JP2005269968A - Lactic acid bacterium producing substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a lactic acid bacterium producing substance providing effect by lactic acid bacteria by intake of a small amount of the substance. <P>SOLUTION: The lactic acid bacterium producing substance is obtained by filtering a culture solution of lactic acid bacterium prepared by using lactic acid bacteria of three genuses of a lactic acid bacterium belonging to the genus Lactobacillus, a lactic acid bacterium belonging to the genus Bifidobacterium and a lactic acid bacterium belonging to the genus Streptococcus, using one or more kinds of lactic acid bacteria selected from these three genuses of lactic acid bacteria to form a plurality of groups, subculturing each of these groups to keep a symbiotic state and mutually subjecting the lactic acid bacteria of the subcultured group unit to symbiosis culture. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a lactic acid bacteria production material obtained by culturing lactic acid bacteria.

Lactic acid bacteria are known to greatly influence the growth of intestinal microorganisms in the intestine and keep the intestinal environment favorable. The lactic acid bacteria inhabit the human intestine as intestinal benign bacteria, so-called good bacteria, and maintain the intestinal environment. Therefore, when the number of lactic acid bacteria decreases or the activity decreases, the intestinal environment is destroyed.
By preparing the intestinal environment, it is expected that immunity will be improved and it will be difficult to suffer from various diseases, or the medical condition will be improved.
Therefore, in recent years, yogurt, beverages and the like containing the live bacteria have been sold in order to supply live bacteria of lactic acid bacteria to the intestines. These yogurts and beverages are taken directly into the body to increase the amount of lactic acid bacteria in the intestines.
Since the applicant of the present application has developed the present invention based on a known technique without conducting a prior art search, there is no prior art document information relating to the present case.

However, even if you ingest food products such as yogurt or beverages that contain the above live bacteria, by the time the live bacteria reach the intestines, they are decomposed or digested by stomach acid, and most of the live bacteria are killed. End up. In other words, even if live bacteria are ingested from yogurt or beverages, only a small amount of lactic acid bacteria reaches the intestines alive.
Therefore, the expected intestinal regulating action by lactic acid bacteria is often not obtained.

Considering the killing of live bacteria as described above, in order to deliver live bacteria to the intestines, a large amount of food containing the lactic acid bacteria must be ingested. However, since the amount of lactic acid bacteria contained in a food and beverage unit such as yogurt (a bottle of yogurt) is limited, it is considerably difficult to take a large amount of lactic acid bacteria at one time.
On the other hand, in recent years, the causal relationship has not been clarified in many parts, but it has been found that even if the ingested lactic acid bacteria are killed, various good effects appear on the body. This effect is thought to be due to the production of lactic acid bacteria.
An object of the present invention is to provide a lactic acid bacterium-producing substance that can obtain the effect of lactic acid bacteria by ingesting a small amount.

The first invention uses lactic acid bacteria belonging to the genus Lactobacillus, lactic acid bacteria belonging to the genus Bifidobacterium, and three lactic acid bacteria belonging to the genus Streptococcus. A plurality of groups are formed using one or two or more lactic acid bacteria selected from lactic acid bacteria, and subculture is performed for each group to maintain a co-cultivated state. It is characterized in that it consists of a filtrate obtained by filtering a lactic acid bacteria culture solution obtained by coculturing.
The use of the three genera of lactic acid bacteria means that at least one lactic acid bacterium belonging to each genus is used.

  As a lactic acid bacterium belonging to the genus Lactobacillus, the second invention is L. Lactobacillus acidophilus, L. Lactobacillus brevis, L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. It is characterized by the use of one or more of Delblicki (Lactobacillus delbrueckii subsp. Delbrueckii).

  A third invention is a lactic acid bacterium belonging to the genus Streptococcus. Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. It is characterized in that one or more of Lactococcus lactis are used.

  The fourth invention is a lactic acid bacterium belonging to the genus Bifidobacterium as described above. Bongidobacterium longum, B. Bifidobacterium bifidum, B. It is characterized in that one or more of Adrecentis (Bifidobacterium adolescentis) is used.

  The fifth invention is premised on the first invention, and is described in L.L. Lactobacillus acidophilus, L. Lactobacillus brevis, L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), S. Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. Lactococcus lactis, B.I. Bongidobacterium longum, B. Bifidobacterium bifidum, B. It is characterized by the use of 16 types of lactic acid bacteria of Bifidobacterium adolescentis.

