MXPA00008508A - Novel preparation of lactic acid bacteria having biopurification activity - Google Patents

Abstract

Even though the novel species known as Lactobacillus Clearans are highly effective for health, these species lack intestinal purification action. In order to produce a powerful specie and mend this disadvantage it was considered the concurrent use of an active substance. This means the preparation of lactic acid bacteria capable of invigorating healthy individuals and giving a renovated sensation of well-being and of recovered health to ill or unhealthy individuals. The invention is related with a preparation of lactic acid bacteria, comprising viable cells of Lactobacillus Clearans and any or both of viable or dead cells of Enterococcus Faecalis capable of reducing one or more triglycerides and cholesterol.

Description

PREPARATION OF NOVEDOSE LACTIC ACID BACTERIA WHICH HAS BIOPURIFICATION ACTIVITY BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a preparation of lactic acid bacteria that is extremely significant for the health of humans and animals, wherein the concurrent use of Lactobacillus clearans, which was first isolated by the inventors, and belongs to the genus of Lactobacillus, has even specific characteristics not found in conventionally known bacteria, and either or both of viable or dead cells of En terococcus faecalis (Enterococcus), which are deeply involved in the metabolism of lipids, allows the advantages of each one they are obtained more clearly and effectively. 2. Description of the Related Art There are approximately 300 species and 100 trillion individual bacteria in the intestines, weighing a total of 1 kg and far surpassing the total of 60 trillion human cells. Years of study and immunological research have gradually elucidated their significant relationship in health and in various diseases of humans and animals, and experts now consider the intestinal flora as a function of a vital organ. The beneficial bacteria that belong to the group of lactic acid bacteria, such as the genus Bifidobacterium and the genus Lactobacillus, coexist in a balanced manner when they predominate. This balance can be broken by a variety of factors, such as changes in the amount and quality of the daily diet, excessive exercise, lack of sleep and mental stress, resulting in the proliferation of harmful bacteria such as Welch bacilli and Veillonella. The expulsion of beneficial bacteria results in increased production of harmful substances, which can lead to a variety of diseases and accelerated general aging, from fecal disorders such as diarrhea and constipation, to those resulting from poisoned blood. , such as chronic fatigue, cracked skin, liver dysfunction, hypertension and arteriosclerosis. The Russian Metchnikoff (1845 to 1916), had the theory that the main cause of aging was the poisoning of toxins formed by intestinal rotten fermentation, and advocated as a remedy the habit of drinking beverages with lactic acid bacteria, such as yoghurt to help prevent aging. With all the vagaries of the history of lactic acid bacteria since then, it has not been possible to grasp the true practical utility of such bacteria. This is why the matter revealed now by the immunological studies and elucidated by the experiments, could not be unraveled. Metchnikoff's hypothesis has been confirmed and is now a common knowledge in the health sciences, as has elucidated recent progress in microbiology, a variety of important functions by beneficial intestinal bacteria, such as the reduction of intestinal acidity and the promotion of intestinal motility, to aid in the digestion of food and 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 a improved immunological power. In addition, along with recent health trends, lactic acid bacteria have established a firm base as beverages and antiflatulent for many people. It can not be denied, however, that such products do not attract people who are looking for positive results, who have failed to have better health and improved symptoms through the ingestion or daily use of lactic acid bacteria. Unfortunately, the products of lactic acid bacteria are still consumed simply as a soda, or even tend to be considered as a preference similar to that of coffee. In view of the above, there is a need for a product, which can be rapidly administered to obtain better effects, not available with conventional lactic acid drinks or bacterial preparations. Although the inventors previously found that novel species designated as ctobacillus clearans are highly effective for health purposes, they noted in particular that these species lacked sufficient intestinal purification action. Efforts to produce a strain with greater potency and the like to remedy this disadvantage, led to the inevitable conclusion that the use of the bacteria by themselves produces limited results. The concurrent use of another active substance was then considered, in the search for a preparation of lactic acid bacteria able to invigorate healthy individuals, and to renew a feeling of well-being and health restored in half ill or half healthy individuals.

BRIEF DESCRIPTION OF THE INVENTION As a result of extensive research to find a compatible biological or non-biological partner to remedy the above disadvantages, the inventors perfected the present invention with the finding, among the En terococcus faecalis belonging to the same group of lactobacilli as the Lactobacillus clearans, a species capable of reducing at least one of triglycerides and cholesterol. That is, the present invention is a preparation of lactic acid bacteria, comprising viable cells of Lactobacillus clearans, and viable cells of En terococcus 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, comprising viable cells of La ctobacillus clearans and dead cells of En terococcus 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, comprising viable cells of Lactobacillus clearans and viable and dead cells of En terococcus faecalis, capable of reducing one or more of at least triglycerides and cholesterol.

