ITRM20000469A1 - NEW PREPARATION OF LACTIC ACID BACTERIA HAVING BIOLOGICAL PURIFICATION ACTIVITIES. - Google Patents

Description

"NEW PREPARATION OF LACTIC ACID BACTERIA WITH BIOLOGICAL PURIFICATION ACTIVITIES"

FOUNDATION OF THE INVENTION

1. Field of the invention

The present invention relates to a preparation of lactic acid bacteria which is extremely significant for the health of humans and animals, in which the concomitant use of Lactobacillus clearans, which was first isolated by the inventors and which belongs to the genus Lactobacillus has still specific characteristics not found in conventionally known bacteria, and either or both viable or killed cells of Enterococcus faecalis (Enterococcus), which are deeply involved in lipid metabolism, allows the advantages of each to be revealed more clearly and effectively.

2. Description of the related technique According to the opinion, there are 300 species and 100 trillion individual bacteria in the intestines, weighing a total of 1 kg, and far exceeding the total of 60 trillion human cells. Years of study and immunological research have gradually elucidated their significant involvement in the health and various diseases of humans and animals, and experts now regard gut flora as a vital organ function. Beneficial bacteria, belonging to the group of lactic acid bacteria, such as the genus Bifidobacteriuw and the genus Lactobacillus, when predominant, coexist in a balanced way. This balance can be disrupted by a variety of factors, such as variations in the quantity and quality of the daily diet, overstrain, lack of sleep and mental stress, resulting in the proliferation of harmful bacteria such as Welch bacillus and Veillonella. The expulsion of beneficial bacteria results in increased production of harmful substances, which can lead to a variety of diseases and general accelerated aging, from stool disorders such as diarrhea and constipation, to those resulting from blood poisoning, such as chronic fatigue. , cracked skin, liver dysfunction, hypertension and arteriosclerosis.

The Russian Metchnikoff (1845 until 1916) posited as a theory that the primary cause of aging was poisoning by toxins formed by putrescent intestinal fermentation and advocated as a remedy the habit of drinking beverages with lactic acid bacteria such as yogurt to help to prevent aging.

Everywhere extravagances of the history of lactic acid bacteria until it was possible to grasp the real practical usefulness of such bacteria. This is because the matters now revealed by immunological studies and employed by experiments could not be clarified.

Metchnikoff's hypothesis has been confirmed and is now common knowledge in the health sciences, as recent advances in microbiology have elucidated a variety of important functions by beneficial intestinal bacteria, such as reducing intestinal acidity and promoting mobility. intestinal to aid in the digestion of food and in the absorption of nutrients, the simultaneous suppression and degradation of harmful substances, the synthesis of vitamins and amino acids, the protection against intestinal infections by pathogens and the increased immunological power.

In addition, along with recent health trends, lactic acid bacteria have established a firm hold as drinks and antiflatulents for many people. It cannot be denied however, that such products are not attractive to people who are seeking positive results, having failed to achieve better health and improved symptoms through ingestion or daily use of lactic acid bacteria. Unfortunately, lactic acid bacteria products are still consumed rather as a refreshing drink, or they still tend to be regarded as a coffee-like preference.

In view of the foregoing, there was a need for a product that could be rapidly administered to bring about improved effects not available in conventional bacterial lactic acid preparations or beverages. Although the inventors had previously found that the new indicated species Lactobacillus clearans was highly effective for health purposes, they particularly noted that this species lacked sufficient intestinal cleansing action. Efforts to produce a higher potency and similar strain to address this disadvantage led to the inevitable conclusion that using the bacterium alone produced limited results. The concomitant use of another active substance was thus considered in the search for a preparation of lactic acid bacteria capable of strengthening healthy individuals, and of renewing a sense of well-being and restored health in semi-ill individuals or individuals. semi-healthy.

SUMMARY OF THE INVENTION

As a result of extensive research to find a compatible biological or non-biological participant to address the disadvantages mentioned above, the inventors have perfected the present invention with the discovery, among Enterococcus faecalis belonging to the same group of lactobacilli as Lactobacillus clearans, a capable species to reduce at least one of triglycerides and cholesterol. That is, the present invention is a preparation of lactic acid bacteria which comprises viable cells of Lactobacillus clearans, and viable cells of Enterococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol.

The second of the inventions is a preparation of lactic acid bacteria which includes viable cells of Lactobacillus clearans, and killed cells of Enterococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol.

The third of the inventions is a preparation of lactic acid bacteria which includes viable cells of Lactobacillus clearans, and viable and killed cells of Enterococcus faecalis capable of reducing one or more of at least triglycerides and cholesterol.

DESCRIPTION OF THE PREFERRED REALIZATIONS TF.

