. Further patent literature relates to reducing the water activity (aw) of bacterial preparations, for example by adding SiO which will bind some water₂. There are also different forms of desiccants used for water removal.

US4956295 discloses dried lactobacilli mixed with a silica gel adsorbent. It is claimed that the mixture can be stored without refrigeration.

US7122370B and US7229818B disclose formulations comprising probiotic bacteria with monovalent alginate salts, wherein the formulations have a water activity between 0.01 and 0.07 and wherein an alginic acid gel is formed when exposed to an acidic environment, protecting the probiotic bacteria from the antibiotic effect of the acidic environment. The alginate is dried to a moisture content of less than 5% prior to mixing with the bacteria.

EP1482811B discloses a method for obtaining a food product comprising the steps of

a. Mixing the viable microbial preparation with other ingredients,

b. drying the mixture to a water activity of less than 0.3,

c. the mixture is compressed and the mixture is compressed,

d. a coating, and

e. mixing with food product.

US2005/0100559 discloses a dry bacterial composition having a water activity of less than 0.5.

US6953592B discloses a matrix based on a non-foaming, water-soluble or water-dispersible carbohydrate, which matrix comprises in closed pores an amount of entrapped gas sufficient to promote dissolution or dispersion of the matrix when contacted with water.

US2005/0266069 claims viable and stable probiotic formulations for intestinal targeting comprising: a plurality of probiotic microspheres, each microsphere comprising: a core comprising one or more probiotic bacteria, a cellulosic excipient, a disintegrant, and one or more additives; and an enteric coating capable of resisting gastric juice having a residual level of less than 5% and a water activity (aw) of between 0.1 and 0.5.

WO 2005/063200 discloses probiotic tablets comprising probiotic micro-organisms (e.g. Lactobacillus GG) and other nutritionally active ingredients in two zones; the first zone comprises said probiotic micro-organisms and the second zone comprises at least one of said other active ingredients. The water activity in the first zone should be no greater than 0.2. Good microbial viability is said to be obtained despite the relatively high total water content.

WO08048731A claims a method of extending the shelf life of a powdered nutritional formulation comprising LGG by reducing the water activity of the formulation comprising LGG to less than about 0.16 and maintaining the formulation temperature at 25 ℃ or below 25 ℃.

US7037708B discloses a composition comprising at least one Lactobacillus plantarum (Lactobacillus plantarum) in a carrier-bound form, which a) has a particle size of at least about 0.1mm and b) comprises from about 10E10 to 10E12cfu/g of at least one microbial species; c) has a water activity (aw) of less than 0.15 and d) is compressed. It is disclosed that the culture may comprise an effervescent additive.

However, none of these bacterial preparations is satisfactory from a commercial point of view, in particular for bifidobacterial cells. Accordingly, there is a need for improved compositions comprising bacterial cells with improved stability and enhanced delivery of viable bacterial cells.

In particular, there is a need to provide stable compositions comprising strains of bifidobacterium, which were previously extremely difficult to store for long periods at room temperature.

Disclosure of Invention

The present inventors have surprisingly found that tablets comprising cells of a Bifidobacterium (Bifidobacterium) strain and sodium bicarbonate carbonate have excellent stability, in particular if stored in an environment which is not exposed to ambient moisture, for example in a sealed aluminium bag or closure, or in a closed glass container. The superior stability is most pronounced when the protected tablet is exposed to high temperatures for extended periods of time.

Furthermore, the inventors have found that the addition of a salt of carbonic acid to a composition comprising cells of a strain of bifidobacterium surprisingly prevents or reduces discolouration (red pigment) which normally occurs during storage.

Based on these surprising findings, the present invention relates to a method for stabilizing (e.g. increasing the stability and/or reducing/avoiding discolouration) a composition comprising viable cells of a strain of bifidobacterium by adding a salt of carbonic acid to the composition. In particular, the present invention relates to a method for improving the color stability of a composition comprising bacterial cells, said method comprising mixing the bacterial cells with a salt of carbonic acid.