DESCRIPTION OF THE PREFERRED MODALITIES The ctoba ci ll cesses referred to in the present invention is a novel strain belonging to the genus Lactobacillus, and has the following biochemical characteristics 1, 2, 3 and 4. Specifically, it comprises strains of the genus Lactobacillus that 1) are able to reduce both the Na2S .9H20 and NH4OH, when 0.5 g of Na2S.9H20 and / or 0.5 mL of NH4OH is or are added to 5 g of meat extract, 5 g of peptone, 1 g of glucose, 1 g of CaCO3, and 1 L of water (neutral pH); 2) does not show an action that promotes growth despite the addition of 0.5 g of Na2S.9H20 and / or 0.5 mL of NH4OH, during the logarithmic growth phase of the bacterial culture in medium comprising 1 g of casamino acid and vitamins ( A: 900 IU; Bi; 1 mg; B2; 1 mg; B6; 1 mg; Bi2; 5?; Nicotinamide; 16 mg; calcium pantothenate: 8 mg; C: 64 mg; and D2: 120 IU) in medium from Stephenson-Wetham ((abbreviated as SW) 1 g KH2P04; 0.7 g MgSO4.7H20, 1 g NaCl, 4 g (NH4) 2HP04, 0.03 FeSO .7H20, and 5 g glucose); 3) has resistance, in the form of naturally isolated strains, to Na2S.9H20 greater than that of known lactic acid bacteria, but less than that of La ctoba ci l l us deodorans; and 4) are gram-positive, rod-shaped, non-motile, and negative for catalase, do not reduce nitrate and do not degrade gelatin, do not produce indole or hydrogen sulfide, have a high capacity for lactic acid form of glucose and lactose, and have a growth promoted by the addition of acetic acid (see Japanese Patent Publication (Kokoku) H4-632.

The advantages of La ctoba cíll us clearans, in addition to the action of common lactobacillus, include a potent intestinal cleansing action, absent in conventional lactobacilli as described in Japanese Patent 1714413 and Japanese Patent Application 11-15177, such as : 1) decomposition of substances with rotten odor in the intestine; 2) increase in beneficial bacteria such as Bifidobacterium and Lactobacillus in the intestinal flora, with a dramatic reduction in harmful bacteria such as the Welch and Veillonella bacilli, thus providing a better balance in the intestinal flora, to improve the intestinal medium; and 3) suppression of growth mainly of etiological agents that cause intestinal infections, and decrease the toxicity. This action will be described later through the tests. In vi tro tests were conducted first. 15 mL of a 10-fold dilution of times were introduced into test tubes (18 x 180 mm). The test tubes were then inoculated with simple bacteria 1) which has been sterilized at high pressure for 15 minutes at 120 ° C and 2) which has not been sterilized. Test tubes capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. One mL of air from the inside of the test tubes was extracted with a syringe and injected into 5 L of odor bags for olfactory tests by a panel of 6 individuals. The odor was evaluated based on the stool odor criterion given in Table 1.

Table 1: Stool smell criterion Three typical strains of Lactobacillus clearans, specifically, FERM BP-6972, FERM BP-6971, FERM BP-6973, were used to test the deodorizing capacity in the aforementioned in vitro tests. The results in Table 2 show that samples sterilized at high pressure were clearly deodorized, with an odor assessed as weak or faint odor. The unsterilized samples, or on the other hand, showed less deodorization due to the fact that smelly substances were produced, perhaps as a result of the activity of numerous putrefied bacteria in the feces, but nevertheless, they were also clearly deodorized. The results for the three typical stages of Lactoba cill us clearans previously mentioned were reproduced in a similar manner with other similar strains.

Table 2: Deodorizing capacity of Lactobacillus clearans (in vitro) In the subsequent in vivo tests, 150 mL of yoghurt prepared using Lactoba cill us clearans FERM BP-6973, was ingested once a day, and the smell of the feces was determined from 20 to 30 days, 50 to 60 days, and 80 90 days after ingestion, with the averages given in Table 3. The results were determined by introducing 0.5 g of stool samples from 10 individuals into 20 L scent bags, which were stored at room temperature for odor evaluation by a panel of 6 individuals after 30 minutes. The average stool odor for 5 individuals who did not eat yoghurt was rated as 100. Although the odor of the feces varied considerably depending on the content of the diet, the average odor for 10 individuals who ingested yoghurt decreased approximately 50% 1 month after the ingestion, 70% after 2 months, and 85% after 3 months. After 3 months, however, continuous ingestion resulted in a peak odor reduction of 80 to 90%.

