Recovering, identifying and describing strains with probiotic activity recovered from faeces of exclusively breast-fed children

FIELD: medicine.

SUBSTANCE: group of inventions refers to biotechnology. There are presented the probiotic strains Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034 and Bifidobacterium breve CNCM I-4035 recovered from faeces of the exclusively breast-fed children. The above strains either taken separately, or mixed are used as ingredients of a probiotic composition.

EFFECT: due to their properties, the presented strains have the positive effect on health of the individuals taking them.

36 cl, 14 dwg, 4 tbl, 12 ex

 

Field of the invention

The main objective of this study lies in the selection of probiotic microorganisms for use in food and pharmaceutical industries, particularly for their application in dairy mixes for baby food. These microorganisms have a high resistance to pH, salts of bile acids and adhesion to intestinal cells, therefore they are particularly suitable for use in the aforementioned industries.

Prior art

The diet passed a very significant development, which has changed its concept in recent decades. Previously it was thought that food plays a role of source of nutrients needed to maintain health, but now from this concept developed the idea that diet may include foods that, in addition to providing power, will contribute to health. On this basis, in the food industry began the development of many products that promote the health and well-being. This area has undergone substantial development of lines of functional food, which daily increases the human consumption of probiotics. A serious problem is the expansion of knowl� about these foods among which of particular interest are products containing probiotics.

There are very old information regarding beneficial effects of foods with a high content of bacteria, such as in the embodiment of ancient healing, where it is said that Abraham binds his long life with the use of milk, or by the Roman historian Pliny, who, in the 76 year BC recommended that the use of fermented milk products for treating gastroenteritis (Senmier and De Vrese 2001).

At the beginning of the last century Russian microbiologist Ilya Mechnikov (1845-1916) confirmed that consumption of fermented milk products modulates the intestinal microbiota, providing a positive impact on human health (Metchnikoff 1908). He focused on the fact that Bulgaria was a surprisingly high number of centenarians, despite the fact that she was one of the poorest countries in Europe. He drew attention to the fact that Bulgarians have consumed large quantities of yogurt. Swordsmen have successfully identified the bacteria, producing yogurt, and used them in their research. This was the beginning of studies of probiotics. Mechnikov became a staunch defender of the idea that food can protect the body from invasion of pathogens and, consequently, prolongs life and improves its quality. He is also the first to develop drugs� with the use of lactobacilli in the form of capsules for oral administration, named lactobacilli.

At the same time, the French microbiologist Tissier drew attention to the fact that the faecal microbiota of newborns fed breast milk contains more bacteria of the genus Bifidobacterium than faecal microbiota of infants receiving artificial milk, and confirmed the useful role of this microorganism.

Later, in 1940, came bifemoral (Bifidus Milk) for the relief of malnutrition in children during the First world war. In 1950 the factory Degusta made Biogur and Bio-garde. In 1989, increased consumption and production of dairy products in Switzerland. In 1993 two researchers, Modler (Modler and Villa-Garcia (Vila-Garcia), developed the first nizkochastotnyi bio yogurt.

In 1965, Lilly (Lilly and Stillwell (Stillwell) first used the term "platinum" to describe the products of gastric fermentation. However, a more reasonable and widely used definition of probiotics was the definition formulated much later by fuller (Fuller 1992, Fuller 1989). Probiotics are defined as follows: "supplements of live microorganisms, which when added to foods exert beneficial effects on the health of those who uses them, because they lead to the improvement of the microbial balance in his/her intestines. For an adult this includes to�to products, derived from fermented milk products and preparations, lyophilized with these bacteria.

In 1998, the international Institute of biomedical Sciences (International Life Science Institute. (ILSI) in Brussels has defined probiotics as live microorganisms which when administered in sufficient quantities, have beneficial effects on health, significantly higher than the impact from usual food. They have a useful contribution to one or more functions of the body. They provide the best state of health and well-being and/or reduce the risk of disease. They can be functional for the General population or to specific groups.

Currently there are the following criteria for the identification of probiotic microorganisms:

1) they originate from a person;

2) they are non-pathogenic in nature;

3) they are resistant to destruction by technical means;

4) they are resistant to destruction by gastric acid and bile;

5) they are capable of adhesion to the intestinal epithelium;

6) they are able to colonize the gastrointestinal tract;

7) they produce antimicrobial substances;

8) they modulate the immune response;

9) they affect metabolic processes (assimilation of cholesterol, the formation of vitamins and so on).

Probiotic bacteria can �influence all the cells of the intestine and mechanisms of functioning of these cells, including effects on microbiota (Backhed and Ley 2005), modulation of immune function (Picard et al. 2004; Kalliomaki 2004) and the strengthening of the barrier function of the epithelium (Madsen et al. 2001; Isolauri & Salminen 2005).

Among bacteria with probiotic activity in the intestines are the most numerous bacteria of the genus Bifidobacterium, and they make up 25% of bacteria in the colon of adults and 95% of the newborn, feeding on mother's milk. Today there are many food products (yogurt and milk), complemented by bacteria of this type. Other strains also possess probiotic activity, are strains of the genus Lactobacillus, which, according to studies in vitro, inhibit the adhesion of other anaerobic bacteria such as Clostridium, Bacteroides, Bifidobacterium, Pseudomonas, Staphylococcus, Streptococcus and Enterobacteriacea (Silva et al., 1987).

The use of probiotics as a medical instrument in certain pathologies taken very well, and evidence of their effectiveness is convincing, mainly as a result of clinical trials and meta-analyses regarding the malabsorption of lactose (Adolfsson et al. 2004; Piaia et al. 2003), gastrointestinal infections (Brownlee et al. 2003) and diarrhoea associated with antibiotic use (D'souza et al. 2002). In addition, the positive effects of probiotic bacteria Bifidobacterium, Lactobacillus and/or mixtures thereof in certain diseases �iseverything system. In the literature there is ample evidence that these positive effects.

Understanding of the relationships existing between the components of the intestinal microbiota and its interaction with the host organism is very complex. Genome facilitates the analysis of the response of the isolated bacteria to the conditions in the gut, in part by showing the metabolic ability of the strains, however, the conditions under which the manifestation of these abilities, as well as the conditions, allowing to allocate the majority of strains that shape the intestinal microbiota, are still largely unknown, thus identifying it is possible only by molecular methods with partial or complete identification of their genome. For this reason, developments in the field of probiotics and functional foods is growing rapidly.

Thus, taking into account that there are different effects between probiotic strains and what species of bacteria belonging to the same species may show different physiological characteristics, which gives them excellent or improved probiotic properties compared with other bacteria, identification and description of effects of new probiotic strains is very important in the light of their medical and industrial value.

The main objective of this study is Thu�to highlight the probiotic microorganisms with improved probiotic properties of resistance to acidic pH, resistance to salts of bile acids and adhesion to intestinal cells for subsequent use in food and pharmaceutical industries, particularly for their application in dairy mixes for baby food.

According to the present invention was proposed and characterized probiotic microorganisms isolated from the feces of children fed exclusively on mother's milk.

Increased resistance of the strains according to the invention to the pH and salts of bile acids attached probiotic microorganisms increased survival as they pass through the stomach and intestines and thus increases their colonize effect and, consequently, their antagonistic effects against other potentially pathogenic bacteria. On the other hand, increased the ability of probiotic strains of the invention for adhesion to the cells of the human intestine provides a stronger impact in modulating the entire immune system.

A brief summary of the invention

According to the present invention proposed probiotic microorganisms isolated from the feces of children fed exclusively on mother's milk. These microorganisms are used in food or pharmaceutical industry, in particular for use in dairy mixes for baby food, due to their probiotic�Kim properties providing beneficial impact on the health of those who take them.

For the allocation of these probiotic microorganisms according to the present invention has the following specific objectives: a) selection of strains of lactic acid bacteria obtained from feces of children fed exclusively on mother's milk; b) evaluation of resistance to pH and salts of bile acids; and b) assessing the capacity for adhesion to the cells of the intestinal epithelium.

Probiotic bacteria are often found in the feces of children. In addition, there is a certain approach, according to which probiotics should have the origin from the man, presumably in view of their better integration in the human intestine. However, many strains do not meet the definition of a probiotic, since they rarely possess or do not possess resistance to the digestive juices, and many of them are capable of adhesion to the intestinal epithelium. In the present invention selected children fed exclusively on mother's milk, to ensure that the selected bacteria are not commercial bacteria. In addition, it is shown that the intestinal microbiota of infants fed formula milk, very rich in bifidobacteria and lactobacilli.

Regarding probiotics, effective probiotic must have the following with�the properties:

1) the ability to exert beneficial effects on the host organism, for example, to provide immunity to disease;

2) does not cause pathogenicity or toxicity;

3) the ability to survive passage through the intestinal tract; for example, resistance to gastric acid and bile acids;

4) the ability of adhesion to the cells of the intestinal wall;

5) permanent short generation time and ability to remain viable for long periods in storage conditions;

6) is derived from person;

7) production of antimicrobial substances against pathogens; anticancer properties;

8) the ability to influence metabolic activity.

The health benefits associated with taking probiotics, includes the following:

1) relief of symptoms arising from the malabsorption of lactose;

2) increasing natural immunity to infectious diseases of the intestinal tract;

3) decrease in the concentration of cholesterol in serum;

4) improvement of digestion;

5) stimulation of immunity in the gastrointestinal tract;

6) the development of immunological tolerance to food antigens and the reduced risk of allergies.

Thus, the present invention relates to new probiotic microorganisms, weathering�enny from the feces of children. The invention in particular relates to microorganisms Lactobacillus rhamnosus HERO 22A (CNCM 1-4036), Lactobacillus paracasei HERO 7 (CNCM I-4034) and Bifidobacterium breve HERO 15B (CNCM 1-4035). These microorganisms are improved probiotic properties in comparison with microorganisms of the same species.

"Composition" refers to compositions or group of one or more of the ingredients, i.e. class and the number of elements present in complex substance (food or drug that, among other things), and the ratio in which they are located.

Under the "media" understand the substance of any type that provides the possibility of the cultivation, transfer, and/or the introduction of strains of the present invention. Depending on the destination and/or for uses for which it is designed for these strains, "media" can be of different nature. The present invention relates to pharmaceutically acceptable "carriers," such as "media", usually used in capsules, tablets or powders, as well as "carriers" formed of the ingredients or food products.

Foodstuffs intended for special nutrition, are food products that in mind their special composition or method of manufacture will undoubtedly differ from a mass of foodstuff which is suitable for the stated purpose of a food and which are submitted to the�titles on the market, with indication, they meet specified objectives (Council Directive 89/398/EEC of 3 may 1989 relating to the approximation of the laws of the participating countries on foodstuffs intended for special nutrition (DO, L series, No. 186 dated 30 June).

Under a special power to understand the power that must satisfy specific nutritional needs:

1) certain classes of people who have the disorder of the process of assimilation or metabolism; or

2) certain classes of people, having certain physiological state and receiving, as a consequence, a special benefit from the controlled intake of certain nutrients; or

3) healthy infants or young children.

Food additive refers to those foods, the purpose of which is to Supplement the normal diet and which consist of concentrated sources of nutrients or other substances that have a nutritional or physiological effect, alone or in combination, and are commercially available in dosage form, i.e. in the form of capsules, pills, tablets, lozenges, and other similar forms, sachets of powder, ampoules of liquids, drop vials and other similar forms of liquids and powders to be taken in small unit quantities (Directive 2002/46/EC of the European Parliament� and of the Council of 10 June 2002, relating to the approximation of the laws of the participating countries in respect of food additives).

Probiotic refers to the products of microbial cells or microbial cells or components of microbial cells that provide beneficial effects on health and well-being of the host.

Under prebiotic understand "nevereverever ingredient of the diet, providing a useful effect and stimulate the growth of intestinal bacteria, improving the intestinal balance of the host organism". Used prebiotics include inulin, fructo-oligosaccharides, galactooligosaccharides and oligosaccharides obtained by hydrolysis of pectins and other resins, and mucilage, and resistant starches and maltodextrins, as well as nucleotides.

The present invention relates to a strain of probiotic microorganism isolated from the feces of children fed exclusively on mother's milk, characterized by the fact that it consists of Lactobacillus rhamnosus HERO 22A (CNCM 1-4036), or Lactobacillus paracasei HERO 7 (CNCM 1-4034), or Bifidobacterium breve HERO 15B (CNCM 1-4035).

In a particular embodiment of the specified strain is a Lactobacillus rhamnosus HERO 22A (CNCM 1-4036).

