KR100398665B1 - Lactic acid bacterial preparation - Google Patents

Abstract

The new species named by the inventors Lactobacillus clearans have been found to have a high effect on health, but in particular, there is a lack of purification in the intestine, and thus high titer for the solution. Although various strains have been tested, such as making strains, it was inevitable to use the strain alone. Therefore, in combination with other effective substances, lactic acid bacteria preparations that can be felt more vividly and healthily to healthy people, and unpleasant subjective symptoms to those who are recovering or unhealthy can be resolved and reliably returned to health. Is required.

Live organisms and microorganisms of Enterococus faecalis, which can degrade at least one or two types of Lactobacillus clearance and triglyceride and cholesterol. It is a lactic acid bacterium preparation which consists of one type or two types of (死 菌体).

Description

Lactobacillus preparations {LACTIC ACID BACTERIAL PREPARATION}

The present invention belongs to Lactobacillus genus (Lactobacillus genus) (Lactobacillus genus), which the inventors have succeeded in separating and collecting for the first time in the world, and Lactobacillus clearans (Lactobacillus clearans) having unique properties that are not known in the conventionally known bacteria. And one or two types of live and dead microorganisms of Enterococcus faecalis, which are deeply involved in metabolism and lipid metabolism. The present invention relates to a lactic acid bacterium preparation which is very meaningful to human and animal health by expressing each characteristic more clearly and effectively.

It is said that there are 300 kinds of 100 trillion bacteria in the intestine, and its total weight is 1kg, which is much more than 60 trillion human cells. And it has become increasingly apparent through long years of epidemiological investigations and studies that there is a profound relationship to human and animal health or to various diseases, leading experts to regard the intestinal bacterial phase as an important organ. have. When healthy, Bifidobacterium genus and Lactobacillus genus, which are representatives of beneficial bacteria belonging to the lactic acid bacteria group, coexist in a balanced state. The balance is broken down by various factors, such as changes in the quantity and quality of daily meals, overwork, lack of sleep, and mental stress, and the fertility of Welchii and Veillonella, the representatives of harmful bacteria, When you start to drive away the beneficial bacteria, the production of harmful substances increases, first of all, from diarrhea or constipation, such as constipation, to chronic fatigue, coarse skin, liver damage, high blood pressure and arteriosclerosis from blood contamination. Will cause illness and aging of the whole body.

Early on, Russia's Mechinikoff (1845-1916) suggested the main cause of aging was poisoning by toxic substances formed by intestinal rot fermentation. He suggested that it would be helpful. After that, the history of lactic acid bacteria was not noticed as soon as the attention was attracted, and then it was impossible to obtain practicality in the true meaning by repeating the attention again. This is because what the epidemiological investigation reveals is not well correlated with what the experiment reveals.

In recent years, due to the development of microbiology, the growth of beneficial bacteria in the intestine, that is, lowering the acidity in the intestine, promoting the movement of the intestine, aiding in digestion of food and absorption of nutrients, and the generation of harmful substances. Mechnikov's conjecture proves that it is clear that it is carrying out such important movements as suppressing and simultaneously degrading, synthesizing vitamins and amino acids, defending the intestinal infections of pathogens, and enhancing immunity. It becomes the common sense of health science now.

In addition, the recent trend of health-oriented, lactic acid bacteria seems to have been completely settled among people as a drink or formal dressing (整腸 劑). However, the reality is that even if you take or take lactobacillus every day without filtering, you can not realize that your health is improved or your symptoms are improved. Like coffee or coffee, it is still a strong character as a favorite food.

In the above situation, there is a need for a product which exhibits an excellent effect which does not exist in the conventional lactic acid bacteria drink or lactic acid bacterium preparation as a short-term administration, but a new species previously named by the present inventors Lactobacillus clearance is healthy. Although it has been found to have a high effect, in particular, it is lacking in intestinal purifying action and has been tested in various ways, such as making high titer strains for the solution, but only using the bacteria alone I had to admit that there was a limit. Therefore, in combination with other effective substances, lactic acid bacteria preparations that can be felt more vividly and healthily to healthy people, and unpleasant subjective symptoms to those who are recovering or unhealthy can be resolved and reliably returned to health. Is required.

In order to solve the above problems, the present inventors intensively searched for a partner having good relevance regardless of living or non-living organisms, and finally, Trigly in Enterococus faecalis belonging to the same lactic acid bacterium group as Lactobacillus clearance. A strain capable of lowering at least one kind of triglyceride and cholesterol has been found to achieve the present invention. That is, a lactic acid bacterium preparation comprising a live cell of Lactobacillus clearance and a live cell of Enterococcus pecharis capable of reducing at least one or two types of triglycerides and cholesterol.

A second object of the present invention is a lactobacillus preparation comprising a live bacilli of Lactobacillus clearance and a bacterium of Enterococcus pecaris capable of reducing at least one or two types of triglycerides and cholesterol.