Table 3: Stool odor after ingestion of yoghurt prepared using Lactobacillus clearans In terms of the correlation between purification and bacteria in the natural world, the inventors limited the tested substances to readily evaluable malodorous substances, which were broadly classified into sulfur compounds, nitrogen compounds and carbon compounds. It was found that bacteria capable of degrading compounds with sulfur with bad odor such as Na2S-9H2 ?, nitrogen compounds with a bad odor such as NH3, and compounds with a bad smell of carbon such as acetic acid, butyric acid and lower fatty acids Likewise, they were able to degrade most of the compounds with a bad odor of sulfur, nitrogen and carbon from their polymers, this is referred to as the SNC theory. The following tests were conducted according to this theory. 0.5 g of Na2S-9H20 or 0.5 mL of water with ammonia (NH40H) was added to 1 mL of synthetic medium comprising 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 Lactoba cill US clearans during 72 hours of anaerobic culture at 37 ° C, and the decrease in the Na2S-9H20 or NH40H that was added was determined with time. Na2S-9H20, was measured by the iodide titration method of JIS K0102-1985, and NH4OH was measured by the phenol indole blue absorbance method of JIS K0102-1985. The results are given in Tables 4 and 5. The tables show that La ctobacillus clearans had the ability to reduce Na2S-9H20 and toxic NH4OH with bad smell, from 40 to 50% in 72 hours. This means that it can degrade and assimilate most other toxic substances with bad odor. Table 4: Degradation and assimilation of sodium disulfide by Lactobacillus clearans Table 5: Degradation and assimilation of ammonia by Lactobacillus clearans Table 5: Degradation and assimilation of ammonia by Lactobacillus clearans (continued) Previous sensory tests in vi tro and chemical analyzes are described as methods for evaluating the deodorizing action of Lactoba cill us clearans. 10-fold dilutions of stool samples prepared at this time were centrifuged before culture and 72 hours after culture, 5 mL of supernatant was collected and diluted 10 times to determine the concentrations of the S2 + and NH4 + ions, and the degree of their decrease was calculated. The results in Table 6 show that the decrease in the S2 + and NH4 + ions in the feces was even greater than the decrease in the aforementioned synthetic medium, indicating that the feces were a more suitable habitat for the La ctobacill us clearans. The S2 + ions were determined by the iodine titration method of JIS K0102-1985, and the NH4 + ions were determined by the phenol indole blue absorbance method of JIS K0102-1985.

Table 6: Decrease in S2 + and NH4 + by Lactobacillus clearans 2 μL of the aforementioned supernatant were introduced into a gas chromatograph to analyze the lower fatty acids. Gas chromatography involved the use of a Column of unisole glass F-200 30/60 (3 fx 3 m), at a carrier gas velocity of 50 mL / mip (He), with 172 kPa (0.55 kg / cm2G) of hydrogen and 152 kPa (0.55 kg / cm2G) of air, at a column temperature of 140 ° C and Inj of 200 ° C. The substances evaluated were acetic acid, propionic acid, isobutyric acid, or n-butyric acid, isovaleric acid, and n-valeric acid, with the calculated concentrations given in Table 7. Table 7 shows that the concentrations of the fatty acids Lower samples decreased from 50 to 75% with samples sterilized at high pressure, and from 40 to 60% with non-sterilized samples.

Table 7: Decrease in lower fatty acids by Lactobacillus clearans Table 7: Decrease in lower fatty acids by Lactobacillus clearans (continued) Lyophilized cells from three typical Lactobacillus clearans strains, ie FERM BP-6972, 6971 and 6973, were prepared, and mixed in equal parts to form a preparation. 2 g of the preparation (5 x 108 cells / q), were taken continuously, and changes in cell counts (cells / 1 g of feces) of Bifidobecterium and Lactobacillus (except for Lactobacillus clearans), which are known as typical beneficial bacteria in the intestinal flora, as well as typical harmful bacteria such as Veillonella and Clostridium perfringes (Welch bacilli), were measured over time. Table 8 shows the results of oral administration to 20 healthy individuals, and Table 9 shows the results of oral administration to 20 constitutionally weak individuals.

Table 8: Effects of oral administration of the preparation of Lactobacillus clearans on the intestinal flora of healthy individuals (average values for 20 healthy individuals) Table 8: Effects of oral administration of Lactobacillus clearans preparation on the intestinal flora of constitutionally weak individuals (mean values for 20 constitutionally weak individuals) Tables 8 and 9 show that prior to the administration of La ctobacill us clearans preparations, the beneficial bacteria among the intestinal flora were an average of 220% higher in healthy individuals than in constitutionally weak individuals, whereas harmful bacteria they were only about 22.5%. This shows that the state of the intestinal flora is an index of the current state of health, that is, how deeply the intestinal flora is involved in health. A new finding was that the ingestion of the first preparation resulted in an increase in beneficial bacteria in healthy individuals, and that with this increase, there was a gradual decrease in the harmful bacteria. In contrast to this pattern, the pattern found in constitutionally weak individuals was first, that harmful bacteria decreased, after a gradual increase in beneficial bacteria. In general, the average changes in beneficial and harmful bacteria reflected an increase in beneficial bacteria and a corresponding decrease in harmful bacteria. This trend accelerated after 3 months, with Bifidoba cteri um increasing 50% after 6 months and 110% after 1 year, with Lactobacillus increasing by 300% after 6 months and 950% after 1 year. In contrast, Clostridium um decreased 70% after 6 months and 85% after 1 year, while Veillonella decreased 67.5% after 6 months and 82.5% after 1 year. Escherichia coli 0-157, Salmonella en teri tidis, and Shigella flexneri were grown alone and together with La ctobacillus clearans (FERM BP-6973) to verify the action of Lactobacillus clearans against pathogens., and the results were compared. The composition of the medium used for the cultures comprised 10 g of meat extract, 10 g of peptone, 2 g of glucose, 2 g of NaCl, 1 g of CaCO3 per liter, with the pH adjusted to 7.2. The anaerobic culture was carried out at 37 ° C, with the subcultures repeated every 72 hours, at which point the plates were diluted and smeared to verify the changes in the cell count, if the colonies were of the S type (original) or they have mutated in type R with diminished toxicity, their proportion, and the like. The pathogens used in the tests were purchased from Medie KK (registered health research institute). Table 10 shows the results of E. coli 0-157 cultivated by itself, and Table 11 shows the results of the mixed culture of. E. coli 0-157 and La ctobacillus clearans. Table 10 shows that the cultures of E. coli 0-157 alone, the cell count was virtually constant at 4 to 5 x 109 cells / g through 20 subcultures, without any type R shown. Table 11 shows that in the mixed cultures of E. coli 0-157 and Lactobacillus clearans, there was a small change in the cellular count of Lactobacillus clearans, but that there were significant changes in the cellular count of E. coli 0-157. The R types were shown in the 5th subculture, and the proportion of R types increased with the accumulation of subsequent subcultures, until all the cells formed R types towards the 18th crop. There was no reversion to type S in subcultures continued subsequently.