  The sixth invention is based on the fifth invention, and E.I. Fesium and L. Helveticas in the first group, E. Fesium and L. Acidophilus in the second group, E.I. Fesium and L. Gasseley is the third group, E.I. Fesium, L.C. Acidophilus and L. Brevis the 4th group, E. Fesium, L.C. Acidophilus and L. Brevis the 5th group, E. Fesium, L.C. Acidophilus, L.H. Brevis, L. Paracasei in the sixth group Adrecentis alone is group 7; Delbricky and L. Gasselie is in the 8th group, L.C. Delbricky alone is the ninth group, E.D. Fesium, L.C. Genseny, L. Paracasei and L. Brevis the 10th group, L. Acidophilus alone is group 11; Fesium and L. Gasserie was placed in the 12th group, L. Paracasei alone was added to the 13th group, L. Gassely, E.M. Fesium and B.I. Bifidum is the 14th group. Longum, S.M. Thermophilus and E. coli. Fecium was added to the 15th group, L.P. Gasserie alone, group 16; Bulgarix and S. Thermophilus in the 17th group, L. Gassely, L.C. Lactis, L. Gasserie and E.C. Fesium was added to the 18th group, L.C. Gassley, S.M. Thermophilus and L. Bulgarix was introduced in the 19th group, L.B. Lactis alone, 20th group, L. Gasserie and E.C. Fecium was added to the 21st group, L.P. In the 22nd group, L. Casei alone, group 23, B.I. Longum alone is used as the 24th group, and subcultured for each group to maintain the co-cultivated state. Among these culture solutions, the first, second groups, the third, fourth groups, the fifth, sixth Groups, 7th, 8th group, 9th, 10th group, 11th, 12th group, 13th, 14th group, 15th, 16th group, 17th, 18th group, 19th, 20th group Primary cultures between the 21st and 22nd groups, the 23rd and 24th groups, the 1st and 2nd groups, the 3rd and 4th groups, the 5th and 6th groups, the 7th and 8th groups, the 9th, 10th group, 11th and 12th group, 13th and 14th group, 15th and 16th group, 17th and 18th group, 19th and 20th group, 21st and 22nd group, 23rd and 2nd group It was subcultured in a group with each other, further characterized in that the mixture to tertiary culturing these respective secondary culture.

  The seventh invention is characterized in that the third cultivated lactic acid bacterium culture solution is sterilized by heating and then filtrated.

According to the first to seventh inventions, a lactic acid bacterium-producing substance can be obtained by culturing a plurality of types of lactic acid bacteria, and the lactic acid bacterium-producing substance can contain many types of metabolites in large quantities.
In particular, since lactic acid bacteria belonging to three genus bacteria are combined and co-cultivated, many types of metabolites are generated rather than metabolites of single genus and single species of lactic acid bacteria.
In addition, since a group of lactic acid bacteria that maintain a symbiotic state is generated and the groups are cultivated together, the culturing of a large number of bacterial species can be performed stably in a short time. As a result, it is possible to stably produce lactic acid bacteria-producing substances containing many types of useful metabolites in a short time.

When this lactic acid bacterium-producing substance is ingested, the intestinal benign bacteria are activated by the metabolite, and the intestinal environment can be improved. That is, the effect of lactic acid bacteria can be obtained without directly ingesting live lactic acid bacteria.
In addition, since the lactic acid bacterium-producing substance contains a large amount of metabolites, it is not necessary to ingest a large amount when ingesting this, unlike ingesting live bacteria.

  The applicant of the present application paid attention to the metabolite of lactic acid bacteria and thought that the metabolite of lactic acid bacteria was useful for adjusting the intestinal environment. That is, it is hypothesized that when the above-mentioned metabolites of lactic acid bacteria are supplied into the intestines, the components contained in these metabolites become nutrient sources for the intestinal benign bacteria and the benign bacteria are activated. He stood up and repeated various experiments.

  Therefore, first, a group in which a plurality of types of lactic acid bacteria coexisted was formed from a large number of lactic acid bacteria, and these groups or single bacteria were combined and cultured together to obtain a lactic acid bacteria production substance. In addition, since the unit at the time of performing the primary cocultivation culture described later is a group of the present invention, a single bacterium is considered as a group consisting of the single bacterium.

Below, the method to produce | generate the lactic acid bacteria production substance of this invention is demonstrated.
In order to produce the lactic acid bacteria-producing substance in this embodiment, the lactic acid bacteria belonging to the genus Lactobacillus, the lactic acid bacteria belonging to the genus Bifidobacterium, or the lactic acid bacteria belonging to the genus Streptococcus A plurality of belonging lactic acid bacteria was used.

  As a lactic acid bacterium belonging to the genus Lactobacillus, L. Lactobacillus acidophilus, L. Lactobacillus brevis, L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), L. One kind or two or more kinds of Lactococcus lactis were used.

  As lactic acid bacteria belonging to the genus Bifidobacterium, B. Bongidobacterium longum, B. Bifidobacterium bifidum, B. One type or two or more types of Adrecentis (Bifidobacterium adolescentis) were used.

  As lactic acid bacteria belonging to the genus Streptococcus, S. Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. One or more of Lactococcus lactis was used.

And the above lactic acid bacteria were cultured as follows.
First, a plurality of lactic acid bacteria were divided into 24 groups and subcultured for each group. The grouping is as follows. That is, as shown in FIG. Fesium and L. The second group consists of E. helveticas. Fesium and L. It consists of acidophilus. The third group is E.E. Fesium and L. The fourth group consists of E. Gasseley. Fesium, L. Acidophilus and L. Consists of Brevis.