Lactobacillus clearans reported in the present invention is a new strain belonging to the genus Lactobacillus and has the following biochemical characteristics 1, 2, 3 and 4. Specifically, it includes strains of the genus Lactobacillus that 1) are able to reduce both Na2S .9H20 and NH4OH when 0.5 grams of Na2S.9H20 and / or 0.5 mL of NH4OH is or is added to 5 g of meat extract, 5 g of peptone, 1 g of glucose, 1 g of CaCO3, and 1 1 of water {neutral pH); 2) show no growth promoting action despite the addition of 0.5 g of Na2S.9H20 and / or 0.5 mL of NH40H during the logarithmic growth phase of bacterial culture in medium containing 1 g of acid casamino and vitamins (A: 900 IU; Bi: 1 mg; B2: 1 mg; B6: 1 mg; Bì2: 5 γ; nicotinamide: 16 mg; calcium pantothenate: 8 mg; C: 64 mg; and D2: 120 IU) in Stephenson-Wetham medium {(abbreviated as S-W) 1 g of KH2P04, 0.7 g of MgSo4.7H20, 1 g of NaCl, 4 g of (NH4) 2HP04, 0.03 g of FeS04.7H20, and 5 g glucose); 3) they have resistance, in the form of naturally isolated strains, to Na2S.9H20 greater than that of conventionally known lactic acid bacteria, but weaker than that of Lactobacillus deodorans; and 4) are gram positive, rod-shaped, non-motile and negative catalase, they are not nitrate reducing agents and they do not degrade gelatin, they do not produce indole or hydrogen sulfide, they have a high capacity to form lactic acid from glucose and lactose , and have growth favored by the addition of acetic acid (see Japanese Patent Publication (Kokoku) H4-632.

The advantages of Lactobacillus clearans, in addition to the action of common Lactobacillus, include powerful intestinal cleansing action absent in conventional Lactobacillus as described in Japanese patent 1714413 and in Japanese patent application 11-15177, such as: 1) decomposition of malodorous intestinal substances , putrefying; 2) increase of beneficial bacteria such as Bifidobacterium and Lactobacillus among the intestinal flora, with very strong reductions of harmful bacteria such as Welch bacillus and Veillonella, thereby providing a better balance in the intestinal flora to improve intestinal conditions; and 3) suppression of growth primarily of etiological agents that cause intestinal infections, and decreased toxicity.

This action will be described below by means of evidence. In vitro tests were first conducted. 15 mL of a 10-fold dilution of stool was introduced into the tubes (18 X 180 mm). The tubes were then inoculated with sample bacteria 1) which were sterilized under high pressure for 15 minutes at 120 ° C and 2) which were not sterilized. The tubes were capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. 1 ml of air was then extracted by syringe from inside the test tubes and injected into 5 liters of odor bags for olfactory tests by a group of 6 individuals. Odor was assessed based on the stool odor criteria given in Table 1.

Table 1: Stool odor criteria

Three typical strains of Lactobacillus clearans, specifically, FERM BP-6972, FERM BP-6971, FERM BP-6973, were used to test the deodorant capacity in the in vitro tests mentioned above. The results in Table 2 show that the high pressure sterilized samples were clearly deodorized, with an odor known to be the faint or mild odor of stool. The unsterilized samples on the other hand, showed less deodorization than the high pressure sterilized samples, because foul-smelling substances had been produced, possibly as a result of the activity of numerous putrefying bacteria in the stool, but still they were not yet clearly deodorized. results of the three typical Lactobacillus clearans strains in the above-mentioned test were reproduced similarly to other similar strains.

Table 2: Deodorant capacity of Lactobacillus clearans (in vitro)

In subsequent in vivo tests, 150 mL of yogurt prepared using Lactobacillus clearans FERM BP-6973 was ingested once daily and stool odor was determined at 20 to 30 days, 50 to 60 days and 80 to 80 days. 90 days post ingestion, with the averages given in Table 3. Results were determined by introducing 0.5 g of stool samples from 10 individuals into 20 1 odor bags which were stored at room temperature for evaluation. odor by a group of 6 individuals after 30 minutes. The average stool odor for 5 individuals who did not ingest yogurt was rated as 100. Although stool odor varied considerably depending on the diet content, the average odor for 10 individuals who ingested yogurt decreased to approximately 50% a month after ingestion, 70% after two months and 85% after three months. After three months, however, continued ingestion resulted in a peak odor reduction of 80 to 90%.

Table 3: Stool odor after ingestion of yogurt prepared using Lactobacillus clearans

In terms of the correlation between purification and bacteria in the natural world, the inventors reduced the analyzed substances into easily testable foul-smelling substances, which were broadly classified into sulfur compounds, nitrogen compounds and carbon compounds. Bacteria capable of degrading foul-smelling sulfur compounds such as Na2S.9H20, foul-smelling nitrogen compounds such as NH3, and foul-smelling carbon compounds such as acetic acid, butyric acid and similar lower fatty acids were found to be able to degrade most of the malodorous compounds of sulfur, nitrogen and carbon, of polymers from these, this being referred to as the CNS theory. The following tests were conducted according to this theory.

