JPWO2005077390A1 - Glucose level lowering agent, diabetes treatment / prevention agent and method for producing the same - Google Patents

Abstract

The present invention relates to one or more microorganisms selected from the group of Enterococcus genus bacteria comprising Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus durans, Enterococcus salivarius, Enterococcus mitis and Enterococcus icinus The present invention relates to a blood sugar level-lowering agent, a diabetes treatment / prevention agent, and a production method thereof, which are contained as active ingredients.

Description

  The present invention relates to a blood sugar level-lowering agent, a diabetes treatment / prevention agent, and a method for producing the same, comprising lactic acid bacteria as an active ingredient.

  In recent years, especially in advanced countries, diabetes, which is one of lifestyle-related diseases and adult diseases, has spread due to excessive caloric intake due to meals. The number of diabetes patients is said to exceed 6.5 million in Japan alone. Furthermore, it is said that the reserve arm of diabetic patients suffering from impaired glucose tolerance is one in ten, that is, over 10 million.

  At present, many types of blood sugar level-lowering agents and anti-diabetic agents are in practical use. However, many of these are not necessarily satisfactory in terms of pharmacological effects and side effects, and the demand for more effective drugs has increased.

  As a highly safe blood sugar level-lowering agent, an aqueous extract of Lactobacillus cells is known (Japanese Patent Laid-Open No. 06-116156). However, there is still a need for safer and more effective drugs.

  An object of the present invention is to provide a blood sugar level-lowering agent and a therapeutic / preventive agent for diabetes that are highly safe and effective.

  The inventor of the present application has found that various microorganisms (lactic acid bacteria) belonging to the genus Enterococcus have an action of reducing blood glucose level very effectively. Furthermore, the present inventor has found that those microorganisms whose origins are so-called enteric bacteria are substantially non-toxic orally and have extremely high pharmacological activity. As a result, the present inventor, without causing the various side effects that the various microorganisms belonging to the genus Enterococcus have had so far various blood sugar level lowering agents and diabetic drugs that reduce various symptoms of diabetes, It has been found that it can be effectively used as a drug having a blood glucose level-lowering effect and a diabetes-treating and preventing effect even if it is taken in duplicate with other drugs, and the present invention has been completed as follows.

  The present invention relates to one or more microorganisms selected from the group of Enterococcus genus bacteria comprising Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus durans, Enterococcus salivarius, Enterococcus mitis and Enterococcus icinus The present invention relates to a blood sugar level-lowering agent contained as an active ingredient.

  The present invention also relates to one or more microorganisms selected from the group of Enterococcus bacteria comprising Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus durans, Enterococcus salivaius, Enterococcus mitis and Enterococcus icinus The present invention also relates to a therapeutic / preventive agent for diabetes containing as an active ingredient.

  Here, the microorganism (bacteria belonging to the genus Enterococcus) may be in any form of a culture, a fungus body, or a treated cell of the microorganism. In addition, the microbial cell or the treated microbial cell may be a live microbial cell or a dead microbial cell, or a mixture thereof, but is preferably a dead microbial cell.

  The present invention also provides a blood glucose level-lowering agent or a diabetes treatment / prevention agent comprising a microorganism belonging to the genus Enterococcus as an active ingredient, comprising preparing a culture by culturing the microorganism belonging to the genus Enterococcus in a Rogosa liquid medium. It also relates to the manufacturing method.

  In this production method, the microorganism is cultured aerobically (for example, static culture) in a Rogosa liquid medium under the conditions of pH 6.5-8.0 and temperature 36-38 ° C. Is preferred.

  In the present invention, it is preferable that the production method further includes recovering the microbial cells from the culture prepared as described above and washing them. The washed cells may be prepared as a cell suspension by suspending in a solvent such as physiological saline.

  Furthermore, in the present invention, it is preferable that the production method further includes heating and washing the washed bacterial cells (or bacterial suspension). The washed cells may be further prepared as a suspension. Here, it is preferable to heat the washed cells (or cell suspension) at 110 to 120 ° C. for 5 to 10 minutes.