  The fifth group is E.I. Fesium, L.C. Acidophilus and L. The sixth group consists of E.Brevis. Fesium, L.C. Acidophilus, L.H. Brevis, L. The seventh group consists of paracasei. It consists of Adrecentis alone.

  The eighth group is Delbricky and L. The 9th group consists of L. Gasseley. The 10th group consists of E.D. Fesium, L.C. Genseny, L. Paracasei and L. The eleventh group consists of L.B. It consists of Acidophilus alone.

  The twelfth group is Fesium and L. The thirteenth group consists of L. The 14th group is composed of L. Paracasei alone. Gassely, E.M. Fesium and B.I. The 15th group consists of B. Longum, S.M. Thermophilus and E. coli. It consists of fesium. The sixteenth group is The 17th group consists of L. Bulgarix and S. It consists of thermophilus.

  The eighteenth group is Gassely, L.C. Lactis, L. Gasserie and E.C. The 19th group consists of L.F. Gassley, S.M. Thermophilus and L. The 20th group consists of L.B. Lactis is a simple substance. Gasserie and E.C. The 22nd group consists of L.F. It consists of Ramonausus alone, and the 23rd group Casei consists of a single case. It is composed of longum alone.

  While subcultured for each group, among those culture solutions, the first and second groups, the third and fourth groups, the fifth and sixth groups, the seventh and eighth groups, the ninth and tenth groups Between the 11th and 12th groups, between the 13th and 14th groups, between the 15th and 16th groups, between the 17th and 18th groups, between the 19th and 20th groups, between the 21st and 22nd groups, between the 23rd and 24th groups Primary culture.

Further, the first, second and third groups, the fourth and fifth groups, the fifth and sixth groups, the seventh and eighth groups, the ninth and tenth groups, the eleventh and twelfth groups, the thirteenth and fourteenth groups, and the fifteenth and sixteenth groups. Secondary culture was performed between the 17th and 18th groups and the 19th and 20th groups, the 21st and 22nd groups and the 23rd and 24th groups, and these secondary cultures were further mixed and subjected to tertiary culture.
Then, the tertiary culture solution obtained as described above was heated to sterilize lactic acid bacteria and filtered to obtain a filtrate.

In addition, the lactic acid bacteria of each said group are subcultured as follows. That is, each lactic acid bacterium in each group is assembled while predicting that it will be able to maintain a symbiotic state even when they are subcultured.
The fact that a plurality of lactic acid bacteria maintain a symbiotic state means that a plurality of types of lactic acid bacteria simultaneously exist and a symbiotic state where each can live is present. In particular, one does not affect the other, or only one survives.

Each said lactic acid bacteria group was formed by selecting the combination which can coexist from the combination of various bacteria.
In addition, among the lactic acid bacteria constituting the 24 groups, there are bacterial species belonging to a plurality of groups at the same time. This is because the microbial species were combined in consideration of the fact that the cultivated state is maintained in the process of culturing up to the third order. In addition, lactic acid bacteria of the same species include those with different strains.

As the culture medium of each lactic acid bacteria group, three types of medium consisting of GAM semi-fluid high layer medium, BL agar medium or modified GAM agar medium manufactured by Nissui Pharmaceutical Co., Ltd. are used according to the lactic acid bacteria, and these mediums are used. Incubated at 32 ° C. for 12 hours. Then, it culture | cultivated at 37 degreeC for 12 hours, and also cultured at 40 degreeC for 24 hours. The grouped lactic acid bacteria were subcultured as described above, and the culture solution was refrigerated at 5 ° C.
In the 24th group, B.I. As the longum, Morinaga BB536, a strain produced by Morinaga Milk Industry Co., Ltd., was used.

  In this way, a group of lactic acid bacteria was subcultured and identified for each group. The identification test was requested from the Japan Food Analysis Center. The outline of the identification test is as follows. Each group of specimens is directly inoculated and cultured on an agar plate medium, and colonies with different shapes that have grown predominantly are isolated and lactic acid bacteria are separated for each group. Observation, physiological property test, and measurement of GC content of intracellular DNA were performed and identified with reference to the following literature.

1. Sneath, P.A. H. A., Mair, N. S., Sharpe, M .; E. and Holt, J .; G. : “Bergey's Manual of Systematic Bacteriology” Vol. 2, (1986) Williams & Wilkins.
2. Holt, J .; G., Krieg, N.A. R., Sneath, P.A. H. A., Staley, J.A. T. and Williams, S. T. “Bergey's Manual of Determinative Bacteriology” Ninth Edition (1994) Williams & Wilkins.
3. Mitsuoka Mitsuoka: “The World of Enteric Bacteria”, (1984) Sobunsha.
4). Yoshino Ogino: Microorganism 6,3-14 (1990).
5). Supervised by Ministry of Health and Welfare, Health Sanitation Bureau: “Food Sanitation Inspection Guidelines-Microorganisms” (1990) Japan Food Sanitation Association.
6). Schleifer, K. H. and Kilpper-Balz, R .: Int. J. Syst. Bacteriol., 34, 31-34 (1984).