0.5 g of Na2S.9H20 or 0.5 mL of ammonia water (NH40H) was added to one mL of synthetic medium containing 5 g of meat extract, 5 g of peptone, 3 g of sodium butyrate, 5 g of glucose and 3 g of CaCo3, the medium was inoculated with Lactobacillus clearans for 72 hours of anaerobic culture at 37 ° C, and the decrease in the added Na2S.9H20 or NH40H was determined over time. Na2S.9H20 was measured by the iodine titration method of JIS K0102-1985, and the NH4OH was measured by the phenol blue indole absorption method of JIS K0102-1985. The results are given in Tables 4 and 5. The tables show that Lactobacillus clearans has the ability to reduce toxic foul-smelling Na2S.9H20 and NH4OH by 40 to 50% in 72 hours. This means that it can degrade and assimilate most of the other toxic foul-smelling substances.

Table 4: degradation and assimilation of sodium disulfide by Lactobacillus clearans

Table 5: degradation and assimilation of ammonia by Lactobacillus clearans

The above in vitro sensory tests and chemical analysis have been described as methods to ascertain the deodorising action of Lactobacillus clearans. The 10-fold dilutions of the stool samples prepared at this time were centrifuged before culture and 72 hours after culture, collected 5 mL of supernatant and diluted 10 times to determine the S2 + and NH4 + ion concentrations, and it was calculated the extent of their decrease. The results in Table 6 show that the decrease in S2 + and NH4 + ions of the stool was even greater than the decrease in the synthetic medium mentioned above, indicating that the stool was a more suitable habitat for Lactobacillus clearans. S2 + ions were determined by JIS K0102-1985's iodine titration method, and NH4 + ions were determined by JIS K0102-1985's phenol blue indole absorption method.

Table 6: decrease in S2 + and NH4 + by Lactobacillus clearans

2 μΐι of the aforementioned supernatant were introduced into a gas chromatograph to analyze the lower fatty acids. Gas chromatography involved the use of Col. Unisole F-200 30/60 glass {30 x 3 m) at a carrier gas regime of 50 mL / min (He), with 152 kPa (0.55 kg / cm2G ) of hydrogen and 152 kPa (0.55 kg / cm2G) of air, at a column temperature of 140 ° C and injection at 200 ° C. The substances tested were acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, nvaleric acid, with the concentrations calculated in table 7. Table 7 shows that the lower fatty acid concentrations decreased by 50 to 75% with high pressure sterilized samples, and 40 to 60% with unsterilized samples.

Table 7: decrease of lower fatty acids by Lactobacillus clearans

Lyophilized cells of three typical Lactobacillus clearans strains, namely FERM BP-6972, 6971 and 6973, were prepared and mixed in equal parts to form a preparation. 2 g of the preparation (5 x 10 8 cells / g) were taken continuously and changes in cell counts (cells / l g of faeces) of Bifidobacterium and Lactobacillus (with the exception of Lactobacillus clearans) were measured over time, which were known as typical beneficial bacteria in the intestinal flora, as well as typical harmful bacteria such as Veillonella and Clostridium perfrìngers (Welch bacillus). Table 8 shows the results of oral administration to 20 healthy individuals, and Table 9 shows the results of oral administration to 20 weakly built individuals.

Tables 8 and 9 show that prior to administration of Lactobacillus clearans preparations, beneficial bacteria in the gut flora were on average 220% greater in healthy individuals than in constitutionally weak individuals, while harmful bacteria were only about 22.5 %. This shows that the state of the gut flora is an index of the current state of health, that is, how deeply the gut flora is involved in health. A new finding was that ingestion of the preparation resulted primarily in an increase in beneficial bacteria in healthy individuals, and that with that increase, there was a gradual decrease in harmful bacteria. In contrast to this configuration, the pattern found in weakly built individuals was that harmful bacteria decreased first, followed by a gradual increase in beneficial bacteria. Generally, the mean changes in beneficial and harmful bacteria reflected an increase in beneficial bacteria and a corresponding decrease in harmful bacteria. This trend accelerated after three months, with Bifidobacterium increasing 50% after 6 months and 110% after one year, and with Lactobacillus increasing to 300% after 6 months and 950% after one year. In contrast, Clostridium decreased by 70% after 6 months and by 85% after one year, while Veillonella decreased by 67.5% after 6 months and 82.5% after one year.

Escherichia coli 0-157, Salmonella enteri tidis, and Shigella flexnerì were grown alone or together with Lactobacillus clearans (FERM BP-6973) to test the action of Lactobacillus clearans against pathogens, and the results were compared. The composition of the medium used for the cultures included 10 g of meat extract, 10 g of peptone, 2 g of glucose, 2 g of NaCl, and 1 g of CaCo3 per liter, with the pH adjusted to 7.2. Anaerobic culture was performed at 37 ° C, with repeated subcultures every 72 hours, at which time the plates were diluted and greased to monitor changes in cell counts, whether colonies were S (original) or colonies were been mutated to type R with reduced toxicity, their ratio, and the like. The pathogens used in the tests were purchased from KK Medium (registered health research institute).