In another specific embodiment of the specified strain is a Lactobacillus paracasei HERO 7 (CNCM 1-4034).

In yet another specific embodiment of the specified strain is a Bifidobacterium breve HERO IN 15 (CNCM 1-4035).

One in�the glossing strain described above is presented in the form of pure biological culture. In another embodiment the strain is selected.

In one embodiment described above, the microorganism strain is presented in the form of viable cells; in another embodiment the strain is presented in the form of non-viable cells.

Another object of the invention relates to compositions containing a microorganism strain as described above. In a particular embodiment said composition contains other probiotic material; in another embodiment, it further comprises a prebiotic material.

In another specific embodiment of the described composition comprises a carrier suitable for oral administration. The specified media is a pharmaceutically acceptable carrier, such as carriers, commonly used in capsules, tablets or powders.

In yet another specific embodiment of the specified carrier is a food product. The specified food product selected from the group consisting of milk and products obtained from milk, in particular dairy products and cheeses; cereals and their derivatives, including the dough for baking bread; soups and other similar products in dehydrated form; fermented meat products; processed products of fruits, juices and non-alcoholic beverages; foodstuffs for use in special diet.

Another object of the invention relative�are consistent with the strain of probiotic microorganism or song, as described above, for use in food. In one embodiment the specified diet related to child and/or adult and/or special diet.

In another embodiment the probiotic strain of microorganism or the composition described above is used for the manufacture of dairy mixes for baby food. In a particular embodiment of the compounds consist of ready-to-drink milk for baby food and/or cereal products for baby food and/or products for baby food.

In yet another embodiment, the strain of probiotic microorganism or a composition as described above is used for the manufacture of food additives.

In yet another embodiment, the strain of probiotic microorganism or composition, described above, is used for making special mixtures for oral and/or enteral nutrition.

In yet another embodiment, the strain of probiotic microorganism or a composition as described above is used for the manufacture of a product for pharmaceutical application, or product, is applicable as a medicament or for use in the manufacture of a pharmaceutical product.

In yet another embodiment, the strain of probiotic microorganism or a composition as described above, suitable for stimulating the immune system, and/or prevention/treatment of asthma, and/or pre�disgust/treatment of gastrointestinal disorders, and/or correcting/modulation main pathogens that infect the digestive system, and/or prevention/treatment of obesity and related diseases, including metabolic syndrome and diabetes, and/or diseases associated with aging.

These gastro-intestinal disturbances include changes of the passage of contents through the intestines, such as constipation, and changes in bioavailability of minerals, infections and malabsorption syndromes.

These malabsorption syndromes include disorders affecting the anatomy of the intestine, such as the syndrome shortened small intestine, and disorders affecting the physiology of the intestine, such as cystic fibrosis pancreas, malabsorption of Sugars, in particular lactose, changes in the absorption of lipids, food Allergy and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

Description of graphic materials

Fig.1 in the form of the table shows the effect of pH on the survival of strains of Lactobacillus rhamnosus CNCM 1-4036 and Lactobacillus paracasei CNCM I-4034 No. to the invention compared with two strains introduced into production. Specifically, the table presents the values of viability (%of colony-forming units) from the studies of stability of the isolated strains Lactobacillus rhamnosus 22A (CNCM I-4036), Lactobacillus paracasei 7 (CNCM I-4034) and� appropriate production controls to pH. The values are expressed as % survival obtained by comparison of the number of bacteria present in the control, with the number of bacteria present at different pH was investigated. The results are presented in percentage units in the column "%". It is seen that at pH 3 strains 7 and 22A have the same or a slightly higher resistance than industrial strains investigated. However, at pH 2 strain 22A has a very high viability compared with the other strains do not survive at that pH.

Fig.2 in table form shows the influence of bile salts (Oxgall) on the survival of strains of Lactobacillus rhamnosus CNCM I-4036 and Lactobacillus paracasei CNCM I-4034 according to the invention compared with two production strains. Thus, in the table presents the values of the viability of research of stability of the isolated strains Lactobacillus rhamnosus 22A (CNCM I-4036), Lactobacillus paracasei 7 (CNCM I-4034) and their respective production controls to the salts of the bile acids. The values are expressed as % survival when comparing the number of bacteria present in the control, with the number of bacteria present investigated at different concentrations of bile salts. The results are presented in percentage units in the column "%". Based on these results it can be concluded that strain 7, and strain 22A demonstrate snecial�but a higher percentage of survival, which is about two times higher percentage of survival was studied industrial strains, as when the concentration of bile salts is 0.3% and the concentration of bile salts is 0.7%. Both strains have a survival rate of over 100%, indicating that they may even multiply in the presence of these salts. Along with their high resistance to pH, this indicates a high colonizing potential of these strains.

Fig.3 in table form shows the adhesion of strains of Lactobacillus rhamnosus CNCM I-4036 and Lactobacillus paracasei CNCM I-4034 according to the invention on the cells of human intestinal HT-29 compared to the two production strains. This ability is expressed through the values of the vitality of research on the adhesion of strains of Lactobacillus rhamnosus 22A (CNCM I-4036), Lactobacillus paracasei 7 (CNCM I-4034) and their respective production controls in the cells of the intestinal epithelium. The values are expressed as % of attached bacteria compared to the number of bacteria present in the control. Both strains show the percent adhesion on cells human intestinal HT-29, far exceeding the percentage of adhesion was studied industrial strains, indicating their potential effect in the modulation of the activity of intestinal cells, including immunomodulation.

Fig.4 in table form shows the influence of pH on the survival shta�mA Bifidobacterium breve CNCM I-4035 according to the invention compared with two production strains. The specified effect is expressed through the values of the viability of sustainability research indicated strain to LV in comparison to his respective production controls. The values are expressed as % survival obtained by comparison of the number of bacteria present in the control, with the number of bacteria present at different pH was investigated. The results are expressed in percentage units in the column "%". It is seen that at pH 3 the strain 15B show a much higher resistance than the other two studied strains of bifidobacteria, while its viability is greater than 100%, indicating that the bacteria can even multiply at this pH.

Fig.5 in table form shows the influence of bile salts (Oxgall) on the survival of the strain Bifidobacterium breve CNCM I-4035 according to the invention compared with two production strains. This influence is demonstrated by studies of stability of the isolated strain of Bifidobacterium breve 15B (CNCM I-4035) and its corresponding production controls to the salts of the bile acids.The values are expressed as % survival obtained by comparison of the number of bacteria present in the control, with the number of bacteria present investigated at different concentrations of bile salts. The results are expressed in percentage units in the column "%". Indicators in�freeze in the presence of bile salts at low concentrations similar to the other two indicators of bifidobacteria. However, the strain 15B shows the best survival at high concentrations.

Fig.6 in table form shows the adhesion of the strain Bifidobacterium breve CNCM I-4035 according to the invention on the cells of the human intestine compared with two production strains. This ability is expressed through the values of the viability of the studies of adhesion of the isolated strain of Bifidobacterium breve 15B (CNCM I-4035) and its corresponding production controls in the cells of the intestinal epithelium. The values are expressed as % of attached bacteria compared to the number of bacteria present in the control. Strain according to the invention has a percentage of adhesion to the cells of the human intestinal HT-29, far exceeding the percentage of adhesion was studied industrial strains, indicating their potential effect in the modulation of the activity of intestinal cells, including immunomodulation.

Fig.7 illustrates the enzymatic activity (in units per ml of culture medium, U/ml) strain of Bifidobacterium breve CNCM I-4035 (Bifidobacterium breve 15B) according to the invention compared with two production strains (controls). The results are presented as described in Example 11. The obtained results allow to conclude that the enzymatic activity CNCM I-4035 coincides with the enzymatic activity of the species of the genus Bifidobacterium, giving the possibility to classify CNCM I-435 as belonging to the specified kind.

Fig.8 shows the enzymatic activity of the strains Lactobacillus rhamnosus CNCM I-4036 (Lactobacillus rhamnosus HERO 22A) and Lactobacillus paracasei CNCM I-4034 (Lactobacillus paracasei HERO 7) according to the invention compared with two production strains (controls). The results are presented as described in Example 11. The obtained results allow to conclude that the enzymatic activity HERO 7 HERO and 22A coincides with the enzymatic activity of species of the genus Lactobacillus paracasei and Lactobacillus rhamnosus, giving the possibility to classify CNCM I-4036 and CNCM I-4034 as belonging to the specified type and kind.

Fig.9 - Fig.9A and 9B show the results on the enzymatic activity of the selected strains CNCM I-4034 (Lactobacillus paracasei HERO 7) and CNCM I-4036 (Lactobacillus rhamnosus HERO 22A) and their controls in relation to carbohydrates and other substrates (API 50 CHL). The results are presented as described in Example 11. The obtained results allow to conclude that the enzymatic activity HERO 7 (CNCM I-4034) and HERO 22A (CNCM I-4036) coincides with the enzymatic activity of species of the genus Lactobacillus paracasei and Lactobacillus rhamnosus, giving the possibility to classify CNCM I-4036 and CNCM I-4034 specified as belonging to the genus.

Fig.10 - Fig.10A, 10B and 10B shows the results regarding the effect of probiotic bacteria of the present invention on L. monocytogenes CECT 4031 and S. sonnei CECT 457. (A) the Effect on L. monocytogenes CECT 4031 10x-concentrated supernatant liquid�Yu, obtained after 17 hours of growth of L. paracasei CNCM I-4034. (B) the Effect on L. monocytogenes CECT 4031 10x-concentrated supernatant obtained after 24 hours of growth of B. breve CNCM I-4035. (B) the Effect on S. sonnei CECT 457 10x-concentrated supernatant obtained after 24 hours of growth of L. rhamnosus CNCM I-4036.

Fig.11 shows the results for the inhibitory effects of 1% and 4% neutralized and neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time when the cultivation of 17 and 24 hours against bacteria Salmonella typhi CECT 725, Salmonella typhimurium CECT 443 and Salmonella typhimurium CECT 4594. (A) Inhibitory effect neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time 17 hours of cultivation in respect of Salmonella typhimurium CECT 443. (B) Inhibitory effect neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time of cultivation 24 hours in respect of Salmonella typhimurium CECT 4594. (B) Inhibitory effect neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time of cultivation for 24 hours against bacteria Salmonella typhi CECT 725. (D) Inhibitory effect neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time of cultivation for 24 hours against bacteria Salmonella typhi CECT 725. (D) Inhibitory effect neutralized supernatant of Lactobacillus paracasei CNCM I-4034 time when cultivation�Oia 17 hours against bacteria Salmonella typhi CECT 725.

Fig.12 shows the inhibitory effects of 1% and 4% neutralized and neutralized supernatant Bifidobactehum breve CNCM I-4035 time when the cultivation of 17 and 24 hours against bacteria Salmonella typhi CECT 725. (A) Inhibitory effects of 1% and 4% not neutralized supernatant Bifidobactenum breve CNCM I-4035 time of culturing for 17 hours against bacteria Salmonella typhi CECT 725. (B) Inhibitory effects of 1% and 4% neutralized supernatant Bifidobactenum breve CNCM I-4035 time of culturing for 17 hours against bacteria Salmonella typhi CECJ 725. (B) Inhibitory effects of 1% and 4% not neutralized supernatant Bifidobactenum breve CNCM I-4035 time of cultivation for 24 hours against bacteria Salmonella typhi CECT 725. (D) Inhibitory effects of 1% and 4% neutralized supernatant Bifidobactenum breve CNCM I-4035 time of cultivation for 24 hours against bacteria Salmonella typhi CECT 725.

Fig.13 shows the inhibitory effects of 1% and 4% neutralized and neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time when the cultivation of 17 and 24 hours against bacteria Salmonella typhi CECT 725, Salmonella typhimurium CECT 4594, Escherichia coli ETEC CECT 501, Escherichia coil ETEC CECT 515, Escherichia coli EPEC CECT 729 and Escherichia coli EPEC CECT 742. (A) Inhibitory effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of culturing for 17 hours against bacteria Salmonella typhi CECT 72. (B) Inhibitory effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of cultivation for 24 hours against bacteria Salmonella typhi CECT 725. (B) Inhibitory effects of 1% and 4% neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of cultivation 24 hours in respect of Salmonella typhimurium CECT 4594. (D) Inhibitory effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of culturing for 17 hours against bacteria Escherichia coli ETEC CECT 501. (D) Inhibitory effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of cultivation for 24 hours against bacteria Escherichia coli ETEC CECT 501. (E) Inhibitory effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of culturing for 17 hours against bacteria Escherichia coli ETEC CECT 515. (G) Inhibiting effects of 1% and 4% not neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of culturing for 17 hours against bacteria Escherichia coli EPEC CECT 729. (3) Inhibitory effects of 1% and 4% neutralized supernatant of Lactobacillus rhamnosus CNCM I-4036 time of cultivation for 24 hours against bacteria Escherichia coli EPEC CECT 742.