Table 10: Results of the cultures of E. coli 0-157 by itself Table 10: Results of the cultures of E. coli 0-157 by itself (continued) Table 11: Results of mixed crop cultures of E. coli 0-157 and Lactobacillus clearans Table 11: Results of cultures of mixed cultures of E. coli 0-157 and Lactobacillus clearans (continued) Table 12 shows that the results obtained with Salmonella in cultivated teri tidis by themselves, and Table 13 shows the results obtained with mixed cultures of Salmonella in teri tidis and Lactobacillus clearans. Table 12 shows that in cultures of Salmonella in teri tidis alone, the cell count was 3 to 5 x 109 cells / g. The R types showed themselves spontaneously in the 14th subculture, reaching a proportion of 5% towards the 20th subculture. The relationship increased in the subsequent subcultures, with a peak in 23% towards the 50th subculture. The proportion of types R after this remained approximately 20%. Table 13 shows that in the mixed cultures of Salmonella in teri tidis and Lactoba cill us clearans, the ratio of types R was 29% in the 5th subculture, and that the ratio of types R increases with the accumulation of subsequent subcultures, reaching 50% in the 10th subculture, 90% in the 20th subculture, and 100% in the 47th subculture. Reversion to type S in the subsequent subcultures was less than 1%. After the 70th subculture, all S types disappeared, without additional reversion.

Table 12: Results of Salmonella enteritidis cultures by themselves Table 12: Results of Salmonella enteritidis cultures by themselves (continued) Table 13: Results of the mixed cultures of Salmonella enteritidis and Lactobacillus clearans Table 14 shows the results of the cultivated Shigella flexneri itself, and Table 15 shows the results of the mixed culture of Shigella flexneri and Lactobacillus clearans. Table 14 shows that the Shigella flexneri cultures alone, the R types showed spontaneously in the 10th subculture, reached a proportion of 9% towards the 20th subculture. The proportion was increased in the subsequent subcultures, reaching a maximum of 20% towards the 40th subculture. The type R ratio subsequently remained approximately 10 to 20%. Table 15 shows that in the mixed cultures of Shigella flexneri and Lactobacillus clearans, the R types were shown in the 5th subculture, and that the proportion of the R types increased with the accumulation of subsequent crops, reaching 100% in the 80th subculture There was a reversion to type S in subsequent crops, but less than 1%. After the 108th subculture, all S types disappeared, without any additional reversion.

Table 14: Results of Shigella flexneri crops by itself Table 14: Results of the Shigella flexneri crops by itself (continued) Table 15: Results of the mixed cultures of Shigella flexneri and Lactobacillus clearans Table 15: Results of the mixed cultures of Shigella flexneri and Lactobacillus clearans (continued) Groups of 10 8-week-old male mice were intraperitoneally administered 1 x 108 cells per animal to study the toxicity of S and R types of E. coli 0-157, Salmonella enteri tidis, and Shigella flexneri. The conserved strains were used for the S types, and the strains that mutated 100% of the R types by Lactobacillus clearans were used at this time for types R. The results in Table 16 show that all the animals died within the 4 days with type S pathogens, while none died with R types except for the Shigella flexneri that resulted in death by 7m0 day.

Table 16: Toxicity of S and R types of E. coli 0-157, Salmonella enteri tidis, and Shigella flexnep in mice The yogurt prepared with Lactobacillus clearans described above was administered orally to humans, and the blood was analyzed once a month to study the changes in the intestinal flora. Cholesterol and triglyceride levels fell from approximately 10% to more than 80% in individuals compared to before administration. Table 17 shows the differences in function between the Lactobacillus clearans of the present invention and the conventional Lactobacillus strains. Table 17: Comparison of functions between Lactobacillus clearans and conventional Lactobacillus strains.