The present invention may be seen as a method for improving the stability/shelf-life of a bacterial composition, and thus the present invention also relates to a method for preparing a bacterial composition with improved increased/improved/extended stability/shelf-life comprising the steps of:

a) providing a powder by mixing bacterial cells, preferably bacterial cells belonging to the genus bifidobacterium, with a carrier comprising a carbonate salt; and

b) the powder is optionally compressed to form tablets or pellets.

The invention also relates to a stabilized composition and to a food product comprising the stabilized composition, the stabilized composition being either incorporated into or packaged with the food product.

Detailed Description

In a first aspect, the present invention relates to a method for preparing a bacterial composition (e.g. for improving the survival of cells and/or reducing discolouration of the composition) comprising the steps of:

a) providing a powder by mixing bacterial cells with a carrier comprising a salt (or carbonate) of carbonic acid; and

b) optionally compressing the powder.

Furthermore, the present invention relates to a method for i) increasing the survival time of bacterial cells in a composition, or ii) avoiding or reducing discoloration of a bacterial composition, or iii) increasing the shelf life of a bacterial composition, or iv) increasing the stability of a bacterial composition, comprising the steps of:

a) providing a powder by mixing bacterial cells with a carrier comprising a salt (or carbonate) of carbonic acid; and

b) optionally compressing the powder.

In an interesting embodiment of the invention, the method is used for improving the stability (including color stability) of a composition comprising bacterial cells by mixing the bacterial cells with a salt of carbonic acid.

It will be appreciated that the purpose of the mixing step a) is to contact the bacterial cells with the salt, and therefore the order of mixing is not important. Thus, the bacterial cells may be mixed with the carrier before and/or after and/or simultaneously with the mixing with the salt. It is currently preferred that all ingredients be mixed simultaneously.

By compressing the powder, the product of the process of the invention will be a bacterial composition in the form of a powder or in a solid form such as a tablet or pellet.

The salt of carbonic acid is preferably selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, potassium carbonate, potassium bicarbonate, potassium sesquicarbonate, magnesium carbonate, sodium glycinate carbonate, L-lysine carbonate, arginine carbonate, amorphous calcium carbonate, ammonium bicarbonate and combinations thereof, and the presently most preferred salt is sodium bicarbonate.

a) The carrier in (1) also contains an acidic component, such as an organic acid, preferably in powder form.

The acidic component is preferably selected from the group consisting of citric acid, tartaric acid, palmitic acid, fumaric acid, adipic acid, lactic acid, succinic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate and combinations thereof.

In an embodiment of the invention, the method further comprises a drying step, such as freeze drying, vacuum drying, drying by desiccant or heating. The drying step may be carried out as follows:

i) prior to the mixing step (i.e., one or more of the bacterial cells or the carrier is dried); and/or

ii) after the mixing step (i.e., the mixture is dried); and/or

iii) after an optional compression step (i.e. the tablets or pellets are dried).

The bacterial cells are preferably lactic acid producing cells and/or probiotic cells, for example bacterial cells belonging to a genus selected from the group consisting of: bifidobacterium, Lactobacillus and Streptococcus (Streptococcus). Presently preferred bacterial cells belong to a species selected from the group consisting of: lactobacillus casei (Lactobacillus casei), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus bifidus (Lactobacillus bifidus), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus plantarum, Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus rhamnosus (Lactobacillus amylovorus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus crispatus (Lactobacillus crispus), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium lactis (Bifidobacterium), Bifidobacterium breve (Bifidobacterium), Bifidobacterium bifidum thermophilum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium lactis), Bifidobacterium breve (Bifidobacterium), Bifidobacterium bifidum (Bifidobacterium), Bifidobacterium lactis (Bifidobacterium), Bifidobacterium (Bifidobacterium) and Bifidobacterium (Bifidobacterium) bacteria (Bifidobacterium longum (Bifidobacterium) are preferably used in the preparation for treating aoccicus thermophilus) and Lactococcus lactis (Lactococcus lactis), for example, bacterial cells belonging to a strain selected from the group consisting of: BB-12LA-1, LA-5, BB-02, Bifidobacterium animalis strain DSM15954, Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.infantis) strain DSM15953, Bifidobacterium longum subspecies longum (Bifidobacterium longum subsp.longum) strain DSM15955, Enterococcus faecalis (Enterococcus faecalis) strain DSM15958, Lactobacillus acidophilus strain DSM13241, Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) strain DSM15956, Lactobacillus helveticus (Lactobacillus helveticus) strain DSM14998, Lactobacillus helveticus strain DSM14997, Lactobacillus lactis strain DSM14797, Streptococcus fermentum strain DSM15957, Lactobacillus rhamnosus strain ATCC55826 and a mutant or variant of any one of them. It is presently most preferred that the bacterial cells belong to Bifidobacterium strain BB-12Or BB-12free。