Table 10 shows the results of E. Coli 0-157 cultured by itself, and Table 11 shows the results of mixed culture of E. Coli 0-157 and Lactobacillus clearans. Table 10 shows that in cultures of E. Coli 0-157 alone, the cell count was virtually a constant 4 to 5 X 10 9 cells / g for all 20 subcultures, without highlighting the R types. 11 shows that in mixed cultures of E. Coli 0-157 and Lactobacillus clearans, there was a small variation in the cell count of Lactobacillus clearans, but that there were significant changes in the cell count of E. Coli 0-157. R types were seen up to the fifth subculture, and the ratio of R types increased with the accumulation of subsequent subcultures, until all cells formed R types from the eighteenth subculture. There was no inversion to type S in subsequently continued subcultures.

Table 10: Results of cultures of E. Coli 0-157 per se

Table II: results of mixed cultures of E. Coli 0-175 and Lactobacillus clearans

Table 12 shows the results obtained with Salmonella enteritidis grown on its own, and Table 13 shows the results obtained with mixed cultures of Salmonella enteritidis and Lactobacillus clearans. Table 12 shows that in cultures of Salmonella enteritidis alone, the cell count was 3 to 5 X 10 9 cells / g. R types showed spontaneously in the 14th subculture, reaching a ratio of 5% from the 20th subculture. The ratio increased in subsequent subcultures, peaking at 23% from the fiftieth subculture. The ratio of the R types after that was about 20%. Table 13 shows that in mixed cultures of Salmonella enteritidis and Lactobacillus clearans, the ratio of R types was 29% in the fifth subculture and that the ratio of R types increased with the accumulation of subsequent subcultures, reaching 50% in the 10th subculture, 90% in the twentieth subculture and 100% in the forty-seventh subculture. S-type inversion in subsequent subcultures was less than 1%. After the 70th subculture all S types disappeared, without further inversion.

Table 12: Results of Salmonella enteri tidis cultures per se

Table 13: Results of mixed cultures of Salmonella enteri tidis and Lactobacillus clearans

Table 14 shows the results of Shigella flexneri grown on its own, and Table 15 shows the results of mixed culture of Shigella flexneri and Lactohacillus clearans. Table 14 shows that in the culture of Shigella flexneri alone, the R types show spontaneously in the 10th subculture, reaching a ratio of 9% from the 20th subculture. The ratio increased in subsequent subcultures, peaking at 20% from the 40th subculture. The ratio of the R types after that was about 10 to 20%. Table 15 shows that in the mixed cultures of Shigella flexneri and Lactohacillus clearans, the R types showed up in the fifth subculture and that the ratio of the R types increased with the accumulation of subsequent subcultures, reaching 100% in the 80th subculture. There was S-type inversion in subsequent subcultures, but less than 1%. After the 180th subculture, all S types disappeared without further inversion.

Table 14: Culture results of Shigella flexneri per se

Table 15: Results of mixed cultures of Shigella flexneri and Lactobacillus clearans

Groups of 10 8-week-old male mice were intraperitonally administered 1 X 10 8 cells per animal in order to investigate the toxicity of types S and R of E. Coli 0-157, Salmonella enteritidis, and Shigella flexneri. Preserved strains were used for S types and strains that were mutated to 100% R types by Lactobacillus clearans were used at this point for R types. The results in Table 16 showed that all animals died within four days with S-type pathogens, while none died with R-types with the exception of Shigella flexneri which resulted in death from day 7.

Table 16: Toxicity of types S and R of E. Coli 0-157, Salmonella enteri tidis and Shigella flexneri in mice

The yogurt prepared with Lactobacillus clearans described above was administered orally to humans and blood was analyzed once a month to study changes in intestinal flora. Cholesterol and triglyceride levels decreased by approximately 10% in more than 80% of individuals compared with prior to administration.

Table 17 shows the differences in function between the Lactobacillus clearans of the present invention and conventional Lactobacillus strains.

Table 17: Comparison of functions between Lactobacillus clearans and conventional Lactobacillus strains

Enterococcus faecalis is a group of bacteria that constitutes the intestinal flora and belongs to the group of lactic acid bacteria, which are normally present in a quantity of about 1 X 10 7 cells per gram of stool. These appear in the form of two spherical or ovoid shapes, or in the form of short chains. They are gram positive cocci with potent resistance to heat, gallic acid chlorine drying and the like, and grow in a wider temperature range of 10 to 45 ° C compared to common streptococci. Although there are pathogenic strains, they are non-toxic. The viable cells act as antiflatulents, and have been commercially available for more than 10 years in Japan. They have been shown to be non-toxic when taken by mouth. Apart from such commercial products, viable and killed cells of some strains have recently been shown to effectively lower blood cholesterol and triglycerides. Thus they promised the prevention or treatment of diseases typically related to adults (diseases that originate from lifestyle habits), such as hyperlipidemia, hypertension and arteriosclerosis. Enterococcus faecalis with reference to the present invention indicates the strains that have such functions.