As a result of the identification, as shown in FIG. In addition to Longum (BB536), 34 types of strains were identified, and it was confirmed that they maintained a symbiotic state within each group.
The culture solution of each group subcultured as described above was subjected to primary co-cultivation. In FIG. 1, (1) to (12) correspond to primary co-cultivation. And in this primary co-cultivation culture, while using three types of medium consisting of a GAM semi-fluid high layer medium, BL agar medium or modified GAM agar medium manufactured by Nissui Pharmaceutical Co., Ltd. as the primary co-culture medium, These primary co-cultivation culture media were selectively used according to lactic acid bacteria.

  Then, the grouped lactic acid bacteria were added to the medium for any one of the three types of primary co-cultivation, and the primary culture was performed. For example, the grouped lactic acid bacteria medium was fluidized as described above. In the case of a medium having high properties, and when the lactic acid bacterium contains bifidobacteria, 10% lactic acid bacterium was added to the primary co-culturing medium. In the case of the group not containing bifidobacteria, 3% lactic acid bacteria were added to the medium for primary co-cultivation.

In addition, when the grouped lactic acid bacteria culture medium is a medium with high solidity, a single amount lifted with a platinum loop was added to the primary co-cultivation culture medium.
These cultures were cultured for about 6 to 12 hours at 37 ° C. until pH 4.6.

  In secondary co-cultivation, soy milk and skim milk are mixed at a ratio of 4: 1 as a medium, and glucose 0.5% (w / w), yeast extract 0.4% (w / w) and the existing product are mixed in this mixture. 1% (w / w) of the third cultivated broth was added. Then, this medium was cultured at 37 ° C. until it reached pH 4.55 for about 6 to 10 hours. In this secondary co-cultivation, the medium and additive elements were all the same for all the bacteria.

  In the tertiary co-cultivation, soy milk and skim milk are mixed at a ratio of 8: 1, and this mixture is mixed with glucose 0.5% (w / w), yeast extract 0.4% (w / w), and already produced tertiary. 1% (w / w) of symbiotic broth was added. The medium thus prepared was first cultured at 32 ° C. for 24 hours, then cultured at 40 ° C. for 48 hours, and further cultured at 37 ° C. for 24 hours.

  Lactic acid bacteria were sterilized by using the high-pressure steam sterilization method for the mixed culture liquid obtained by the third co-cultivation as described above. That is, in the sterilization apparatus heated to about 120 ° C., the above-mentioned third cultivated broth was kept at 65 ° C. for 40 minutes to sterilize lactic acid bacteria in the third cultivated broth. The sterilized final culture solution was cooled to 15 ° C. to coagulate unnecessary substances such as fat. The reason why the fat is coagulated in this manner is to facilitate removal of the fat by filtration. If the fat is removed in this manner, the filtrate when filtered can be prevented from being suspended. Moreover, it can also prevent that an oxide increases in a filtrate, when fat content oxidizes.

  The final culture solution cooled as described above was added with 12% absolute ethanol and crushed with a high-speed homogenizer. By crushing the final culture solution in this manner, the subsequent filtration can be performed for a short time. Will be able to. Moreover, the component in the lactic acid bacteria can be extracted by adding the absolute ethanol.

And after naturally filtering the said sterilized final culture solution using a 3 micrometer filter paper, while applying a 10-kg weight to it and further squeezing, this squeezed liquid is filtered with a 0.1-0.2 micrometer hollow fiber membrane. Impurities were removed to obtain a filtrate.
In addition, although the said filtrate is heated for sterilization as mentioned above in the production | generation process, the component contained in lactic acid bacteria can be heat-extracted by performing heat processing in this way. . Moreover, since it is sterilized by heat, live bacteria do not act in the product using the lactic acid bacteria-producing substance, and the quality is not changed.

A product obtained by removing fat and dead bacteria from the final culture solution after the third co-cultivation as described above by filtration is the lactic acid bacterium-producing substance of the present invention.
In addition, the analysis result of the lactic acid bacteria production substance produced | generated as mentioned above is shown in Table 1, Table 2, and Table 3.

As shown in Tables 1 and 2, this lactic acid bacterium-producing substance contains a large number of metabolites of lactic acid bacteria.
Table 2 shows the results of amino acid analysis. As is clear from Table 2, the lactic acid bacteria-producing substance of the present invention contains many amino acids.
These are microbial trophic factors.
Furthermore, physiologically active substances as shown in Table 3 have also been detected from the lactic acid bacteria-producing substance.