A third object of the present invention is a lactic acid bacterium preparation comprising a live bacterium of Lactobacillus clearance and a live cell and a dead cell of Enterococcus pecaris capable of reducing at least one or two types of triglycerides and cholesterol. .

(Example)

Lactobacillus clearans in the present invention is a new strain of the genus Lactobacillus having the following biochemical properties ①, ②, ③ and ④. That is, when 1 g of meat extract, 5 g of peptone, 1 g of glucose, 1 g of CaCO _{3, and} 1 L of water (pH neutral) add 0.5 g of Na ₂ S · 9H ₂ O and / or 0.5 ml of NH ₄ OH, Na ₂ S · 9H _{Both 2} O and NH ₄ OH can be reduced. ; ② Stephenson-Wetham badge (abbreviated as “SW”); 1 g of KH ₂ PO ₄ , 0.7 g of MgSO ₄ · 7H ₂ O, 1 g of NaCl, (NH ₄ ) ₂ HPO _{4 4} g, FeSO ₄ · 7H ₂ O 0.03 g, glucose 5 g] to vitamin [A: 900 international units, B ₁ : 1 mg, B ₂ : 1 mg, B ₆ : 1 mg, B ₁₂ : 5γ, nicotinic acid amide: 16 mg, calcium pantothenate: 8 mg, C: 64 mg, D ₂ : 120 international units] and 1 g of kasamino acid The bacteria were cultured in the medium, and 0.5 g of Na ₂ S · 9H ₂ O and / or 0.5 ml of NH ₄ OH were added to the logarithmic growth phase. ; ③ The natural isolate is stronger than Na ₂ S · 9H ₂ O than conventionally known lactic acid bacteria and weaker than Lactobacillus deodorans. ; ④ It does not decompose gelatin without reducing gram-positive, rod, non-kinetic, catalase-negative, nitrate, indole and hydrogen sulfide Its ability to form lactic acid is higher than that of glucose and lactose without producing citrate; acetic acid promotes development by addition; A strain of the genus Lactobacillus (see Japanese Patent Application Laid-Open No. 4-632).

The characteristics of Lactobacillus clearance are, in addition to the action of Lactobacillus in general, as described in Japanese Patent No. 1714413 and Japanese Patent Application No. 15177, Japanese Patent Publication No. 15177. Reduce and disassemble. ; ② Participate in the increase of Bifidobacterium and Lactobacillus, which are representative of beneficial bacteria in the intestinal bacterial phases, and on the contrary, significantly reduce the intestines of Welch and Baylonella, which are representative of harmful bacteria. Good balance of bacterial flora improves the intestinal environment. ; ③ mainly inhibit the proliferation of pathogens that cause intestinal infection, and weaken the toxicity; It has a powerful intestinal purifying action not found in conventional Lactobacillus.

Next, the effect of these is demonstrated through a test. First, an in vitro test is performed. 15 ml of a 10-fold dilution of feces was added to a test tube (18 × 180 mm), and ① inoculated with autoclave at 120 ° C. for 15 minutes, ② not sterilized, and each sample was inoculated with test bacteria ( Iv), sealed with rubber stopper and incubated anaerobicly at 37 ° C. for 72 hours, inhaling 1 ml of air in a test tube with a syringe and injecting it into a 5 L odor bag. Conduct olfactory sensory test. In addition, the degree of odor is in accordance with the odor evaluation criteria of feces shown in Table 1.

Using three representative strains of Lactobacillus clearance, that is, FERM P-17148, FERM P-17149 and FERM P-17150, the deodorizing ability is tested by the in vitro test described above. As a result, as shown in Table 2, high-pressure sterilization is deodorized reliably to change from mild smell of feces to weak smell of feces. On the other hand, non-sterile ones are active in a large number of decaying bacteria inhabiting feces, and odorous toxic substances are increasingly produced, so the degree of deodorization is weaker than high-pressure sterilization. Deodorized. The above test is a representative three weeks of Lactobacillus clearance, but similar results were obtained for other strains of the same species.

Next, as an oral administration test in vivo, 150 ml of yogurt prepared by Lactobacillus clearance FERM P-17150 was taken once daily, followed by 20-30 days, 50-60 days, 80 The smell of feces excreted daily for ˜90 days is measured and the average is shown in Table 3. In the measurement method, 0.5g of feces excreted by 10 persons ingested were placed in a 20-ml odor bag and left at room temperature to determine the odor after 30 minutes by six evaluators. Here, the average value of the stool smell of five people who did not ingest is 100. The smell of feces was significantly different depending on the contents of the meal, but the average smell of ten people who tasted yogurt decreased by 50% after one month, 70% after two months, and 85% after three months. However, after three months, the odor level was reduced by 80-90% even after ingestion.