The following is an example of a method for preparing viable Enterococcus faecalis cells for use in the present invention. Specifically, cells that have been cultured and centrifuged by an ordinary method are suspended in physiological saline, washed, centrifuged again and harvested. They can be freeze-dried using soluble starch as a preservative. As an example of a method of preparing killed Enterococcus faecalis cells, soluble starch and the washed and centrifuged cells obtained when the above-mentioned viable cells have been collected, can be treated for 30 minutes in very hot water at 100 ° C and then freeze-dried.

Although Enterococcus faecalis has no in vitro ability to reduce sodium sulfide (Na2S.9H20) or ammonia, it has a weak deodorizing ability against 10-fold dilution of stool. Table 18 gives the deodorizing capacity of two typical Enterococcus faecalis strains, namely FERM BP-7230 and FERM BP-7231. The test was the same as that for Lactobacillus clearans, and the criteria were the same as those given in Table 1.

Table 18: Deodorant capacity of Enterococcus faecalis (in vitro)

The two typical strains of Enterococcus faecalis were administered orally in quantities of 1 X 10 9 cells / person per day. Stool odor before administration was rated as 100. Stool odor was determined for 20 to 30 days, 50 to 60 days, and 80 to 90 days after ingestion, with results given in Table 19 Table 19 shows that odor was reduced by Enterococcus faecalis, albeit weakly.

Table 19: Stool odor during the ingestion of Enterococcus faecalis

Viable and killed cells of two typical strains of Enterococcus faecalis, namely, FERM BP-7230 and BP-7231, were prepared and mixed in equal parts of viable cells and killed to produce a preparation. The preparation was taken in an amount of 1 X 10 9 cells / day / person for six months, and changes in intestinal flora cell count were determined over time by measuring changes in cell count (cells / l gram of stool ) of Bifidobacterium and Lactobacillus (with the exception of Lactobacillus clearans) which are known as typical beneficial bacteria, as well as typical harmful bacteria such as Veillonella and Clostridium perfringens. Table 20 shows the results of oral administration to 10 healthy individuals. Table 20 shows that viable cells had a slightly higher improvement regime than killed cells. However, the results were far lower than those of Lactobacillus clearans.

Table 20: Effect of oral administration of the Enterococcus faecalis preparation on intestinal flora in healthy individuals

Mixed cultures of Escherichia Coli 0-157, Salmonella enteri tidis and Shigella fi exneri were carried out to study the action of Enterococcus faecalis against pathogens. The relationships in which pathogens mutate from S to R types were virtually the same as the relationships that occur spontaneously. Pathogenic cell counts gradually decreased with each subculture, until Salmonella enteri tidis disappeared from the 25th subculture, Shigella flexneri disappeared from the 33rd subculture E. Coli 0-157 disappeared from the 35th subculture. Although this suggested that the growth of Enterococcus f secali s outweighed the pathogens, the potential production of some physiologically active substances that suppressed the growth of pathogens could not be ruled out.

The above-mentioned preparations of viable and killed cells of Enterococcus faecalis were added to the feed in quantities of 1 X 10 9 cells / l feed gram each. Mice raised for three months on this feed and mice raised on conventional feed alone were compared for triglycerides and serum cholesterol. The mice were divided into groups of 10 allowing for free feeding. Triglycerides and cholesterol were measured by means of an E-Test Wako and C-Test Wako respectively. The mean level for the reference mice was 100%. The results are given in Table 21. Table 21 shows that mice that continuously ingested either viable or killed cells had a 20 to 40% reduction in the levels of both parameters, leaving no room for doubt about their efficacy.

Table 21: Effect of oral administration of Enterococcus faecalis preparation on serum triglyceride and cholesterol levels

Fifteen 8-week spontaneously hypertensive (SHR) rats were divided into three groups. The groups were fed feed containing viable and killed cell preparations of Enterococcus faecalis in quantities of 1 X 10 9 cells / gram of feed. No cell preparation was administered to the reference group. The animals were kept for three months. Table 22 gives the blood pressure after three months. Table 22 shows an approximately 10% reduction in blood pressure.

Table 22: Effects of oral administration of the Enterococcus faecalis preparation on rat blood pressure

EXAMPLES

The combined action of Lactobacillus clearans and Enterococcus faecalis surprisingly allowed effective results to be obtained quickly. The manufacturing methods and examples of the preparations in the present invention are described below, but the scope of the present invention is not limited by these manufacturing examples and processing examples.

(Manufacturing example 1)

Production of the preparation of Lactobacillus clearans: 10 liters of medium containing 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 and 2 g of yeast extract per liter of medium, was inoculated with Lactobacillus clearans for 72 hours of anaerobic culture at 37 ° C. The resulting culture was centrifuged, giving 10 g of biomass. This was washed with 500 mL of physiological saline and centrifuged 2 times. The washed biomass was introduced into 500 mL of solution containing 50 g of skimmed milk, 30 g of trehalose, and 0.5 g of taurine, and was completely stirred. The mixture was lyophilized by a common method, giving 82.5 g of cell preparation (3 X 10 9 cells / gram). This was mixed with 330 g of fully dried skim milk to give a preparation of Lactobacillus clearans containing 5 X 10 8 viable cells per gram.