Fig.14 shows the reduction in the formation of infectious foci 1x-concentrated supernatant fluids strains really hard�after the acquisition (A) 17 hours of growth, and (B) 24 hours of growth when infected line HT-29 by viruses Ito, Wa and VA70.

Detailed description of the invention

According to the present invention proposed probiotic microorganisms with improved probiotic properties of resistance to pH, salts of bile acids and adhesion. Specifically, according to the present invention isolated and characterized bacteria Lactobacillus rhamnosus HERO 22A (CNCM I-4036), Lactobacillus paracasei HERO 7 (CNCM I-4034) and Bifidobacterium breve HERO 15B (CNCM I-4035), isolated from the feces of children.

It is known that the surface of the mucous membranes inhabited by a large variety of microorganisms. In adults the number of prokaryotic cells is greater than the number of eukaryotic cells, indeed, by some estimates 90% of the cells in the human body are microbial cells, while only 10% correspond to eukaryotic cells (Savage 1977). The impact of this microbial community in the human physiology is probably more obvious in the intestine, in view of the fact that this body contains most of these organisms. Their density in the proximal and middle sections of the small intestine is relatively small, but in the distal small intestine is significantly increased and may reach 108colony forming units (CFU) per ml of intestinal contents, and in the colon up to 1011-1012/g.

During the first few days of life, the composition of the intestinal microbiota significantly �smenyaetsya. At birth, the intestine is sterile and in the first few hours of life in the faeces begin to appear bacteria. First comes the colonization of the gastrointestinal tract maternal vaginal and fecal bacterial flora. The first microorganisms colonizing the intestine, are microorganisms with high reducing ability, including varieties such as enterobacteria, streptococci and staphylococci. The oxygen consumption of these bacteria gradually changes the environment in the intestine, which allows the growth of anaerobic bacteria, including lactobacilli and bifidobacteria. These bacteria colonize the body of the newborn, originate mainly from the mother and from the environment, and the type of delivery is one of the main determining factors for intestinal microbiota (Bezirtzoglou 1997).

The intestinal ecosystem is formed during the interaction between the microbiota, the intestinal epithelium, immune system, mucous membranes and nervous system of the intestine (Gordon et al. 1997). Comparison of normal rats and rats with sterile intestines revealed a number of anatomical, biochemical and physiological differences. For example, the presence of the microbiota increases epithelial metabolism, it also provides conjugation and removal of the hydroxyl group of bile acids, metabolize bilirubin and vos�tanalised cholesterol to coprotein.

Thus, the relationship between intestinal microbiota and are very close, and they can be seen as a symbiotic relationship, as, for example, the microbiota can break down carbohydrates, which is split in the intestine impossible due to the lack of enzymatic mechanism. The products obtained by this splitting, are used mainly as nutrients for the intestinal epithelium, as is the case with short-chain fatty acids. Moreover, the presence of the microbiota exerts immunomodulatory effect, since the main physiological characterization of the intestinal epithelium is the ability to run a strong response against invasive pathogens that can colonize the intestinal epithelium, and at the same time the lack of response to the bacteria included in the composition of food or the resident microbiota. This lack of response is an active process involving several mechanisms, known as oral tolerance. This process is necessary for in the host organism did not develop an inflammatory response to the presence of any microorganism and, consequently, to different microorganisms in the host organism have evolved different responses, thereby promoting stability of intestinal flora. For this reason� the presence of normal microbiota composition may contribute to the physiological, immune and metabolic development of the host body. This ecosystem balances, and any reason that violates the balance may lead to the development of pathology (diarrhea, inflammatory diseases).

It is important to understand the significance of this ecosystem, functioning of non-pathogenic strains or "good bacteria". On the basis of this idea, developed the concept of probiotics as mediators of human health. From the different studied areas, the influence of probiotics on the modulation of gene expression in various situations seems to be the most interesting.

As described above, according to the present invention the selected lactic acid bacteria and bifidobacteria with improved probiotic properties from the feces of children fed exclusively on mother's milk. To do this, the bacteria isolated were evaluated for the resistance to pH, resistance to bile salts of acids and their capacity for adhesion to the cells of the intestinal epithelium. The main results of the present invention indicate that in the feces of these children there are bacteria that are highly resistant to gastric pH, salts of bile acids and capacity for adhesion to the cells of the intestinal epithelium, which can be used as probiotics.

To test the probiotic activity of bacteria first neo�should be subjected to a series of in vitro tests with simulated conditions which will affect these bacteria in the body, they should remain viable under these conditions, and, consequently, to maintain their healthy properties. In the present invention used control bacteria (Example 12), against which bacteria according to the invention demonstrate improved their probiotic properties, are, for bifidobacteria: Bifidobacterium bifidum and Bifidobacterium longum supplied Him Espana S. A.; and for lactobacilli used 2 producing lactobacilli: Lactobacillus casei (Danone®) and Lactobacillus rhamnosus GG (LGG) (VALIO®).

Taking into account the usefulness of the bacteria constituting the present invention, the stimulation of the immune system and their impact on the major pathogens affecting the digestive system, control these bacteria were chosen because they currently are on sale at the international level in the form of fermented milk products in other dosage forms, and there are a number of publications about their probiotic effects, especially in the prevention of acute diarrhea in children and modulation of the immune system in both animals and humans.

Thus, these in vitro tests are as follows.

Resistance to gastric acidity

Before you reach the intestinal tract, probiotic bacteria must survive in their �rogojeni through the stomach (Henriksson et al. 1999). Secretion of acid gastric juice in the stomach forms the first defense mechanism against the most part of the microbial load supplied by mouth. For this reason, the survival of bacterial strains in acid gastric juice is the most accurate indicator of their ability to pass through the stomach. Research group on probiotics University College Cork (University College of Corkbased Probiotic Research Group) successfully isolated and identified lactic acid bacteria, demonstrating the perfect probiotic properties (Dunne et al. 1999). Conducted preliminary experiments to determine the degree of initial resistance strains of lactobacilli and bifidobacteria isolated from the ileum of a human. Human gastric juice was obtained from healthy individuals by aspiration through a nasogastric tube. Because pH changes in the stomach (he can reach 1.5), it was measured before use. This acid was added in Wednesday RSM (Wednesday Romance-Sharpe). Initial survival of the strains was estimated as 108% and 106% for lactobacilli and bifidobacteria, respectively, in the environment of the RSM (De Man et al 1960) with changing pH values from 2.0 to 3.4 by HCl. The results demonstrated a significant sensitivity of bifidobacteria to the acidity (Thornton 1996). Thus, the strains constituting the present izobreteny�, are sustainability indicators, exceeding the indicators of the strains investigated in these experiments, i.e., they have greater resistance to acidity (Fig.1 and 4).

Resistance to salts of bile acids

As explained above, for the characterization of the probiotic potential requires that the strain had a resistance to salts of bile acids (Lee and Salminen 1995). Bile acids are synthesized from cholesterol in the liver and are secreted into the duodenum from the gall bladder in the form of conjugates (500-700 ml/day), these acids undergo further chemical modification (deconjugation, dehydration, dehydrogenation and deconjugation glucuronic acid) in the colon, mainly only as a result of microbial activity. Both conjugated and dekonyugirovannye acids possess antibacterial activity, inhibiting the growth of strains of Escherichia coli, Klebsiella sp., and Enterococcus sp. in vitro (Lewis et al 1972; Stewart et al 1986). Dekonyugirovannye forms have greater inhibitory activity, and gram-positive microorganisms are more sensitive than gram-negative (Floch et al. 1972; Percy-Robb 1972). The group Dunne (Dunne et al. 1999) have chosen to use in the first analysis of solid nutrient medium with the addition of bovine, swine and human bile acids to a final concentration of 0.3% and 7.5% to assess the resistance of strains to bile salts to�slot. After the growth of lactobacilli and bifidobacteria the result was that they showed resistance to bovine bile acid, and that result from the use of porcine bile acids was significantly stronger inhibition against both groups of bacteria (Thornton 1996). Thus, regarding the search for a possible probiotic use in humans, the most significant result is their ability to grow in human bile. Taking into account that the human bile has a standard structure, and that the bile acid content are subject to considerable individual fluctuations, the use of ox bile with standard content of bile acid (OXGALL) as a replacement of human bile is a common practice in this area and makes it possible to record reproducible analyses. This method is used in the present invention for determining the resistance of bacteria of the present invention to salts of bile acids. The results obtained demonstrate that these bacteria have a higher resistance to salts of bile acids, their production controls (Fig.2 and 5).

The ability of probiotic strains for adhesion to the intestinal epithelium When breeding is also necessary to evaluate the adhesion of strains, adhering to epithelial�Oh tissue of the intestine, and the ability to colonize the gastrointestinal tract. The importance of this action is based on the fact that after breeding many probiotics are no longer able to colonize their target host. Indeed, currently available probiotics, apparently, only L rhamnosus GG remains in the gastrointestinal tract for a considerable period of time (Berg et al. 1998; Goldin et al. 1992). L. rhamnosus GG is able to adhesiveness to cells CACO-2, cells HT-29 cells and CACO-2 belonging to the cell lines of the human intestine, they exhibit morphological and physiological properties of normal colonocyte person, and they are used for studies of the mechanisms that mediate the adhesion of enteropathogens (Bernet 1994). In recent studies they have been used for breeding and, thus, to assess the potential of lactic acid bacteria or of bifidobacteria for adhesion (Coconnier et al 1992; Bernet et al. 1993; Greene &Klaenhammer 1994; Crociani et al 1995; Sarem et al. 1996; Tuomola &Salminen 1998).

On the basis of studies using these cell lines, it is concluded that the ability of Lactobacillus strains of the present invention for adhesion, comprising about 9% of the cells CACO-2 and about 5% on cells HT-29 (Tuomola et al, 1998; Dunne et al, 2001; Botes et al, 2008), very high (7,5% for L. rhamnosus CNCM I-4036 and 15.5% for L. paracasei CNCM I-4034 (Fig.3)) with�avanyu with well described by the strain Lactobacillus rhamnosus GG. In the art it is understood that the adhesion of bifidobacteria small compared to lactobacilli, regardless of their species (Dunne et al, 2001). However, for strains of Bifidobacterium bifidum and B. longum, used HERO Espana addressed in the present invention as controls, indicators of adhesion on cells HT-29 close to 9%. Similarly, for the bifidobacteria of the present invention, the values of adhesion in these cells is much higher, at 16.7 per cent (Fig.6).

Breeding bacteria

Thus, in the present invention carried out the breeding of bacteria with the use of special culture media (Example 4), both for bifidobacteria and lactobacilli. When you select used three new culture media that have been described as specific for bifidobacteria, they are: Wednesday BFM (Nebra and Blanch 1999), modified Colombian medium (Modified Columbia) and Wednesday Berens (Beerens) (Beerens 1991; Examples 4.1, 4.2 and 4.3), whereby upon receipt of the colonies of bifidobacteria was obtained the best result.For selection of lactobacilli used in the culture medium consisted of agar medium Cattail (Example 4.4).

In the present invention were incubated 4680 colonies of bacteria from different children and subjected these colonies breeding tests. After a first analysis on resistance to pH 3.0 and salts VC�tions acids remained 758 colonies with a viability of 90%, after studies of adhesion on the intestinal epithelium cells were only 90 colonies (Examples 5, 6 and 7).

These colonies were separated on lactobacilli and bifidobacteria on the basis of the culture medium from which they were obtained. Performed direct molecular identification (Example 10) amplification of 16S ribosomal RNA (rRNA) of each colony for further sequencing and searching for homologues in the BLAST database of the National center for biotechnology information (NCBI).

In total, there were 29 bacterial strains isolated from the environment Berens, 13 strains isolated from the environment Cattail, and 10 strains isolated from Colombian modified environments that have successfully passed the selection. Taking into account the number of selected colonies, as it was explained, were their direct molecular identification by 16S rRNA gene amplification of each colony for further sequencing and searching for homologues in the NCBI database (BLAST).

Strains classified as lactobacilli, sequenced, and these sequences were aligned relative to each other to determine the presence of bacteria with identical 16S rDNA gene, and was found 41 bacterium, which can be divided into the following 2 groups.