In the present invention, a method for producing a blood glucose level-lowering agent or a diabetes treatment / prevention agent containing a microorganism belonging to the genus Enterococcus as an active ingredient, including the following steps (a) to (e) is particularly preferred:
(A) inoculating a Rogosa liquid medium with live cells of microorganisms belonging to the genus Enterococcus;
(B) preparing a culture by statically culturing the inoculated medium aerobically under conditions of pH 6.5 to 8.0 and a temperature of 36 to 38 ° C;
(C) collecting the cells by centrifuging the prepared culture,
(D) The step which wash | cleans the collect | recovered microbial cell with a physiological saline, and the step which obtains a microbial suspension by suspending the washed microbial cell in a physiological saline.

Further, the manufacturing method may further include the following steps (f) to (h):
(F) A step of heating the bacterial suspension obtained in step (e) at 110 to 120 ° C. for 5 to 10 minutes,
(G) a step of washing the microbial cells in the heated bacterial solution with physiological saline, and (h) suspending the washed microbial cells in physiological saline to remove dead cells of microorganisms belonging to the genus Enterococcus. Preparing a bacterial suspension containing;

  In the production method, the microorganism belonging to the genus Enterococcus is preferably selected from the group of bacteria consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus dulans, Enterococcus salivaius, Enterococcus mitis and Enterococcus equinus One or more microorganisms.

  The blood sugar level lowering agent and the diabetes treatment / prevention agent according to the present invention are intestinal bacteria, and therefore do not cause side effects. It does not cause problems, is very effective in lowering blood glucose levels, and in the treatment and prevention of diabetes.

  This specification includes the contents described in the specification of Japanese Patent Application No. 2002-190616 on which the priority of the present application is based.

  Hereinafter, the microorganisms (lactic acid bacteria), the cell treatment method, the pharmacological effects and the like used in the present invention will be described in detail.

1. Microorganisms For the production of the blood sugar level lowering agent and the diabetes treatment / prevention agent of the present invention, it is preferable to use one or more microorganisms among microorganisms belonging to the genus Enterococcus (lactic acid bacteria). Microorganisms belonging to the genus Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus dulans, Enterococcus salivarius, Enterococcus mitis and Enterococcus icinus To do.

  These microorganisms belonging to the genus Enterococcus can be obtained not only as commercial products but also from various biological depositary institutions. Examples of such biological depository institutions include the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Japan), ATCC (American Type Culture Collection; USA), and the like. For example, as Enterococcus faecalis, the Enterococcus faecalis KRI-68 strain listed in Table 1 described below has been deposited with the ATCC (American Type Culture Collection; USA) under the accession number “ATCC Number 70000802.” In the present invention, Enterococcus faecalis KRI-68 strain can be obtained from ATCC based on this accession number and can be used preferably.

  Specific strains that are particularly useful in the present invention are illustrated in Table 1.

In the table, the numbers with “KRI” are reference numbers for identifying the strains. These strains can be obtained from Kawai Lactococcus Institute (Tokyo, Japan).

Table 2 shows the mycological properties of the strains exemplified in Table 1.

2. Culture Method and Cell Treatment Method The microorganism belonging to the genus Enterococcus used in the present invention is not limited, but is preferably cultured in the following Rogosa liquid medium.

  In the present invention, a blood glucose level-lowering agent and a diabetes treatment / prevention agent can be produced using the microorganism belonging to the genus Enterococcus. In the present invention, microorganisms belonging to the genus Enterococcus cultivated (proliferated), preferably in a Rogosa liquid medium, can be used for the production of a hypoglycemic agent and a therapeutic / preventive agent for diabetes. In the present invention, the thus obtained culture of microorganisms belonging to the genus Enterococcus may be directly used for the production of a blood glucose level-lowering agent and a diabetes treatment / prevention agent. In the present invention, the culture supernatant separated from the culture (including a small amount of microbial cells, a part of the microbial cells, secretions from the microbial cells, etc.) is also used for the production of an agent for lowering blood glucose level and treating or preventing diabetes. be able to.

  In the present invention, it is more preferable to use the microbial cells isolated from the culture for the production of a blood glucose level lowering agent and a diabetes treatment / prevention agent. Particularly preferably, the microorganism belonging to the genus Enterococcus is cultured, for example, in a Rogosa liquid medium (for example, static aerobic culture under conditions of pH 6.5 to 8.0 and a temperature of 36 to 38 ° C.). The cells obtained by centrifuging the obtained culture and collecting the precipitate are used for the production of a blood glucose level-lowering agent and a diabetes treatment / prevention agent. The bacterial cells (mainly viable cells) thus obtained may be used directly for the production of a drug, but are washed with an isotonic solution such as physiological saline and then suspended in an isotonic solution such as physiological saline. It may be used after becoming cloudy.