As shown in Tables 1 to 3 above, it is considered that many types of nutrient factors and physiologically active substances are contained in the lactic acid bacterium-producing substance as a result of using various lactic acid bacteria. Various types of metabolites are also produced from a single lactic acid bacterium, but if the number of types of lactic acid bacteria increases, the number of types may increase. The amount of the above-mentioned nutritional factors and physiologically active substances can also be increased.
Therefore, in comparison with the case of ingesting raw lactic acid bacteria, if a small amount of the lactic acid bacteria producing substance is ingested, many of the above-mentioned nutritional factors and physiologically active substances can be taken into the body. As a result, it can be expected that intestinal benign bacteria are activated in the intestine, the amount thereof increases, and the intestinal environment is improved.

In particular, since the lactic acid bacterium-producing substance of the present invention is composed of a plurality of types of lactic acid bacteria and is cultivated together with each other, the lactic acid bacteria finally contain a metabolite produced from a very large number of types of lactic acid bacteria. Production material can be obtained.
In particular, since a group is formed by combining multiple bacteria that are easy to maintain a co-cultivated state, compared to repeating co-cultivation by combining two types suitable for co-cultivation from among many bacterial species, There is also an advantage that the final culture can be reached with a small order.

(Experimental example 1)
The lactic acid bacteria-producing substance obtained as described above was orally ingested by 100 people under the following conditions. That is, each person ingested 7 ml continuously for 8 weeks on an empty stomach between morning, noon, and night. As a result, as shown in Table 4, 86% of the respondents said that they had an effect on subjective sensations such as numbness, constipation, abdominal distension, and headaches, and most people showed improvement in their subjective sensations. It was.

Thus, the improvement in the sense of consciousness was observed regardless of the survival of the bacteria, because many metabolites in the lactic acid bacteria-producing substance were supplied into the intestine, which became a nutrient source for benign bacteria in the intestine. This is probably because the intestinal benign bacteria were activated.
In addition, the above experimental example 1 was requested from the Beijing China-Japan Friendship Clinic.

(Experimental example 2)
In Experimental Example 2, 50 diabetic patients were orally ingested the same lactic acid bacteria-producing substance as in Experimental Example 1 under the following conditions. In other words, 7 ml of each person was orally ingested continuously for 16 weeks during fasting between meals in the morning, noon and evening. As a result, a significant decrease in blood glucose level was observed. Table 5 shows the experimental results. Table 5 shows the average value of the above 50 people. The experimental results were displayed as “reference value ± standard value”.

As shown in Table 5 above, it can be seen that both the venous blood glucose level (GLU) and the glycated hemoglobin level (HbA1c) were clearly reduced by ingesting the lactic acid bacteria-producing substance. Therefore, it can be said that the lactic acid bacteria-producing substance has a remarkable effect on venous blood glucose level (GLU) and glycated hemoglobin level (HbA1c).
In addition, even after one month from stopping the intake of the lactic acid bacteria-producing substance, this did not rise again, and there were people who fell further.

The lactic acid bacterium-producing substance as described above is considered to maintain a good intestinal environment and to improve the absorption efficiency of nutrients in the intestine and maintain homeostasis by maintaining the intestinal environment.
In addition, the above experimental example 2 was requested from the Chinese medicine department in Beijing China-Japan Friendship Hospital.

(Experimental example 3)
In Experimental Example 3, 100 diabetic patients were orally ingested the lactic acid bacterium-producing substance of the present invention under the same conditions as in Experimental Example 2, and venous blood glucose levels (GLU) and glycated hemoglobin levels before and after oral intake ( HbA1c) was measured. The results are shown in the graphs of FIGS. FIG. 2 is a graph showing the venous blood glucose level (GLU), and FIG. 3 is a graph showing the glycated hemoglobin value (HbA1c).
The 100 diabetic patients were 5 patients with type I diabetes (insulin dependent diabetes) and 95 patients with type II diabetes (insulin independent diabetes).

2 and 3, it can be seen that venous blood glucose level (GLU) and glycated hemoglobin level (HbA1c) are clearly reduced by ingesting the lactic acid bacteria-producing substance. Therefore, it can be said that the lactic acid bacteria-producing substance has a remarkable effect on venous blood glucose level (GLU) and glycated hemoglobin level (HbA1c).
The glycated hemoglobin value (HbA1c) is an index representing a long-term blood glucose state, and the improvement of this value is very innovative in the field of diabetes treatment.
In addition, the above experimental example 3 was requested from the Beijing China-Japan Friendship Clinic.

(Experimental example 4)
In Experimental Example 4, 50 digestive system tumor patients were orally ingested the same lactic acid bacteria-producing substance as in Experimental Example 1 under the following conditions. That is, each person ingested 7 ml once a day. The intake period was one course of 40 days. As a result, there were 23 subjective symptoms of increased appetite, accounting for 46% of the total. There were 24 subjective symptoms that mental fatigue improved, accounting for 48% of the total.

