JPH0769907A - Composition for preventing opportunistic infective disease - Google Patents

Abstract

PURPOSE:To obtain a safe composition effective for preventing opportunistic infective diseases and absolutely free from adverse reaction. CONSTITUTION:This composition for preventing opportunistic infective diseases contains concentrated or dried material of culture liquid of a microbial strain belonging to the genus Bifidobacterium at a concentration of >=10<10> cells of the microorganism per 1g of the composition.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a composition for protecting opportunistic infections.

[0002]

2. Description of the Related Art In recent years, due to various social, economic, and iatrogenic factors, many hosts have an abnormality in the defense mechanism against infection, and normally, they are not enough to stimulate an immune response. It is known that infectious diseases (opportunistic infectious diseases) caused by so-called plain harmless bacteria, which can never be pathogenic, are becoming more common (bacteriology is here, supervised by Yoshie Hachisuka, Nanae Publishing). p305 (1986)). That is, an opportunistic infection is an infection caused by a bacterium that is considered to be harmless in a healthy host in a host whose resistance to infection has decreased, and a host in which such resistance has decreased, For example, patients who have been exposed to radiation, excision of immune system, diabetic patients, patients whose immunocompetence has been reduced by the administration of immunosuppressive drugs during organ transplantation, and patients who have suffered serious trauma such as burns. There is. Intestinal bacteria (especially Gram-negative bacilli) are considered as one of the potential sources of opportunistic infections. Therefore, it is known that in the above-mentioned patients, a part of the resident enterobacteria migrate (translocate) to tissues outside the intestinal tract, and colonize and proliferate.

Bacterial translocation is defined as a phenomenon in which intestinal resident bacteria pass from the intestinal tract through the mucosal barrier of the intestinal epithelium and migrate into various organs such as mesenteric lymph nodes, liver and the blood. (Infection and Immunity, 2
3, 403 (1979)). Further, unlike general intestinal infectious diseases, bacterial translocation is caused by bacteria that do not have cell invasiveness, and can cause the so-called opportunistic infections described above. The causes of such bacterial translocation include, for example, abnormal growth of certain bacteria due to disruption of intestinal bacterial homeostasis, damage to the immune system, and mucosal ba
Physical breakdown of rrier is considered (sterile organisms,
20 , 38 (1990)).

[0004] As a method of protecting against opportunistic infections, treatments such as antibiotics and intravenous administration of antibodies, interferons and the like to eliminate the causative bacteria have been carried out. Oral administration of antibodies in secretory milk produced by cows immunized with bacterial antigens prevents opportunistic infections.
A treatment method (Japanese Patent Laid-Open No. 4-216613) has also been devised.

[0005]

Among the opportunistic infection protection methods shown in the prior art, the method of administering an antibiotic is as follows:
In addition to having to select an effective drug, there are problems that resistant bacteria may appear and side effects may be observed. Further, when an antibody or the like is administered intravenously or orally, there is a problem that the time and labor required for selecting or producing an antibody effective against the bacterium is enormous. All of these conventional techniques are essentially the same in that after the onset of the disease, the causative bacterium is identified and then a substance having a specific antibacterial activity is administered, and they have a symptomatic side to opportunistic infections. Is. The present invention is obtained by a simple process of culturing a Bifidobacterium strain, concentrating it, or drying it, and no side effects are observed even if a small amount of this is added to the diet on a daily basis, It is intended to provide a safe composition for protecting opportunistic infections.

[0006]

[Means for Solving the Problems] The present inventors have conducted extensive studies using an animal experimental model for the inhibition of bacterial translocation of intestinal resident bacteria as the first step in the development of opportunistic infections. As a result, it was found that the administration of the culture (concentrate or dried product) of the Bifidobacterium strain with food suppresses the translocation to the abdominal cavity, mesenteric lymph nodes, etc. The present invention has been completed. That is, the present invention is a concentrate or dried product of a culture solution of a strain of the genus Bifidobacterium,
The present invention relates to a composition for protecting opportunistic infections, which contains 10 ¹⁰ or more of the bacterium per 1 g as an active ingredient.

In the present invention, a Bifidobacterium strain is first cultivated in a nutrient medium. The microorganism used in the present invention may be any microorganism as long as it is a microorganism belonging to the genus Bifidobacterium, for example, Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium infantis (B.infantis) Bifidobacterium breve (B. breve), Bifidobacterium adrescentis (B. adolescentis), Bifidobacterium bifidum (B. bifidum) strains belonging to, such as Bifido Bacterium longum
No.7 is mentioned.