Early on, the present inventors narrowed the range of symmetrical substances to odorous substances that are easy to test in the correlation between natural purification and microorganisms, so that these compounds were sulfur compounds, nitrogen compounds, and carbon compounds. Divided into two. And lower fatty acids such as Na ₂ S · 9H ₂ O as a representative of odorous sulfur compounds, NH ₃ as a representative of odorous nitrogen compounds, and acetic acid and butyric acid as representatives of odorous carbon compounds. The microorganisms reducing and decomposing the microorganisms were found to reduce and decompose a large amount of the high molecular odorous sulfur compounds, the odorous nitrogen compounds and the odorous carbon compounds, and were called SNC-theories. According to this theory, the following various tests are carried out.

As a synthetic medium, lactobacillus clearance was added by adding 5 g of meat extract, 5 g of peptone, 5 g of sodium butylate, 5 g of glucose, and ₃ g of CaCO ₃ to 0.5 g of Na ₂ S.9H ₂ O or 0.5 ml of ammonia water (NH ₄ OH) in 1 L. Anaerobic incubation at 37 ° C. for 72 hours and the rate of reduction of added Na ₂ S · 9H ₂ O or NH ₄ OH were measured over time. The measurement method employed is iodine titration method of JIS K0102-1985 for Na ₂ S · 9H ₂ O and indophenol blue absorbance method of JIS K0102-1985 for NH ₄ OH. . The measurement results are shown in Tables 4 and 5. As is clear from these tables, Lactobacillus clearance has the ability to reduce odorous toxic Na ₂ S.9H ₂ O and NH ₄ OH by 40-50% over 72 hours. This means reducing and breaking down many other odorous toxic substances.

As a method for evaluating the deodorizing effect of Lactobacillus clearance, the sensory test and chemical test in vitro were described first, but the 10-fold dilutions of feces prepared at that time were centrifuged immediately before and 72 hours after incubation, respectively. 5 ml of the liquid floating on the surface of the liquid is taken, diluted 10-fold, and the concentration of S ²⁺ ions and NH ₄ ⁺ ions is measured to calculate the degree of reduction. As a result, as is apparent from Table 6, it can be said that the reduction rate of S ²⁺ ions and NH ₄ ⁺ ions contained in the feces is slightly higher than the reduction rate in the synthetic medium described above. This indicates that the stool is suitable as a habitat for Lactobacillus clearance. The measurement method adopted is the urea titration method of JIS K0102-1985 for S ²⁺ ions, and the indophenol blue absorbance method of JIS K0102-1985 for NH ₄ ⁺ ions.

2 µl of the liquid floating on the surface of the liquid is injected into gas chromatography to analyze lower fatty acids. Gas chromatography was performed on Col. unisole F-200 30/60 glass 3ø × 3m, carrier gas 50ml / min (He), hydrogen 0.55kg / cm ² G, air 0.55kg / cm ² G, column temperature 140 ℃, Used at Inj 200 ° C. Measured materials include acetic acid, propionic acid, iso-butyric acid, n-butyl acid, iso-valeric acid, and n-giric acid. Appears. As can be seen from Table 7, the concentration of lower fatty acids is reduced by 50 to 75% when autoclaved, and by 40 to 60% even when not sterilized.

Representative strains of Lactobacillus clearance, that is, FERM P-17148 strain, FERM P-17149 strain, making it a method of preparing a freeze-dried cells of FERM P-17150 strain respectively yangssik mixture of these, and 2g (5 × 10 ⁸ gae / g) daily and do not include Bifido bacterium and Lactobacillus, the strains inherent in the intestine, known as representatives of beneficial bacteria in the intestinal bacterial phase, lactobacillus clearance administered ), The disappearance and growth of Veillonella and Clostridium (Clostridium perfrigens = Welch) as representatives of the other harmful bacteria are measured over time. The results of oral administration to 20 healthy persons are shown in Table 8, and the results of oral administration to 20 sick persons are shown in Table 9.

As can be seen from Table 8 and Table 9, intestinal bacterial images before taking Lactobacillus clearance preparations show that healthy people have an average of 220% more beneficial bacteria than sick ones, whereas harmful bacteria only 22.5. It was only%. This is a clear indication of how intestinal bacterial shape is indicative of the current state of health, ie, how deeply intestinal bacterial shape is involved in health. In addition, as a new discovery, by taking the present formulation, healthy people first found that the beneficial bacteria increased and the harmful bacteria gradually decreased with the increase. The sick person knew first that the harmful bacteria began to decrease, and then the beneficial bacteria took a gradually increasing pattern. As a whole, the average disappearance and growth of beneficial and harmful bacteria is increased immediately after taking the beneficial bacteria, and conversely, harmful bacteria are reduced. So from 3 months the trend accelerates, with Bifidobacterium growing 50% after 6 months, 110% after 1 year, Lactobacillus 300% after 6 months, and 950% after 1 year. On the other hand, Clostridium decreased 70% after 6 months, 85% after 1 year, and Baylonella decreased 67.5% after 6 months, and 82.5% after 1 year.