Furthermore, 24 cases improved the feeling of collapse of the legs and waist, accounting for 48% of the total. Weight gain was 22 cases, accounting for 44% of the total. In addition, 26 cases were observed to have improved quality of life (QOL), accounting for 52% of the total. There were 17 dry mouth improvement cases, accounting for 34% of the total.
These results are considered to indicate that the lactic acid bacteria-producing substance is beneficial to the liver and spleen.
Table 6 shows the blood test results.

As shown in Table 6, increased hemoglobin (Hb), increased NK cell activity, increased T cell subgroup CD3, and increased CD4 / CD8 value were confirmed. Both rates of increase were significant.
Furthermore, it was confirmed that the values of white blood cells (WBC) and platelets (BPC) were also constantly increased. There was also a tendency for improvement in blood glucose level.
Note that BUN (urea nitrogen) and Cr (quatin) in Table 6 are both indicators of renal function, and are problematic indicators if their values are too large.
This Experimental Example 4 was commissioned to Nanjing Oncologist, Beijing Zhongxi Medical Association, Cancer Research Fund Association Xing Tumor Clinic, Beijing Chunichi Friendship Clinic Chugaku Oncology.

(Experimental example 5)
In Experimental Example 5, the same lactic acid bacteria-producing substance as in Experimental Example 1 was orally administered to 30 patients with chronic nephritis under the following conditions. That is, each person ingested 7 ml once orally before morning, noon, and evening meals. The experimental period was 2 months. The results are shown in Tables 7 and 8.

Table 7 above shows the number of people who have symptoms of swelling, high blood pressure, low back pain, weakness, and night urine. As is apparent from Table 7, the number of people who have the above symptoms is reduced by the intake of the lactic acid bacteria-producing substance. That is, symptom improvement was observed. Table 8 shows the urine protein quantification by reference value ± standard difference. As shown in Table 8, the urine protein quantification was clearly reduced.
Experimental Example 5 was requested from the Beijing-China-Japan Friendship Clinic, Hebei Province Tamadaggu Clinic, Beijing Zhongcheng Clinic, and the Beijing China Pharmaceutical Development Association Interview Department.

(Experimental example 6)
In Experimental Example 6, the same lactic acid bacterium-producing substance as in Experimental Example 1 was orally administered to streptozotocin (STZ) -induced diabetic rats under the following conditions. First, streptozotocin was dissolved in 15 rats with a citrate buffer solution at 1 ml / kg and then administered through the tail vein, and it was confirmed that the blood glucose level of the rats was 180 mg / dl or more. And this was divided into three groups of 5 animals each, and these were made into groups 1-3.

  Group 1 was orally administered with 4 ml of distilled water, Group 2 with 2 ml of lactic acid bacteria producing substance + 2 ml of distilled water, and Group 3 with 4 ml of lactic acid bacteria producing substance. Oral administration was performed once a day for 28 days. Then, the plasma glucose concentration of these rats was measured. The measurement results are shown as average values and standard errors.

As a result of Experimental Example 6, in Group 1, a high value of 200 mg / dl or more was maintained throughout the test period. On the other hand, in group 2 to which the lactic acid bacteria production substance was administered, compared to 274.4 ± 17.4 before administration, it decreased by 30% or more in the first week to 178.2 ± 27.6, and after 2 weeks it was 159.3 ± 9.8. A significant difference was seen over 1.
Further, in group 3 to which the lactic acid bacteria-producing substance was administered, 153.8 ± 7.6 at the first week of administration, showing a significant difference compared to group 1. In the second week, the effect was sustained at 160.1 ± 5.5. Furthermore, it was 95.2 ± 2.2 at 3 weeks and 103.2 ± 11.1 on the last day of the study. This was almost the same value as that of healthy rats not treated with streptozotocin.
From the above, it was found that the lactic acid bacteria-producing substance clearly improves hyperglycemia to normal values.

Moreover, after the said test, the liver and duodenum of each group were formalin fixed, the tissue section was created, and histological evaluation was performed.
As a result, in Group 1, the degranulation of the islets of Langerhans β cells, that is, the decrease of the granules was observed, and the cavitation of the cells was confirmed. The number of β cells was 19 or more in rats not treated with streptozotocin, but in this group 1, it was 7.0 ± 2.0, and a decrease in the number of β cells was also confirmed.
On the other hand, in the groups 2 and 3 to which the lactic acid bacteria-producing substance was administered, suppression of the decrease in the number of β cells was recognized in a dose-dependent manner. The number of β cells at this time was 10.3 ± 2.3.
In group 2, β-cell granule reduction and cell cavitation were observed, but the number was smaller than in group 1.

From the above results, the lactic acid bacteria-producing substance lowers the blood glucose level predominantly in a concentration-dependent manner and improves it to a normal value, but the mechanism is not a single action mechanism but a plurality of action mechanisms. This is because histologically it improved the degeneration of Langerhans islet β cells, but this alone is considered insufficient to restore blood glucose levels to almost normal levels. That is, it is considered that administration of a lactic acid bacterium-producing substance has some effect on glucose metabolism, and as a result, the blood glucose level is improved to a normal level.
Note that Experimental Example 6 was requested from the Drug Safety Testing Center Co., Ltd.