The No. 7 strain has the following mycological properties. A. Morphological properties (1) Cell shape: Bacillus or branched polymorphism (2) Motility: None (3) Presence or absence of spores: None (4) Gram stainability: Positive B. Growth state on medium When this bacterium is spread on a BL agar medium (Eiken) plate and cultured by a steel wool method at 37 ° C. for 48 hours, colonies having an opaque circular hemispherical gloss are formed. C. Physiological properties (1) Nitrate reduction: negative (2) Indole formation: negative (3) Gelatin liquefaction: negative (4) Catalase: negative (5) Attitude toward oxygen: obligate anaerobic bacterium

(6) Degradability of various carbohydrates 1. L-arabinose: + 2.D-xylose: + 3.D-ribose: + 4.D-glucose: + 5.D-fructose: + 6.D- Galactose: + 7. Sucrose: + 8. Maltose: + 9. Lactose: + 10. Mellibiose: + 11. Raffinose: + 12. Melletitose: + 13. D-mannose: -14. Cellobiose: -15. Trehalose: − 16. Glycogen: − 17. Mannitol: − 18. Sorbitol: − 19. Inositol: − 20. Esculin: − 21. Salicin: − 22. Amygdalin: −

Based on the above mycological properties, Bergey's Manu
When identified by al of Systematic Bacteriology (Vol.2) (1986), this strain was identified as Bifidobacterium Longum No. 7 because it was in agreement with the mycological properties of Bifidobacterium Longum. This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Technology as FERMP-13610.

As a medium for culturing the above-mentioned strain, a medium usually used for culturing lactic acid bacteria is used. That is, any medium can be used as long as it contains a main carbon source as well as a nitrogen source, an inorganic substance and other nutrients.
Carbon sources include lactose, glucose, sucrose,
Fructose, starch hydrolysates, molasses, etc. can be used depending on the assimilation of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, soybean protein hydrolyzate and whey protein hydrolyzate can be used. In addition, meat extract, fish meat extract, yeast extract and the like are used as growth promoters.

The culture is carried out under anaerobic conditions. Therefore, a known method such as a method of culturing while aerating carbon gas can be applied. Generally, the culturing temperature is preferably 35 to 40 ° C, but other temperature conditions may be used as long as the temperature allows the bacterium to grow. It is preferable to maintain the pH of the culture medium at 6.0 to 7.0. The culture time is usually 10 to 20 hours. After completion of the culture, the culture solution is concentrated or further dried to a bacterial cell concentration of 10 ¹⁰ cells or more per 1 g of the concentrate or the dried product. Although the maximum number of bacteria is not particularly limited here, it is usually about 4 × 10 ¹⁰ cells / g in the concentrated solution and about 5 × 10 ¹¹ cells / g in the dried product. Concentration can be performed by a conventional method, for example, vacuum concentration. Drying can also be performed by an ordinary means, for example, vacuum drying, spray drying, freeze drying and the like.

The concentrate or dried product of the present invention, as it is, or after oral administration after heating or radiation sterilization, is an intestinal resident bacterium, for example, Escherichia coli, Pseudomonas aeruginosa, Klebsiella which are known to cause opportunistic infections. , It is possible to prevent migration (bacterial translocation) to extra-intestinal tissues such as Enterobacter, for example, abdominal cavity, mesenteric lymph node, liver and the like, and thus to prevent opportunistic infections in mammals including humans. Expected to be useful for prevention and treatment. As is clear from the above, the present concentrate or dried product is useful even when the cells therein are not only live cells but dead cells.

The concentrate or dried product is used as such or in a pharmaceutical composition with at least one pharmaceutical auxiliary. The concentrate or dried product is usually orally administered, and can be formulated into a suitable form. Tablets, capsules, granules, fine granules, powders are commonly used pharmaceutical auxiliaries such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose), excipients (lactose). , Sucrose, corn starch, calcium phosphate, sorbit, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.) Can be included. The tablets can be coated by a conventional method. The oral liquid preparation can be an aqueous solution or a dry product. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).