In terococcua faecalis is a group of bacteria that constitutes in the intestinal flora and belong to the group of lactic acid bacteria, which normally occur in an amount of 1 x 107 cells per gram of feces. They appear in the form of two spherical or ovoid shapes, or in the form of short chains. They are gram-positive cocci with a strong resistance to heat, drying, chlorine, gallic acid, and the like, and grow in a wider temperature range of 10 to 45 ° C, compared to common streptococci. Although there are some pathogenic strains, they are not toxic. Viable cells act as antiflatulents, and have been commercially available for more than 10 years in Japan. It has been proven that they are not toxic when taken orally, apart from such commercial products, the viable and dead cells of some strains have recently been shown to effectively lower cholesterol and triglycerides in the blood. Therefore they hold a promise in the prevention or treatment of diseases typically related to adults (diseases arising from lifestyle habits), such as hyperlipidemia, hyperton, and arteriosclerosis. The In terococcus faecalis referred to in the present invention indicate strains having such functions. The following is an example of a method for preparing viable cells of En terococcus faecalis for use in the present invention. Specifically, cells that have been cultured and centrifuged by a common method are suspended in physiological saline, washed, centrifuged again and harvested. They can be lyophilized using soluble starch as a preservative. As an example of a method for preparing dead Enterococcus faecalis cells, the soluble starch and the washed and centrifuged cells obtained when the above mentioned viable cells were harvested, can be treated for 30 minutes in hot water at 100 ° C and then lyophilized. Although En terococcus faecalis has no ability to reduce sodium sulfide (Na2S * 9H20) or ammonia, it has a weak deodorizing capacity against 10-fold dilution of faeces. Table 18 gives the deodorizing capacity two typical strains of En terococcus faecalis, namely, FERM BP-7230 and FERM BP-7231. The test was the same as for the Lactobacillus clearans, and the criterion was the same as that given in Table 1. Table 18: Deodorizing capacity of En terococcus faecalis (in vi tro) The two typical strains of En terococcus faecalis were administered orally in amounts of 1 x 109 cells / person per day. Stool odor before administration was scored as 100. Stool odor was determined 20 to 30 days, 50 to 60 days, and 80 to 90 days after ingestion, with the results given in Table 19 Table 19 shows that the odor was reduced by En terococcus faecalis, although weakly.

Table 19: Odor of stool during ingestion of Enterococcus faecalis The viable and dead cells of two typical strains of En terococcus faecalis, namely, FERM BP-7230 and BP-7231 were prepared and mixed in equal parts viable and dead cells to produce a preparation. The preparation was taken in an amount of 1 x 109 cells / day / person for 6 months, and changes in cell count of intestinal flora were determined over time by measuring changes in cell count (cells / 1 gram of stool ) of Bifidobacterium and Lactobacillus (except for Lactobacillus clearans), which is known to be a typical beneficial bacterium, 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 the viable cells had a slightly higher rate of improvement than the dead cells. However, the results were much lower than those of Lactobacillus clearans.

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

The mixed cultures of Escheri chia coli 0-157, Salmonella enteri tidis, and Shigella flexneri, were carried out to study the action of En terococcus faecalis against pathogens. The proportions in which the pathogens mutated from types S to R were virtually the same as the proportions that occur spontaneously. The cellular counts of the pathogens gradually decreased with each subculture, until the Salmonella in teri tidis disappeared towards the 25th crop, the Shigella flexneri disappeared towards the 33th crop, and the E. coli 0-157 disappeared towards the 35th crop. Although this suggests that the growth of Enterococcus faecalis outweighs pathogens, the potential production of some physiologically active substances that suppress the growth of pathogens can not be regulated. The above-mentioned viable and killed cell preparations of En terococcus faecalis were added, in amounts of 1 x 109 cells / 1 g in each feeding or feeding. Mice bred for 3 months with this or mice bred with conventional feed were only compared for serum triglycerides and cholesterol. The mice were divided into groups of 10, and were allowed to feed freely. Triglycerides and cholesterol were measured by means of an E-Test Wako and C-Test Wako, respectively. The average level for the control mice was 100%. The results are given in Table 21. Table 21 shows that mice that continuously ingested either viable or dead cells had 20 to 40% reduction in levels for both parameters, leaving no room for doubt of their effectiveness .

Table 21: Effect of oral administration of Enterococcus faecalis preparation on triglyceride and serum cholesterol levels in mice Fifteen spontaneously hypertensive rats of 8 weeks of age (SHR) were divided into three groups. The groups were fed with food containing preparations of viable and killed cells of En terococcus faecalis in amounts of 1 x 109 cells / gram per feed. The control group was not administered with any cell preparation. The animals were reared for 3 months. Table 22 gives blood pressure after 3 months. Table 22 shows a decrease of approximately 10% in blood pressure.

Table 22: Effects of oral administration on the preparation of Enterococcus faecalis in rat blood pressure EXAMPLES The combined action of Ctobacillus clearans and Enterococcus faecalis allowed surprisingly effective results to be obtained rapidly. The manufacturing methods and examples of the preparations of the present invention are described below, but the scope of the present invention is not limited by these examples of manufacture and working examples.

(Example of manufacture 1) The production of the preparation of Ctobacillus clearans: 10 L of medium comprising 5 g of meat extract, 5 g of peptone, 3 g of sodium acetate, 1 mL of ammonia water, g of glucose, 0.5 g of cystine, and 2 g of yeast extract per liter of medium were inoculated with La ctoba cill us clearans for 72 hours of anaerobic culture at 37 ° C. The resulting culture was centrifuged, yielding 10 g of biomass. This was washed with 500 L of physiological saline and centrifuged twice. The washed biomass was introduced into 500 mL of solution comprising 50 g of skimmed milk, 30 g of trihalose and 0.5 g of taurine, and stirred well. The mixture was lyophilized by a common method, giving 82.5 g of cell preparation (3 x 109 cells / g). This was mixed with 330 g of dry skim milk, giving a preparation of La ctobacillus clearans containing 5 x 108 viable cells per gram.