  Furthermore, in the present invention, the obtained bacterial cells are subjected to any treatment including cell destruction treatment such as heat treatment, ultrasonic treatment, freeze-thawing treatment, etc. to prepare a treated bacterial cell product, which is used for reducing blood glucose level. You may use for manufacture of an agent or diabetes treatment and prevention agent. The treated bacterial cell may contain live cells, but is preferably substantially dead cells (for example, a composition containing dead cells such as a dead cell suspension). Examples of dead cells include destroyed cells obtained by destroying the cells obtained above by ultrasonic treatment, heat-treated cells obtained by heat-treating the cells obtained above, and the like. The dead cells contained in the processed microbial cells used in the production of the blood glucose level lowering agent and the diabetes treatment / prevention agent may be whole cells or a part of the cells.

  The microorganism belonging to the genus Enterococcus used for the production of the blood glucose level lowering agent and the diabetes treatment / prevention agent of the present invention may be prepared, for example, as a microbial cell or a processed microbial cell as follows.

1) Preparation Example of Viable Cell (Live Cell Suspension) A microorganism belonging to the genus Enterococcus is inoculated into 5 liters of the above-mentioned Rogosa liquid medium, and the temperature condition is 37 ° C. in the range of PH 6.5 to 8.0 (36 to 38 ° C.) is aerobically statically cultured for 5 hours to prepare a culture having a viable cell count of 10 ⁹ / ml. The obtained culture was subjected to continuous centrifugation at 12000 rpm to collect viable bacteria, which were washed twice with physiological saline, suspended in physiological saline, and then suspended in physiological saline to give 50 ml of bacterial suspension (10 ¹¹ cells). / Ml).

2) Example of preparation of dead cell (heat-treated cell) 50 ml (10 ¹¹ cells / ml) of the cell suspension obtained according to the preparation example of 1) above is heated at 115 ° C. for 10 minutes. 80 mg of the cells thus heat-treated are washed with 0.5 ml of physiological saline and then suspended in physiological saline to obtain a dead cell suspension.

3. Microorganisms belonging to the genus Enterococcus and their use in blood glucose level lowering agents and diabetes treatment / prevention agents , microorganisms belonging to the genus Enterococcus prepared as described above (preferably a culture, cell or treatment of the microorganism) ) Shows a good blood glucose level lowering action. The blood glucose level-reducing action of these microorganisms (preferably culture, fungus body or treated product thereof) is determined by using a blood glucose level measurement method known to those skilled in the art, for example, as described in Examples below. Can be confirmed. A microorganism belonging to the genus Enterococcus used in the present invention (preferably a culture, a microbial cell, or a processed microbial cell of the microbial organism) can effectively reduce blood glucose level, but is not limited thereto. The blood glucose level can be reduced by 10% to 40% compared to the group.

In addition, the acute toxicity of the microorganism of the present invention (preferably a culture, a microbial cell, or a processed microbial cell of the microbial cell) is very low. For example, the LD ₅₀ value (50% lethal dose) shown in the Examples described later is 9.9 × 10 ^{8 to} 1.3 × 10 ¹⁰ cells / mouse (intraperitoneal administration) in the case of a living cell suspension. In the case of a dead cell suspension, it is 5400 mg / kg body weight (oral administration) and 920 mg / kg body weight (intraperitoneal) regardless of the bacteria. In particular, in the case of oral administration, virtually no acute toxicity is observed in both live and dead cell suspensions.

  Therefore, a drug containing the microorganism according to the present invention (preferably, a culture, a microbial cell or a processed microbial cell product of the microorganism) as an active ingredient is very effective as a blood glucose level reducing agent, It can be effectively used as a therapeutic agent for diabetes and a prophylactic agent for diabetes that are closely related.

  The blood sugar level-lowering agent and diabetes treatment / prevention agent of the present invention may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin. , Propylene glycol, paraffin, stearyl alcohol, stearic acid, sorbitol, lactose and the like. The additive used is selected appropriately or in combination depending on the dosage form of the drug. The blood sugar level-lowering agent and diabetes treatment / prevention agent of the present invention include other pharmacologically generally used fillers, diluents, binders, lubricants, disintegrants, wetting agents, stabilizers, buffers, and corrigents. Further, additives such as preservatives and coloring agents may be further contained.