To see the effects of Lactobacillus clearance on pathogens, E. coli O-157 (Escherichia coli O-157), Salmonella enteritidis and Shigera flexneri (Sigma) and Shigella flexneri alone Incubate and mix culture with Lactobacillus clearance (FERM P-17150) and compare the results. The medium composition used for the culture was adjusted to pH 7.2, containing 10 g of meat extract, 10 g of peptone, 2 g of glucose, 12 g of NaCl, and 1 g of CaCO _{3 in} 1 L. Cultivation is anaerobic at 37 ° C, repeated subcultures every 72 hours, and then diluted and applied to the plate every time, so that the disappearance and growth of bacteria and whether the newly emerged bacteria are S-type (round). Observe whether the type R is weakened due to mutations, and what is the ratio. Pathogens used in the experiment were obtained from Medic.

Table 10 shows the results of the single culture of E. coli O-157 and Table 11 shows the results of the mixed culture of E. coli O-157 and Lactobacillus clearance. As can be seen from Table 10, in the single culture of Escherichia coli O-157, the number of bacteria was almost constant from 4 to 5 x 10 ⁹ / g through passages in the 20s, and no R-form appeared between them. On the other hand, in the mixed culture of Escherichia coli O-157 and Lactobacillus clearance, as shown in Table 11, the variation in the number of bacteria of Lactobacillus clearance is small, but the variation in the number of bacteria of Escherichia coli O-157 is large, and R-type from the fifth generation With the subsequent passages, the proportion of R-type increases, and in the 18th generation, all bacteria form R-type. Since then, S-type has never been resurrected even after continuing the passage.

Table 12 shows the results of the single culture of Salmonella enteritidis and Table 13 shows the results of the mixed culture of Salmonella enteritidis and Lactobacillus clearance. As can be seen from Table 12, in the single culture of Salmonella enteritidis, the number of bacteria was 3-5 × 10 ⁹ / g, and R-type spontaneously appeared in the 14th generation, and the ratio became 5% in the 20s. In the fifties, the highest score was 23%, while the proportion of R-type afterwards was about 20%. On the other hand, in the mixed culture of Salmonella enteritidis and Lactobacillus clearance, as shown in Table 13, only 5 passages had an R-type ratio of 29%, and as the passage was repeated, the ratio of R-types increased to 10th generation. In the 50s, 90% in the 20s, and 100% in the 47s. Subsequent passages continue to resurrect the S-type in some cases, but the proportion is less than 1%. After the 70s, the S-type is completely lost and never resurrected.

Table 14 shows the results of the single culture of Shigera Plexelli (Erythrax) and Table 15 shows the results of the mixed culture of Shigera flexnelly (Lactobacillus) and Lactobacillus clearance. As can be seen from Table 14, in the single culture of Shigera flexnelly (Rhizobacteria), R-type spontaneously appeared in the tenth generation, and the ratio became 9% in the twenties. Although the highest value was recorded at 20%, the proportion of the R-type after that ends at around 10-20%. On the other hand, in the mixed culture of Shigera flexnelly and Lactobacillus clearance, as shown in Table 15, the proportion of R-types increased as the R-types appeared in only five passages and increased in the passage, finally reaching the 80s. It became 100%. Subsequent passages continue to resurrect S-types in some cases, but the proportion is less than 1%. After 108 units, S-types are completely lost and never resurrected.

For each of S and R types of Escherichia coli O-157, Salmonella enteritidis and Shigera flexelli (Rice), 10 per group to examine its toxicity, 1 per 8-week-old male mouse (mice) a × 10 ⁸ of bacteria is administered intraperitoneally. For strain S, strains were used. For strain R, strains at the time when the strain was transformed into strain 100% by Lactobacillus clearance were used. As a result, as shown in Table 16, all of the S-type pathogens died within 4 days, but in the R-type, no cases were found except in the case of Shigera flexnelly (Rice) on the 7th day.

When a yogurt prepared with Lactobacillus clearance described above is administered orally to humans and the intestinal bacterial changes are examined, blood is tested at a rate of once / month. As a result, it was found that cholesterol and triglyceride (triglyceride) were reduced by about 10% in more than 80% of the people before administration.

The characteristic of the Lactobacillus clearance of this invention and the difference in function and bacteria of the genus Lactobacillus of the prior art are shown in Table 17.