(Example of manufacture 2) Production of the preparation of In terococcus faecalis with viable cells: 10 L of medium comprising 5 g of meat extract, 5 g of peptone, 2 g of sodium chloride, 2 g of yeast extract and 10 g of glucose per liter of medium were inoculated with En terococcus faecalis during 72 hours of aerobic culture at 37 ° C. The resulting culture was centrifuged, yielding 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 comprising 20 g of skimmed milk, 30 g of soluble starch and 0.5 g of sodium glutamate, and stirred well. The mre was lyophilized by a common method, yielding 54 g per cell preparation (5 x 109 cells / g). This was mixed with 486 g of completely dry soluble starch, giving a viable cellular preparation of En terococcus faecalis containing 5 x 10 8 viable cells per gram.

(Example of manufacture 3) Production of the preparation of In terococcus faecalis with dead cells: The washed biomass obtained in the example of manufacture 2 was suspended in 500 mL of saline, 50 g of soluble starch were then introduced, the solution was I sterilize thermally during minutes at 110 ° C, and the suspension was lyophilized by a common method, yielding 53 g per cell preparation (approximately 5 x 109 cells / g). Preparations with dead cells can also be produced by ultrasonically disrupting cells or the like.

(Example of manufacture 4) Production of the mre preparation of Lactobacillus clearans and Enterococcus faecalis: The preparation of the Ctobacillus clearans produced in the manufacturing example 1 and the preparation of En terococcus faecalis with viable cells produced in the manufacturing example 2 or the preparation of Enterococcus faecalis with dead cells produced in the manufacturing example 3, 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 10 8 cells / g and In terococcus faecalis in an amount of 2.5 x 10 8 cells / g, but the relationship between the preparations of Lactobacillus clearans and In terococcus faecalis can be varied during the manufacturing process to produce mres containing any desired cell count. The preparation can be made 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 feces was introduced into test tubes (18 x 180 mm). The test tubes were then inoculated with sample bacteria 1) that have been sterilized at high pressure for 15 minutes at 120 ° C and 2) that they have not been sterilized. The test tubes were capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. 1 mL of air was extracted with a syringe from the inside of the test tubes and injected into 5 L smell bags for the olfactory tests by a panel of 6 individuals. The odor was evaluated based on the stool odor criterion given in Table 1. For the preparations of En terococcus faecalis with dead cells, 0.1 g of the preparation produced in the manufacturing example 3 was added to the test tubes. The test results in Table 23 show that the smell of faeces was considerably weakened, particularly in the case of diluted faeces treated with samples sterilized at high pressure. This will be evident in a comparison with the results for the test batches inoculated with Lactobacillus clearans alone in Table 2.

Table 23: Deodorization test with a mre of Lactobacillus clearans and Enterococcus faecalis (1) (Example 2) A combination of 1 g of a viable cell preparation of Enterococcus faecalis (FERM BP-7230) or 1 g of a preparation of killed cells of En terococcus faecalis and 100 ml of yoghurt was prepared with La ctoba cillus clearans FERM BP-6973 in a medium comprising 100 g of skim milk, 50 g of sucrose, and 2 g of agar per liter, was administered continuously to 10 individuals, and the smell of the feces was determined continuously from 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 the 10 individuals into 20 L odor bags, which were stored at room temperature for odor evaluation by a panel of 6 individuals after 30 minutes. The average stool odor for 5 individuals who did not eat yoghurt was rated 100. The average values in Table 24 show that oral administration increased the odor of faeces over time. After 90 days, the odor decreased considerably and it was not unpleasant anymore. The appreciable effects will be evident when compared with the results obtained with the administration of La ctoba cillus clearans only in Table 3. Table 24: Deodorization test with a mixture of Lactobacillus clearans and Enterococcus faecalis (2) (Example 3) In the same manner as in Example 1, the 10-fold diluted test tubes were inoculated with sample bacteria that 1) were sterilized at high pressure for 15 minutes at 120 ° C and 2) that They have not been sterilized. The test tubes were capped with rubber stoppers for 72 hours of anaerobic culture at 37 ° C. The cultures were centrifuged, 5 mL of supernatant was collected from each of the 10-fold dilutions, and the concentrations of the free sulfide ions (S2 +) and ammonium ions (NH4 +) were determined in the resulting 50 mL solution. The concentration of the lower fatty acids was also determined by gas chromatography. Tables 25 and 26 give the measured results. The tables show that the typical substances in the faeces were reduced to a high proportion of 70 to 90% by both sterilized and unsterilized samples. The proportion of the reduction will be evident in a comparison with the results obtained with the inoculation with La ctobacillus clearans only in Tables 6 and 7.

