WO2000007571A2

WO2000007571A2 - Formulations having probiotically active microorganisms

Info

Publication number: WO2000007571A2
Application number: PCT/AT1999/000192
Authority: WO
Inventors: Helmut Viernstein
Original assignee: Helmut Viernstein
Priority date: 1998-08-06
Filing date: 1999-07-29
Publication date: 2000-02-17
Also published as: WO2000007571A3; AU5270499A; AT407008B; ATA136098A; EP1102580A2

Abstract

The invention relates to a formulation containing stabilized microorganisms and buffering substances. The inventive formulation facilitates the passage of the microorganisms through the stomach after their dispersion and their adsorption in the digestive tract while preserving their vitality.

Description

Formulations with probiotic microorganisms

The invention relates to formulations with probiotic microorganisms.

According to a modern definition (Havenaar et al., "Probiotics: A General View" in: "The Lactic Acid Bacteria, Volume 1", BJB Wood ed. (1992), the probiotic microorganisms (common synonym "probiotics") are , Elsevier Applied Science, 155/156) describes individual or mixed cultures of living microorganisms which are applied to humans and animals and which have a beneficial effect on the host by supporting the properties of the naturally present intestinal microflora. This means that only products that contain living or viable microorganisms, e.g. as freeze-dried cells, improve the health status of humans and animals and which can develop their effects in the mouth or the gastrointestinal tract.

From the group of lactic acid bacteria (MSB), in particular, after intensive research, special germs that have valuable properties in terms of human and food technology have become of scientific interest.

According to the current state of knowledge and current taxonomy, lactic acid bacteria of the genera Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Enterococcus, Bifidobacterium, Carnobacterium and Sporolactobacillus (Bergey's Manual of Systematic Bacteri) are in pharmaceutical preparations and food supplements according to the current state of knowledge and current taxonomy. increasingly found. Many subspecies of the genera Lactobacillus, Bifidobacterium and Enterococcus are considered to be necessary natural intestinal inhabitants of humans and animals. They occur - depending on nutritional status, age, state of health and intestinal tract - in dynamically changing amounts.

Lactic acid bacteria are assessed according to their abilities in a similar way to other microorganisms. These are: 1. biochemical (eg the utilization of carbohydrate utilization)

2. physiological (e.g. stimulation of intestinal peristalsis)

3.antimicrobial (e.g. inhibition of other microorganisms)

4. competitive (e.g. inhibition or degradation of enterotoxins).

(Volker Rusch, "Microbiological Therapy", Therapeutikum 6 (1992), pages 292-298; K. Zimmermann, "Gastrointestinal Passage Microbiological Preparations and Their Access to the Immune System with Oral Administration", top-medicine 8 (1994), Pages 29-31; Petra Kolb-Jaeckel, "Microbiological Therapy and Its Possibilities for Inflammatory Bowel Diseases", empirical medicine 43 (9) (1994), pages 502-504.

The microflora of the intestine can be specifically supported by the application of certain MSB via medical indications or the way of supplementing food. Today, the focus is on both dietary and prophylactic and therapeutic indications, such as for traveler's diarrhea, treatment of osteipation, restitution of a disturbed intestinal flora after taking antibiotics, regulating function in stressful situations, lowering the cholesterol level, cancer therapy, gingivitis, supplementing diet forms, etc.

The functionality of viable or living MSB is only ensured if the preferred germs get into the intestine in high density. Natural physiological barriers can kill microorganisms in unprotected forms of intake. Especially with the direct oral administration form of viable microorganisms, it should be noted that, depending on the genus or strain, the majority of the bacteria are already destroyed in the stomach, which with its acidic milieu is a natural barrier for almost all types of bacteria.

In addition, living microorganisms are difficult to stabilize. In preparations, they are usually used dried as viable, lyophilized microorganisms. The display is the responsibility of the culture producers. The continuous production of the microorganisms in the required qualities, as well as their further processing into physiologically effective forms. Definitions and the subsequent storage period determine the biological activity of the material (Brennan et al., J. Food Prot. 46 (1983), 887-892).

The administration of such probiotic preparations in liquid form, for example in beverages, has not previously been considered, since such cultures are inactivated in the stomach area in such a way that they can no longer produce positive effects in the intestine. Furthermore, ready-to-use probiotics cannot be stored in liquid form for sufficiently long periods. The provision of microorganisms in mixtures with (dry) effervescent powders (tartaric acid or citric acid; development of C0 ₂ from sodium hydrogen carbonate) has not been considered until now, since acidic reactions can occur during the dissolution process of effervescent powders, in which the microorganisms - analogous to the gastric passage - are irreversibly damaged.