(Experimental example 7)
In Experimental Example 7, an experiment was conducted in which the lactic acid bacterium-producing substance of the present invention was given to a diabetic patient and clinically observed.
Observed subjects were 60 patients who were diagnosed with diabetes surely in hospitals, patients aged 32 to 78 years, and the duration of disease was 1 to 5 years for 20 cases and 6 to 10 years for 20 cases, respectively. , 20 cases are over 10 years.
These 60 patients were divided into 2 groups of 30 patients. One group was orally ingested 10 ml of lactic acid bacteria-producing substance twice daily, and the other group 15 g of Tamakimaru twice daily. This was continued for 3 months and the effects before and after treatment were compared. The results are shown in Table 9. In addition to administering other than the above-mentioned lactic acid bacteria-producing substance or Tamaizumimaru, each patient is simultaneously treated according to the needs of individual patients, such as administration of an antihypertensive agent. The above-mentioned Yusenmaru is a traditional Chinese medicine for improving the symptoms of diabetes.
In the table below, the group of lactic acid bacteria-producing substances is expressed as an experimental group, and the group of Tamaizumi Maru is referred to as a control group.

表９に示すように、上記乳酸菌生産物質、玉泉丸ともに改善が見られた。
特に、乳酸菌生産物質を摂取したグループでは、有効率が８６．７％と非常に高く、玉泉丸のグループの有効率６６．６％に比べても優位であった。

As shown in Table 9, both the lactic acid bacteria-producing substance and Tamaizumi Maru were improved.
In particular, the effective rate was 86.7% in the group that ingested the lactic acid bacteria-producing substance, which was superior to the effective rate of 66.6% in the Tazumizumi group.

The “significant effect” shown in Table 9 means that the fasting venous blood glucose level (GUL) decreased by 1 to 2 (mmol / l) and the glycated hemoglobin value (HbA1C) decreased by 1% to 2%. It is in the state. In other words, the above biochemical indices are clearly improved.
In addition, “effective” means that the fasting blood glucose level is less than 1 (mmol / l) or the glycated hemoglobin value is decreased by less than 1%, and the improvement in the biochemical index is not clear, Subjective symptoms are improved.
On the other hand, “no effect” is a case where neither the biochemical index nor the subjective symptoms improved.
That is, from Table 9 above, it was proved that the lactic acid bacteria-producing substance of the present invention was more effective than Diazumimaru for diabetic patients.

Moreover, since indexes other than the blood glucose level and the glycated hemoglobin level were also measured before and after the treatment, the measurement results are shown in Table 10.

From Table 10 above, the following was found.
Regarding blood glucose level and glycated hemoglobin level, both groups show a significant difference before and after treatment. That is, although it is the same as the result of the said Table 9, both lactic-acid-bacteria production substances and Tamaizumimaru have the therapeutic effect with respect to diabetes.
In addition, the group that ingested the lactic acid bacteria-producing substance has decreased triglycerides (TG) and cholesterol (CHO), but the group that has ingested Tamaizumi Maru has not decreased these values. The difference between the two is not a significant difference, but the intake of lactic acid bacteria may be effective for patients with hyperlipidemia with high neutral fat and cholesterol as complications of diabetes. There is.

Furthermore, blood pressure has decreased after treatment in both groups, but both groups include patients using blood pressure lowering agents, which is considered to be the effect.
In addition, lactic acid bacteria-producing substances have the effect of reducing body weight, whereas Tamaizumi Maru has no effect.

  In addition, Table 11 shows the results of comparing the blood glucose level decrease values of individual patients for the group that ingested the lactic acid bacteria-producing substance of the present invention and the group that ingested Tamaizumi Maru. The results are shown in Table 12.

上記表１１に示すように、実験組、対照組ともに、血糖値が下がっているが、実験組の方が、より低下数値が大きく、その効果には差が有ることが分かる。

As shown in Table 11 above, both the experimental group and the control group have lower blood glucose levels, but it can be seen that the experimental group has a larger decrease value and there is a difference in its effect.

上記表１２に示すように、実験組は対照組に比べて、糖化ヘモグロビン値を下げる効果が大きいことが分かる。

As shown in Table 12 above, it can be seen that the experimental group has a greater effect of lowering the glycated hemoglobin value than the control group.

この表１３から、実験組では、発病年数による効果の現れ方には、特に差がないことが分かった。これに対し、実験組では、発病年数によって効果に差が有り、発病年数が長くなると顕著な効果が現れにくくなることが分かった。
この結果から、上記乳酸菌生産物質は、発病年数が長く、玉泉丸では明らかな効果が現れないような糖尿病患者に対しても有効であることが分かった。
なお、この実験例７は、中日友好医院および北京中医薬大学に依頼したものである。 Furthermore, Table 13 shows the results of examining the correlation with the age of onset for the effects before and after treatment shown in Table 9 above. The age of onset is the number of years since the onset of diabetes.

