HK1056315B - Use of bifidobacteria in manufacture of medicaments for preventing diarrhea - Google Patents

Description

Application of bifidobacterium in preparation of medicament for preventing diarrhea

The present invention relates to the use of non-pathogenic microorganisms of the genus Bifidobacterium for the preparation of a carrier and for the incorporation in food or pharmaceutical products for the treatment or prevention of rotavirus induced diarrhoea, and to food or pharmaceutical compositions containing such microorganisms.

Organisms producing the main metabolic composition lactic acid have long been known. These bacteria are also found in the intestines of humans and animals in milk or milk processing plants, in living or decaying plants. These microorganisms, which are summarized under the term "lactic acid bacteria", represent a group of quite different classes of bacteria including, for example, Lactococcus (Lactococcus), Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Pediococcus (Pediococcus).

Lactic acid bacteria are used as leaven for preserving food at low pH and the action of the leaven inhibits the growth of harmful bacteria. In addition, lactic acid bacteria are also used in the manufacture of different dairy products such as cheese, yogurt and other fermented dairy products.

Recently, some strains of lactic acid bacteria have attracted considerable attention, showing valuable properties for human and animal uptake. In particular, particular strains of lactic acid bacteria or bifidobacteria have been found to be able to colonize the intestinal lining and to contribute to the maintenance of human and animal health.

In this respect, european patent EP0768375 discloses specific strains of bifidobacteria which are capable of being infused into the intestine and adhering to intestinal cells. These bifidobacteria are reported to contribute to immune function, to competitively inhibit adhesion of pathogenic bacteria to intestinal cells, and to maintain the health of the individual organism.

Research in recent years has focused on the potential use of lactic acid bacteria as probiotic agents. Probiotics are considered to be viable microbial preparations that promote the health of an individual organism by maintaining the intestinal natural microflora. Microbial preparations are generally considered probiotics, i.e. such potent microorganisms, and their mode of action is known. Probiotics adhere to the intestinal mucosa, colonize the intestinal tract and prevent the adhesion of harmful microorganisms thereon. A crucial prerequisite for their action in the intestine is that they must adhere to the mucous membranes of the viscera in a suitable viable manner and not be destroyed in the upper part of the stomach, in particular not by the low pH region of the stomach.

In this respect, as such a probiotic, WO97/00078 discloses a specific strain, called Lactobacillus GG (ATCC 53103). The use of such microorganisms in particular in a method for preventing or treating food-induced hypersensitivity reactions, i.e. the administration to a subject of such microorganisms together with food products which have been subjected to a treatment with pepsin and/or insulin and hydrolysis. The selected lactic acid bacteria strain shows adhesion and growth properties and a protease system so that the proteins contained in the food to be administered are further hydrolyzed by proteases secreted by this particular lactic acid bacteria. The process discussed in this document eventually leads to the digestion of the protein by the viscera, without showing the presence of any allergenic substances.

Furthermore, in the reference EP 0577903, lactic acid bacteria are used which have the ability to replace helicobacter pylori, which is known to be the cause of ulcers, and the above preparation can treat and prevent ulcers caused by helicobacter pylori.

The particular strains of lactic acid bacteria exhibit valuable properties and the prior art aims at the desire to find other properties of bacterial strains that can be beneficial to the health of humans and/or animals.

The problem to be solved by the present invention is therefore to provide additional lactic acid bacteria which have a beneficial effect on the feeding function of the organism.

In the course of the research leading to the present invention, the properties of microorganisms of the Bifidobacterium class which have not been identified have now surprisingly been found. The present invention provides the use of a microorganism belonging to the genus Bifidobacterium and being capable of substantially preventing rotavirus from infecting intestinal cells in a carrier for the treatment or care of diarrhea.

The Bifidobacterium is preferably selected from Bifidobacterium adolescentis (Bifidobacterium adolescentis) or Bifidobacterium longum (Bifidobacterium longum), preferably Bifidobacterium adolescentis, more preferably Bifidobacterium CNCM I-2168.

The microorganism can be used in the preparation of an ingestible support, such as milk, yogurt, curd, fermented milk, milk-based fermented products, fermented cereal products, milk powders (milk based powders), infant food or pet food, and is present in an amount of about 10⁵cfu/g to 10¹¹The amount of cfu/g is contained in the carrier. The abbreviation cfu as used herein means "colony forming unit", defined as the number of bacterial cells expressed as the microbial count on an agar plate.