The object of the present invention is to provide formulations containing probiotic microorganisms which, when properly administered in suspension form, guarantee that physiologically relevant germ contents enter the human or animal intestine and which can therefore be highly effective in the intestinal physiology. Linked to this is the task of maintaining the viability of the probiotics, especially the MSB, in the product itself.

The formulations themselves can be produced on a large scale and reproducibly as medical formulations for therapeutic indications or as an additional or preventive food supplement for humans and animals.

This object is achieved according to the invention by a formulation comprising buffer substances and microorganisms stabilized by lyophilization and. optionally technologically necessary auxiliaries, wherein the microorganisms stabilized by lyophilization are dry incorporated into the matrix-forming buffer substances and these formulations can be dry, for example either in powder form or as tablets. The preparations according to the invention develop their effect, which are predominantly in powder form or as a tablet, after appropriate dissolution and ingestion by the patient or consumer. Surprisingly, with the help of the buffer substances, the phase of the disintegration of the formulation can be carried out, for example, in water without a substantial loss of the number of bacteria, even if acidic reactions occur when dissolved. Apparently, the (spatial) close contact between microorganisms and buffer substances is sufficient to survive even the acidic processes during the dissolution process if the formulation according to the invention is formulated as effervescent powder and substances such as organic acids (e.g. tartaric acid, citric acid), acidifying agents, various (acidic) ) Flavors, etc. Furthermore, the buffer substances are also able to exert this protective effect when passing through the stomach.

After dispersion in water or other suitable liquids, the formulation according to the invention guarantees protection of the microorganisms stabilized by lyophilization from the natural acid barrier of the stomach, the microorganisms being protected by the action of the buffer substances of the formulation after their reconstitution. With the formulations according to the invention, after administration and subsequent gastric passage, the microorganisms therefore enter the intestine in a viable form, where they can develop their effectiveness.

The formulations according to the present invention are outstandingly suitable for the production of preparations for a wide variety of medical indications and as an additional or preventive food supplement for humans and animals.

The microorganisms stabilized by lyophilization preferably contain auxiliaries for lyophilization, as well as residues from the nutrient medium. These lyophilisate compositions determine the hygroscopic properties of the lyophilisate. Preferred lyophilisates have a _w values in the range from 0.2 or below, in particular from 0.1 to 0.2, after the freeze-drying process. As a result, they are long-lasting, but they are also able to absorb large quantities of water quickly from the ambient air, which of course must be avoided during storage got to. This problem can be technologically solved by further processing - tableting, filling, packaging - immediately after the introduction of lyophilized microorganisms into the effervescent matrix, so that contact with the ambient air is minimized. The packaging must have an a _w value of 0.1 to 0.2 in the long term so that free water is not available for active life processes in cells. Packaging in composite films, sachets or for tablets in aluminum sleeves with desiccants are particularly suitable for this. The storage temperature should be chosen so that 20 ° C is not exceeded.

Accordingly, the formulation according to the invention advantageously has an a _w value of 0.1 to 0.2, in particular 0.1 to 0.15. Therefore, buffer matrices which are composed of completely anhydrous mineral constituents are preferably also used according to the invention. In principle, these are easy to prepare, but have so far not been used in the production of probiotic formulations.

Preferred buffer systems in the context of the present invention are phosphate buffers, citrate buffers, carbonate buffers, malate buffers and tartrate buffers with alkalis and alkaline earths (pH 4-7.5).

Strains from the families Lactobacillus, Enterococcus, Bifidobacterium and Enterobacteriaceae, preferably from the genera Enterobacter and Escherichia, are used in the formulations according to the invention as microorganisms stabilized or preserved by lyophilization, in particular strains from the genera Lactobacillus delbrückii subs. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei GG, Lactobacillus casei subsp. casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium infantis, Bififobacterium longum (as well as other Bifidobacterium species) and physiologically important subspecies of Escherichia coli and mixtures of two or more species of these microorganisms are particularly preferred. Furthermore, yeast strains, in particular Saccharomyces bulardii for the human field, or other probiotic yeasts, for example Kluyveromyces marxianus for animals, are suitable for the production of the formulations according to the invention.

The physiologically significant effects ("probiotic" properties) of the species mentioned have already been sufficiently described. Their importance makes them preferred microorganisms in the context of the present invention.

Also suitable as microorganisms are those whose effects can be used for oral active immunization.