From Table 13, it was found that in the experimental group, there is no particular difference in the appearance of the effect depending on the age of the disease. On the other hand, in the experimental group, there was a difference in effect depending on the age of onset, and it became clear that the remarkable effect became difficult to appear as the age of onset became longer.
From this result, it was found that the above-mentioned lactic acid bacteria-producing substance is effective even for diabetic patients who have a long-onset disease and do not show obvious effects in Tamakimaru.
This Experimental Example 7 was requested from Chunichi Friendship Clinic and Beijing Chugoku Medical University.

It is the figure which showed the order of the cocultivation culture | cultivation for producing | generating the lactic acid bacteria production substance of this invention. 10 is a graph showing changes in venous blood glucose levels in the experimental results of Experimental Example 3. 10 is a graph showing changes in glycated hemoglobin value (HbA1c) in the experimental results of Experimental Example 3.

Claims (7)

  Lactobacillus belonging to the genus Lactobacillus, lactic acid bacteria belonging to the genus Bifidobacterium, lactic acid bacteria belonging to the genus Streptococcus and one or two selected from these three genera A lactic acid bacterium culture solution obtained by forming a plurality of groups using the above lactic acid bacteria, subcultured for each group to maintain a co-cultivated state, and further coculturing the lactic acid bacteria of the subcultured group units. A lactic acid bacteria production substance consisting of a filtrate obtained by filtration.   As lactic acid bacteria belonging to the genus Lactobacillus, L. Lactobacillus acidophilus, L. Lactobacillus brevis, L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. The lactic acid bacterium-producing substance according to claim 1, wherein one or two or more of Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii) is used.   As lactic acid bacteria belonging to the genus Streptococcus, Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. The lactic acid bacteria-producing substance according to claim 1, wherein one or more of Lactococcus lactis is used.   As lactic acid bacteria belonging to the genus Bifidobacterium, Bongidobacterium longum, B. Bifidobacterium bifidum, B. The lactic acid bacterium-producing substance according to claim 1, wherein one or more of Adrecentis (Bifidobacterium adolescentis) is used.   L. Lactobacillus acidophilus, L. Lactobacillus brevis, L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), S. Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. Lactococcus lactis, B.I. Bongidobacterium longum, B. Bifidobacterium bifidum, B. The lactic acid bacteria-producing substance according to claim 1, wherein 16 types of lactic acid bacteria of Bifidobacterium adolescentis are used.   E. Fesium and L. Helveticas in the first group, E. Fesium and L. Acidophilus in the second group, E.I. Fesium and L. Gasseley is the third group, E.I. Fesium, L.C. Acidophilus and L. Brevis the 4th group, E. Fesium, L.C. Acidophilus and L. Brevis the 5th group, E. Fesium, L.C. Acidophilus, L.H. Brevis, L. Paracasei in the sixth group Adrecentis alone is group 7; Delbricky and L. Gasselie is in the 8th group, L.C. Delbricky alone is the ninth group, E.D. Fesium, L.C. Genseny, L. Paracasei and L. Brevis the 10th group, L. Acidophilus alone is group 11; Fesium and L. Gasserie was placed in the 12th group, L. Paracasei alone was added to the 13th group, L. Gassely, E.M. Fesium and B.I. Bifidum is the 14th group. Longum, S.M. Thermophilus and E. coli. Fecium was added to the 15th group, L.P. Gasserie alone, group 16; Bulgarix and S. Thermophilus in the 17th group, L. Gassely, L.C. Lactis, L. Gasserie and E.C. Fesium was added to the 18th group, L.C. Gassley, S.M. Thermophilus and L. Bulgarix was introduced in the 19th group, L.B. Lactis alone, 20th group, L. Gasserie and E.C. Fecium was added to the 21st group, L.P. In the 22nd group, L. Casei alone, group 23, B.I. Longum alone is used as the 24th group, and subcultured for each group to maintain the co-cultivated state. Among these culture solutions, the first, second groups, the third, fourth groups, the fifth, sixth Groups, 7th, 8th group, 9th, 10th group, 11th, 12th group, 13th, 14th group, 15th, 16th group, 17th, 18th group, 19th, 20th group Primary cultures between the 21st and 22nd groups, the 23rd and 24th groups, the 1st and 2nd groups, the 3rd and 4th groups, the 5th and 6th groups, the 7th and 8th groups, the 9th, 10th group, 11th and 12th group, 13th and 14th group, 15th and 16th group, 17th and 18th group, 19th and 20th group, 21st and 22nd group, 23rd and 2nd group Was subcultured in a group with each other, further, lactic acid production material of claim 5 in which the tertiary culture as a mixture thereof each secondary culture.   The lactic acid bacterium-producing substance according to claim 6, comprising a filtrate obtained by subjecting a cultivated lactic acid bacteria culture solution to heat sterilization and then filtration.

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