CN1065567C - Lactobacillus for Immunity Boost - Google Patents

Abstract

Compositions containing Lactobacillus brevis subsp. coagulans are used to enhance the immune function of patients, especially with regard to increasing interferon production, 2-5A synthetase activity and natural killer cell activity.

Description

Lactobacillus for Immunity Boost

The invention relates to an immune function enhancer, which can increase the production of human interferon and enhance human immune function.

Interferon (IFN) is produced by a variety of cells, such as leukocytes, fibroblasts and T lymphocytes, which are produced upon viral infection and protect other cells from viral infection. Therefore, interferon has the effect of inhibiting virus reproduction.

Among several interferons, it is reported that the production of IFN-α is decreased in patients suffering from cancer, diabetes, tuberculosis, etc., and the ability to produce IFN-α is negatively correlated with the susceptibility to infection.

In addition, it has also been reported that the production of IFN-γ is decreased in cancer patients and increased in patients suffering from Hashimoto's disease, autoimmune diseases and the like.

It is also known that the production of IFN-γ contributes differently to the immune function of the human body than that reflected by the production of IFN-α.

Therefore, the ability to produce IFN-α and IFN-γ is used as a useful parameter for evaluating human immune function.

Enhancing these immune functions is thought to improve the general condition and quality of life of immunocompromised patients.

In order to enhance immune function, it has been suggested to take IFN or use strong immune booster such as OK 432.

It is known that the activity of natural killer cells (NK) is related to the condition of cancer patients, and it is well known that taking IFN can enhance the activity of NK cells.

However, repeated use of IFN or immune boosters such as OK 432 has been found to cause side effects. Such as internal environment disorder, fever and discomfort.

Therefore, instead of taking IFN, immune function enhancers have been developed, which can increase the production of interferon in the body and enhance immune function. In fact, double-helix RNA, pyran copolymer, anionic polymer and various polysaccharides (see Japanese Patent Laid-Open No. Hei. 3-9882) have been suggested as immune function enhancing agents.

However, because these well-known enhancers of immune function do not naturally occur in the human body, these enhancers are toxic and can cause side effects in humans when used to treat infections or tumors.

One object of the present invention is to provide an immune function enhancing agent, which can increase the production of interferon and enhance the immune function of human body. Infections and tumors can be treated or prevented with immune boosters.

In this regard, the present inventors have discovered an immune function enhancer comprising Lactobacillus brevis subsp. coagulans CCTCC M94025 bacterial powder. Described bacterial strain was preserved on May 29, 1994 in China Center for Type Culture Collection (in the campus of Wuhan University, Hubei, China), and the preservation number is CCTCC M94025.

Lactobacillus brevis subsp. coagulans bacteria powder can be produced, for example, by the following steps:

First, the starting strain as a source of Lactobacillus is cultured in the following medium A, B or C for 18-40 hours at 25-35° C. and stirring at 100 rpm.

Then, the culture solution is concentrated or centrifuged, and then freeze-dried to obtain Lactobacillus brevis subsp. coagulans powder. Medium A

①Yeast paste 0.5%

② Glucose 1.5%

③ Potassium dihydrogen phosphate 0.5%

④ Sodium hydrogen phosphate 2.0%

⑤Sodium chloride 0.425%

⑥Sodium hydroxide 0.0375%

⑦Defatted soybean extract 8.75%

⑧Calcium carbonate precipitation 0.1% medium B

Peptone (polypeptone) 0.5-1.0%

Yeast paste 0.5-1.0%

Glucose 0.5-1.0%

pH 6.5-7.0 Medium C

Yeast paste 0.55%

Glucose 1.10%

Peptone 1.25%

Sodium dihydrogen phosphate 0.025%

Sodium hydrogen phosphate 0.025%

Magnesium Sulfate 0.01%

Manganese sulfate 0.0005%

pH 6.5-7.0

To prepare medium A, components ①-⑦ were dissolved in water under heating. Then, after adding component ⑧ to the solution, adjust the pH of the solution to 6.8-7.0, and then sterilize at 121° C. for 15 minutes by high-pressure steam. In addition, the defatted soybean extract is prepared by the following steps:

After defatting, soak the soybeans in dilute hydrochloric acid (about 0.25N), add pepsin, and then keep the mixture at 37°C for 40-48 hours for digestion with occasional stirring. After digestion, sodium hydroxide was added to neutralize the solution. This solution was then centrifuged, and the supernatant was taken out as a defatted soybean extract.