The general characteristics of Enterococcus faecalis, belonging to the lactic acid bacteria group and make up the intestinal bacterial phase, usually inhabit about 1 × 10 ⁷ per 1g of feces. It is spherical to egg-shaped and appears in two or short chains. The growth temperature range is 10 to 45 ℃ wider than general streptococci, and it is resistant to heat, dry conditions, chlorine, and bile acids. This is a strong Gram positive cocci. Some strains have pathogens, but most of them are harmless, and their living organisms have been marketed in Japan for decades as formal dressings. Apart from these commercially available products, it has recently been found that in some strains, their live or dead cells effectively lower the values of cholesterol and triglycerides (triglycerides) in the blood. Therefore, it is expected to prevent or treat typical adult diseases (lifestyle diseases) associated with them, that is, diseases such as hyperlipidemia, hypertension, and atherosclerosis. In addition, enterococcus pecarris referred to in the present invention refers to a strain having such a capability.

An example of a method for preparing live cells of Enterococcus pekaris used in the present invention is as follows. In other words, the strain cultured by the conventional method and suspended in physiological saline is suspended in physiological saline, washed and centrifuged once again to collect the bacteria, and lyophilized using soluble starch as a protective agent. In addition, one example of a method for preparing Enterococcus pecaris microorganisms is to treat the centrifuged cells and the soluble starch after washing for 30 minutes in hot water at 100 ° C and freeze-dry them.

Enterococcus pecharis was not recognized for its ability to reduce sodium sulfide (Na ₂ S.9H ₂ O) and ammonia in vitro, and had a weak deodorizing ability in 10-fold dilutions of feces. Table 18 shows the deodorizing ability of two representative strains of Enterococcus pecharis, that is, FERM P-17151 and FERM P-17152. In addition, the test method is the same as in the case of Lactobacillus clearance, the evaluation criteria are shown in Table 1.

Two representative strains of Enterococcus pecaris are orally administered daily at 1 × 10 ⁹ / person and the smell of feces before ingestion is 100, and 20 to 30 days, 50 to 60 days, 80 to 80 days after ingestion. The smell of feces of 90 days was measured and shown in Table 19. As can be seen from Table 19, there was a slight, but slight decrease in odor caused by Enterococcus pekaris.

Two representative strains of Enterococcus pecharis, ie, FERM P-17151 and FERM P-17152, were prepared, respectively, and prepared by mixing the same amount of live cells and dead cells as 1 × 10 ⁹ / Bifidobacterium and Lactobacillus, known as representatives of beneficial bacteria, for the intestinal bacterial extinction and growth taken for 6 months as a person / person (His own strain, lactobacillus clearance administered) are not included. The disappearance and production of 1 g of bacteria / stool of stool is measured over time as the representative of the harmful bacteria on the other, and the disappearance and growth of 1 g of bacteria / stool of Clostridium. The results of oral administration to 10 healthy persons are shown in Table 20. As can be seen in Table 20, the live cells showed a slightly higher improvement effect than the dead cells. However, the effect of Lactobacillus clearance was not significantly lower.

In order to see the action of Enterococcus pecaris on the pathogen, a mixed culture with Escherichia coli O-157, Salmonella enteritidis and Shigera flexneri was performed. However, the rate of SR mutation in these pathogens was little different from the rate of spontaneous occurrence, but the number of pathogens gradually decreased with each passage, and Salmonella enteritidis was in the twenties, while Shigera flexelli (33) was 33 In the third generation, Escherichia coli O-157 disappeared at the 35th generation. While this suggests that the growth of Enterococcus pecharis is fighting against pathogens, the possibility of producing any bioactive substances that inhibit the growth of pathogens cannot be denied.

Enterococcus page to each 1 × 10 ⁹ pieces / feed 1g was added to Charis the raw cell preparation and use feed the cell preparation in mice bred three months, and the triglyceride and cholesterol in serum of the mice bred only with ordinary feed Compare. In addition, mice should have 10 groups in each group, and feed should be taken at will. Triglycerides are measured by E-test table, and cholesterol is measured by C-test table, and the average value of the control mice is shown in Table 21 as 100%. As can be seen from Table 21, both mice, which continued ingestion irrespective of live and dead cells, decreased by 20 to 40%, and there was no question about their effectiveness.

15 8-week-old rats (SHR) naturally occurring in high blood pressure were divided into three groups, and the biobacterial and bacteriostatic formulations of Enterococcus pecaris were fed into the feed 1 × 10 ⁹ / feed 1g, respectively. The group was added and the control group (사 照 사) not administered with the mycelium preparations was raised for 3 months. Blood pressure after 3 months is shown in Table 22. As shown in Table 22, about 10% around the blood pressure lowering action was confirmed.

Surprising efficacy and effectiveness can be found in a short time by combining the excellent action of Lactobacillus clearans and Enterococus faecalis as described above. Hereinafter, although the manufacturing method and Example of each formulation of this invention are demonstrated, it should not be limited to the meaning of this invention and its manufacture example and Example.