The amount of the concentrate or dried product in the composition for protection against opportunistic infections can be variously changed, but is usually 5 to 100% (w / w), particularly 10 to 100% (w / w). Is appropriate. The appropriate dose of the opportunistic infectious disease protective composition is 1 to 1,000 mg / kg / day as an active ingredient. In addition, since the concentrate or dried product has the advantage that it does not adversely affect the living body even if it is ingested in a large amount, it can be used as it is, or by adding various nutrients, etc., or by including it in food and drink to protect against opportunistic infections. It may be eaten as a functional food or health food having the function of. That is, for example, by adding nutrients such as various vitamins and minerals, for example, liquid foods such as nutritional drinks, soy milk, soups and solid foods of various shapes, and further as a powder as it is or added to diet or various foods. It can also be used. Such functional food,
The content and ingestion amount of the active ingredient in the composition for protection against opportunistic infections as a health food may be the same as the content and administration dose in the above-mentioned pharmaceutical products, respectively.

[0016]

EXAMPLES The present invention will now be described with reference to examples. Example 1 (Culture solution concentrate) Bifidobacterium longum No. 7, FERM P-13610 was added to casein hydrolyzate 3.0% (w / v), lactose 4.0% (w
/ v), yeast extract 0.5% (w / v) in a medium that was aerobically cultivated at 37 ° C, pH 6.0 for 15 hours while aerating carbon dioxide, and concentrated the culture under reduced pressure to obtain a 10-fold concentrate (cell concentration). 2 × 10 ¹⁰ cells / g)
Got The composition of this concentrate is protein 19.2% (w / v), lactose 10.9% (w / v), acetic acid 6.4% (w / v), lactic acid 6.5% (w / v).
/ v), ash 12.8% (w / v), galactose 6.5% (w / v),
The water content was 31.7% (w / v) and the other was 6.0% (w / v).

Example 2 (Suppression of Bacterial Translocation) Using 80 male 4-week-old Slc; ICR mice (Japan SLC), the concentrate of Example 1 was used as a basic feed (CMF: Oriental Yeast Co., Ltd. )) To which 1% (w / v) was added (MB group), and as a control, a group to which only basic feed was given (CMF group). The feed used in the experiment was γ
A line-sterilized (50 kGy) was used. Each group was allowed to freely ingest food and water for 4 weeks until the end of the experiment. Three weeks after the start of the experiment, in order to sterilize the intestines of the mice in both groups, the drinking water containing streptomycin 2 g / L and penicillin 1 g / L was added 3 times.
Given for days. Next, Brain Heart Infusion (BH
I) Escherichia coli C25 (E.coli C25, streptomycin resistant) strain cultured overnight in liquid medium (manufactured by Difco)
Orally administered 0.5 mL each, drinking water streptomycin 1 g / L
It was turned on and given until the end of the experiment. 4 weeks after the start of the experiment,
Mouse zymosan both groups (Zymosan, hereinafter Zy yeast cells Kabeara fraction off-white powder xanthine oxidase by activating superoxide anion (O ₂ ^-..) And a large amount generated, inflamed the intestinal wall.) Was dissolved in physiological saline and intraperitoneally administered at 0.1 mg / g-body. As a control, 1 mL of physiological saline was intraperitoneally administered.

These mice were killed by cervical dislocation 24 hours after Zy treatment, and aseptically dissected. After sterilizing the abdomen with 80% alcohol, the abdomen was opened with sterile scissors. Wipe the abdominal cavity with a sterile cotton swab, transfer it to BHI liquid medium, and
The cells were cultured aerobically at ℃ for 24 hours and examined for bacterial translocation to the abdominal cavity. Subsequently, the mesenteric lymph nodes (MLN) were aseptically removed, and BHI liquid medium (1 mL) was used.
Was homogenized by transferring to a glass homogenizer containing. A part of this homogenate is DHL containing streptomycin 1g / L
The agar medium was inoculated. Further, the cecum was excised aseptically, and similarly transferred to a glass homogenizer containing BHI liquid medium (9 mL) for homogenization. The homogenate is diluted with sterile phosphate buffer, and a part of the diluted solution is streptomycin 1 g / L.
Added DHL agar medium was inoculated. These agars are at 37 ° C
After culturing aerobically for 24 hours, MLN
The number of bacteria translocated and the number of bacteria in the cecum were calculated (Table 1).