The group with 99% homology to the fragment of 16S rDNA length 1474 base pairs (BP):

Lactobacillus ramnosus, strain R-11;

Lactobacillus rhamnosus, strain La;

Lactobacillus rhamnosus strain MNFLM01;

Lactobacillus rhamnosus strain IDCC 3201;

Lactobacillus rhamnosus, strain YIT 0105 (corresponds to ATS 7469);

Lactobacillus rhamnosus, Lcr35 strain 16S.

Given these results from this group were selected bacteria with the best performance in the tests of stability (Examples 7 and 9), and they were identified as Lactobacillus rhamnosus HERO 22A (which later at the Institut Pasteur (CNCM, national collection of cultures of microorganisms (National Collection of Microorganism Culture); PASTEUR INSTITUTE; 25, Rue du Docteur Roux F-75724 Paris) was assigned a number of Lactobacillus rhamnosus CNCM I-4036, where they were deposited on 2 July 2008).

Another group with 100% homology to the fragment of 16S rDNA length 1276 p. O.:

Lactobacillus paracasei, strain T11-9;

Lactobacillus paracasei, strain T7-10).

Lactobacillus casei, strain KLDS 1.0720;

Lactobacillus casei, strain L5;

Lactobacillus casei, strain YIT 0209 (corresponds NCDO 151);

Lactobacillus casei, strain YIT 0180 (corresponds to ATS 334);

Lactobacillus paracasei, strain IMPC 2.1;

Lactobacillus paracasei, strain NRIC 1944;

Lactobacillus paracasei, strain NRIC 1942;

Lactobacillus paracasei, strain NRIC 1938;

Lactobacillus paracasei, strain NRIC 1934;

Lactobacillus paracasei, strain NRIC 0638;

Lactobacillus casei of ATS 334;

Lactobacillus paracasei, strain DJ1;

Lactobacillus casei, strain Ru2-2i;

Lactobacillus paracasei, isolate 3C;

Lactobacillus paracasei, isolate 2C;

Lactobacillus paracasei;

Lactobacillus casei, strain MCRF-284;

Lactobacillus sp. L02;

Lactobacillus paracasei;

Lactobacillus paracasei, strain SM20;

Lactobacillus casei strain BL23;

Lactoacillus paracasei, subspecies paracasei;

Lactobacillus paracasei, subspecies paracasei;

Lactobacillus casei;

Lactobacillus paracasei, SSP. tolerans.

Given these results from this second group were selected bacteria with the best performance in the tests of stability (Examples 7 and 9), and initially they were identified as Lactobacillus paracasei HERO 7 (which later at the Institut Pasteur (CNCM, national collection of cultures of microorganisms; PASTEUR INSTITUTE; 25, Rue du Docteur Roux F-75724 Paris) was assigned a number of Lactobacillus paracasei CNCM I-4034, where they were deposited on 2 July 2008).

Subsequently, the same procedure was conducted with a group of bifidobacteria, it was revealed only one group with 100% homology to the fragment of 16S rDNA length 1136 p. O.:

uncultivated bacterial clone rRNA235;

Bifidobacterium breve strain of ATSS 15700.

Given these results from the group of Bifidobacterium bacteria were selected with the best results in terms of resistance (139,6% at pH 2,5) (Examples 7 and 9), and initially they were called Bifidobacterium breve HERO IN 15 (which was subsequently assigned another name for Bifidobacterium breve CNCM I-4035 at the Pasteur Institute (CNCM, national collection of cultures of microorganisms; PASTEUR INSTITUTE, 25, Rue du Docteur Roux F-75724 Paris), where they were deposited on 2 July 2008).

Thus, these bacteria are classified as:

Lactobacillus rhamnosus HERO 22A;

Lactobacillus paracasei HERO 7;

Bifidobacterium breve HERO 15B;

were sent to the Institute �Astaire for their deposition, where they were found to be unique, and they were awarded following the final designation.

Initial designationFinal designation
Lactobacillus paracasei HERO 7CNCM I-4034
Bifidobacterium breve HERO 15BCNCM I-4035
Lactobacillus rhamnosus HERO 22ACNCM I-4036

The results in terms of resistance to pH. salts of bile acids and adhesion on cells

As indicated above, in order for the probiotic strains exerted beneficial effects on the intestine, they must survive the passage through the stomach, showing resistance to its acidity (pH 2.5 to 3.5) (Holzapfel et al. 1998), and, on the other hand, they must be resistant to the salts of the bile acids present in the small intestine to reach the large intestine (Oties et al. 2003).

In the present study, bacteria were incubated at pH 3.0 for 3 hours, although it was described that 90 minutes should be enough for playing time from receipt in the stomach to the exit from the stomach (Jin et al. 1998). In this case, the selected strains and controls demonstrated the viability close to 100% (Example 12/Fig.1 and 4), but the impact of pH of 2.5 showed, Thu� it was very selective, since no control is not demonstrated viability, and only strain Lactobacillus rhamnosus 22A (CNCM I-4036) proved to be viable. In other words, the strain Lactobacillus rhamnosus 22A according to the invention are considerably more stable to acid than the control bacteria, which therefore facilitates its passage in the gastrointestinal tract and subsequent colonization. On the other hand, the strain L. rhamnosus 7A according to the invention shows higher viability at pH 3.0 than the strains used as controls (Example 12, Fig.1, 4), which means a more efficient passage into the small intestine.

The viability of probiotic cultures at pH 3.0 for 2 hours in a medium containing 500-1000 mg (0,05-0,1%) of bile salts per liter, is considered as standard tests tolerability acid and salts of bile acids (Snelling 2005), although the appropriate concentration of bile salts for selection of probiotics 0.3 percent. In tests for resistance to salts of bile acids, carried out at different concentrations (0.3% and 0.7%), for bacteria according to the invention in all cases had received a higher than 100%, exceeding the indicators of production control bacteria (Example 12, Fig.2 and 5). In conclusion, strains of lactobacilli according to the invention is more resistant to pH and salts of bile acids than other bacteria, currently used as� probiotics.

It has been described that the lactobacilli generally demonstrate greater resistance to the conditions of the gastrointestinal tract, especially in relation to acidity and bile salts (Ross et al. 2005). The results obtained are consistent with this description, because the sustainability indicators to the conditions of the gastrointestinal tract of lactobacilli slightly higher than those of bifidobacteria.

As also mentioned above, another very important aspect for the introduction of probiotics in the intestinal microbiota is the ability to adhesion to the cells of the intestinal epithelium, because they prevent the elimination of probiotic strains due to peristaltic movements and other bacteria constituting the intestinal microbiota. In addition, adhesion is the first stage of colonization, and probably a prerequisite for competitive displacement of enteropathogens (Forestier et al. 2001; Lee et al. 2003) and immunomodulation of the host organism (Ouwehand et al. 1999; Plant and Conway 2002).

In the present invention, the properties of adhesion of different strains was studied using cells HT-29 as a model of intestinal epithelium in vitro (Example 12, Fig.3 and 6). Painting adhesion demonstrated specificity for each strain, as they were obtained very different values, although they belong to the same species. This can be understood by comparing the ability of various described blood collection tube�ticks for adhesion, for example, Lactobacillus casei (Fyos®) index of adhesion is 14.4%, while for Lactobacillus casei (Lactophilus®), it is 2.6% (Morata De Ambrosini et al., 1999). As mentioned above, the performance of adhesion of Lactobacillus strains of the present invention is significantly higher (7,5% for L. rhamnosus CNCM I-4036 and 15.5% for L. paracasei CNCM I-4034) than other bacteria such as Lactobacillus rhamnosus GG, which they make up about 5% on cells HT-29 (Dunne et al., 2001) (table 3). Similarly, a Bifidobacterium according to the invention B. breve CNCM I-4035 also demonstrates adhesion to these cells with a figure far in excess of 16.7% (Fig.5), compared with 9% in bacteria, used as controls.

These data confirm the results of the present invention, demonstrating the viability of different probiotic strains. In this particular case controls of lactobacilli demonstrate adhesion (4%), corresponding to half of the indicators adhesion controls bifidobacteria (8%), however, isolated strains of the present invention exhibit higher adhesion than their controls, because the alignment of the 16S rRNA of the different groups of strains were selected with the best rates of adhesion to epithelial cells.

Thus, in the case of lactobacilli strains were selected, demonstrating the adhesion of the order of 7.48 and for 11.55% compared to 4.80 and of 4.09% in their controls (Fig.3), and in case�e bifidobacteria strains were selected, demonstrating the adhesion of the order of 16.7% compared to 8.8 and 9.1% in their controls (Fig.6). In other words, the bacteria, which is a different object of the present invention, have a higher ability to colonize and delay in the intestines and therefore will provide the best probiotic effect.

Results characterization of isolated bacteria

The study of 16S RNA

Have developed various molecular methods of identification, determining the composition and quantity of the total bacterial community of the intestine, most of which are based on the study of 16S ribosomal RNA (rRNA), because in the last ten years, the 16S rRNA gene has revolutionized the ways in which experts on the taxonomy classify and identify bacteria. 16S rRNA gene contains both visokomoralniye and high-conservative region, and differences of sequences is used to determine phylogenetic relationships and to separate bacteria species and strains. Available database with over 200000 16S rRNA genes, such as, for example, NCBI/BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), a database project ribosomal RNA (ribosomal database project, RDP; http://rpd.cme.msu.edu/htlm) and EMBL (http://www.embl-heidelberg.de/); in these databases compare existing 16S rRNA gene sequences obtained with the new sequences. As shown � Example 2 isolated strains of the present invention show high homology with Lactobacillus rhamnosus, Lactobacillus paracasei and Bifidobacterium breve, which is consistent with the documents of the prior art, in which it is determined that the child is fed breast milk, high levels of bifidobacteria in the feces, accounting for 40-60% of the total microbiota, and Bifidobacterium breve detected in a large percentage of cases (Harmsen et al. 2000). Also, a high percentage of lactobacilli, mainly L. casei, L paracasei, L. acidophilus, among others (Heiling et al. 2002, Satokari et al. 2002).

The percentage homology of the 16S rRNA gene of the isolated strains was very high (99-100%) according to the database NCBI/BLAST (Example 2). The 16S rRNA gene fragments have a length of approximately 1400 p. O., and secondary fragments have a length of 1474 p. O. for Lactobacillus rhamnosus HERO 22A (CNCM I-4036), 1274 p. O. for (CNCM I-4034) 7 HERO and 1118 p. O. for Bifidobacterium breve 15B HERO (CNCM I-4035). More precisely, the latter demonstrates 100% homology Bifidobacterium breve ATCC 15700 and uncultivated clone Bifidobacterium, for this reason, the sequenced amplification of a fragment of the 16S rRNA gene would be interesting, but in this case getting larger fragment was not possible because the oligonucleotides 27F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2), used to amplify a fragment approximately 1400 p. O., were not suitable for amplification of the 16S rRNA gene of the strain Bifidobacterium breve 15B HERO (CNCM I-4035). Therefore used other multi�e oligonucleotides, such as 39F (SEQ ID NO: 3) and 1391 R (SEQ ID NO: 4), which amplificateur smaller fragments.

The sequenced fragments of the 16S rRNA gene of the isolated strains of lactobacilli demonstrated 99% homology to the group of Lactobacillus rhamnosus, and other strains showing 100% homology to a number of Lactobacillus paracasei and a small number of Lactobacillus casei. Performed alignment of 16S rRNA gene fragments of controls, strains Lactobacillus rhamnosus HERO 22A (CNCM I-4036) and Lactobacillus paracasei 7 HERO (CNCM I-4034) and fragment of L. paracasei, which had high homology to the selected strain. This shows that the control strains L. rhamnosus HERO 22A (CNCM I-4036) and L. paracasei 7 HERO (CNCM I-4034) 4 different bases. Controls LGG and L. casei also 4 different bases. The strain Lactobacillus rhamnosus 22A, HERO (CNCM I-4036) demonstrates the difference between both of the controls are on 1 base, contrast control LGG on 1 base and distinct from L. casei on 3 bases.

8 this case, the differences between leveled a few strains, indicating a significant conservatism of the 16S rRNA gene of these strains.