The invention also provides a food or pharmaceutical composition comprising at least one bifidobacterium strain capable of substantially preventing infection of intestinal cells by rotavirus.

The food composition prepared by the invention uses at least one bifidobacterium strain and has a ratio of about 10⁵cfu/g to 10¹¹The amount of cfu/g, and a suitable carrier are mixed together, preferably in an amount of about 10⁶cfu/g to 10¹⁰cfu/g, more preferably about 10⁷cfu/g to 10⁹cfu/g.。

The pharmaceutical product can be prepared in the form of tablet, liquid bacterial suspension, oral supplement (oral supplement), wet oral supplement, dry tube feeding or wet tube feeding, etc., and the content of Bifidobacterium strain is up to 10¹²cfug, preferably about 10⁷cfu/g to 10¹¹cfu/g, more preferably 10⁷cfug to 10¹⁰cfu/g.

In summary, the technical solution provided by the present invention is:

use of a lactic acid bacterium belonging to the genus bifidobacterium selected from the group consisting of bifidobacterium longum or bifidobacterium adolescentis capable of preventing rotavirus from infecting intestinal cells for the preparation of a carrier for the treatment and prevention of diarrhea. The Bifidobacterium is preferably Bifidobacterium CNCMI-2168.

The bifidobacteria may be contained in an absorbable carrier.

The bifidobacteria may be contained in the carrier in an amount of from 10⁵To 10¹²cfu/g vector.

The carrier is a food composition selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice cream, fermented cereal products, milk powder, baby food.

The invention also provides a food or pharmaceutical composition comprising at least one strain of bifidobacterium capable of preventing rotavirus from infecting intestinal cells.

The composition is selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice creams, fermented cereal products, milk powders, baby foods, tablets, liquid bacterial suspensions, dry oral supplements, wet oral supplements, dry tubular feeds or wet tubular feeds.

The composition is in the form of a tablet, a liquid bacterial suspension, a dry oral supplement, a wet oral supplement, a dry tubular feed or a wet tubular feed.

The microorganisms may further be formulated in a carrier to obtain a desired release profile such as capsules and the like. Suitable excipients and/or additives may be selected according to the purpose of those skilled in the art.

The activity of the microorganisms in the intestine of the organism depends of course on the dosage. I.e. the more microorganisms that are ingested by the food or pharmaceutical composition, respectively, the higher the protective and/or therapeutic effect. Since the microorganisms used are harmless to humans or animals and are actually isolated from the natural environment, i.e. from the faeces of infants, the amount added is high and the proportion of the organisms responsible for the intestinal microbial colonization is high.

In these figures

FIG. 1 shows a schematic illustration of cell cultures used to evaluate the protective properties of bacterial strains against rotavirus.

In the course of extensive research, the inventors investigated different bacterial strains isolated from infant faeces or obtained from the American tissue and cell Collection (ATCC 15704). These different strains were tested for their ability to prevent intestinal cells from diarrhea caused by rotavirus.

Due to its rotavirus inhibiting properties, several bacteria including Bifidobacterium, lactococcus and Streptococcus species were screened. Inhibition performance tests were performed with three rotavirus serotypes representing the major causative agent of human viral diarrhea (serotypes G1, G3, and G4).

The various lactic acid bacteria are grown in a suitable medium, such as MRS, Hugo-Jago or M17 medium, with an optimal growth temperature of 30 to 40 ℃. After the bacteria had stably grown, they were collected by centrifugation and resuspended in a physiological saline solution. Bacterial cells between these different experiments were stored frozen (-20 ℃).

Various rotavirus stocks were prepared by infection of a confluent cell monolayer. These rotaviruses were cultured prior to infection. These cells were infected with 20 tissue culture infectious doses.

Two different protocols were used to evaluate the anti-rotavirus properties. One protocol directly tests these different bacterial strains that act directly on rotaviruses, while another protocol allows these strains to interact with cellular rotavirus receptors and screen for these bacteria.

The first protocol involves the use of different rotavirus strains in contact with respective bacterial suspensions and cultured in a suitable medium. This virus-bacteria mixture was then applied to a monolayer of human undifferentiated colon adenoma cells HT-29 (intestinal epithelial cell line) and cultured continuously. The virus was then tested for replication.