  The blood glucose level-lowering agent and diabetes treatment / prevention agent of the present invention are not limited, but can be administered orally or intravenously. The blood glucose level lowering agent and diabetes treatment / prevention agent of the present invention are dissolved in solid preparations such as tablets, granules, pills, capsules, powders including freeze-dried powder, gels, physiological saline, etc. Ordinary dosage forms such as liquid preparations including liquids, suspensions, and syrups.

  Subjects to which the blood glucose level-lowering agent and the diabetes treatment / prevention agent of the present invention are administered are mammals including humans, domestic animals, pets, experimental (test) animals and the like. In particular, mammals such as humans having a high blood glucose level or mammals such as humans having a predisposition to suffering from diabetes are preferred as administration subjects.

  The dosage of the blood glucose level-lowering agent and the diabetes treatment / prevention agent of the present invention varies depending on the animal species, age and weight, administration route, number of administrations, and the like of the administration target, and can be varied widely within the discretion of those skilled in the art. For example, the oral dose of a microorganism as an active ingredient (preferably a culture, a microbial cell or a treated microbial cell of the microbial organism) can be preferably about 80 to 900 mg / kg body weight per day. The blood glucose level-lowering agent and diabetes treatment / prevention agent of the present invention are not limited, but it is preferable to administer continuously (for example, one month or more) at least once a day.

  The present invention provides a blood glucose level comprising administering a microorganism belonging to the genus Enterococcus (preferably a culture, a microbial cell or a processed microbial cell of the microorganism) or a pharmaceutical composition containing the same as described above. And a method of treating and / or preventing diabetes.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Example 1]
1. Breeding of experimental animals and measurement of blood glucose level and urine sugar level In all the examples described later, experiments using experimental animals were performed as follows.

  As experimental animals, diabetic mice KK-Ay (male) were obtained from Claire Japan at 4 weeks of age, and used for experiments after 2 weeks of pre-breeding.

(1) Breeding conditions Seven animals were bred in one cage at room temperature 22 ± 1 ° C., humidity 55.0 ± 10%, and illumination cycle 12 hours. For the feed, tap water passed through the solid feed was placed in a water supply bottle and allowed to be freely ingested.

(2) Blood collection method Blood was collected once every 5 days and collected from the tail artery of the mouse.

(3) Measurement of blood glucose level For the measurement of blood glucose level, the advantage test strip S was set in Advantage II marketed by Roche Dyacstick Co., Ltd., and the collected blood was poured to measure the blood glucose level.

(4) Urine collection and measurement of urine sugar level Urine collection was performed once every 5 days. A net was set between the stick bottle and the transparent cup, a mouse was placed on the stick bottle side, and urine was collected on the transparent cup. Uropaper IIG purchased from Eiken Chemical Co., Ltd., was soaked in the collected urine, and was judged in six stages:-, ±, +, ++, ++++, and ++++. The urine determined to be + or more was determined as the onset of diabetes.

2. Results of preparation of live cell suspension and administration to mice According to the above preparation example of live cells, Enterococcus faecium KRI-622 strain was inoculated into Rogosa liquid medium and PH 6.5-8.0. Then, static culture was carried out aerobically at a temperature of 36 to 38 ° C., and then a bacterial suspension (viable cell) was prepared in physiological saline. Further, from this bacterial suspension, heat-treated dead cells (dead cell suspension) were prepared according to the above-mentioned preparation example of dead cells.

Next, the prepared viable cell suspension (bacteria: 10 ¹¹ cells / day) or heat-treated dead cells (80 mg / 0.5 ml / day) was used for the diabetic mouse KK-Ay (male 6 weeks). Ordinary breeding, average body weight 31 g, 7 animals in each group) were orally administered daily, and the breeding was continued for 85 days. During the breeding period, blood was collected from the tail artery of the mouse once every 5 days. Table 4 shows the results of measuring the blood glucose level in the blood by the above method.

  As shown in Table 4, the microorganism Enterococcus faecium KRI-622 showed a marked decrease in blood glucose level of 37.4% at 80 days and 37.5% at 85 days in dead cells. The viable cells also showed a decrease of 16.6% and 17.1% on the 80th and 85th days, respectively.