(Manufacturing example 2)

Production of preparation of viable cells of Enterococcus faecalìs: 10 liters of medium containing 5 g of meat extract, 5 g of peptone, 2 grams of sodium chloride, 2 g of yeast extract and 10 g of glucose per liter of medium, they were inoculated with Enterococcus faecalìs for 72 hours of aerobic culture at 37 ° C. The resulting culture was centrifuged, giving 16 g of biomass. This was washed with 800 mL of physiological saline and centrifuged twice. The washed biomass was introduced into 500 mL of solution containing 20 g of skimmed milk, 30 g of soluble starch and 0.5 g of sodium glutamate, and was stirred thoroughly. The mixture was lyophilized by a common method, giving 54 g of cell preparation (5 X 10 9 cells / gram). This was mixed with 486 g of completely dried soluble starch, giving an Enterococcus faecalis viable cell preparation containing 5 X 108 viable cells per gram.

(Manufacturing example 3)

Production of preparation of killed cells of Enterococcus faecalis: the washed biomass obtained in manufacturing example 2 was suspended in 500 mL of physiological saline solution, 50 g of soluble starch was then introduced, the solution was thermally sterilized for 15 minutes at 110 ° C and the suspension was lyophilized by means of a common method, giving 53 g of cell preparation (approximately 5 X 10 9 cells / gram). Preparations of killed cells can also be produced by disrupting the cells with ultrasound or the like.

(Manufacturing example 4)

Production of preparation of mixture of Lactobacillus clearans and Enterococcus faecalis: the preparation of Lactobacillus clearans produced in manufacturing example 1 and the preparation of viable cells of Enterococcus faecalis produced in manufacturing example 2 or the preparation of killed cells of Enterococcus faecalis produced in manufacturing example 3 they were mixed in equal parts to produce a mixed preparation. The preparation in this case contained Lactobacillus clearans in an amount of 2.5 X IO8 cells / g and Enterococcus faecalis in an amount of 2.5 X IO8 cells / g, but the ratio between the preparations of Lactobacillus clearans and Enterococcus faecalis can be varied during the manufacturing process to produce mixtures containing any desired cell count. The preparation can be in the form of powders, granules, capsules or other common formulations with a suitable excipient.

(Example 1)

15 mL of a 10-fold dilution of stool was introduced into tubes (18 X 180 mm). The tubes were then inoculated with sample bacteria 1) which were sterilized under high pressure for 15 minutes at 120 ° C, and 2) which were not sterilized. The tubes were capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. 1 mL of air was then extracted by syringe from inside the tubes and injected into 5 liter odor bags for olfactory testing by a group of six individuals. The odor was ascertained on the basis of the stool odor criteria given in Table 1. For preparations of killed cells of Enterococcus faecalis, 0.1 g of the preparation produced in Manufacturing Example 3 was added to the tubes. The test results in Table 23 show that stool odor was considerably reduced particularly in the case of stool diluent treated with high pressure sterilized samples. This will be evident in a comparison with the results for Lactobacillus clearans inoculated test fillers in Table 2 only.

Table 23: Deodorization test with a mixture of Lactobacillus clearans and Enterococcus faecalis (1)

(Example 2)

A combination of 1 g of Enterococcus faecalis viable cell preparation (FERM BP-7230) or 1 g of Enterococcus faecalis killed cell preparation (FERM BP-7230) and 100 mL of yogurt prepared with Lactobacillus clearans FERM BP-6973 in medium containing 100 g of skimmed milk, 50 g of sucrose and 2 g of agar per liter was continuously administered to 10 individuals, and stool odor was continuously determined at 20 to 30 days, 50 to 60 days and 80 to 90 days after administration. The results were determined by introducing 0.5 g of stool samples from 10 individuals into 20 liter odor bags, which were stored at room temperature for odor assessment by a group of 6 individuals after 30 minutes. The mean stool odor for 5 individuals who had not ingested yogurt was rated as 100. The mean values in Table 24 show that oral administration significantly reduced stool odor over time. After 90 days, the odor had considerably diminished and was no longer unpleasant. The appreciable effects will be evident when compared with the results obtained with the administration of Lactobacillus clearans alone in Table 3.

Table 24: Deodorization test with a mixture of Lactobacillus clearans and Enterococcus faecalis (2)

(Example 3)

Similarly as in Example 1, the 10-fold diluted stool tubes were inoculated with sample bacteria 1) which had been sterilized under high pressure for 15 minutes at 120 ° C and 2) which had not been sterilized. The tubes were capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. The cultures were then centrifuged, 5 mL of supernatant was collected from each and diluted 10 times, and the concentrations of free sulfur ions (S2 +) and ammonium ions (NH4 +) were determined in the resulting 50 mL solution. The concentration of lower fatty acids was also determined by gas chromatography. Tables 25 and 26 give the measured results. The tables show that typical substances in faeces were reduced at a high rate of 70 to 90% by weight of both sterilized and unsterilized samples. The reduction regimen will be evident in a comparison with the results obtained by inoculation with Lactobacillus clearans alone in Tables 6 and 7.