To extend information about the genome of the studied bacterial strains were amplified intergenic space that is present between genes 16S and 23S rRNA, the amount of which, as is known, varies significantly (Barry et al. 1991, Navarro et al. 1992), and which is also used to distinguish species of prokaryotes (Barry et al. 1991). The selected strains length of fragments migennes� space varies as follows: Lactobacillus rhamnosus HERO 22A (CNCM I-4036) - 579 p. O.; Lactobacillus paracasei 7 HERO (CNCM I-4034) - 512 p. O.; and Bifidobacterium breve 15B HERO (CNCM I-4035) - 182 p. o. Intergenic fragments of the 16S-23S compared with the same fragments in the database NCBI/BLAST, and the results revealed 100% homology of the strain Lactobacillus rhamnosus HERO 22A (CNCM I-4036) selected Lactobacillus rhamnosus TS1 and Lactobacillus rhamnosus PS1 16S. When comparing the results of the homology shown for 16S rRNA, the results are completely different, therefore, this strain appears to be missing in the database or in the database NCBI/BLAST for more info on 16S rRNA and not about the intergenic space 16S-23S. These results show that isolated bacteria according to the invention is unique, as confirmed by the Pasteur Institute, namely when they were given the name Lactobacillus rhamnosus CNCM I-4036.

In the case of the strain Lactobacillus paracasei 7 HERO (CNCM I-4034) the results of the study the intergenic space 16S-23S demonstrated 100% homology to Lactobacillus casei ATCC 334. Database NCBI/BLAST contains a complete genome, and it is presented in the list L. casei, showing 100% homology to the 16S rRNA gene, therefore, it is highly likely that the selected strain is a L. casei ATCC 334; in any case, when the sequencing of other genomes, such as genes 23S or 5S or others can confirm or refute the idea that it corresponds to this strain. In this regard, after the appropriate tests at the Pasteur Institute this strain was recognized as unique.

In the case of the strain of Bifidobacterium fragment is quite small, thus, the intergenic space controls were amplified with obtaining fragment length 165 p. O. for Bifidobacterium longum and 298 p. O. for Bifidobacterium bifidum, which confirms the presence of a wide variety of block sizes. Conducted alignment control strains isolated strain of Bifidobacterium breve IN 15 HERO (CNCM I-4035) and the strain showed 99% homology (NCBI/BLAST) strain according to the invention, it was observed significant differences between the controls and the strains Bifidobacterium breve; however, the strain of the invention and the strain is showing 99% homology (Bifidobacterium breve, internal transcribed spacer (ITS) 16S-23S, strain Y8) differ by only one base. This strain is completely different from the strain with 100% homology to the 16S rRNA gene, therefore, it can be a strain that is not entered into the database.

All these results demonstrate that three of the isolated strain of the present invention are new, because they were not described in the prior art.

Phenotypic identification

As shown in Example 11, the present invention is used set to evaluate the fermentation of carbohydrates (API 50CH) (Fig.8) and kit for determination of enzyme activities (API Zym) (Fig.9) for the analysis of biochemical abilities of selected species. They are quick and theoretical�cally reproducible method phenotypic identification of pure bacterial cultures. These tests were used for description and identification of lactobacilli in milk (Medina et al. 2001), yogurt and other dairy products (Andrighetto ef al. 1998) and cheeses (Andrighetto et al. 1998, Bouton et al. 1998 and De Angelis et al. 2001). However, doubts arose as to the reliability of these tests, especially the API 50CH, because it was originally developed for identification of strains of lactobacilli for clinical use, and because the database of the manufacturer has not been updated for some species of lactobacilli, which leads to questionable results identification (Andrighetto et al. 1998 and Collins et al. 1993), however, provides information has value to obtain the phenotypic characteristics of the isolated strains.

As can be seen from Fig.8, the selected strains and the controls show low proteolytic activity, functioning as trypsin and α-chymotrypsin, although they showed activity different from leucine, valine they demonstrate the minimal activity of bifidobacteria and very high activity of lactobacilli.

Bifidobacteria also show a high activity against α - and β-galactosidase and α-glucosidase. α-Galactosidase and α-glycosidase the activity of other lactic acid bacteria may differ from the activity of Bifidobacterium, as described by Desjardins et al. (1990).

You can see that bifidobacteria and selected strain of Lactobacillus ramnosus HERO 22A (CNCM I-4036) high α-galactosidase activity. In other words, this activity is important because the hydrolysis of certain Sugars, such as α-D-galactosyl-oligosaccharide, makes possible the selective proliferation of bifidobacteria in the intestinal tract (Gopal et al. 2001; Gulewicz et al. 2002), as bifidobacteria can use galactooligosaccharides. Evaluation of phenotypic properties of bifidobacteria confirmed their previous classification of genetic methods and to determine their enzymatic activity make possible the formation of lactic acid using a range of hydrocarbon substrates, preferably polymers of glucose with α-bonds.

It has been described that a small number of strains from other sources and genera, including Lactobacillus, demonstrate β-glucuronidase activity (Gopa et al. 2001; Hopkins et al. 1998). Thus, strains of lactobacilli and bifidobacteria of the present invention, as well as their controls, do not exhibit the levels of β-glucuronidase activity that is seen as a favorable property. The lack of R-glucuronidase activity is a property that should have all probiotics are considered as good, as this enzyme is formed by the enzymes of feces (nitroreduktaza, azoreductase) of microbial origin, which are responsible for the conversion of procarcinogens to carcinogens (Kurman 1988). Nanno et al. (1986) trademonster�grown, the extracts of β-glucuronidase-positive bacteria enhance the mutagenic activation of biliary metabolites of benzo [a] pyrene, while the extracts of β-glucuronidase-negative bacteria do not exhibit such activity. In conclusion, it can be noted that because these bacteria exhibit low activity against other carbon sources such as mannose, fucose and glucuronides, they prefer lactose and glucose as sources of carbon for metabolism, although selected strain of Lactobacillus rhamnosus HERO 22A (CNCM I-4036) demonstrates the enzymatic activity of a-fucose, which entails a positive effect on the fermentation fucosyl-lactose derivatives such as those present in breast milk, providing a known beneficial effect on the growth of bifidobacteria. This property means that the probiotics of the present invention is particularly applicable in a child's diet, although they can also be used in the diets of adults and special meals.

Another aspect to be mentioned is the result of the API SN (Fig.9B), where the strain Lactobacillus paracasei 7 HERO (CNCM I-4034) aged inulin, that is, has the ability which is lacking in its controls. Ability to ferment inulin is not a common property of lactobacilli (Cebeci and Gurakan 2003; Makras et al. 2005). This UK�shows, what strain according to the invention has the ability to ferment fructooligosaccharides (FOS), widely used as prebiotics in infant feeding, stimulating the development of the intestinal microbiota dominated by lactobacilli and bifidobacteria. This property bifidobacteria according to the invention is confirmed by the fact that the probiotics of the present invention is particularly applicable in a child's diet, although they can also be used in the diets of adults and special food. Was shown the ability of inulin and FOS to increase the number of bifidobacteria in the colon (Roberfroid et al. 1998; Van Loo et al. 1999).

Probiotic activity

In accordance with the above requirements regarding resistance to pH, resistance to salts of bile acids, the ability of adhesion to the cells of the intestinal epithelium and sequencing 16S get allocated and a specific probiotic microorganism. The next stage is to obtain the characteristics of its probiotic activity, more specifically, it is necessary to describe the probiotic properties of Lactobacillus rhamnosus HERO 22A (CNCM I-4036), and/or Lactobacillus paracasei HERO 7 (CNCM I-4034), and/or Bifidobacterium breve HERO 15B (CNCM I-4035).

Taking into account that the bacteria according to the invention meet the requirements for probiotics, relative resistance to pH, salts of bile acids and abilities � adhesion, these bacteria can be applied in various fields in which the use of probiotics is a well-known part of modern art, among other things, in the treatment and prevention of various pathologies, such as malabsorption of lactose, reduced levels of cholesterol in plasma, various types of diarrhea, inflammatory bowel disease, cancer, disorders caused by pathogenic bacteria, and so on. Thus, these probiotics are applicable in various areas such as, inter alia, in the fight against the main pathogens affecting the digestive system and stimulate the immune system.

Pathogenic bacteria

From different types of studies conducted with the use of probiotics, of particular interest are studies involving pathogenic bacteria affecting the digestive system. When choosing a pathogenic bacteria is necessary to take into account the mechanism of their pathogenicity, which varies with different bacteria. The bacterium of interest is enterotoxigenic Escherichia coli, since the formation of enterotoxin is an important cause of diarrhea in humans. In recent studies described that certain lactic acid bacteria have antagonistic effect against enterotoxigenic Escherichia coli (Gopal et al. 2001; Todoriki et al. 001; Chu et al. 2005; Tsai et al. 2007) and in relation to other pathogenic bacteria such as Salmonella typhimorium and Shigella flexneri (Tien et al. 2006; Jankowska et al. 2008).

Another interesting aspect of probiotic strains is a possible formation of substances called bacteriocins, which secrete some bacteria to compete with other microorganisms growing in the same niche. These substances can inhibit the growth or adhesion of pathogenic bacteria on the intestinal epithelium cells, and can produce some lactic acid bacteria (Klaenhamer 1993; Jack et al. 1995; Sablon et al. 2000).

An important aspect is the knowledge of the interaction of probiotic or pathogenic bacteria with the intestinal epithelium cells, because all the bacteria present in the human microbiota interact with them.

It has been described that the presence of pathogenic bacteria in the cells of the intestinal epithelium stimulates the profile of Pro-inflammatory response (Th1) with the release of cytokines such as tumor necrosis factor-a (TNF-α) and interleukin-8 (IL-8), the activation of NF-κb (Tien et al. 2006; Nagy et al. 2006) and increased their expression. The answer in most cases is partially reduced in the presence of probiotic bacteria, suggesting a useful effect (music from servin 2004).

So, some probiotics can modulate the properties of dendritic cells (DC), including their ability to activate specific�th immune response (Kelsall et al. 2002). The balance of stimulation and tolerance after exposure to probiotic bacteria in the gut may be important for maintenance of homeostasis and the ability to perform their useful protective function against pathogenic bacteria in the digestive system of the host.

It is shown that the probiotic strains of the present invention have an inhibitory activity against the growth of intestinal pathogenic microorganisms such as pathogenic bacteria, among others, Helicobacter pylori, Listeria monocytogenes, Shigella sonnei, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, intestinal Salmonella and enteric viruses such as Rotavirus.

Malabsorption of lactose

Mammals are born with enough lactase activity to use lactose-free milk. After the cessation of breastfeeding, this activity gradually decreases with age, and the use of food containing lactose, leads to signs and symptoms associated with lactose intolerance (abnormal increase gas formation, abdominal pain, diarrhea and the like). It is known that the use of probiotics, releasing lactase, promotes the absorption of lactose in the intestinal lumen, eliminating the symptoms of lactose intolerance.

Lowering cholesterol levels

The result is high levels of lipids, such as cholesteryl nom� and triglycerides, in the blood is a high risk to human health because they are associated with heart disease. Because foods low in fat or microorganisms involved in the metabolism of lipids, is very useful to prevent these States were described strains of lactobacilli regulating the levels of lipids in serum. This probiotic effect is closely related to the hydrolysis of bile salts.

In various studies in animals (Akalin et al. 1997; Fukushina and Nakano 1996) and in humans (Lin et al. 1989) observed that the introduction of probiotics can reduce the concentration of cholesterol in serum.

Diarrhoea

Really, it is best confirmed by documentary evidence of the clinical application of probiotics in the treatment of acute diarrhea. Clinical trials have demonstrated the efficacy of probiotics in the treatment to prevent and/or treat some intestinal disorders, including diarrhea caused by antibiotic use (McFarland et al. 1995), diarrhoea in adults (Hocter et al. 1990), diarrhoea in children (Cetina-Sauri and Sierra 1994) and travelers ' diarrhea (Kollaritsch et al. 1993).

In these cases, the probiotic used as a biotherapeutic agent that affects the expression and activity of a large number of enzymes and proteins that regulate intestinal epithelium and possibly the microbiota.

In from�Oseni diarrhea, associated with the use of antibiotics, taking probiotics when prescribing antibiotics may weaken the expression and/or decrease the duration of diarrhea. The most widely used microorganisms: Enterococcus faecium SF6 (Wunderlich et al. 1989), Lactobacillus GG (Siitonen et al. 1990; Vanderhoof et al. 1999), Lactobacillus acidophilus, L. bulgaiicus and Saccharomyces boulardii. These agents help to reduce changes in the microbiota, changes the consistency and frequency of stool.