The second protocol involved first culturing the respective bacterial suspension together with human undifferentiated colon adenoma cells HT-29 and then adding the virus. Virus replication was examined after continuous culture.

Rotavirus replication is readily detected by tissue immunostaining of rotavirus proteins in infected cells.

When the number of infected cells in the cell culture medium cultured with rotavirus plus the indicated bacteria was reduced to 90% compared to cells cultured with rotavirus alone, it was evident that inhibition of rotavirus was exerted on the given bacteria.

Only 4 of the different bacterial strains with a total of over 260 species had the effect of inhibiting rotavirus replication. These different bacteria belong to the sub species Bifidobacterium adolescentis or Bifidobacterium longum of the genus Bifidobacterium. Belonging to Bifidobacterium adolescentis, designated Bad4, was preserved according to the Budapest treaty and obtained the preservation number CNCM I-2168. This strain proved to be extremely effective in preventing infection of human cells by rotaviruses. The invention will now be described by way of example.

Culture media and solutions

MRS(Difco)，

Hugo-Jago (peptone Difco 30g/l, yeast extract Difco 10g/l, lactose Difco 5g/l, KH)₂PO₄5g/l, beef extract Difco 2g/l, agar Difco 2g/l)

M17(Difco)

M199(Seromed)

Ringer solution (Oxoid)

PBS(NaCl 8g/l，KCl 0.2g/l，Na₂HPO₄ 1.15g/l，KH₂PO₄ 0.2g/l))

Peptone phosphate broth (fluid)

Pancreatin-ethylenediaminetetraacetic acid solution (mucus)

Human rotavirus Wa (serotype G1) and simian rotavirus SA-11 (serotype G3) were obtained from the united states philadelphia children hospital p.a. DS-1xRRV reassortant (reassiortant) virus was obtained from the national institute of health, Bethesda, A.Kapikian. Serotype human rotavirus Hochi was obtained from bachmann, university of munich, germany.

Example 1

Separation of lactic acid bacteria from infant faeces

Fresh faeces were collected from diapers of 15 to 27 days old healthy infants. 1g of fresh faeces was sent to the laboratory in the absence of oxygen and after serial dilution of the samples with Ringer solution, microbiological analysis was performed within 2 hours and plated on selective media. For isolation of lactic acid bacteria, MRS agar (fosfomycin 80. mu.g/ml, sulfamethoxazole neomycin 93pg/ml, trimethoprim 5ug/ml) supplemented with antibiotics was cultured at 37 ℃ for 48 hours. Randomly extracting and purifying thalli. Analysis of the physiological and genetic characteristics of the isolates was performed. Another strain obtained from ATCC (ATCC15704), corresponding to the preferred strain Bad4 of the present invention, was also used in this experiment.

Example 2

Strain assay for anti-rotavirus activity in cells

Bacteria of several genera consisting of Bifidobacterium, lactococcus and Streptococcus were selected and tested for members thereof showing anti-rotavirus activity in a cell culture medium inhibition assay (see the following 1)^stAnd 2^stScheme). The genus lactococcus employs a species (lactococcus lactis) consisting of two subspecies (lactococcus lactis subspecies lactococcus lactis and lactococcus lactis cremoris) (lc. A total of 30 strains were tested. Streptococcus genus employs 45 strains of one species (streptococcus thermophilus). Leuconostoc (Leuconostoc) and Propionibacterium (Propionibacterium) are represented by one species (6 strains), while enterococcus and Staphylococcus (Staphylococcus) are represented by two species each, for a total of 17 strains.

A total of 260 bacterial strains were tested for rotavirus activity.

First scheme：

30ul of the respective bacterial suspension (containing an average of 3X 106 bacteria) and 70u.l M199 medium supplemented with 10% peptone phosphate broth (fluid) and 5% trypsin EDTA solution (mucus) were mixed, and 100 ul of virus in the supplemented M199 medium was added. This virus-bacterium mixture was incubated at 4 ℃ for 1 hour and at 37 ℃ for 1 hour. Human undifferentiated colon tumor cells HT-29 grown as confluent monolayers in 96-well microtiter plates (supplemented 1: 4 with 10% peptone phosphate broth (fluid) and 5% insulin-EDTA solution (mucus) diluted in PBS in M99 medium) were washed three times with phosphate buffer saline (PBS, Ph7.2). The virus and bacteria mixture mixed by the above process is transferred to these cells and in microtiter plates in CO₂The culture was carried out in an incubator for 18 hours. Replication of the virus was tested as described below.