[Example 2]
Experiments were carried out in the same procedure as in Example 1 using Enterococcus faecalis KRI-68 strain instead of Enterococcus faecium KRI-622 strain. The prepared viable cell suspension (bacteria: 10 ¹¹ cells / day) or heat-treated dead cells (80 mg / 0.5 ml / day) were used for the diabetic mouse KK-Ay (male 6-week-old breeding, An average body weight of 31 g, 7 mice in each group) was orally administered daily and kept for 85 days. During the breeding period, blood was collected from the tail artery of the mouse once every 5 days. Table 5 shows the results of measuring the blood glucose level in the blood by the above method.

  As shown in Table 5, the microorganism Enterococcus faecalis KRI-68 showed a marked decrease in blood glucose level of 39% on day 80 and 36% on day 85 in dead cells. The viable cells also showed a decrease of 16% and 17% on the 80th and 85th days, respectively.

[Example 3]
Experiments were performed in the same procedure as in Example 1 using Enterococcus abium KRI-1223 strain instead of Enterococcus faecium KRI-622 strain. The prepared viable cell suspension (bacteria: 10 ¹¹ cells / day) or heat-treated dead cells (80 mg / 0.5 ml / day) were used for the diabetic mouse KK-Ay (male 6-week-old breeding, An average body weight of 31 g, 7 mice in each group) was orally administered daily and kept for 85 days. During the breeding period, blood was collected from the tail artery of the mouse once every 5 days. Table 6 shows the results of measuring the blood glucose level in the blood by the above method.

  As shown in Table 6, the microorganism Enterococcus ebium KRI-1223 showed a decrease in blood glucose level of 11% on the 80th day and 12% on the 85th day in the dead cells. In viable cells, the decrease rate was 1.8% on the 80th day and 8% on the 85th day.

[Example 4]
Experiments were performed in the same procedure as in Example 1 using Enterococcus durans KRI-614 strain instead of Enterococcus faecium KRI-622 strain. The prepared viable cell suspension (bacteria: 10 ¹¹ cells / day) or heat-treated dead cells (80 mg / 0.5 ml / day) were used for the diabetic mouse KK-Ay (male 6-week-old breeding, An average body weight of 31 g, 7 mice in each group) was orally administered daily and kept for 85 days. During the breeding period, blood was collected from the tail artery of the mouse once every 5 days. Table 7 shows the results of measuring the blood glucose level in the blood by the above method.

  As shown in Table 7, the microorganism Enterococcus durans KRI-614 showed a 14% decrease in blood glucose level on the 80th and 85th days for dead cells. The viable cells showed a 9% reduction rate on the 80th and 85th days.

[Example 5]
Instead of Enterococcus faecium KRI-622 strain, Enterococcus mitis KRI-814 strain, Enterococcus salvarius KRI-1216 strain, and Enterococcus equinus strain 120 Enterococcus kuincoccus Were used in the same procedure as in Example 1. The prepared viable cell suspension (bacteria: 10 ¹¹ cells / day) or heat-treated dead cells (80 mg / 0.5 ml / day) were used for the diabetic mouse KK-Ay (male 6-week-old breeding, An average body weight of 31 g, 7 mice in each group) was orally administered daily and kept for 85 days. During the breeding period, blood was collected from the tail artery of the mouse once every 5 days. Table 8 shows the results of measuring the blood glucose level in the blood by the above method.

  As shown in Table 8, the microorganisms Enterococcus mitis KRI-814 and Enterococcus ikuinus KRI-120 showed a decrease in blood glucose level of 8.1% on the 85th day in dead cells.

[Example 6]
ICR strain mice (male 6 weeks old, average body weight 30 g ± 0.7 g) were used to examine the acute toxicity of the cells according to the present invention or the treated cells.

In the same manner as in Example 1, a live cell suspension of each strain was prepared according to the above-mentioned preparation example of live cells. From the resulting viable cell suspension, a physiological saline suspension is prepared so as to contain three stages of viable cell doses of 9 × 10 ⁹ , 9 × 10 ⁸ , and 9 × 10 ⁷ per mouse. And 0.5 ml of the physiological saline suspension was intraperitoneally administered to ICR mice, and the mice were observed for life or death for 14 days (10 mice in each group). The LD ₅₀ values measured according to the BEHRENS-KARBER method are shown in Table 9.