Table 25: Test for the decrease in toxic malodorous substances by a mixture of Lactobacillus clearans and Enterococcus faecalis (1) (dilution of sterile feces) Table 26: Proof of the decrease in toxic malodorous substances by a mixture of Lactobacillus clearans and Enterococcus faecalis (2) (dilution of sterile feces) (Example 4) 1 g of preparation of Ctobacillus clearans (5 x 108 cells / g), comprising a mixture of equal parts of three typical strains of La ctobacillus clearans, specifically, FERM BP-6972, FERM BP-6971, FERM BP-6973, and 1 g of viable cell preparation of Enterococcus faecalis (5 x 108 cells / g) of Enterococcus faecalis FERM BP-7230 or 1 g of preparation of dead cells of Enterococcus faecalis FERM BP-7231 (5 x 108 cells / g), of Enterococcus faecalis, was administered continuously for 6 months to 20 healthy individuals and 20 constitutionally weak individuals. The intestinal flora was verified over time, with the results given in Tables 27 and 28. The tables show that the oral administration of viable or dead cells of En terococcus faecalis mixed with La ctobacill us clearans, increased the beneficial bacteria more quickly and decreased harmful bacteria when Lactobacill us clearans was administered alone, as shown in Tables 8 and 9. That is, improvements were approximately 25% faster in healthy individuals, with 10 to 50% higher cell count of beneficial bacteria and a 20 to 40% lower cell count of harmful bacteria. In constitutionally weak individuals, improvements were approximately 30% faster, with 10 to 30% of a higher cell count of beneficial bacteria and 20 to 50% of the lower cell count of harmful bacteria. It can be concluded, therefore, that the improvement in intestinal flora was more effective in constitutionally weak individuals.

Table 27: Effects of the mixture of Lactobacillus clearans and Enterococcus faecalis on the intestinal flora (healthy individuals) Table 27: Effects of the mixture of Lactobacillus clearans and Enterococcus faecalis on the intestinal flora (constitutionally weak individuals) (Example 5) Viable or dead Enococcus faecalis cells were added to mixed cultures of La ctobacillus clearans, E. coli 0-157, Salmonella enteri tidis, or Shigella flexneri for anaerobic culture at 37 ° C, and subcultures they were repeated every 72 hours, at which point the plates were diluted and spread on a slide to verify changes in cell count and the SR mutation ratio of E. coli 0-157, Salmonella in teri tidis, and Shigella flexneri. The results are given in Tables 29, 30, 31, 32, 33 and 34. Strains FERM BP-6973 of Lactobacillus clearans were used, as the strain FERM BP-7231 of En terococcus faecalis. The dead cell preparation of En terococcus faecalis mentioned above was added in an amount one gram per L of medium. The composition of medium used for the crops comprised 10 grams of meat extract, 10 grams of peptone, 2 grams of glucose, 2 grams of NaCl, and 1 gram of CaCO3 per liter, with the pH adjusted to 7.2, and sterilized at high pressure for 15 minutes at 120 ° C. The subsequent Tables reveal that inoculation of pathogens such as E. Coli 0-157, Salmonella in teri tidis and Shigella flexneri with viable Lactobacillus clearans cells and In terococcus faecalis rapidly decreased cell counts of such pathogens and resulted in their mutation in R types over the course of subculture. The e . Coli 0-157 mutated 100% to type R towards 13av0 and disappeared towards the 15th subculture. Salmonella in teri tidis and Shigella flexneri disappeared before the 100% R-type mutation towards the 13th and 15th subcultures, respectively. When Lactobacillus clearans was used by itself, as shown in Tables 11, 13 and 15, these pathogens mutated towards the R types over the course of numerous subcultures, but the bacteria maintained a constant cell count of 1 to 3 x. 109 cells / ml, without disappearing. Although the addition of dead cells of Lactobacillus clearans and Enterococcus faecalis to the aforementioned pathogens, showed the same tendencies as those prevailing with the inoculation of viable cells, the mutation to the R types was slower than that prevailing with the inoculation of cells. viable, and cell counts decreased but did not disappear. However, the number of subcultures that presented 100% of mutations to type R was 15 with E. Coli 0-157, 30 with Salmonella in teri tidis, and 15 with Shigella flexneri, which were much faster than when Lactobacillus clearans was used as the inoculum by itself.