Diarrhea caused by Clostridium difficile, which is an opportunistic pathogen that uses changes in the intestinal microbiota due to antibiotic therapy, leading to a wide range of clinical symptoms ranging from mild uncomplicated diarrhea to severe colitis with toxic megacolon, abdominal and systemic complications, which may lead to death of the patient. Diarrhea usually begins a few weeks after the start of antibiotic therapy. The pathogenetic chain begins with changes in the intestinal bacterial microbiota caused by antibiotics, which makes possible the colonization of C. difficile, when the agent enters the human body. Then the bacteria release toxins, causing tissue damage. Pathogenic strains of C. difficile produce toxins, called A and B. S. boulardii inhibits toxins A and b by releasing proteases 54 kDa,�ora breaks down these toxins and their membrane receptors (Castagliuolo et al. 1999).

Observed that the oral administration of Lactobacillus rhamnosus GG and Saccharomyces boulardii is effective in restoring normal microbiota in patients.

The travelers ' diarrhea occurs due to bacterial, viral or parasitic infection. It is caused by many microorganisms, and they probably differ in different countries. According to the frequency they include: E coli, Shigela, Salmonella, Campylobacter, Rotavirus and Giardia lamblia. The travelers ' diarrhea affects half of travelers heading to the high-risk areas. Bacteria used as probiotics in various studies, are: lactobacilli, bifidobacteria, streptococci and enterococci. Regarding travelers ' diarrhea is the most effective probiotic is Lactobacillus GG.

Inflammatory bowel disease

One of the main indications for the use of probiotics is the imbalance of the microbiota and the immune system. Following this practical interest, the study of inflammatory bowel disease is one of the most interesting of the main issues for the possible use of probiotics as clinical therapeutic agents.

Research conducted in this area, has provided important information about the clinical use of probiotics and gene expression of different mediators involved in �year of the disease.

Inflammatory bowel diseases (IBD) are chronic inflammatory bowel disorders of unknown origin (ulcerative colitis, Crohn's disease), their pathogenesis is complex and includes at least 3 important elements: the factors of genetic predisposition, intestinal microflora and immune-mediated damage. It has been hypothesized that inflammatory bowel diseases arise from an abnormal response of T-cells in the microbiota, also discussed that these diseases may lead to the presence of pathogenic organisms.

In biopsies of the colon of patients with IBD reduced levels of lactobacilli and bifidobacteria (Fabia et al. 1993; Favier et al. 1997). Traditional methods for the treatment of IBD have focused on the suppression or modulation of the host immune system, and among these treatment methods, the use of antibiotics is an effective way to treat Crohn's disease. This indicates that the use of probiotics to modify the microflora may be important in the treatment of IBD.

In the prior art there is a recent study where it was found that in the faeces of patients with Crohn's disease during active periods of the disease reduced the amount of β-galactosidase. This decrease correlates with the decrease in the content of bifidobacteria, which is the source of β-galactosidase (Favier et al. 1997).

R�to

Colorectal cancer is one of the most severe complications of IBD, including ulcerative colitis and Crohn's disease (Eaden et al. 2001). The exact mechanism by which GCS may lead to the process of carcinogenesis are not well understood. Guessing he may be the cause of the chronic inflammatory process (Weitzman & Gordon 1990), which in some experimental models can act as a stimulator of tumor development.

Intestinal microbiota and the immune system play an important role in the regulation of carcinogenesis. Probiotics can affect those skills, so to combat colorectal cancer in this area, significant efforts were made. It was found that probiotics can reduce the concentration of enzymes, mutagens, secondary bile salts, which may be involved in the process of carcinogenesis in the colon (Wollowski et al. 2001). Epidemiological data confirm that daily consumption of fermented foods has a protective effect against adenomas or colon cancer (Rafter and Glinghammar 1995).

Symbiotic combination comprising a mixture of a probiotic and prebiotic, was used to study cancer prevention. This combination increased the levels of short chain fatty acids, which are the main products of bacterial enzyme�tion, the basic meaning of which is that they act as source of nutrients for intestinal epithelium. They are associated with induction of differentiation, inhibition of proliferation and increased apoptosis in vitro (Heerdt et al. 1997; Medina et al. 1997), and they can play a role in preventing certain diseases, such as gastrointestinal disorders and cancer (Julia et al. 2006).

The present invention not only demonstrates the ability of selected strains to inhibit the growth of pathogenic bacteria and intestinal enteroviruses, but it also has outstanding characteristics that define probiotic properties of microorganisms, such as resistance to pH, salts of bile acids and adhesion in the intestine, compared with the control probiotic bacteria, known in the art.

In all cases, the results showed that the probiotic properties of bacteria according to the invention is better than that of the specified control bacteria. So, it is known that the activity of bacteria, used as controls, are concentrated in the following areas:

L. casei inmunitas from Danone

The beneficial effects associated with probiotic compositions containing L. casei inmunitas (Actimel®), include better immune response to various infectious agents, increasing the level of activation of immune cytokines si�topics the improvement of the proliferative response of T cells and modulation of expression of NK-cells. According to observations, the consumption of Actimel® improves the prognosis of diarrhea in children-related infections, reducing their severity and duration.

Compositions containing L. casei inmunitas, in turn, have a positive anti-inflammatory effect on the mucosa of the human colon because they enhance the immune response in the host organism, which is useful for individuals with inflammatory bowel disease and to prevent colon cancer.

In the light of the above, and various comparative experiments showed the best probiotic properties of Lactobacillus paracasei HERO 7 (CNCM I-4034) compared to L. casei inmunitas, Lactobacillus paracasei HERO 7 (CNCM I-4034) is particularly applicable, inter alia, in the prevention of various pathologies, such as malabsorption of lactose, reduced levels of cholesterol in plasma, various types of diarrhea, inflammatory bowel disease, cancer and so on, to improve the immune response to various infectious agents, improvement in diarrhea in children as an antiinflammatory agent to the mucosa of the human colon (and thus in the prevention of colon cancer).

LGG

Lactobacillus GG is capable of adhesion to the intestinal cells by stimulating them�unny response and preventing diarrhea, caused by pathogens. Various studies have demonstrated that the use of compositions containing LGG, such as Bioactif from Kaiku®, inhibits the competitive colonization of the intestine by pathogenic microorganisms. These microorganisms, in turn, produce antimicrobial compounds that inhibit the growth of pathogenic strains, resulting in the inhibition of growth of pathogenic strains. Thus, the use of compositions with LGG maintains or restores the balance of intestinal microflora, optimizing the processes of absorption as a function of the intestinal mucosa.

In the light of the above, and various comparative experiments showed the best probiotic properties of Lactobacillus rhamnosus HERO 22A (CNCM I-4036) compared with Lactobacillus GG, Lactobacillus rhamnosus HERO 22A (CNCM I-4036) is particularly applicable to, inter alia, stimulation of the immune response and prevention of diarrhea caused by pathogens, and also maintaining or restoring the balance of intestinal microflora.

Bifidobacterium longum and Bifidobacterium bifidum

From the prior art it is known that Bifidobacterium longum is resistant to antibiotics, for this reason their use in those periods when individuals receive treatment with antibiotics, prevents patients diarrhea, which sometimes results treatment with antibiotics. Other applications of these microorganisms is aimed at �reduces cholesterol levels, relief of symptoms of lactose intolerance, stimulation of the immune system and cancer prevention.

The use of compositions with B. bifidum relieves the symptoms associated with diarrhoea. In turn, they are microorganisms that enhance the immunological response of the individual by increasing phagocytic activity in peripheral blood.

In the light of the above, and various comparative experiments showed the best probiotic properties of Bifidobacterium breve HERO 15B (CNCM I-4035) in comparison with Bifidobacterium longum and B. bifdum, Bifidobacterium breve HERO 15B (CNCM I-4035) is particularly applicable to, inter alia, stimulation of the immune response and prevention of diarrhea caused by antibiotics, to reduce levels of cholesterol, improvement of symptoms of lactose intolerance, prevents cancer and so on.

Probiotics in foods, beverages, medicines and the like

The practice of including viable microorganisms in food products has long existed. Yogurt and other dairy products are the foods traditionally include live microorganisms. The development of functional foods in recent years has led to the development of new approaches based on the use of microorganisms capable of exerting beneficial effects on the body.

Region children's power�I was no exception in the field of functional foods, and in recent years the ingredients of this type include steel in various types of products for baby food. Probiotics are one of the main lines of development, and they are used mainly in the field of dairy mixes, primarily in mixtures for continuous feeding and growth stimulation. The purpose of inclusion of probiotics in foods is their implementation in the large intestine of the host organism and obtain a number of beneficial effects (reduction of pathogens, the formation of vitamins and other nutrients, decrease in pH and so on).

All probiotic bacteria of the present invention is considered as species belonging to the group of lactic acid bacteria, and they have been known for some time at the international level in mind they have no pathogenic properties. Thus, they are suitable for use in the fermentation of dairy products, among other things, by themselves or in combination with other lactic acid bacteria, e.g., Streptococcus thermophilus, Lactococcus lactis, Streptococcus lactis, and so on. Likewise, their use in milk for baby food, as the application of any other lactic acid bacteria, does not involve any potential problems for food safety.

The inclusion of probiotics in foods and beverages by using a suitable method� mixing (or fermentation, where this is suitable) should provide a certain number of live bacteria in the final product upon expiry of the maximum period of storage of the product.

Probiotics of the present invention can be used in baby food, as well as in the diets of adults and special food. These Probiotics can be used in powder form, alone or in mixture with other excipients known in the art, such as sugar, proteins, milk powder and so on, or as active ingredients for the fermentation, preferably product based on milk. Thus, these Probiotics can be included, in powder form or in liquid form, in the food products used by the General population, especially milk and products obtained from milk, in particular dairy products and cheeses; cereals and their derivatives, including the dough for baking bread; soups and other similar products in dehydrated form; fermented meat products; processed fruits, juices and soft drinks; food products for use in special food, including milk for baby food, cereal and baby foods ready-to-eat baby food and so on. They can also be found in food additives and special mixtures for TRANS�General and enteral nutrition for clinical use.

If they are in a powder product (milk for baby food, cereal and so on), probiotics will be incorporated by mixing in dry form with the final product. Thus, the probiotics of the present invention may be included in powder, food intended for dilution with water or another liquid, such as milk (dry milk for baby food, cereal and so on).

When they are used for fermentation of milk or dairy products and the manufacture of dairy products probiotics added to liquid milk based on the intermediate stage of the method, conducting the fermentation at a controlled temperature for a controlled time with obtaining a fermented milk product.

Probiotics according to the invention may also be used in food additives and even in pharmaceutical products that may be in the form of powder preparations, tablets, coated with sugar-coated, and so on. The scope of these products is the treatment of inflammatory bowel disease, stomach ulcers, acute diarrhea and other diseases of the gastrointestinal tract.

EXAMPLES

Example 1. Amplification of intergenic fragments of the 16S-23S

Were amplified and sequenced the intergenic segments of the selected strains, and the search for homologues was performed in the basis given�'s NCBI (BLAST), which led to the following results.

Lactobacillus rhamnosus 22A (CNCM I-4036) has 100% homology to the fragment length 579 p. O.:

Lactobacillus rhamnosus, isolate TS1;

Lactobacillus rhamnosus, isolate PS1 16S.

- Lactobacillus paracasei 7 (CNCM I-4034) has 100% homology to the fragment length 512 p. O.:

Lactobacillus casei, strain ATS 334.

- Bifidobacterium breve IN 15 (CNCM I-4035) has 99% homology to the fragment of the intergenic space 16S-23S length 182 p. O.:

- Bifidobacterium breve (ITS), the strain Y8;

- Bifidobacterium longum (ITS), the strain Y10.

Example 2. Alignment of the sequenced segments

For alignment of the sequenced segments of the selected strains and controls used online software tool Clustalw (http://www.ebi.ac.uk/clustalw/).

The overall alignment of the sequences of 16S rDNA of strains and controls demonstrated the presence of differences in the sequences of controls and samples L. rhamnosus 22A and L. paracasei 7, and shows the full homology of the sample 7 and the selected sequence of L. paracasei.

With respect to the selected sample B. breve 15B, the difference becomes obvious sequences of controls and sample B. breve 15B and 100% homology of B. breve 15B and the selected sequence of B. breve.

The overall alignment of the sequences of the intergenic space 16S-23S strain 15B and controls made it possible to see a significant difference between the sequence of controls and sample B. breve 15B.

Indeed, sequencing the intergenic space 16S-23 is unique and does not match with previous descriptions for bifidobacteria, by showing the uniqueness of the strain according to the invention was recognized by the Pasteur Institute, where he also was assigned a unique designation.