The starting strain is best obtained from foods such as Lactobacillus (Tsukemono) fermented vegetables. Each medium A, B and C 501 produces 100-500 g of bacterial powder, which contains 2-50 billion bacteria per g (2×10 ⁹ -5×10 ¹⁰ /g).

The bacterial powder is preferably administered orally in the form of powder, tablet, capsule or granulated powder.

When preparing tablets, bacterial powder can be mixed with L-sodium glutamate (10%) or with a mixture of skimmed milk powder (10%) and L-sodium glutamate as a dispersion medium, and dry potato starch as an excipient mix.

Preferred embodiments of the present invention are described in detail below.

The starting strain was obtained from the Japanese Brassica genus Suigukina variety (Bras-sica J a ponica Var. Suigukina) fermented by Lactobacillus. Starting Strain Bacteria were grown in medium A for 30 hours at 25°-35°C with stirring at 100 rpm. Then about 10000rpm centrifugal culture fluid, and freeze-drying becomes bacterial powder, claims it as Lactobacillus brevis PK.

The bacterial powder obtained in this way had the following characteristics (1)-(6) characteristic of Lactobacillus, and this bacterial powder was also identified as Lactobacillus brevis subsp. coagulans based on its fermentation characteristics shown in Table 1 below.

(1) It is a Gram-positive bacillus.

(2) It decomposes calcium carbonate in the medium.

(3) It uses glucose in the medium to produce lactic acid.

(4) The ability to reduce nitrate is negative.

(5) The ability to liquefy gelatin was negative.

(6) The ability to digest casein was negative.

Then, dry potato powder is added to the bacteria powder obtained by the above method, and the bacteria powder is adjusted to contain 10 ⁸ -10 ⁹ bacteria per g. Then add anhydrous lactose as excipient. Tablets of 250 mg are thus prepared.

4 healthy men and 6 healthy women aged 25-65 were orally administered 6 tablets per day for 4 weeks. Before administration and 2 weeks and 4 weeks after administration, peripheral blood was collected in heparin-containing test tubes. The production of IFN-α and IFN-γ, the activity of natural killer cells (NK) and the synthesis activity of 2-5A (2′,5′ oligoadenylic acid) were measured, and the measurement results are shown in Table 2 and Table 3.

IFN-[alpha] and -[gamma] were determined using FL-cells (from human amnion) and Sindobis virus with a 50% CPE (cytopathogenic effect) bioassay.

An example of a method for measuring IFN-α is described below:

Heparinized peripheral blood was processed for IFN production using the whole blood method. Then 2ml of whole blood was collected in a centrifuge tube. HVJ (Sendai virus) was added at a final concentration of 500 HA/ml. Incubate at 37°C for 20 hours, and then centrifuge at 3,000 rpm. Supernatants were collected to measure IFN-α production.

An example of a method for assaying IFN-γ is described below:

Heparinized peripheral blood was diluted 4 times in Eagle's MEM, and PHA-P (purchased from Sigma) was added to make the final concentration 25 μg/ml. After culturing at 37°C for 48 hours, centrifuge at 3,000 rpm, and collect the supernatant as an IFN-γ sample.

When measuring NK cell activity, peripheral mononuclear cells were isolated from peripheral blood by the Ficoll-Paque method, thus obtaining effector cells. K562 cells labeled with Cr ⁵¹ were used as target cells and effector cells were mixed at an E/T (effect/target) ratio of 20:1. Cytotoxicity was determined by conventional methods.

The activity of 2-5A synthetase in unstimulated plasma was too low to be detected, and the activity of 2-5A synthetase was measured after the plasma was stimulated with HVJ (Sendai virus) for 20 hours to evaluate the production capacity of IFN-α. The activity of 2',5'-oligoadenylic acid (2-5A) synthetase was measured using a radioimmunoassay kit (manufactured by Eiken Chemical Co. Tokyo).