Table 29: Effects of viable cells of Lactoba cillus clearans (FERM BP-6973) and In terococcus faecalis (FERM BP-7231) in E. coli. 0-157 Table 30: Effects of viable cells of Lactobacillus clearans (FERM BP-6973) and In terococcus faecalis (FERM BP-7231) on Salmonella in teri tidis Table 31: Effects of viable cells of Lactobacillus clearans (FERM BP-6973) and In terococcus faecalis (FERM BP-7231) on Shigella flexneri Table 32: Effects of dead cells from Lactobacillus clearans (FERM BP-6973) and In terococcus faecalis (FERM BP-7231) in E. Coli 0-157 Table 33: Effects of dead cells of Lactobacillus clearans (FERM BP-6973) and Enterococcus faecalis (FERM BP-7231) on Salmonella enteritidis Table 34: Effects of dead cells of Lactobacillus clearans (FERM BP-6973) and on Teroccoccus faecalis (FERM BP-7231) in Shigella flexneri Example (6) A viable cell preparation of Lactobacillus clearans FERM BP-6972 or Lactobacillus clearans FERM BP-6972 was prepared by the method in Manufacturing Example 1, a preparation of viable cells of Enterococcus faecalis FERM BP-7230 or En terococcus faecalis 7231 was prepared by the method in Manufacturing Example 2, and a preparation of killed cells of En terococcus faecalis FERM BP-7230 or En terococcus faecalis 7231 was prepared by the method of Example of Manufacture 3, were combined and added to the feed, which was given to a group of five male mice of 10 weeks of age. One gram of food contained 5 x 108 cells of Lactobacillus clearans and 5 x 108 of Enterococcus faecalis (2.5 x 108 viable and dead cells when used in combination). The animals were allowed to feed freely for 3 months. Triglycerides and cholesterol in serum were measured for comparison with control levels fed only with normal food. Triglycerides were measured with a Triglyceride E-Wako test, while cholesterol was measured with a Triglyceride C Wako test. The average levels for each group were obtained. The average level for the mice in the control group was 100%. The results are given in Table 35. Tables 35 and 21 show that the addition of viable or dead, as well as viable and dead cells of En terococcus faecalis, which have the action in decreasing triglycerides and cholesterol, Lactoba cillus clearans was much more effective than when Enococcus faecalis alone was administered. Unlike the results obtained when administered alone, the combined use of dead cells resulted in greater effects than with viable cells. Compared to the control group, the levels decreased by as much as 1/2. Table 35: Effect of the mixture of ctobacillus clearans and Enterococcus faecalis on triglycerides and serum cholesterol in mouse serum (Example 7) 7 groups comprising a total of thirty-five spontaneously hypertensive rats of 8 weeks of age (SHR), were bred for 7 months with feeding containing the cell preparations of Example 6. Blood pressure was measured before and after from administration to the groups given each type of cell preparation, and to the control group, with the results given in Table 36. Tables 36 and 22 reveal that the blood pressure was improved, an average of about 15% after 3 months. The dead cells of En terococcus faecalis had a more effective action than the viable cells in the same manner as in Example 6. The drop in blood pressure was about 10 to 20%, results which were better than when they were administered in Enterococcus faecalis by itself.

Table 36: Effect of the mixture of Lactobacillus clearans and In terococcus faecalis on the blood pressure of rats.

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

Table 73: Comparative summary Health care and related approaches have gradually changed in our aging society. Our time, where the treatment of acute diseases and chronic diseases have been considered with the greatest importance, is witness to a change, now or in the near future, to "preventive medicine", and even "nutritional therapy" in light of our previous society. There are no treatments that are underestimated after the disease is acquired. Instead the approaches to create health in oneself through a more educated consumption of nutrition, is now increasing in the mainstream in order to naturally prevent the disease. It is not an exaggeration of the case to suggest that the prevention of disease to avoid the need for treatment at all is at the forefront of treatment in the 21st century. Oral administration of the lactic acid bacteria preparation of the present invention gradually extends the influence of the beneficial intestinal bacteria, constantly protects the intestinal mucosa, produces vitamins and synthesizes amino acids, while suppressing the growth of foreign bacteria and foreign pathogens, decreasing their toxicity, and activating the immune function under the guidance of the lactic acid bacteria of the preparation. Harmful intestinal bacteria are markedly reduced in this way, and the production of putrefied substances with bad odor is suppressed, resulting in significantly deodorized feces. As if under attack, the lactic acid bacteria in the preparation actively feed the putrefied intestinal substances, resulting in a cleaner intestinal environment, and normalization of the intestinal mucosa and the surrounding vessels and nerves. The purification of the intestines, which are the source of human vitality, energy, blood and meat result in a better absorption of vital substances and vice versa, in a lesser absorption of toxic substances through the intestines. This inevitably results in a better liver function, and therefore in lower triglycerides and cholesterol in the serum, as well as in cleaner blood. 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 therefore improved, and all systemic functions are invigorated. That is, the contaminated intestines are cleaned, and they are allowed to return to their pristine condition, resulting in a general improvement and in a vital state of health without the appearance of disease. It can be said that Metchnikoff's doctrine of longevity has now been realized a century later. The preparation of lactic acid bacteria of the present invention can be stored without any loss of title for 2 or 3 years, allowing it to be produced in the form of portable goods, which can be easily used anywhere and at any time, from this mode is of immeasurable value. In societies with older populations that are increasing more and more older individuals, they are likely to remain in bed, although waste disposal can become a problem, deodorization of such waste would make such a task much less unpleasant, and could be of help to the people who care.

Claims (3)

1. CLAIMS 1. A preparation of lactic acid bacteria, characterized in that it comprises viable cells of La ctobacillus clearans, and viable cells of En terococcus faecalis capable of reducing one or more of at least triglycerides and cholesterol.
2. 2. A preparation of lactic acid bacteria, characterized in that it comprises viable cells of Lactobacillus clearans, and dead cells of En terococcus faecalis capable of reducing one or more of at least triglycerides and cholesterol.
3. 3. A preparation of lactic acid bacteria, characterized in that it comprises viable cells of La ctobacillus clearans, and viable and dead Enterococcus faecalis cells capable of reducing one or more of at least triglycerides and cholesterol.