Example 3. The preparation and handling of samples sampling

The faeces of children aged 2 to 4 months, fed exclusively on mother's milk, obtained under anaerobic conditions in the pediatric clinic JM, which is the developer of the present invention. Parents are asked to bring their children to the clinic early in the morning, after stimulation was waiting for the babies will have a bowel movement, and after defecation feces was collected in a sterile container with a plastic spoon attached to the lid of the container. At the end of the collection container with the sample was placed in the anaerobic jar, polycarbonate (Anaerojan®, Oxoid, Hampshire, United Kingdom) with a sachet, providing an anaerobic atmosphere (Anaerogen®, Oxoid, Hampshire, United Kingdom), and the container is sealed closed and transported to the laboratory where samples were processed within 2 hours.

Processing and seed samples

After harvest or prior to cooling at -80°C, samples can be kept in a designated accordingly the tubes of Eppendorf.

Thus, receive a 10% suspension of feces in PBS (phosphate-buffered saline (PBS, Sigma-Aldrich, Madrid, Spain)) and the hydrochloride of L-cysteine (Scharlau CEIME, Barcelona, Spain) (0.05 per cent). This drug is produced 7 breeding from 10sup> 1up to 107finally , 50 µl of each dilution were sown in two selected culture medium and incubated under anaerobic conditions for cultivation of bifidobacteria (Anaerogen®) and is rich in CO2environment for lactobacilli (CO2Gen) for 72 hours at 37°C.

Example 4. Preparation of culture media

Described below are 3 specific culture medium for bifidobacteria and the culture medium specific for lactobacilli.

1. Wednesday Berens (Beerens 1990). This environment is used for determination of bifidobacteria.

For its preparation in the Erlenmeyer flask with a volume of one liter is mixed with 47 g of agar with cardio-cerebral extract, 5 g of D-(+)-glucose, 0.5 g of iron citrate(III), 0.5 g of L-cysteine and one liter of distilled water. This mixture was heated with constant stirring is heated for several minutes to the boil, then leave to cool at room temperature. Upon reaching 55°C add 5 ml of propionic acid and 2.2 ml of 2 mEq/l of sodium hydroxide, and then the pH was adjusted to 5.0.

2. Wednesday BFM (Nebra & Blanch 1999). This medium is specific for bifidobacteria. This environment includes the following components in the indicated ratios per liter of solution:

2 g of meat extract;

- 7 g of yeast extract;

2 g of starch;

- 0.5 g of the hydrochloride of L-cysteine;

- 5 g of sodium chloride;

5 g peptone;

2 g of tryptone;

5 g of lactulose;

1 mg of Riboflavin*;

- 1 mamtimin*;

- 16 mg methylene blue;

2 g of lithium chloride;

5 ml of propionic acid;

- 15 diver.

The relevant quantities used to prepare 500 ml of this selective medium for bifidobacteria. This mixture was heated with constant stirring is heated for several minutes to boil, and then the solution by autoclaving.In conclusion, prepare concentrated solutions of vitamins (*) (stock solution, 1 mg/ml), then filtered and added along with propionic acid in a culture medium when the solution has cooled to approximately 55°C.

3. Modified Colombian medium (pH 5.0). This medium is specific for bifidobacteria. This environment includes the following components in the indicated ratios per liter of solution:

- Columbia agar medium (Oxoid, Hampshire, United Kingdom);

- glucose (5 g/l);

- cysteine (0.5 g/l);

agar (15 g/l).

The above number is used for preparation of 1000 ml of this selective medium for bifidobacteria. The mixture was heated with constant stirring is heated for several minutes to melt, then the solution by autoclaving.In conclusion, in culture medium was added propionic acid, when the solution has cooled to approx�till then 55°C, and the pH was adjusted to 5.0 using 1 n NaOH.

4. Agar medium Romance. This environment is used for determination of lactobacilli. For its preparation follow the descriptions provided by the business firm.

It is prepared according to the descriptions provided by the business firm.

Example 5. The seed samples

After receiving breeding each of them seeded in triplicate using a bacteriological loop. All tablets with different culture media and then incubated in an incubator with controlled temperature at 37°C.

Tablets from the culture medium for bifidobacteria in advance is placed in the anaerobic jar, polycarbonate with a sachet Anaerogen® (a system providing an anaerobic atmosphere), and the plates with the culture medium for lactobacilli placed in a container with a sachet, CO2Gen® (the system that provides the atmosphere with CO2), in the end, they were incubated for 72 hours at 37°C.

Example 6. Determining the number of colony-forming units

After incubation, the selected dilution of more than 10 colony forming units (CFU) and SOME values in each of the environments are counted using an electronic counter of colonies (Colony counter model 608702, Bio Co., Kobe, Japan). Finally, calculate the total number of CFU by the following formula:

CFU = number of colonies × dilution factor × dilution.

After receiving�Oia crops remaining samples of faeces were stored at -80°C until further molecular-biological research.

Example 7. Determination of resistance to pH and salts of bile acids

After 72 hours of incubation were selected 100 colonies from each culture medium at the child, taking into account the morphology, visible to the naked eye. These colonies of lactobacilli and bifidobacteria, were incubated in a liquid medium Mann-Romance-Sharpe (Man Rogosa Sharpe (RSM)) and under anaerobic conditions for 48 hours. Then, from each culture had immediate source glycerol solutions (RSM+10% glycerol).

Simultaneously with the initial glycerol solutions of various colonies to assess their viability at pH 3.0 and 3% concentration of bile salts (Oxgall, Sigma-Aldrich, Spain). To do this, perform the following steps:

1) the colony was centrifuged at 5000 rpm for 5 minutes.

2) the supernatant is removed and pellet resuspended in sterile PBS;

3) then perform centrifugation in the same conditions as described above;

4) stage 1-3 is repeated three times;

5) in conclusion, resuspension in 1 ml sterile PBS;

6) to make 100 ml of the above suspension in 900 ml of PBS at pH 7.0 and at pH of 3.0 and 0.3% Oxgall, dissolved in PBS;

7) incubation under anaerobic conditions for 3 hours at 37°C;

8) receive different dilutions (from 101up to 105for each of the conditions in which incubation was carried out;

9) seeding 50 cloggage breeding;

10) incubation for 72 hours at 37°C under anaerobic conditions;

11) determination by counting the number of colonies present in the control and pH data and Oxgall;

12) determining the viability of each colony using the index:

viability = ((number of colonies at these pH/Oxgall)/(number of colonies in the control))×100.

All the colonies that demonstrate viability at pH 3.0 and 0.3% Oxgall above 90% are considered to be positive and keep for the remaining studies. The rest of the colony expelled.

Example 8. Control bacteria

The colony, used as positive controls consisted of the following.

For bifidobacteria: Bifidobacterium bifidum and Bifidobacterium longum supplied Hero Spain S. A.

For lactobacilli used 2 production echobelly, Lactobacillus casei (Danone®) and Lactobacillus rhamnosus GG (LGG) (KAI KU®).

After the initial screening, selected colonies was performed a second screening.In this case, checking the viability at pH 2.5 and 2.0 and at 0.5% and 0.7% Oxgall. Followed Protocol, similar used in the first analysis. After determining the viability was determined ranges of vitality. In this second screening bacterial production controls showed a lower than zero, so control of the percent of viability of more than 4% was Oprah�Elen initially as the optimal range for selection of positive colonies. The colonies were divided into 3 groups:

group 1 - colony with a viability of more than 66%;

group 2 - colony with vitality from 33 to 66%;

group 3 - colony with a viability of more than 4%.

Fig.1 and 4 show the results of the sustainability and survival of the strains according to the invention at different pH. For strains Lactobacillus rhamnosus 22A (CNCM I-4036) and Lactobacillus paracasei 7 (CNCM I-4034), the results shown in Fig.1, and they show that at pH 3.0 these strains have resistance similar to or slightly higher than the resistance of the examined production strains. However, at pH 2.0 and strain 22A has a very high viability compared with the other strains that do not survive at that pH. For the strain of Bifidobacterium breve CNCM I-4035 results shown in Fig.4, where it is seen that at pH 3.0 the strain In 15 demonstrates stability far exceeding the stability of the two other studied bifidobacteria, with a viability greater than 100%, indicating the ability of these bacteria to multiply when the pH.

Fig.2 and 5 show the results of the influence of bile salts on the survival of strains of the present invention. Fig.2 presents the results for the strains CNCM I-4036 and CNCM I-4034. It is seen that both strains show a survival rate far exceeding the percentage of survival was studied industrial strains, with� is more than 100%, indicating that they may even multiply in the presence of these salts. Fig.5 presents the results for the strain CNCM I-4035. As shown in this graphic material, at higher concentrations this strain shows greater survival compared to the control bifidobacteria in the art.

Example 9. The study of adhesion of the cells of the intestinal epithelium

Analysis of the adhesion of the cells is carried out using colonies selected for resistance to pH and salts of bile acids.The analysis was performed using the cells of the intestinal epithelium NT29. At first I made several attempts of determination of adhesion on cells through a variety of staining: gram staining, staining methylene blue staining by Giemsa and so on. It was observed that the percentage of adhesion of the cells NT29 using these methods is very difficult.

The question arose about what would be the best way to determine the percentage of adhesion, thus it was concluded that the best way would probably be a way to cover all of attached bacteria. For this reason was chosen as the method of trypsinization, and to this end were as follows:

1) incubation of cells NT29 at 37°C and 5% CO2to confluently in 24-well plates;

3) bringing the bacteria into contact with the cells according to the stages described below:

(a) centrifuging the bacteria at 5000 rpm for 5 minutes.

b) removal of supernatant and resuspension of bacteria in 1 ml of sterile PBS;

C) repeating stages (a) and (b) two more times;

g) determining the optical density (OD) for each sample of bacteria at 600 nm;

d) dilution of the bacterial culture to an OD of 0.8 in previously prepared environment for the cultivation of cells without FBS (fetal calf serum) and antibiotics (1 to 5×106CFU/ml);

(e) detachment of cells from the culture medium;

g) repeated washing with sterile PBS to remove residual FBS and antibiotics;

h) add 250 ml of bacterial suspension into each well; the experiment was conducted three times;

and) incubation at 37°C and 5% CO2within 90 minutes.

4) after incubation of bacteria with cells is carried out as follows:

a) remove medium by aspiration with a Pasteur pipette;

b) washing 4 or 5 times with 1x PBS (pH 7.0);

C) add 100 ál of trypsin and incubation for 10-15 minutes at 37°C;

g) selection of only the contents of the wells and its transfer to Eppendorf tube;

d) washing the wells with 150 ál of PBS and introduced into the same Eppendorf tube;

(e) preparing multiple dilution of each sample (4 or 5);

g) seeding 50 μl of each dilution;

h) �incubate under anaerobic conditions at 37°C for 72 hours;

I) counting the number of colonies.

5) definition of % adhesion:

% adhesion = ((the number of attached colonies)/(number of inoculated colonies))×100.

Fig.3 shows the results for the adhesion of the strains CNCM I-4036 (strain 22A) and CNCM I-4034 (strain 7) according to the invention. Both strains have the percent adhesion, greatly exceeding the interest demonstrated by the control strains (in the case of strain 22A more than two times), indicating their potential effect in the modulation of the activity of intestinal cells, including immunomodulation.

Fig.6 shows the results for the adhesion of strain CNCM I-4035 (strain 15V), which also shows the percentage of adhesion of the cells, significantly higher than the percentage of adhesion control strains.

Example 10. Identification of lactic acid bacteria

DNA extraction, amplification and sequencing of a fragment of the 16S rRNA

Given the number of colonies sampled in the study of adhesion, spend their easy identification by amplification of a fragment of the 16S rRNA gene of each colony, sequencing and searching for homologues in the database of the National center for biotechnology information (National Center of Biotechnology Information (NCBI)).

First, the selected colonies were incubated in the environment of the RSM in 48 hours at 37°C under anaerobic conditions. They were then washed with PBS. To do this, hold the following:

1) the colony was centrifuged at 500 rpm for 5 minutes;

2) the supernatant was removed and the bacterial pellet resuspended in 1 ml of sterile PBS;

3) triple repetition of stages (1) and (2);

4) in conclusion, the sediment resuspension in 1 ml sterile PBS.

Then isolated genomic DNA of the bacteria that is carried out as follows:

1) centrifuging the previously obtained suspension at 5000 rpm for 5 minutes and remove the supernatant;

2) the bacterial pellet resuspended in 567 ml of buffer hydroxyethylaminomethyl (Tris) and ethylenediaminetetraacetic acid (EDTA) (TE-buffer);

3) add 30 ml of 10% sodium dodecyl sulphate (SDS) and 3 ml of proteinase K (20 mg/ml);

4) incubation of the mixture at 37°C for 1 hour;

5) add 100 ml of 5 M NaCl and 80 ml of the bromide cetyltrimethylammonium (CTAB)/NaCl;

6) mixing and incubation at 65°C for 10 minutes.