(Production Example 1)

The production method of the Lactobacillus clearance preparation includes 5 g of meat extract, 5 g of peptone, 3 g of sodium acetate, 1 ml of ammonia water, 10 g of glucose, 0.5 g of cystine, in 1 L of medium. Lactobacillus clearance is inoculated into 10 L of medium containing 2 g of yeast extract and incubated anaerobicly at 37 ° C. for 72 hours. After incubation, the culture solution is centrifuged to obtain 10 g of biomass. Then rinse with 500 ml saline and repeat centrifugation twice. The obtained bacterium was placed in 500 ml of a solution consisting of 50 g of skim milk, 30 g of trihalose and 0.5 g of taurine, mixed thoroughly, and lyophilized by the usual method. (Iii) 82.5 g (3 x 10 ⁹ bacteria / g) are obtained. It is mixed with 330 g of completely dried scheme milk to make a Lactobacillus clearance formulation containing 5 x 10 ⁸ live cells per gram.

(Production Example 2)

The production method of the probiotic preparation of Enterococcus pecaris is Enterococcus pecaris in a medium 10L containing 5 g of meat extract, 5 g of peptone, 2 g of sodium choride, 2 g of yeast extract, and 10 g of glucose in 1 L of medium. Inoculated and incubated at 37 ° C. for 72 hours. After incubation, the culture solution is centrifuged to obtain 16 g of bacteria. Then rinse with 800 ml of physiological saline and repeat centrifugation twice. The obtained bacterium was added to 500 ml of a solution consisting of 20 g of skim milk, 30 g of soluble starch, and 0.5 g of sodium glutamate, mixed thoroughly, and lyophilized by a conventional method. ) 54 g (5 × 10 ⁹ bacteria / g) are obtained. It is mixed with 486 g of completely dry soluble starch to make Enterococcus pecaris probiotic formulation containing 5 × 10 ⁸ probiotics per gram.

(Production Example 3)

In the method for preparing the bacteriophage preparation of Enterococcus pecaris, the bacterium obtained by washing according to Preparation Example 2 was suspended in 500 ml of physiological saline solution, 50 g of soluble starch was added thereto, and then heated at 110 ° C. for 15 minutes to sterilize. The suspension is lyophilized in the usual manner to obtain 53 g of bacteria (about 5 × 10 ⁹ / g). In addition, in the production of dead cells, the cells can be prepared by destroying the cells by ultrasonic waves or other methods.

(Production Example 4)

The preparation method of the mixed preparation of Lactobacillus clearance and Enterococcus pecaris is a viable cell preparation of Lactobacillus clearance prepared in Preparation Example 1 and Enterococcus pecaris prepared in Preparation Example 2, or Preparation Example 3 The prepared microbial preparation of Enterococcus pecaris is mixed in the same amount to obtain a mixed preparation. In this case the number of bacteria contained in the formulation, galacto the Bacillus clearance 2.5 × 10 ⁸ gae / g and Enterococcus page Charis 2.5 × 10 ⁸ gae / g but the ratio of the Lactobacillus clearance formulation and Enterococcus page Charis agents in the production process It is also possible to manufacture the mixed formulation containing arbitrary bacterial numbers by changing. In addition, the formulation of each formulation can be suitably selected from conventional formulations such as powder form, granule form, capsule, etc. with appropriate excipients.

Example 1

15 ml of a 10-fold dilution of feces is placed in a test tube (18 × 180 mm), ① autoclaved at 120 ° C for 15 minutes, ② not sterilized, and each sample is inoculated with test bacteria and sealed with a rubber stopper. After anaerobic incubation at 37 ° C. for 72 hours, 1 ml of air in the test tube was aspirated with a syringe and injected into a 5 L odor bag. An olfactory sensory test was performed by six evaluators. The degree of odor is based on the evaluation criteria of feces odor shown in Table 1. In the case of Enterococcus pecharis microbial preparation, the preparation prepared in Preparation Example 3 is added to a 0.1 g test tube. As shown in Table 23, the stool smell is remarkably weak and the effect is remarkable especially when autoclaving the diluent of feces. This is evident compared to the case of inoculation with Lactobacillus clearance shown in Table 2 alone.

Example 2

Lactobacillus clearance FERM P-17150 in 100 ml of yogurt and enterococcus pecharis (FERM P-) prepared in a medium consisting of 100 g of skim milk, 50 g of sucrose and 2 g of agar. 17151 microorganisms) 1 g of live cell preparation or 1 g of enterococcus pecharis (FERM P-17151) microbial preparations were used in combination with daily administration to 10 people, followed by 20-30 days, 50-60 days, 80 Measure the smell of feces discharged daily for ˜90 days. In the measurement method, 0.5 g of each of the 10 excreted feces ingested were put in a 20 L odor bag and left at room temperature to determine the odor by six evaluators after 30 minutes. The stool odor mean value of five people who did not administer here is 100. The average value of the stool, as apparent from Table 24, was oral administration, and the smell of feces became weaker as the days passed, and the smell after 90 days was considerably weaker, and there was no discomfort. This remarkable effect is apparent when compared to the case of administering the Lactobacillus clearance shown in Table 3 alone.