The carrier may further comprise an ingredient selected from the group consisting of: inorganic acids or salts thereof, organic acids or salts thereof, carbohydrates, lactose, sugar alcohols, soluble fibers and starch.

The process of the invention may further comprise additional steps such as a coating step wherein for example the tablets/pellets of the invention are coated in a manner known in the art, and/or a packaging step, for example comprising placing the powder or compressed powder (tablets/pellets) in a sealable container, for example an aluminium and/or polymer container. 2. The method of claim 1 for preparing a bacterial composition with no or reduced discoloration, comprising the steps of:

a) providing a powder by mixing bacterial cells with a carrier comprising a salt of carbonic acid; and

b) the powder is optionally compressed to form tablets or pellets.

3. The method of claim 1 for preparing a bacterial composition with improved cell survival and/or increased shelf life, and/or increased stability, comprising the steps of:

a) providing a powder by mixing bacterial cells with a carrier comprising a salt of carbonic acid; and

b) the powder is optionally compressed to form tablets or pellets.

4. The method of claim 1 for improving cell survival in a composition comprising bacterial cells, comprising mixing the bacterial cells with a salt of carbonic acid.

5. The method of claim 1 for improving the color stability of a composition comprising bacterial cells, comprising mixing the bacterial cells with a salt of carbonic acid.

In a second aspect, the present invention relates to a composition obtainable by the process of the invention. The compositions of the invention comprise bacterial cells and a carrier comprising a salt (or carbonate) of carbonic acid. The bacterial cells may belong to any of the above species or strains. In a currently interesting embodiment, the cells belong to Bifidobacterium strain BB-12The composition may comprise at least 10E5CFU/mg, for example at least 10E7CFU/mg or at least 10E9CFU/mg of bacterial cells, CFU being a cell forming unit of bacterial cells.

In a third aspect, the present invention relates to the use of the composition of the invention as a food, feed additive, medicament, dietary supplement or probiotic. Food or feed products comprising the composition of the invention are also an aspect of the invention and include products such as: dairy products, such as milk or fermented milk, and fruit juices, such as smoothies.

The food/feed product may be mixed with the composition of the invention or mixed at a food/feed processing plant or mixed by the consumer. Thus, the invention also relates to a kit of parts comprising a food/feed product and a composition according to the invention, e.g. a kit comprising a container with a food product and a container with a composition according to the invention.

In a further aspect, the present invention relates to the use of a salt of carbonic acid (such as the above mentioned salt) as a bacterial composition, in particular a bacterial composition comprising bacterial cells belonging to the genus bifidobacterium (such as belonging to a species selected from bifidobacterium longum, bifidobacterium bifidum, bifidobacterium lactis, bifidobacterium breve, bifidobacterium animalis, bifidobacterium adolescentis, bifidobacterium infantis), i) a stabilizer, such as a color stabilizer, ii) for improving its shelf life, iii) for increasing its stability, or iv) for increasing the survival of cells therein.

Definition of

In the present context, the term "tablet" refers to a compressed powder. The term includes all physical forms and all sizes, such as pills, pellets, tablets and the like.