In the case of dead cells, the LD ₅₀ value was 6 × 10 ¹³ cells / mouse or more (administered intraperitoneally) in any of the bacteria. In addition, all the bacteria were substantially non-toxic after oral administration.

[Formulation example]
80 mg (corresponding to 3 × 10 ¹¹ dead cells) of the freeze-dried Enterococcus faecium KRI-622 obtained according to Example 1 is uniformly mixed with 920 mg of purified starch powder, tableted and tableted for oral administration It was. This tablet corresponds to a dead cell dose of 6 × 10 ⁹ cells / kg body weight in an adult weighing 60 kg.

In addition, 500 mg of the above lyophilized product was mixed with 500 mg of purified starch powder and tableted to obtain tablets for oral administration. Similarly, the dead cell dosage in adults weighing 60 kg was 3.3 × 10 ¹⁰ cells / kg. Equivalent to.

  In this way, the blood sugar level-lowering agent and the diabetes-preventing agent of the present invention are prepared by mixing the microbial cells and a pharmaceutically acceptable carrier based on the standard dose and the like to obtain a desired dosage form having the above-mentioned medicinal effect. can do.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

  The blood sugar level-lowering agent and the diabetes treatment / prevention agent comprising the enterococcus bacterium of the present invention (preferably a culture, a microbial cell or a treated product of the bacterium) as an active ingredient have a high blood sugar level-lowering action and high safety. Therefore, for example, it can be suitably used for administration to diabetic patients. Moreover, those production methods can be advantageously used for producing a drug having a high blood glucose level-lowering effect.

Claims (12)

Contains as an active ingredient one or more microorganisms selected from the group of Enterococcus bacteria consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus dulans, Enterococcus salivarius, Enterococcus mitis and Enterococcus icinus A hypoglycemic agent. The blood sugar level-lowering agent according to claim 1, wherein the microorganism is a culture, a microbial cell, or a processed microbial cell product of the microorganism. The blood sugar level-lowering agent according to claim 2, wherein the microbial cell or the microbial cell-treated product is a living microbial cell and / or a dead microbial cell. Contains as an active ingredient one or more microorganisms selected from the group of Enterococcus bacteria consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus dulans, Enterococcus salivarius, Enterococcus mitis and Enterococcus icinus A diabetes treatment / prevention agent. The diabetes treatment / prevention agent according to claim 4, wherein the microorganism is a culture, a microbial cell, or a processed microbial cell of the microbial. The agent for treating or preventing diabetes according to claim 5, wherein the microbial cell or the microbial cell-treated product is a living microbial cell and / or a dead microbial cell. A method for producing a blood glucose level-lowering agent or a diabetes treatment / prevention agent comprising a microorganism belonging to the genus Enterococcus as an active ingredient, comprising preparing a culture by culturing a microorganism belonging to the genus Enterococcus in a Rogosa liquid medium. The method according to claim 7, wherein the culture is aerobically performed under conditions of pH 6.5 to 8.0 and a temperature of 36 to 38 ° C. The method according to claim 7 or 8, further comprising collecting and washing the cells from the culture. The method according to claim 9, further comprising heating and washing the washed cells. A method for producing a blood glucose level-lowering agent or a diabetes treatment / prevention agent containing a microorganism belonging to the genus Enterococcus as an active ingredient, comprising the following steps (a) to (h):
(A) inoculating a Rogosa liquid medium with live cells of microorganisms belonging to the genus Enterococcus;
(B) preparing a culture by statically culturing the inoculated medium aerobically under conditions of pH 6.5 to 8.0 and a temperature of 36 to 38 ° C;
(C) collecting the cells by centrifuging the prepared culture,
(D) washing the collected cells with physiological saline;
(E) A step of obtaining a bacterial suspension by suspending the washed bacterial cells in physiological saline.
(F) A step of heating the bacterial suspension obtained in step (e) at 110 to 120 ° C. for 5 to 10 minutes,
(G) a step of washing the cells in the heated suspension with physiological saline, and (h) containing the dead cells of the microorganism by suspending the washed cells in physiological saline. Preparing a bacterial suspension. The microorganism belonging to the genus Enterococcus is one or more selected from the group of bacteria consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ebium, Enterococcus dulans, Enterococcus salivaius, Enterococcus mitis and Enterococcus ichinus The method according to any one of claims 7 to 11, which is a microorganism.