Example 3

As in Example 1, each of the 10-fold dilutions of feces and high-pressure sterilization at ①120 ° C. for 15 minutes and ② not sterilized were inoculated with test bacteria, sealed with a rubber stopper, and stored at 37 ° C. for 72 hours. After cultivating miraculously, the solution was centrifuged, and 5 ml of the liquid floating on the surface of the liquid was collected, diluted 10-fold, and free sulfur ion (S ⁺² ) and ammonium ion contained in 50 ml of liquid. Measure the concentration of (NH ₄ ⁺ ). In addition, the concentration of lower fatty acids is measured by gas chromatography. These measurement results are shown in Tables 25 and 26. As can be seen from these tables, the reduction rate of the representative substances contained in the feces eventually shows a high rate of 70 to 90% regardless of sterilization. This high reduction rate is evident compared to the case of inoculation with Lactobacillus clearance shown in Tables 6 and 7 alone.

Example 4

1 g of lactobacillus clearance preparations (5 × 10 ⁸ / g) containing three equal strains of Lactobacillus clearance, namely, FERM P-17148, FERM P-17149 and FERM P-17150 ) And 1 g (5 × 10 ⁸ cells / g) of enterococcus pecaris by Enterococcus pecharis or microbial preparation of Enterococcus pecaris by Enterococcus pecaris FERM P-17152 1g (5 × 10 ⁸ dogs / g) was administered daily to 20 healthy and 20 ill persons daily for 6 months to track and examine bacterial phase in the intestine over time. And Table 28. As can be seen from these tables, when lactobacillus clearance is mixed with oral administration of live or dead strain of Enterococcus pecaris, compared with the case of single administration of lactobacillus clearance as shown in Tables 8 and 9 Beneficial bacteria increase rapidly, while harmful bacteria decrease. In the case of healthy people, the improvement rate is increased by about 25%, the number of beneficial bacteria is increased by 10-50%, and the number of bad bacteria is reduced by 20-40%. By increasing the percentage of beneficial bacteria is 10-30%, and the number of harmful bacteria is reduced by 20-50%, it is said that the weaker the improvement in the intestinal bacteria, the better the efficiency.

Example 5

Lactobacillus clearance, Escherichia coli O-157, Salmonella enteritidis or Shigella flexneri were mixed and cultured, and then enterococcus pecaris live or dead cells were added. Anaerobic incubation at 37 ° C, repeated passages every 72 hours, diluted and applied each time, followed by the extinction and growth of bacteria, E. coli O-157, Salmonella enteritidis and Shigera flex The ratio of the SR variation of Neri (erypibacteria) was observed and the results are shown in Tables 29, 30, 31, 32, 33 and 34. Lactobacillus clearance uses FERM P-17150 strains, and Enterococcus pecharis uses FERM P-17152 strains. In addition, the microorganism of Enterococcus pecaris adds 1 g of the microbial agent to 1 L of the medium. The medium used for the culture was autoclaved at 120 ° C. for 15 minutes, containing 10 g of meat extract, 10 g of peptone, 2 g of glucose, 2 g of NaCl, and 1 g of CaCO _{3 in} 1 L. As can be seen from these tables, when inoculated live organisms of Lactobacillus clearance and Enterococcus pecharis to pathogens such as Escherichia coli O-157, Salmonella enteritidis and Shigera flexneri, the passage is repeated. Therefore, each pathogen is rapidly transformed to R-type while reducing the number of bacteria. Escherichia coli O-157 is 100% mutated to R-type at the 13th generation and the bacterium is extinguished at the 15th generation. Salmonella enteritidis is in the thirteenth generation, and Shigera flexneri (lye bacteria) are in the fifteenth generation, and the germ disappears before 100% mutation. Lactobacillus case of using Bacillus clearance alone Table 11, Table 13 and each of the pathogens as shown in Table 15 is shifting to the R type according to the number of passages, rather than pointers' re bacteria is destroyed always 1~3 × 10 ⁹ gae The number of bacteria is maintained at / ml. In addition, the addition of Lactobacillus clearance and Enterococcus pekaris to the pathogen showed a similar tendency to the inoculation of live cells, but the transition to R-type was slightly slower than the inoculation of live cells. Is reduced but not destroyed. Nevertheless, 100% of R-type subtypes were E. coli O-157 in the 15s, Salmonella enteritidis in the 30s, and Shigera flexneri (15) in the 15s, and in any case, inoculated with Lactobacillus clearance alone. Compared to the case is achieved significantly faster.