The term "bacterial composition" is to be understood as a composition comprising bacterial cells or cell cultures. Preferably the cells are viable or dormant, and further preferably the composition comprises at least 10E5 cell forming units/g. The bacterial cells may belong to a single strain or a mixture of cells belonging to different strains.

In the present context, the term "mutant" is to be understood as a strain derived from a strain of the invention by, for example, genetic engineering, irradiation and/or chemical treatment. Preferably the mutant is a functionally equivalent mutant, e.g. a mutant having substantially the same or improved properties as the parent strain, e.g. probiotic. Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenesis treatment, including treatment with a chemical mutagen such as Ethyl Methanesulfonate (EMS) or N-methyl-N' -nitro-N-Nitroguanidine (NTG), ultraviolet light, or to spontaneous mutation.

In the present context, the term "variant" is to be understood as a strain which is functionally equivalent to the strain of the invention, e.g. has essentially the same or improved properties as e.g. a probiotic. Such variants that can be identified using appropriate screening techniques are part of the present invention.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Illustration of the drawings

FIG. 1: BB-12 in powders H1069 and H1071 stored at 37 deg.C for 3 monthsThe survival situation of (1).

FIG. 2: pictures of powder mixtures H1184 a (a), H1184 b (b), H1184 c (c), and H1184 d (d) are shown after 2 weeks at room temperature.

Examples

Example 1:

making two mixtures comprising milled BB-12HA (5.1% (w/w) and excipients (mannitol; 10% (w/w), lactose; 84.4% (w/w), magnesium stearate; 0.5% (w/w), sodium bicarbonate free powder mixture H1069, and containing milled BB-12HA (5.1% (w/w) and vehicle (mannitol; 10% (w/w), lactose; 54.4% (w/w), magnesium stearate; 0.5% (w/w) powder mix with sodium bicarbonate; 30% (w/w) H1071. sodium bicarbonate concentration was obtained by replacing the lactose portion of the vehicle with sodium bicarbonate, all other ingredients in the powder remaining at the same concentration.

The powder was packed in aluminum foil bags (20-30g) and stored at 37 ℃ for 3 months. Colony forming units (cfu/g) and water activity (aw) were analyzed for each bag immediately after packaging (0 months), after 1 month of storage and after 3 months.

As a result: the water activity of powders H1069 and H1071 immediately after packaging was the same, with aw (H1069) being 0.19 and aw (H1071) being 0.19. After 1 and 3 months of storage, the water activity of the powder increased to aw of 0.27-0.31. The results of colony-forming units showed comparable cfu/g at 0 and 1 month, but showed BB-12 after 3 monthsThe survival rate of (c) was very different (fig. 1).

And (4) conclusion: replacement of dextrose excipient with sodium bicarbonate results in BB-12 during storageImprovement in survival. The results show that sodium bicarbonate improves the survival of bifidobacterium animalis in the powder during storage.

Example 2:it was tested whether sodium bicarbonate affected the red coloration of the bifidobacterium formulation containing ascorbate.

Content of powder mixture (w/w)

-H1184A: BB-12 at 24% grindHA culture, 51% mannitol and 25% inulin

-H1184B: BB-12 at 24% grindHA culture, 51% mannitol and 25% sodium bicarbonate

-H1184C: BB-12 at 12% grindHA cultures, 63% mannitol and 25% inulin

-H1184D: BB-12 at 12% grindHA cultures, 63% mannitol and 25% sodium bicarbonate

Milled BB-12HA cultures contain ascorbate as a cryoprotectant. The ascorbate reacts with the fermentation residue and develops a red color. Increasing the temperature and humidity accelerates the reaction.

The powder was placed in an air impermeable aluminum foil bag. For testing, a quantity of 5-8g of powder was removed from the aluminum foil bag and placed in a small plastic container (made by Rotronic for aw measurement). The plastic container was left at room temperature and a difference in color development was seen after 2 weeks, as shown in fig. 2. The powder had a similar aw of about 0.4. Fig. 2 shows that powders a and C turn reddish in color, while powders B and D retain their original yellowish color.