(Example 4)

1 g of Lactobacillus clearans preparation (5 X 10 8 cells / g) containing a mixture of equal parts of three typical Lactobacillus clearans strains, specifically FERM BP-6972, FERM BP-6971, FERM BP-6973, and 1 g of preparation viable Enterococcus faecalis cells (5 X 10 8 cells / g) of Enterococcus faecalis FERM BP-7230 or 1 g of killed Enterococcus faecalis cell preparation (5 X 10 8 cells / g) of Enterococcus faecalis FERM BP-7231 were administered continuously for 6 months to 20 healthy individuals and 20 individuals with a weak constitution. The intestinal flora was monitored over time, with the results given in Tables 27 and 23. The tables show that oral administration of viable cells or killed cells of Enterococcus faecalis mixed with Lactobacillus clearans increased beneficial bacteria and reduced harmful bacteria faster than when Lactobacillus clearans was administered alone, as shown in Tables 8 and 9. That is, improvements were approximately 25% faster in healthy individuals, with a beneficial bacterial cell count greater than 10 to at 50% and a 20 to 40% lower harmful bacteria cell count. In individuals with weak constitution, improvements were about 30% faster, with a cell count of beneficial bacteria 10 to 30% higher and a cell count of harmful bacteria 20 to 50% lower. It can thus be concluded that the improvement in the intestinal flora was more effective in individuals with a weak constitution.

(Example 5)

Viable or killed cells of Enterococcus faecalis were added to mixed cultures of Lactobacillus clearans, E. Coli 0-157, Salmonella enteri tidis, or Shigella flexneri for anaerobic culture at 37 ° C and subcultures were repeated every 72 hours, at which time plates were diluted and greased to monitor changes in cell count and S-R mutation ratio of E. Coli 0-157, Salmonella enteritidis and Shigella flexneri. The results are given in Tables 29, 30, 31, 32, 33 and 34. Lactobacillus clearans strain FERM BP-6973 was used as was Enterococcus faecalis strain FERM BP-7231. The above-mentioned preparation of killed cells of Enterococcus faecalis was added in an amount of 1 g per liter of medium. The composition of the medium used for the cultures included 10 g of meat extract, 10 g of peptone, 2 g of glucose, 2 g of NaCl and 1 g of CaCo3 per liter, with the pH adjusted to 7.2, and was sterilized under high pressure for 15 minutes at 120 ° C. The tables below reveal that inoculation of pathogens such as E. Coli 0-157, Salmonella enteri tidis and Shigella flexneri with viable Lactobacillus clearans and Enterococcus faecalis cells rapidly reduced the cell counts of these pathogens and resulted in the their mutation to R types during subculture. E. Coli 0-157 mutated 100% to form type R from the thirteenth subculture, and disappeared from the fifteenth subculture. Salmonella enteritidis and Shigella flexnerì disappeared before the 100% mutation to form type R from the thirteenth and fifteenth subculture respectively. When Lactobacillus clearans was employed on its own, as shown in Tables 11, 13 and 15, these pathogens mutated into R types over the course of numerous subcultures, but the bacteria maintained a constant cell count of 1 to 3 X 10. cells / mL, without disappearance. Although the addition of killed cells of Lactobacillus clearans and Enterococcus faecalis to the aforementioned pathogens showed the same trends as those that prevailed with the inoculation of viable cells, the mutation to form R types was slower than that prevalent with the inoculation of viable cells, and cell counts decreased but did not disappear. Nonetheless, the number of subcultures resulting in the 100% mutation to R types was 15 with E. Coli 0-157, 30 with Salmonella enteritidis, and 15 with Shigella fiexneri, which were quite faster than when used as an inoculum. Lactobacillus clearans by itself.

Table 29: Effects of viable cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on E. Coli 0-157

Table 30: Effects of viable cells of Lactobacillus clearans (FERM BP-6973) and Enfcerococcus faecalis (FERM BP-7231) on Salmonella enteri tidis

Table 31: Effects of viable cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on Shigella fi exneri

Table 32: Effects of killed cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on E. Coli 0-157

Table 33: Effects of killed cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on Salmonella enteritidis

Table 34: Effects of killed cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on Shigella fi exneri

(Example 6)