Example 6

Lactobacillus clearance FERM P-17148 strain or Lactobacillus clearance FERM P-17149 strain produced by the preparation method of Preparation Example 1 Probiotic, Enterococcus pecaris FERM P-17151 strain prepared by the preparation method of Preparation Example 2, or Enterococcus pecaris FERM P-17152 strain Probiotic preparation, Enterococcus pecaris FERM P-17152 strain or Enterococcus pecaris FERM P-17152 strain were added to feed. Five rats are grouped and administered to 10 week old male mice. The content of Lactobacillus clearance in 1 g of feed is 5 × 10 ⁸ , and the content of Enterococcus pecharis is 5 × 10 ⁸ (2.5 × 10 ⁸ when using live and dead organisms respectively). The triglyceride (cholesterol) and cholesterol (triglyceride) and the cholesterol (cholesterol) in the serum was raised for 3 months, and compared with that of the control mice (사 照 통상). In addition, triglyceride was measured by triglyceride E-test table and cholesterol was measured by triglyceride C-test table, and the average value of each group was shown in Table 35 with the average value of the control mice as 100%. As is apparent from Tables 35 and 21, the effects of adding the live, dead, live and dead cells of Enterococcus pecaris having a lowering effect of triglycerides and cholesterol on Lactobacillus clearance were determined by Enterococcus pecaris alone. It is significantly improved compared to the case of administration. Moreover, the difference with the case where it was administered alone shows that the use of a dead microbe more than a living microbe shows a higher effect. It is also reduced to about 1/2 level compared to the control.

Example 7

35 8-week-old rats naturally developing high blood pressure were divided into 7 groups, and the bacteria were used in Example 6 and added to the feed for 7 months. The blood pressure before and after the administration of the control group and the various cell preparations is measured and shown in Table 36. As apparent from Tables 36 and 22, blood pressure after 3 months improved on average by 15%. As in Example 6, in the enterococcus pecaris, the dead cells act more effectively than the living cells. Compared with the administration of Enterococcus pecharis alone, the blood pressure lowering effect is improved by about 10 to 20%.

Table 37 shows the effects of the Lactobacillus clearance, Enterococcus pecharis, and both used in the present invention in terms of their respective functions.

As human society matures, the way in which health concerns are also gradually thought is changing. After the age when the response to acute disease and chronic disease is of paramount importance, the aging society is now faced. Now, as a preventive medicine, it is also called nutrition therapy. In order to prevent illness from happening and to prevent disease in advance, it is becoming mainstream to think that it is to eat nutrition evenly and make one's own health. In other words, if disease can be prevented, no treatment is necessary, so it is no exaggeration to say that this is really the preoccupation of medical care in the 21st century.

Lactobacillus preparations of the present invention by oral administration (經 口 投 與), the beneficial bacteria in the intestine gradually expand the forces to protect the gut membrane (하고) and produce vitamins to synthesize amino acids (劑) led by lactic acid bacteria to inhibit the proliferation of foreign bacteria and foreign pathogens to weaken the toxicity and also contribute to the activation of immune function. Therefore, the harmful bacteria in the intestines are significantly reduced and the production of decaying substances is suppressed, so the smell of discharged feces is significantly reduced. Furthermore, by accelerating it, the lactic acid bacteria of the present invention actively eat the decayed substances in the intestine, thereby improving the intestinal environment cleanly and contributing to the normalization of the blood vessels and nerves of the mesenteric membrane and surroundings.

If the intestines, which are the source of human vitality, energy, blood and body, are purified, the essential substances of the living body are not wasted and the toxic substances absorbed from the intestines are reduced, which inevitably improves liver function. Triglycerides and cholesterol are lowered to cleanse the blood. The activated blood lowers blood pressure, and vital substances such as hormones, enzymes, antibodies, and immune substances are not blocked in every corner of the body, so metabolism is smoothed, and the function of the whole body is improved. Is activated. In other words, if you clean the contaminated intestine and return it to a clean intestine, it means that the whole body improves and you can enjoy a disease-free life completely. It can be said that Mechinkov's suggestion of Boulognace was implemented as a rain after a century.

In addition, the lactic acid bacterium preparation of the present invention can be stored as it is, and can be easily used anywhere and anytime, while maintaining its titer over a long period of two to three years, so its value can be measured. It is not enough. In addition, in the aging society, the elderly who can't lie down in proportion to the aging population have soared, and the treatment of the excreta is a problem, but when the smell is reduced, the disgust is lost, so it is good news for those who are taking care of the elderly.

Claims (3)

A lactic acid bacterium preparation comprising a live cell of Lactobacillus clearance and a live cell of Enterococcus pecharis capable of lowering at least one or two types of triglycerides and cholesterol. A lactic acid bacterium preparation comprising a live cell of Lactobacillus clearance, and a bacterium of Enterococcus pecharis capable of lowering at least one or two types of triglycerides and cholesterol. A lactic acid bacterium preparation comprising a live cell of Lactobacillus clearance, and a live cell and a dead cell of Enterococcus pecharis capable of reducing at least one or two types of triglycerides and cholesterol.