Second embodiment：

30ul of bacterial suspension and 70ul of M199 medium supplemented with 10% peptone phosphate broth (fluid), 5% trypsin-EDTA solution (mucilage) were mixed, added directly to the growing HT-29 cells and treated as per 1^stProtocol the methods described were pre-treated in a microtiter plate. After 1 hour incubation at 37 ℃, 100 μ l of virus in M199 medium was added to the cells in the microtiter plates. In CO₂The culture was continued in an incubator (Heraeus) for 18 hours. Replicating virus according toThe assay was performed as follows.

Replication of rotavirus was examined by a rotavirus protein tissue immunostaining method of infected cells which will be described later.

One day after infection, the cell culture medium was removed from the microtiter plate and the cells were fixed with absolute ethanol for 10 minutes. The ethanol was removed and the plates were washed three times with PBS buffer. (mainly directed against VP6 protein) 50ul of anti-rotavirus serum (obtained from ISREC, university of Rosemory) from rabbits was diluted 1: 2000 fold in PBS and added to each well and covered with a lid to prevent drying and incubated at 37 ℃ for 1 hour. The antibody sera were then removed and washed three times with PBS. Goat anti-serum anti-rabbit immunoglobulin G (IgG) and peroxidase (GAR-IgG-PO; Nordic) diluted 1: 500 times in PBS were then added to each well and the plates incubated at 37 ℃ for 1 hour. Serum was removed and the plate was washed three more times with PBS. Then 100pLI of the following mixture was added to each well: 0.05M Tris-HCl (pH7.8), 1ml H₂O₂(30% supra, 1: 600 fold diluted with water, Merck) and 200. mu.l of 3-amino-9-ethylcarbazole (0.1g/10ml ethanol stored at-80 ℃ in 200. mu.l aliquots; A-5754; Sigma). These plates were incubated at room temperature for at least 30 minutes. The medium was removed and then 200ul of H was injected₂O to stop the reaction. Infected foci were counted using an inverted microscope (Diavert; Leitz).

Only very few bacterial strains and rotaviruses acted. 4 of the 260 bacterial cells inhibited rotavirus replication in at least the first protocol. Bifidobacterium adolescentis CNCM I-2168(Bad4) has very strong activity against serotype 1 rotavirus, serotype 3 rotavirus SA-11 and serotype 4 rotavirus Hochi.

According to the general of lactic acid bacteria ", Ed.B.J.B.Wood and W.H.Holzapfel, Blackie A&P. the process disclosed, Bad4 is a gram-positive and catalase-negative bacterium, does not produce CO on fermentation₂And L (+) lactic acid is produced.

Claims (10)

1. Use of a lactic acid bacterium belonging to the genus bifidobacterium selected from the group consisting of bifidobacterium longum or bifidobacterium adolescentis capable of preventing rotavirus from infecting intestinal cells for the preparation of a carrier for the treatment and prevention of diarrhea.

2. The use according to claim 1, wherein the bifidobacterium is bifidobacterium CNCM I-2168.

3. The use of claim 1, said bifidobacteria being contained in an absorbable carrier.

4. The use of claim 2, said bifidobacteria being contained in an absorbable carrier.

5. Use according to claim 3, the bifidobacteria being contained in the carrier in an amount of from 10⁵To 10¹²cfu/g vector.

6. Use according to claim 4, said Bifidobacterium being contained in a carrier in an amount of from 10⁵To 10¹²cfu/g vector.

7. Use according to any one of claims 3 to 6, the carrier being a food composition selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice-creams, fermented cereal products, milk powders, infant formulae.

8. Food or pharmaceutical composition comprising at least one strain of bifidobacterium capable of preventing rotavirus from infecting intestinal cells, wherein said bifidobacterium is selected from the group consisting of bifidobacterium longum or bifidobacterium adolescentis.

9. The composition according to claim 8, which is selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice creams, fermented cereal products, milk powders, baby foods, tablets, liquid bacterial suspensions, dry oral supplements, wet oral supplements, dry tube feeds or wet tube feeds.

10. A composition according to claim 8, which is in the form of a tablet, a liquid bacterial suspension, a dry oral supplement, a wet oral supplement, a dry tubular feed or a wet tubular feed.