The formulations according to the invention advantageously comprise further formulation auxiliaries or technically necessary combinations of adjuvants. Depending on the type of microorganism, antioxidants and amino acids may be necessary. The substance classes mentioned are typically part of all natural systems of organic origin and are therefore also obtained on an industrial scale and used as additives. They are present in the concentrations used for the formulation according to the invention below the limit values permitted in medications and nutritional supplements and are completely harmless.

Auxiliaries used with preference are L-ascorbic acid and L-cysteine.

The decisive technological step for the formulations according to the invention lies in the use of buffer substances which, after being dissolved in a suitable solvent, such as water, fruit juices, mineral waters, etc., offer the microorganisms, which were also dry until then, optimal conditions for cell wall reconstitution and these then allow passage through the upper digestive tract, in particular the stomach, with the hydrochloric acid present in the stomach being buffered for a short time. This ensures that the microorganisms absorbed with the solution pass through the small intestine quickly and without damage. The formulation according to the invention is preferably dissolved into an approximately isotonic solution (between 150 and 350 mosmol, in particular 300 mosmol).

When dispersing the pharmaceutical form, a composition should preferably be produced which corresponds to the currently valid WHO recipe for rehydration during or after diarrhea.

According to a preferred embodiment, the formulation according to the invention is made available as a shower, the buffer substances being part of the dry shower bases. When the formulation according to the invention disintegrates, CO _{2 is formed} , which displaces the oxygen present in the solution, and thus leads to more favorable survival conditions for anaerobic and microaerophilic germs. The effervescent basics are preferably divided as effervescent granules (cf., for example, European Pharmacopoeia, 3rd edition (1997), 1847/1848 and Hagers Handbook of Pharmaceutical Practice, 5th edition, page 723).

The buffer granules are manufactured using the technologically customary granulation methods, e.g. by thermal granulation in heatable mixers, by granulation with reactive or non-reactive liquids in suitable commercial devices, whereby granulation can also be carried out in a vacuum (e.g. evacuable mixer with chopper with subsequent drying and screening, the TOPO® process or fluidized bed granulation) . Spiked with the intended microorganisms, these granules can then be tableted or filled into sachets.

The formulation according to the invention preferably contains one or more active pharmaceutical ingredients, in particular an antidarrheal active ingredient, as a result of which a superadditive, anti-diarrhetic effect results from the combination of these substances while at the same time restoring a healthy intestinal flora. Pharmaceutical active ingredients for the formulation according to the invention can be, for example, loperamide, domperidone and ofloxacin, which themselves have no germ-damaging effect on lactic acid bacteria. The active formulation according to the invention preferably preferably comprises

0.1 - 30% by weight of microorganisms stabilized by lyophilization, in particular 0.1 to 5% by weight, and - 10 - 99.9% by weight, in particular 70 to 99% by weight of buffer substances (shower base) and, if necessary, 0 - 80% by weight, in particular 0 to 2% by weight, of auxiliaries. The buffer substances, but also auxiliary substances and lyophilisates, are preferably in granular form or in powder form.

A particularly preferred formulation contains microorganisms of the genus Lactobacillus casei GG stabilized by lyophilization.

According to a further aspect, the present invention relates to the use of the formulations of the intestinal physiologically active according to the invention for the preparation of preparations for medical indications, in particular for gastrointestinal disorders, or as a food supplement and animal food supplement.

The formulations according to the invention are particularly suitable in all cases in which the natural intestinal flora has been impaired or destroyed, for example in the effective accompanying and aftertreatment during or after administration of antibiotics.

Furthermore, formulations according to the invention can also include glucose and / or oligosaccharides and / or other saccharides which are favorable for the growth of germs in the intestinal lumen, as well as polysaccharides and related substances (inulins, dietary fibers).

The invention is illustrated by the following examples, to which it is of course not intended to be limited.

Example 1: Stable formulations of effervescent granules (tablets) with lactic acid bacteria as an effective principle

Mix 1.1 .: Lactobacillus casei GG (LGG, Valio, Finland) approx. 10 ⁸ CfU (colony forming units) / dose

Sodium bicarbonate 984 mg

Potassium hydrogen carbonate 900 mg

Citric acid 1416 mg

Disodium hydrogen phosphate 180 mg

Sodium carbonate 600 mg

Aroma 120 mg

Mix 1.2:

Enterococcus faecium (Ef) (strain M74, Medipharm, Sweden) approx.

10 ⁸ CfU / dose

Sodium bicarbonate 984 mg

Potassium hydrogen carbonate 900 mg

Citric acid 1416 mg

Disodium hydrogen phosphate 180 mg

Sodium carbonate 600 mg

Aroma 120 mg

Mix 1.3:

Bifidobacterium lactis (strain Bb 12, Hansen 's Biosystems / Denmark) approx. 10 ¹⁰ CfU / dose

Glucose 2953 mg

Sodium carbonate 1107 mg

Citric acid 1036 mg potassium carbonate 203 mg

Disodium hydrogen phosphate 584 mg aroma 94 mg

Example 2: Incubation of the Formulations According to the Invention in Artificial Gastric Juice (aMS)

In the context of the present examples, the buffer substances are mixed with bacterial lyophilisates in a weight ratio of 249: 1. One part of lyophilisate is sufficient to achieve a germ density of approximately 10 ⁸ -10 ⁹ CfU / g of formulation mass. With modern lyophilisates, germ densities in ranges above 10 ¹² CfU / g are achieved, which are of course preferred for commercial products according to the invention. The incubation is carried out at a body temperature of 37 ° C to simulate as physiological conditions as possible.

The incubation in aMS is started by mixing the preparations according to the invention with 150 ml of water and 100 ml of aMS (= 100 ml of 0.1 normal HCl) and carried out with agitation in a shaking water bath (SWB).

A sample that is only incubated in water, but not in aMS, serves as a reference sample (REF).

Example 2.1:

The preparation containing Lactobacillus casei GG (formulation mass = 4.20 g) according to mixture 1.1 is examined for its stabilizing effect against the microorganisms during incubation in aMS. The course of the reduction in the number of bacteria is shown in Table 1.

Table 1 Survival rates of Lactobacillus casei GG bacteria after incubation in artificial gastric juice

Column 2 of Table 1 shows the course of the bacterial count as it results from the use of the buffer substance and LGG according to the invention in the case of incubation in aMS. Column 3 contains the REF data. Column 4 shows how aMS affects unprotected lactic acid bacteria in the form of a lyophilisate (Lyo).

Example 2.2:

Mix containing Enterococcus faecium

1.2 (formulation mass 4.20 g) is effect on the microorganisms during incubation in aMS was examined. The course of the bacterial count reduction is shown in Table 2.

Table 2 Survival rates of Enterococcus faecium bacteria after incubation in artificial gastric juice

As in Mix 2.1 for LGG, Ef behaves in a comparable way. In column 1 it is clear from the bacterial count that practically no bacterial loss occurred after incubation in aMS for 2 hours. Without buffer substances (column 4), Ef germs cannot survive in aMS.

Example 2.3:

The Bifidobacterium ssp. Preparation containing (formulation mass 5.98 g) according to mixture 1.3 is examined for its stabilizing effect against the microorganisms during incubation in aMS. The course of the bacterial count reduction is shown in Table 3.

Table 3 Survival rates of Bifidobacterium ssp. After incubation in artificial gastric juice

Also for Bifidobacterium ssp. there is no significant loss of germs over 2 hours when incubated in aMS.

Example 2.4: (Comparison with conventional shower formulations)

Composition of the effervescent mixture (according to Bauer et al. "Pharmaceutical Technology", 5th edition, page 316):

1560 mg citric acid 940 mg NaHC0 ₃ 10 mg LGG

Application amount: 2510 mg

For the investigation in aMS, 2.510 g of bacteria-containing effervescent mixture were suspended in 150 ml of water and a further 100 ml of artificial gastric juice were added; the incubation then took place in the SWB (37 ° C.).

For the reference investigation, 2.510 g of bacteria-containing effervescent mixture were suspended in 250 ml of water and then incubated at 37 ° C in the SWB.

Table 4 below lists the survival germ counts of LGG after incubation in artificial gastric juice and in parallel after incubation in water (REF).

Table 4 Survival rates of Lactobacillus casei GG after incubation in artificial gastric juice and pure water

The incubation of lactic acid bacteria in solutions that only contain effervescent mixtures shows that these auxiliary components alone do not offer any protection against artificial gastric juice at all. In addition, there is also insufficient stabilization of the germs after incubation in pure water.

Example of the effect of Lopera id on the survivability of lactic acid bacteria

For the investigation in aMS, 6.0 g of effervescent mixture (mixture 1.3; however instead of Bifidobacterium lactis LGG) and 2 mg of loperamide were dispersed in 150 ml of water and 100 ml of artificial gastric juice were added; the incubation then took place in the SWB (37 ° C.).

6.0 g of effervescent mixture and 2 mg of loperamide were dispersed in 250 ml of water for the reference sample; the incubation then took place in the SWB (37 ° C.).

Table 5 Survival rate of Bifidobacterium lactis after incubation in artificial gastric juice in the presence of loperamide

From Table 5 it can be seen that the active ingredient loperamide has no negative influence on the survivability of the lactic acid bacteria during the incubation in pure water. The combination of loperamide and artificial gastric juice also has no negative influence on the number of bacteria.

Example 4: Effect of domperidone on the survivability of lactic acid bacteria

For the investigation in aMS, 6.0 g of effervescent mixture (mixture 1.3; however instead of Bifidobacterium lactis LGG) and 10 mg domperidone were dispersed in 150 ml water and 100 ml artificial gastric juice were added; the incubation then took place in the SWB (37 ° C.).

For the reference sample, 6.0 g of effervescent mixture and 10 mg of domperidone were dissolved in 250 ml of water, followed by incubation in the SWB (37 ° C).

Table 6 Survival rates of LGG after incubation in artificial gastric juice in the presence of domperidone

Table 6 shows that the active ingredient domperidone has very little influence on the survivability of the lactic acid bacteria during the incubation. -The combination of domperidone and artificial gastric juice causes a slightly greater decrease in the number of bacteria than water. E.g. Effect of ofloxacin on the survivability of lactic acid bacteria

For the investigation in aMS, 6.0 g of effervescent mixture (mixture 1.3; however instead of Bifidobacterium lactis LGG) and 200 mg ofloxacin were dispersed in 150 ml of water and 100 ml of artificial gastric juice were added; the incubation then took place in the SWB (37 ° C.).

For the reference sample, 6.0 g of effervescent mixture and 200 mg ofloxacin were dispersed in 250 ml of water and then incubated in the SWB (37 ° C).

Table 7 Survival rates of LGG after incubation in artificial gastric juice in the presence of ofloxacin

The results in Table 7 show that the active ingredient ofloxacin only slightly influences the lactic acid bacteria during the incubation in artificial gastric juice.

Claims

Patent claims:

1. Formulation comprising buffer substances and stabilized microorganisms, the microorganisms being incorporated dry into matrix-forming buffer substances.

2. Formulation according to claim 1, characterized in that the buffer matrix is composed of dry shower bases.

3. Formulation according to claim 1 or 2, characterized in that the microorganisms stabilized by lyophilization are selected from the families Lactobacteriaceae, preferably from the genera Lactobacillus, Streptococcus, Enterococcus and / or Bifidobacterium, and Enterobacteriaceae, preferably from the genera Enterbacter and / or Escherichia.

4. Formulation according to one of claims 1 to 3, characterized in that the stabilized by lyophilization microorganisms are selected from the species Lactobacillus delbrückii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei GG, Lactobacillus casei subsp. casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, physiologically significant subsp. from Escherichia coli, the yeasts Saccharomyces boulardii and Kluyveromyces marxianus and mixtures of two or more of these species.

5. Formulation according to one of claims 1 to 4, characterized in that it

0.1 - 30% by weight, in particular 0.1 to 5% by weight, microorganisms stabilized by lyophilization and - 10 - 99.9% by weight, in particular 70 to 99% by weight, buffer substances and

0 - 80 wt.%, In particular 0 to 2 wt.%, Auxiliaries includes.

6. Formulation according to one of claims 1 to 5, characterized in that it contains buffer substances and microorganisms of the genus Lactobacillus casei GG stabilized by lyophilization and auxiliaries, preferably L-ascorbic acid and / or L-cysteine.

7. Formulation according to one of claims 1 to 6, characterized in that the concentration of buffer substances is dimensioned such that an isotonic solution is formed after decay.

8. Formulation according to one of claims 1 to 7, characterized in that the formulation contains glucose and / or oligosaccharides and / or other saccharides which are favorable for the growth of germs in the intestinal lumen.

9. Formulation according to one of claims 1 to 8, characterized in that the formulation contains one or more active pharmaceutical ingredients, in particular an antidiarrheal active ingredient, preferably loperamide.

10. Use of a formulation according to any one of claims 1 to 9 for the manufacture of preparations for the treatment of gastrointestinal diseases or as a food supplement, undesired germs being inhibited in their growth or suppressed or eliminated.

11. Medical preparations which comprise a formulation according to one of claims 1 to 9, in particular for oral active immunization.

12. Formulation according to one of claims 1 to 10, for simultaneous and / or post-treatment with anti-infectives for stabilizing the intestinal flora.

13. Foodstuffs or luxury foods, beverages, in particular reform products, which comprise a formulation according to one of claims 1 to 9.