And (4) conclusion: the presence of sodium bicarbonate in the powder mixture has an effect on the development of the red color. No red coloration developed when sodium bicarbonate was present in the powder mixture.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible modifications thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Reference to the literature

US2004175389，WO03075676，US2005100559，US6953592，WO03012819，WO05266069，WO05063200

All references cited in this patent document are hereby incorporated by reference in their entirety.

Claims (25)

1. A method for preparing a bacterial composition comprising the steps of:

a) providing a powder by mixing bacterial cells belonging to the genus bifidobacterium with a carrier comprising a salt of carbonic acid; and

b) the powder is optionally compressed to form tablets or pellets.

2. The method of claim 1, wherein the bacterial composition is in the form of a powder, granule, tablet or pellet.

3. The method of claim 1, wherein the salt of carbonic acid is selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, potassium carbonate, potassium bicarbonate, potassium sesquicarbonate, magnesium carbonate, sodium glycinate carbonate, L-lysine carbonate, arginine carbonate, amorphous calcium carbonate, ammonium bicarbonate, and combinations thereof.

4. The method of claim 1 wherein the carrier in a) further comprises an acidic component.

5. The method of claim 1, further comprising a drying step.

6. The method of claim 1, wherein the drying step is performed prior to the mixing step.

7. The method of claim 1, wherein the drying step is performed after the mixing step.

8. The method of claim 1, wherein the drying step is performed after the optional compressing step.

9. The method of claim 1, wherein the bacterial cell belongs to a species selected from the group consisting of: bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, and Bifidobacterium infantis.

10. The method of claim 1, wherein the carrier further comprises an ingredient selected from the group consisting of: inorganic acids or salts thereof, organic acids or salts thereof, carbohydrates, lactose, sugar alcohols and soluble fibers.

11. The method of claim 1, further comprising a packaging step.

12. A composition obtainable by the process of any one of claims 1 to 11.

13. The composition of claim 12, wherein the bacterial cell is of a species selected from the group consisting of: bifidobacterium longum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium breve (Bifidobacterium breves), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium adolescentis (Bifidobacterium adolescentis), and Bifidobacterium infantis (Bifidobacterium infantis).

14. A powder composition comprising bacterial cells belonging to the genus bifidobacterium and a carrier, the carrier comprising a salt of carbonic acid.

15. The composition of claim 14, wherein the bacterial cell is of a species selected from the group consisting of: bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, and Bifidobacterium infantis.

16. The composition of any one of claims 12-15, wherein the bacterial cells are of a strain of bifidobacterium

17. The composition of any one of claims 12-15, comprising at least 10E5CFU/mg of bacterial cells belonging to the genus bifidobacterium.

18. Use of a composition according to any one of claims 12 to 15 as a food or feed additive, medicament, dietary supplement or probiotic.

19. A food or feed product comprising the composition of any one of claims 12-17.

20. A kit-of-parts comprising the composition of any one of claims 12 to 15 and a food article.

21. Use of a salt of carbonic acid as a colour stabilizer in a bacterial composition provided as a powder, wherein the bacterial composition comprises bacterial cells belonging to the genus bifidobacterium.

22. Use of a salt of carbonic acid for increasing cell survival in a bacterial composition provided as a powder, wherein the bacterial composition comprises bacterial cells belonging to the genus bifidobacterium.

23. Use of a salt of carbonic acid for increasing the shelf life of a bacterial composition provided as a powder, wherein the bacterial composition comprises bacterial cells belonging to the genus bifidobacterium.

24. Use of a salt of carbonic acid to improve the stability of a bacterial composition provided as a powder, wherein the bacterial composition comprises bacterial cells belonging to the genus bifidobacterium.

25. The use of any one of claims 21-24, wherein the bacterial cell belongs to a species selected from the group consisting of: bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, and Bifidobacterium infantis.