From the results shown in Table 2 and Table 3, it can be clearly seen that the bacterial powder prepared by the above method improves the production of IFN-α, the activity of NK cells and the activity of 2-5A synthetase.

In addition, after administration of the bacterial powder, blood tests showed no large difference from the standard value in any individual.

Table 1 Experiment 1 Experiment 2 Experiment 1 Experiment 2 Arabinose xylose rat season sugar sorbose ribose glucose (gas) mannose sugar galactose sucrose maltose ++--±++-++-+ ++--+++-++-+ Cellulosebiose Lactose Trehalose Biose Raffinose Melezitose Starch pH Mannitol Sorbitol Esculin pH Salicin Amygdalin ---±±---- ---+--------

Table 2 1FNα production 10/ml 1FNT Yield 1u/ml 0 2 weeks later 4 weeks later 0 2 weeks later 4 weeks later female 1 5518100.0 9545173.0 9884179.1 84100.0 145172.6 358426.2 female 2 6457100.0 10784167.0 21986340.5 323100.0 338104.6 705218.3 female 3 5254100.0 15073286.9 10584201.4 424100.0 40294.8 35383.3 female 4 7243100.0 416257.5 571278.9 243100.0 18576.1 309127.2 male 1 5208100.0 5511105.8 6141117.9 506100.0 960189.7 27053.4 male 2 7240100.0 12693175.3 9999138.1 234100.0 11047.0 11047.0 male 3 6495100.0 7229111.3 10617163.5 69100.0 6087.0 137198.6 female 5 4418100.0 9083205.6 4416100.0 75100.0 3141.3 6282.7 female 6 3168100.0 7345231.8 10320325.8 32100.0 87271.2 98306.3 male 4 11620100.0 22075190.0 993985.5 126100.0 129.5 4132.5 average 6262.1100.0 10350165.3 9959.8159.0 211.6100.0 233110.1 244.3112.8

table 3 NK activity % 2-5 A active pmol/shed 0 2 weeks later 4 weeks later 0 2 weeks later 4 weeks later female 1 26100.0 50192.3 47180.8 29.4100.0 32.0108.8 22.175.2 female 2 43100.0 62144.2 59137.2 49.3100.0 41.584.2 45.993.1 female 3 42100.0 3378.6 42100.0 10.0100.0 61.0610.0 45.2452.0 female 4 42100.0 51121.4 51121.4 10.0100.0 22.6226.0 66.3663.0 male 1 24100.0 30125.0 31129.2 22.8100.0 48.4212.3 45.0197.4 male 2 33100.0 89269.7 36109.1 84.9100.0 102.5120.7 119.8141.1 male 3 53100.0 62117.0 63118.9 47.5100.0 61.8130.1 103.2217.3 female 5 36100.0 44122.2 62172.2 54.8100.0 323.5590.3 792.41446.0 female 6 42100.0 79188.1 53126.2 45.0100.0 53.1118.0 118.9264.2 male 4 54100.0 79146.3 63116.7 84.0100.0 99.8118.8 108.7129.4 average 39.5100.0 57.9146.6 50.7128.4 43.8100.0 58.1132.6 75.0171.2

Claims (5)

1. composition, it contains the short lactobacillus subspecies CCTCC M94025 bacterium powder that condenses.

2. the composition of claim 1, the wherein said short lactobacillus every gram of subspecies bacterium powder that condenses contains 2 * 10 ⁹-5 * 10 ¹⁰Individual bacterium.

3. claim 1 or 2 composition, it forms pulvis, tablet, capsule or granulation pulvis.

4. claim 1 or 2 composition, wherein the short lactobacillus subspecies of condensing derive from the Japanese Btassica Suigukina mutation of lactobacillus ferment.

5. the composition of claim 3, wherein the short lactobacillus subspecies of condensing derive from the Japanese Btassica Suigukina mutation of lactobacillus ferment.