The number of E. coli C25 bacteria translocated to MLN in untreated mice was significantly reduced in the control mice in the MB group compared to the control mice in the CMF group. I found out that On the other hand, Table 2 shows the incidence of bacterial translocation of E. coli C25 into the abdominal cavity when Zy was administered. Zy administration significantly increased the incidence of bacterial translocation in the CMF and MB groups. In contrast,
Compared with the CMF group, the MB group was found to have significantly suppressed bacterial translocation into the abdominal cavity of both control mice and Zy-administered mice. Also, MLN
The number of E. coli C25 cells that had undergone hebacterial transfection (Table 3) was also MB in both control and Zy administration.
The group was significantly more suppressed than the CMF group. Furthermore, comparing the number of bacteria translocated to MLN between the control mouse of each group and the Zy-administered mouse, Zy
The degree of bacterial translocation induced by MB was lower in the MB group than in the CMF group.

[0020]

[Table 1] Table 1 Effect of Bifidobacterium longum No. 7 culture on bacterial translocation to mouse mesenteric lymph nodes ────────────────── ─────────────────── Experiment CMF ^a) Number of times ─────────────────────────── ─ Mesenteric lymph node Cecum ──────────────────────────────────── 1 214.7 ± 88.33 ^c) ( n = 18) 9.6 ± 0.15 ^d) (n = 17) 2 146.1 ± 75.47 (n = 19) 9.7 ± 0.21 (n = 20) 3 179.0 ± 146.8 (n = 15) 10.1 ± 0.10 (n = 16) ── ────────────────────────────────── Experiment MB ^b) Number of times ──────────── ──────────────── Mesenteric lymph nodes Cecum ────────────────────── ─────────────── 1 169.5 ± 124.8 (n = 19) 9.4 ± 0.30 (n = 19) 2 86.32 ± 39.80 (n = 19) ** 9.5 ± 0.27 (n = 20 ) 3 82.11 ± 44.08 (n = 19) * 10.1 ± 0.22 (n = 18) ─────────────────────────────── ───── * 5% significant for CMF ** 1% significant for CMF ^a) Bifidobacterium longum No.7 Culture-free feed ^b) Bifidobacterium longum No.7 Feed containing culture ^c) cfu / mesenteric lymph node ^d) log (cfu / g-cecum); cfu: colony forming unit

[0021]

[Table 2] Table 2 Effect of Bifidobacterium longum No. 7 culture on bacterial translocation into mouse peritoneum ───────────────────── ──────────────── CMF ^a) MB ^b) ───────────────────────────── ──────── Control 4/20 ^c) 1/21 ** Zymosan administration 11/20 7/22 * ────────────────────── ────────────── * 5% significant to CMF ** 1% significant to CMF ^a) Bifidobacterium longum No.7 culture-free feed ^b) Feed containing Bifidobacterium longum No. 7 culture ^c) Bacterial translocation-induced mouse / tested mouse

[0022]

[Table 3] Table 3 Effect of Bifidobacterium longum No. 7 culture on bacterial translocation of zymosan-treated mice ──────────────────── ───────────────── CMF ^a) Treatment ─────────────────────────── Membrane lymph node Cecum ──────────────────────────────────── Control 180.0 ± 72.4 ^c) (n = 17 ) 9.9 ± 0.23 ^d) (n = 17) Zymosan administration 269.0 ± 225.0 (n = 19) 9.7 ± 0.20 (n = 20) ────────────────────── ─────────────── MB ^b) Treatment ─────────────────────────── Mesenteric lymph Section Cecum ───────────────────────────────── ─── Control 99.5 ± 66.7 (n = 20) ** 9.9 ± 0.21 (n = 21) Zymosan administration 127.5 ± 133.1 (n = 20) * 9.7 ± 0.17 (n = 22) ────────── ─────────────────────────── * 5% significance for CMF ** 1% significance for CMF ^a) Bifidobacterium・ Feed not containing Longum No.7 culture ^b) Bifidobacterium ・ Feeding containing Longum No.7 culture ^c) cfu / mesenteric lymph node ^d) log (cfu / g-cecum)

Example 3 (Capsule) Lyophilized product of the concentrate of Example 1 200 g Corn starch 150 g Talc 80 g Magnesium stearate 30 g The above ingredients were thoroughly mixed and passed through a wire mesh of 60 mesh to adjust the particle size, Fill 1,000 gelatin capsules.

[0024]

EFFECTS OF THE INVENTION The present invention provides a safe composition for protecting opportunistic infections without any side effects.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kikuji Ito 2-1-7-207 Shiki City Kan Saitama

Claims (1)

[Claims] 1. A concentrated or dried product of a culture solution of a strain of Bifidobacterium, which is 10 ¹⁰
A composition for protecting opportunistic infectious diseases, which comprises one or more as an active ingredient.