A viable cell preparation of Lactobacillus clearans FERM BP-6972 or Lactobacillus clearans FERM BP-6972 prepared by the method in Manufacturing Example 1, a viable cell preparation of Enterococcus faecalis FERM BP-7230 or Enterococcus faecalis 7231 prepared by the method in manufacturing example 2 and a preparation of killed cells of Enterococcus faecalis FERM BP-7230 or Enterococcus faecalis 7231 prepared by the method of manufacturing example 3, were combined and added to the feed, which was administered in groups of 5 10-week-old male mice. 1 gram of feed contained 5 X 10B Lactobacillus clearans cells and 5 X 10B Enterococcus faecalis cells (2.5 X 10B cells each viable and killed when used in combination. The animals were fed freely for three months. Triglycerides and serum cholesterol were measured by comparison with levels in reference mice fed only normal food. Triglycerides were measured with an E. Wako test for triglycerides while cholesterol was measured with a C. Wako test for triglycerides . Mean levels were obtained for each group. The mean level for mice in the reference group was 100%. Results are given in Table 35. Tables 35 and 21 show that addition of viable or killed as well as viable cells and killed of Enterococcus faecalis, which have an action in lowering triglycerides and cholesterol, Lactobacillus clearans was completely more effective than when was fed Enterococcus faecalis alone. Unlike the results obtained when administered alone, the combined use of killed cells resulted in greater effects than viable cells. In comparison with the reference group, the levels were reduced to almost half.

Table 35: Effect of the mixture of Lactobacillus clearans and Enterococcus faecalis on triglycerides and cholesterol in the serum of mice

(Example 7)

7 groups containing a total of 35 8-week-old spontaneously hypertensive (SHR) mice were reared for seven months on a feed containing the cell preparations of Example 6. The blood pressure was measured before and after administration to the groups fed with each type of cell preparation and the reference group, with the results given in Table 36. Tables 36 and 22 reveal that the blood pressure had increased, on average, about 15% after three months. The killed cells of Enterococcus faecalis had a more effective action than the viable cells, in the same way as in Example 6. The drop in blood pressure was about 10 to 20%, results which were better than those obtained when it was administered. Enterococcus faecalis by itself.

Table 36: Effect of Lactobacillus clearans and Enterococcus faecalis mixture on blood pressure in rats

Table 37 shows the action in terms of various functions with the use of Lactobacillus clearans, Enterococcus faecalis, and both.

Table 37: comparison summary

*: excellent; 0: good; 0: common; A: weak; X: none

Healthcare and related endeavors have gradually varied in our society with increasing aging. Our time, in which the treatment of acute illnesses and chronic illnesses had been considered with the utmost consistency, testifies to a shift, now or in the near future, towards "preventive medicine" and also towards "nutritional therapy", in the light of our oldest company. There are no treatments that are undertaken after the attacks of the disease. Rather, attempts to create health per se through more educated food intake are now increasingly on trend in order to naturally prevent disease. It is by no means an exaggeration to suggest that disease prevention to avoid the need for treatment is under consideration for treatment in the twenty-first century.

The oral administration of the lactic acid bacteria preparation of the present invention gradually extends the influence of beneficial intestinal bacteria, firmly guards the intestinal mucosa, produces vitamins and synthesizes amino acids, all while suppressing the growth of foreign bacteria and agents. foreign pathogens, decreasing their toxicity and activating the immunological functions under the guidance of the lactic acid bacteria of the preparation. Harmful intestinal bacteria are thus markedly reduced, and the production of foul-smelling putrefactive substances is suppressed, resulting in significantly deodorized stools. As if under attack, the lactic acid bacteria of the preparation actively act on intestinal putrefactive substances, resulting in a cleaner intestinal environment and normalization of the intestinal mucosa and surrounding vessels and nerves.

Purification of the intestines, which are the source of human vitality, energy, blood and flesh, results in better absorption of vital substances and inversely a reduction in toxic substances absorbed through the intestines. This inevitably results in improved liver function, and thus the lowering of serum triglycerides and cholesterol, as well as cleaner blood. Such revitalized blood results in lower blood pressure, so that vital substances such as hormones, enzymes, antibodies and immunological substances are not prevented from being distributed throughout the entire body system. The metabolism is thus improved, and all systemic functions are strengthened. That is, the infected intestines are purified and returned to their original condition, resulting in a general improvement and a vital state of health without a trace of disease. It can be said that Metchnikoff's doctrine of longevity is now being realized a century later.

The lactic acid bacteria preparation of the present invention can be stored without any loss of titer for two to three years, allowing it to be produced in the form of bearable goods that can be used easily anywhere and at any time, and is thus of unmeasurable value. In societies with greatly increased age populations, older individuals are prone to becoming bed-ridden, although waste disposal may become a problem, deodorising such waste will make such a goal much less unpleasant, and it may apply. for assistants.

Claims (3)

CLAIMS 1. Preparation of lactic acid bacteria containing viable cells of Lactobacillus clearans and viable cells of Enfcerococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol. 2. Preparation of lactic acid bacteria containing viable cells of Lactobacillus clearans and killed cells of Enterococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol. 3. Preparation of lactic acid bacteria, containing viable cells of Lactobacillus clearans, and viable and killed cells of Enterococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol.