WO1997016198A1

WO1997016198A1 - Probiotically acting formulation

Info

Publication number: WO1997016198A1
Application number: PCT/AT1996/000214
Authority: WO
Inventors: Helmut Viernstein; Diether Polheim; Svend Laulund
Original assignee: Chr. Hansen Biosystems A/S
Priority date: 1995-11-02
Filing date: 1996-11-04
Publication date: 1997-05-09
Also published as: AU7265796A; AT405235B; ATA181095A

Abstract

The invention relates to a probiotically acting formulation which comprises micro-organisms stabilised by drying and matrix constituents resisting gastric juice; this formulation is in the form of dry granules. The invention also relates to the use of said formulation.

Description

Probiotic formulation

The invention relates to probiotic compositions.

The general term "lactic acid bacteria" encompasses a category of microorganisms which, due to their human-specific and food-technological properties, has become the focus of scientific interest in recent years. The term "probiotics" as a growth promoter originally limited to the area of animal feed refers to living microorganisms which, as a food additive, favorably influence the balance of the digestive organisms. Today, however, probiotics are generally relevant for a wide range of predominantly lactic acid-containing preparations, substrates, food and feed, and are used both in human and veterinary medicine and in animal nutrition. Taxonomically, the spectrum of lactic acid bacteria currently essentially includes the genera Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Enterococcus, Bifidobacterium, Carnobacterium and Sporolactobacillus (Bergey's Manual of Systematic Bacteriology, IX. Ed.). Some of these representatives (Lactobacillus, Bifidobacterium and Enterococcus) are considered natural and useful intestinal dwellers of humans (and also of animals) and occur there - depending on the intestinal segment, age and eating habits - in different numbers of germs. In general, the effects of lactic acid bacteria are divided into four main effects:

1. Biochemical (including improvement of nutrient utilization)

2. Physiological (especially stimulation of the intestinal peristalsis)

3.Antimicrobial (e.g. growth inhibition of pathogenic germs)

4.Competitive (e.g. inactivation of enterotoxins).

The application of certain lactic acid bacteria, either via the food route or by taking special pharmaceutical medicinal preparations, therefore appears to be useful for the reasons mentioned above. The focus today is on dietary, prophylactic and therapeutic indications, such as for travel diarrhea, regulating function in stressful situations, cancer therapy, treatment of constipation, gingivitis, vaginitis, various diets, etc.

For an efficient effect of preparations containing lactic acid, it is absolutely necessary that high concentrations of the microorganisms reach the intestine. As a result, conventional foods with living microorganisms cannot be used in all cases, since they often contain the microorganisms in a relatively low concentration and the total amount which would then have to be added is too large (Laulund, "Commercial Aspects of Formulation, Production and Marketing of Probiotic Products "in" Human Health: The Contribution of Microorganisms ", SAW Gibson Ed. (1994), Springer Verlag, page 160).

Living microorganisms are difficult to stabilize, which is why they are usually in dried form in preparations; the representation can be carried out by different drying processes, e.g. lyophilization, spray drying, etc.

Furthermore, when oral microorganisms are administered orally, it should be noted that, depending on the type or strain of bacteria, some of the bacteria are already destroyed in the stomach, which is a natural barrier for microorganisms, and only a small number of surviving germs in the Darm arrives (Füller, J.Appl.Bact. 66, 1989, 365-378). Processing into tablets with enteric film coatings is problematic in that the number of viable microorganisms is usually already greatly reduced by the tabletting process due to the shear forces acting on them. It is even known from the literature that a considerable reduction in the number of bacteria is brought about by the tabletting process, whereby a certain decontamination can be achieved (Fassihi et al., Zbl. Pharm. 116 (1977), 1267-1271; Plumpton et al., Int.J. Pharmac. 30 (1986), 237-240; Plumpton et al., Int.J. Pharmac. 30 (1986), 241- 246). Due to the special and expensive technology, the administration in gastric juice-resistant hard gelatin capsules remains limited to the pharmaceutical sector. The processing of probiotic formulations with enteric film coatings as in the pharmaceutical industry (e.g. with coating

® tensile material such as Eudragit) is otherwise not possible because these substances are not approved for food products.

Finally, there is a considerable fluctuation in quality in many of the products on the market, which is mainly due to the fact that the large-scale production and processing of probiotically active formulations are delicate and difficult to reproduce due to their biological nature (Brennan et al., J. Food Prot. 46 (1983), 887-892).

The object of the present invention is therefore to provide a probiotically effective formulation which has a high degree of gastric juice resistance, is easy to process as a food additive and can also be produced on a large scale in a reproducible manner.

This object is achieved according to the invention by a probiotic formulation comprising microorganisms stabilized by drying and enteric matrix components which formulation is present in dry granulated form.

According to the invention, the prerequisites for the effectiveness of bacteria are created by producing formulations which are made from viable microorganisms which have been preserved by drying and from matrix components which are both indispensable for the preparation of the formulations and also ensure a high degree of gastric juice resistance , consist. It is only because of the embedding of microorganisms in suitable enteric matrix components that it is possible that a sufficiently high number of desired surviving microorganisms can get into the intestine. The manufacturing process according to the invention sets up a granulation of powder masses dry way, whereby the processing process itself causes only a slight reduction in the number of survival germs.

Only embedding in suitable enteric matrix components enables a surprisingly large number of living cells to get into the intestine. It is important that the microorganisms stabilized by drying are in dry granulated form. Dry granulation is a sufficiently gentle process in which a high percentage of the microorganisms used survive the manufacturing process, and in the present case is surprisingly suitable to also ensure adequate gastric juice resistance. Furthermore, the presence of the microorganisms in dry granulated form greatly facilitates any further processing which may be connected, or makes it possible for use as a food additive in the first place (in contrast to the enteric coatings such as Eudragit known from the pharmaceutical industry) ).

Particularly preferred gastric juice-resistant matrix components are alginates (eg sodium alginate), cellulose or their derivatives (eg hydroxypropylmethyl cellulose (HPMC), HPMC acetate succinate, HPMC phthalate, methacrylic acid derivatives, shellac, galactomannans and mixtures of these components. These substances are found in pharmaceuticals - and / or the food industry has already been tried and tested, is considered harmless and also shows excellent properties with regard to the microorganisms stabilized by drying. Particularly advantageous examples of HPMC or HPMC derivatives include HPMC-50SH 4000, HPMCP-HP 55 (according to US - Pharmacopeia XXIII, NF18, No. 2910 and No. 200731), and HPMC acetate succinate LF (HPMC-AS-LF) (Shin-Etsu, Japan).

Lactic acid bacteria, preferably from the genera Streptococcus, Lactobacillus, Enterococcus and Bifidobacterium, but also Enterobacteriaceae, preferably the genera Enterobacter and Escherichia, are used as microorganisms in the formulations according to the invention, the genes or species Lactobacillus delbrückii subsp. bulgaricus, lactobacil Ius acidophilus, Lactobacillus casei subsp. casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lac tobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, etc., physiological Escherichia coli and mixtures of these microorganisms are particularly preferred.

The probiotic effect of the species mentioned has already been described sufficiently; their importance in terms of food technology and pharmaceuticals makes them preferred microorganisms in the context of the present invention.

The formulation according to the invention advantageously comprises technically necessary auxiliaries, e.g. pharmaceutical carriers and / or formulation auxiliaries, such as tableting agents, consistency-increasing agents, stabilizers, food additives, etc.

The probiotic formulation according to the invention generally comprises

10 to 90% by weight of microorganisms stabilized by drying,

- 10 to 90 wt .-% enteric matrix components and

0 to 50 wt .-% technically necessary auxiliary components

Formulations which have 30 to 70, in particular 30 to 55% by weight, of stabilized microorganisms are particularly preferred.

Formulations which are particularly preferred according to the invention are those which comprise microorganisms of the species Enterococcus faecium and / or Lactobacillus acidophilus stabilized by drying, and sodium alginate and / or hydroxypropylmethyl cellulose or one of its derivatives and magnesium stearate.

With these formulations, the highest resistance was achieved in vitro against gastric juice (0.1 N HCI).

According to a further aspect, the present invention relates to the use of the probiotically active formulations according to the invention as additives for foods and for the manufacture of medicaments, in particular for the manufacture of preparations for the treatment of disorders of the intestinal flora.

Another aspect of the present invention is a method for producing probiotic formulations, which comprises the following steps:

Providing microorganisms stabilized by drying,

Mixing the stabilized microorganisms with gastric juice-resistant matrix components, dry-granulating the mixture obtained, wherein according to a particular embodiment more than one granulation step is provided.

The invention is explained in more detail with reference to the following examples, to which, however, it should not be restricted.

Example:

Production of granules (dry granulation):

The microorganisms are mixed with the corresponding gastric juice-resistant matrix components and, if appropriate, with further auxiliary components, in particular those necessary for compacting (cube mixer, ERWEKA company) and with an eccentric press (type EK 0, KORSCH company) to give compressed products with a diameter of 10 mm pressed. The pressing pressure applied is 15 kN. The compacts obtained are subsequently processed into granules using a dry granulating device (device type TG 2 / S, company ERWEKA) and this is then classified using a sieve tower (company RETSCH). Granules with particle diameters of 2.0 to 4.0 mm are Further examinations, such as the determination of initial germ counts, survival germ counts, etc., are supplied.

Since the introduction of the granules into a 0.9% NaCl solution causes a strong swelling of the auxiliary substances, glass beads are shaken before the bacterial count is determined in order to destroy the swollen auxiliary substance shell and thereby release the bacteria from the dry granule cores guarantee. For this, sterile glass beads are added to the bacterial suspensions and shaken several times in a horizontal movement. The number of bacteria is then determined using Koch's plate casting method.

Determination of the number of survival bacteria after incubation in 0.1 N HCl:

To simulate a gastric passage, 1.00 g of the sample is mixed with 94.0 ml of 0.1 N HCl and incubated for 1 h at 37 ° C. in a shaking water bath (company GFL, type 1083). Then with 2 ml of 5 N NaOH and 3 ml of an aqueous 1 M Na _? HP0 ₄ solution neutralized (approx. PH 7) and the bacterial count determined according to the Koch plate casting method.

When examining pure lyophilisates with regard to gastric juice resistance, the following circumstance can lead to falsified results: The lyophilisate can already adhere to the glass wall when the dilution bottles are loaded, as a result of which the bacteria partially survive in the dry film under a moist protective layer. For examinations with pure lyophilisates, the dilution bottle must therefore be subjected to an even shaking motion right from the start to prevent the lyophilisate from settling on the bottom.

1. Processing of Enterococcus faecium M74 bacteria

A commercially available preparation of the enterococcus faecium M74 strain (Medipharm AB, Sweden) - Lactiferm L-50 - was used for the present investigations and a granulate according to the invention according to the above-described a process with different auxiliary components. The culture of live germ count was determined for the starting lyophilisate, the starting granulate, and for the preparations after incubation in 0.1 N HCl ("survival germ count"). Detection was carried out by plating on entero- cocci selective agar (Slanetz and Bartley; medium: aerobically for 48 h at 37 ^β C).

1.1 .: Granules with sodium alginate

Formulation recipe:

10.0 g lyophil. Enterococcus faecium M74 10.0 g sodium alginate

1.0 g magnesium stearate 10.0 g CaHP0 ₄

1.2 .: Granules with HPMC phthalate

Formulation recipe:

10.0 g lyophil. Enterococcus faecium M74 10.0 g HPMC phthalate 1.0 g magnesium stearate

1.3 .: Results of the test regarding gastric juice resistance:

Initial number of survivors after incubation in 0.1 N HCI

Lyophilisate 2.8 x 10 10 4.0 x 10 ^!

Granules with

Sodium alginate 9.5 x IO ⁹ 6.0 x IO ⁶ HPMC phthalate 1.2 x 10 ¹ ° 3.0 x 10 ⁷

Table 1

Colony forming units (CFU) / g The results in Table 1 show that the preparations according to the invention significantly increased the survival numbers of the bacteria (in some cases by more than 2 powers of ten).

2. Processing of Enterococcus faecium SF-68 bacteria

A preparation of the strain Enterococcus faecium SF-68 (APP 396) commercially available from Chr. Hansen's bio Systems, Horsholm, Denmark, was used for the tests described below and a granulate according to the invention according to the above described manufacturing process with different matrix components. The number of microbes was determined for the starting lyophilisate, the starting granules, and for the preparations after incubation in 0.1 N HCl (“survival number”).

2.1 .: Granules with HPMC acetate succinate

The components for a first compacting process are mixed and compressed. The compressed products are then granulated into granules A as an intermediate product. The granulate A is compressed again with other necessary components (e.g. with the same parts by weight of HMPC acetate succinate granulate) and granulated to an end product (granulate B). This manufacturing technology optimizes the embedding of the microorganisms in the stabilizing matrix by means of several process steps which can be carried out easily.

Formulation recipe: (Granules A)

12.0 g Hansen APP 396 (Ent.faec. SF-68)

8.0 g HPMC acetate succinate LF 12.0 g magnesium stearate

The powder mixture prepared in accordance with the mixing ratio given above was granulated dry, the granules subsequently mixed 1: 1 with HPMC-AS-LF granules of the same average grain size and subjected to the dry granulation process again. 2.2 .: Granules with HPMC phthalate

Formulation recipe:

10.0 g Hansen APP 396 (Ent. Faec. SF-68) 10.0 g HPMC phthalate

1.0 g magnesium stearate

2.3 .: Granules with sodium alginate and HPMC

Formulation recipe:

10.3 g Hansen APP 396 (Ent. Faec. SF-68) 12.8 g HPMC 60SH-4000

5.1 g sodium alginate 1.4 g magnesium stearate

2.4 .: Results of the test regarding gastric juice resistance:

Initial number of survivors after incubation in 0.1 N HCI

Lyophilisate 1.4 x 10 12 8 x IO ⁹

Granules with

HPMC-AS-LF 3.7 x 10 ¹ ° 3.2 x IO ⁸ HPMC phthalate 1.2 x 10 ¹¹ 1.4 x IO ⁹ sodium alginate + HPMC60SH-4000 2.4 x 10 1 1 1.7 x 10 10

Table 2

Figures in CFU / g

3. Processing of Lactobacillus acidophilus germs

A preparation of the Lactobacillus acidophilus strain (Wiesby L. acidophilus no.145) commercially available from Wiesby was used for the present investigations and a granulate according to the invention was produced with different auxiliary components in accordance with the processes described above. The cultural live germ count determination was for the starting lyophilisate, the starting granules and for the preparations after incubation in 0.1 N HCl ("survival germ count"). Detection was carried out by plating on Rogosa agar (medium: anaerobically for 72 h at 37 ⁰ C; Lit .: Mitsuoka, Zbl.Bakt.I. Orig, 210 (1969), 32-51.); the survival germ numbers were determined in the same way.

3.1 .: Granules with sodium alginate and HPMC

Formulation recipe: 61.5 g L. acidophilus lyophilisate

25.6 g HPMC 60 SH-4000

10.2 g sodium alginate

2.7 g magnesium stearate

3.2 .: Granules with HPMC acetate succinate

Formulation recipe: 49.5 g L. acidophilus lyophilisate

49.5 g HPMC acetate succinate - LF 1.0 g magnesium stearate

3.3 .: Granules with larger portions of HPMC acetate succinate ("granulate mixture")

Production: see 2.1.

Formulation recipe: 20 g L.acidophilus lyophilisate

20 g magnesium stearate 60 g HPMC acetate succinate - LF

3.4 .: Studies on gastric juice resistance:

Granule or lyophilisate samples were drawn after 0, 15, 30, 45 and 60 min incubation in 0.1 N HCl and their bacterial count was determined. The results of the tests are shown in Table 3.

For comparison purposes, pure bacterial lyophilisate and granulated lyophilisate were included in the tests. Time Germ content Germ content Germ content Germ content Germ content (min) Na alg. + HPMC HPMC-AS-LF HPMC-AS-LF Lyophilisate Lyophilisate

Granules Granules Mixed granules-granules

0 5.0 X IO ⁷ 2.3 x IO ⁷ 1.0 x IO ⁷ 4.4 x IO ⁸ 2.2 x IO ⁹

15 1.1 X 10 ⁶ nbnbnbnb

30 4.0 X IO ⁵ 5.2 x IO ⁵ 2.3 x IO ⁶ nbnb

45 3.6 X IO ⁵ nbnbnbnb

60 3.4 X IO ⁵ 1.1 x IO ⁵ 1.7 x IO ⁶ 0 0

n.b. not determined Data in CFU / g

Table 3

These results clearly demonstrate that the formulations according to the invention ensure a comprehensive gastric juice resistance of the microorganisms administered, so that a satisfactory effect in the intestinal area can be achieved. It was also shown that the formulations according to the invention also enable increased storage stability compared to conventional formulations.

Claims

Patent claims:

1. Probiologically active formulation, comprising microorganisms stabilized by drying and enteric-resistant matrix components, which formulation is present in dry granulated form.

2. Probiologically active formulation according to claim 1, characterized in that the enteric matrix components are selected from sodium alginate, hydroxypropylmethyl cellulose and their derivatives, methacrylic acid derivatives and galactomannans and in particular mixtures of these components.

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

4. Probiologically active formulation according to one of claims 1 to 3, characterized in that the microorganisms stabilized by drying are selected from the species Lactobacillus delbrückii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum and physiological Escherichia coli and mixtures of these microorganisms.

5. Probiologically active formulation according to one of claims 1 to 4, characterized in that it further contains technically necessary auxiliaries, such as pharmaceutical carriers and / or formulation auxiliaries.

6. Probiologically active formulation according to one of claims 1 to 5, characterized in that it

10 to 90% by weight of microorganisms stabilized by drying,

- 10 to 90 wt .-% enteric matrix components and

0 to 50 wt .-% technically necessary auxiliaries.

7. Probiologically active formulation according to one of claims 1 to 6, characterized in that it comprises by drying sta¬ bilized microorganisms of the species Enterococcus faecium and / or Lactobacillus acidophilus, sodium alginate and / or hydroxypropylmethyl cellulose or one of its derivatives and magnesium stearate.

8. Use of probiotically active formulations according to one of claims 1 to 7 as additives for food.

9. Use of probiotically active formulations according to one of claims 1 to 7 for the manufacture of a medicament.

10. Use of probiotic formulations according to one of claims 1 to 7 for the preparation of a preparation for the treatment of disorders of the intestinal flora.

11. A method for producing probiotic formulations comprising the steps:

Providing microorganisms stabilized by drying,

Mixing the stabilized microorganisms with gastric juice-resistant matrix components,

Dry granulation of the mixture obtained, preferably more than one granulation step being provided.