7) the addition of an equal volume (780 ml) mixture of chloroform and isoamyl alcohol (24:1);

8) mixing and centrifugation for 5 minutes at 10000 rpm;

9) removing the upper aqueous phase and transfer to a new Eppendorf tube;

10) the addition of an equal volume mixture of phenol, chloroform and isoamyl alcohol(25:24:1);

11) mixing and centrifugation for 5 minutes at 10000 rpm;

12) removing the upper aqueous phase and transfer to a new Eppendorf tube;

13) add 0.6 volume of isoprop�Ola;

14) centrifuged for 13 minutes at 13000 rpm at 4°C;

15) remove the supernatant and add 1 ml of 70% ethanol to the precipitated DNA;

16) centrifugation for 5 minutes at 13000 rpm at 4°C;

17) remove the supernatant and precipitated DNA, and then drying at room temperature;

18), resuspension in 20-50 ál of water;

19) measurement of the concentration with a spectrophotometer at 260 nm and obtaining the ratio of 260/280 to check its purity.

Amplification of 16S rDNA and the intergenic space 16S-23S polymerase chain reaction (PCR)

- Used the oligonucleotides

For amplification of 16S rDNA was used the following sets of universal oligonucleotides:

27F 5'-AGAGTTTGATCMTGGCTCAG-3' (M=A+C) (SEQ ID NO: 1).

1492R 5-TACGGYTACCTTGTTACGACTT-3' (Y=C+T) (SEQ ID NO: 2).

A fragment approximately 1450 p. O. amplificateur at the hybridization temperature of 55 ºc, time amplification 90 seconds and the number of cycles 35.

39F 5'-TGGCTCAGRWYGAACGCTRG-3' (R=A+G, W=A+T, Y=C+T) (SEQ ID NO: 3).

1391 R 5'-GACGGGCGGTGWGTRCA-3' (SEQ ID NO: 4).

A fragment approximately 1350 p. O. amplificateur at the hybridization temperature of 52°C, time amplification 90 seconds and the number of cycles 35.

In addition, oligonucleotides were designed that are specific in respect of bifidobacteria, which are the following:

Bif, 250 p. O., F 5'-CTCGTAGGCGGTTCGTCG-3' (SEQ ID NO: 5);

Bif, 250 p. O., R 5'-AACGGGCCCACATCCAG-3' (SEQ ID NO: 6).

A fragment approximately 250 p. O. amplificateur at the temperature of hybridization of 65°C, time of 20 seconds and amplification the number of cycles 30.

For amplification of the intergenic regions of 16S-23S lactobacilli and bifidobacteria used the following sets of oligonucleotides:

LactoF 5-ACACCGCCCGTCACACCATG-3' (SEQ ID NO: 7).

LactoR 5'-CCHSTTCGCTCGCCGCTACT-3' (H=A+T, S=G+C) (SEQ ID NO: 8).

Oligonucleotides that are specific against lactobacilli. A fragment approximately 600 p. O. amplificateur at the temperature of hybridization of 65°C, amplification for 30 seconds and the number of cycles 30.

ISBif F 5'-GGGATGCTGGTGTGGAAGAGA-3' (SEQ ID NO: 9).

ISBif R 5-TGCTCGCGTCCACTATCCAGT-3' (SEQ ID NO: 10).

Oligonucleotides that are specific in respect of bifidobacteria. A fragment approximately 240 p. O. amplificateur at the hybridization temperature of 60 ºc, time amplification 30 seconds and the number of cycles 30.

For amplification of 16S rDNA and the intergenic space 16S-23S in one PCR reaction used 50-100 ng DNA in the final volume of 50 μl in each cycle used a denaturation temperature of 94°C for 30 seconds. Then was the program in accordance with the conditions for each set of oligonucleotides as indicated above.

The result of amplification was monitored in a 1.3% agarose gel, samples were stained with ethidium bromide and visualized in a transilluminator with UV radiation.

Amplification with negative �esultats repeated, if the result is positive amplification was performed using the kit from GE Healthcare "Ilustra™ GFX™ PCR DNA and gel Band Purification Kit, following the manufacturer's instructions. After cleaning, the samples were resuspended in 25 ml of water and the purification was monitored by visualization in a new a 1.3% agarose gel.

Then the samples were transferred to the service of DNA sequencing (DNA Sequencing Service of the Institute of Parasitology and Biomedicine (Institute of Parasitology and Biomedicine) "Lopez-Neyra" (CSIC).

Example 11. Identification using enzymatic tests

Used system API ZYM and API 50 CHL (bioMerieux's). System API ZYM is a semi-quantitative method of measuring enzymatic activity. This system includes 20 wells, 19 of which contain dehydrated substrate to detect the activity of 19 enzymes (Fig.7 and 8), get the result of colorimetry which indicates the degree of enzymatic activity, which was measured on a scale of 0-5 in comparison with the control. Also used test strips API 50 CH and API environment CHL (bioMerieuxs), which is a method of producing a fermentation profile in respect of 49 carbohydrates (Fig.9 and 10). Get the results of colorimetry, but in this case they are classified only as positive (+), negative (-) and intermediate (V) in comparison with the control. In all tests used the control bacteria.

Example 12. Evaluation of antimicrobial activity of strain�in L. paracasei CNCM I-4034, S. breve CNCM I-4035 and L rhamnosus CNCM I-4036

The investigated strains and culturing conditions and storage

In the present study were analyzed in a total of 3 strains belonging to the genera Lactobacillus and Bidifobaterium (table 1).

Table 1
The strains used in this study and the conditions of cultivation
StrainThe culture mediumTemperatureAeration
L. paracaseiCNCM I-4034RSM37°CAnaerobic conditions
B. breveCNCM I-40350.05% of RSM-cysteine37°CAnaerobic conditions

L. rhamnosusCNCM I-4036RSM37°CAnaerobic conditions

Evaluated the antimicrobial activity of these strains in related�and bacterial pathogenic agents, damaging the digestive system {Helicobacter pylori, Listeria monocytogenes, Shigella sonnei, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Salmonella enterica), and viruses (virus Ito, Wa and Va70) shown in Tables 2 and 3.

Table 2
Used strains of pathogenic microorganisms
TypesHelicobacter pyloriListeria monocytogenesShigella sonneiRotavirus
StrainsLMG 8775CECT4031CECT 457Wa
LMG 18041CECT 935CECT 4887Va70
LMG 19449CECT911CECT 413Ito

Table 3
Used strains of pathogenic microorganisms
TypesEnterotoxigenic Escherichia coli Enteropathogen Escherichia coliSalmonella enterica
StrainsCECT 434CECT 443CECT 443
CECT 501CECT 729CECT 725
CECT515CECT 742CECT 4594

In the case of bacteria, they kept in the RSM solution with the addition of 20% (wt./about.) of glycerol and freezing at -80°C. Viruses were stored frozen in MEM medium at -190°C. Obtain free from cell supernatant for research

To obtain the concentrated supernatant for various analyses, strains were cultured in liquid medium for 17 hours and 24 hours in the environment of the RSM (CNCM I-4034 and CNCM I-4036) or RSM with 0.05% cysteine (CNCM I-4035) at 37°C. the Supernatant of each of the strains were collected by centrifugation and liofilizirovanny. The resulting concentrate was dissolved to obtain a solution with a tenfold concentrated, neutralized to pH value of 6.0 and the resulting solution was sterilized by filtration through a pore size of 0.22 μm. Aliquots of the neutralized and sterilized supernatant was stored frozen at -20°C until used.

Analyses of activity in about�wearing of bacterial pathogens, affecting the digestive system, in a liquid medium

For analysis of inhibition in liquid medium used a modification of the Protocol Spinier et al. (2008). Briefly, multi-well tablets individually contributed volumes of supernatants obtained in 250 μl of increasing percentages (from 0.2% to 4%) (about./about.) to a culture medium inoculated with each pathogen at 5%, which were grown overnight. Growth curves were obtained by monitoring by measuring the OD at 595 nm using a tablet reader Multiskan 5 Ascent. On the basis of the results obtained for different samples are shown inhibition was assessed quantitatively as the percentage of growth inhibition with respect to control without the addition of supernatant inhibitory strain.

Activity against viral pathogens. affecting the digestive system, in a liquid medium

Analyses of the inhibition of viral infection of the supernatant liquids was investigated strains were performed according to the Protocol published by Ana-Marin et al. (2007), with modifications to adapt it to the work undertaken in this project. In this case, these analyses used a cell line of human intestinal HT-29.

The results of activity assays in liquid medium against bacterial pathogens. affecting piwevaritel�th system: Listeria monocvtoaenes, Shiaella sonnei u Helicobacter pylori

To evaluate the effect of supernatants obtained by cultivation of strains of L paracasei, L rhamnosus and B. breve, used neutralized and concentrated 10 times in the supernatant obtained by cultivation of strains for 17 and 24 hours, respectively.

The results varied significantly depending on both probiotic and pathogenic strains. In the case of L. monocytogenes adding the supernatant obtained after cultivation of L. paracasei for 17 hours, showed inhibitory effect (Fig.10A). In the case of L. rhamnosus best results were obtained when adding the supernatant of a 24-hour culture. In the case of B. breve inhibition of L. monocytogenes CECT 4031 T (Fig.10B) was evident. The results obtained for S. sonnei, were similar to that obtained for L. monocytogenes, as L. paracasei best results were obtained when adding the supernatant obtained after cultivation for 17 hours, and for L. rhamnosus best results were obtained when adding the supernatant of a 24-hour culture (Fig.10B). In the case of N. pylori, a significant decrease in growth of the pathogen was obtained with the use of supernatants of L. paracasei and B. breve, obtained after 17 hours and 24 hours of incubation, the highest inhibition was provided by the supernatant of 24-hour cultures. Rece�the percent inhibition shown in the following table (table 4).

L. paracaseiL. rhamnosusB. breve
0.4% of (about./about.)2% (vol./about.)4% of (about./about.)0.4% of (about./about.)2% (vol./about.)4% of (about./about.)0.4% of (about./about.)2% (vol./about.)4% of (about./about.)
17 h24 h17 h24 h17 h24 h17 h24 h17 h24 h17 h24 h17424 h17 h24417 h24 h
L. monocytogenes CECT 9350,00 0,0033,096,64ML)1,891,513,21Of 8.4313,862,4530,310,690,001,240,000,030,00
L. monocytogenes CECT 40310,000,000,000,009,480,000,000,001,24Of 10.678,2115,293,3216,631,06For 9.88Of 4.54To 28.05
L. monocytogenes CECT 9112,605,737,319,9225,6416,818,4 5.1811,9918,3615,5328,134,220,000,000,000,000,00
S. sonnei CECT 4570,000,002,639,95The 9.2520,531,780,410,0016,04Of 6.7933,800,0012,940,00Of 25.752,4419,36
S. sonnei CECT 41328,720,0032,513,8880,9982,100,00Of 5.175,4916,3280,9582,06 8,050,00Of 7.190,0014,830,00
S. sonnei CECT 48870,000,002,639,95The 9.2520,530,000,000.00Of 10.053,8029,070,000,000,000,000,000,00
H. pylori LMG 4081T0,000,000,000,000,009,240,009,702,064,190,000,000,000,0014,970,006,330,00
H. pylori LMG 19499Of 21.1513,2451,7041,0575,8070,89Of 17.3636,6754,8461,1383,4885,2812,56Of 37.8037,7971,92To 57.3377,56
H. pylori IMS 8775At 13.8431,3434,6174.04 WUXGA33,6595,660,000,000,0022,5070,7744,2225,7720,3835,3858,5538,5085,00

The results of activity assays in liquid medium against bacterial pathogens that infect the digestive system: Salmonella typhi, Salmnella thvohimurium u Eschehchia coli

For the supernatant of L. paracasei bacteria, there is a significant growth inhibition against Salmonella, the study groups (Fig.11). This effect is especially pronounced when using a non-neutralized supernatant, confirming that it is due to the formation at fermentation acid that limits the growth of the pathogen. In the case of Salmonella typhi CECT 725 inhibitory effect at 1% and 4% of the supernatant see regardless of whether neutralized or not, confirming that the inhibition is due to the bacteriocins of some type or another factor of unknown nature, rendering this effect against the pathogen.

The supernatant of the bacteria B. breve inhibits the growth of Salmonella typhi (CECT 725). This effect is observed using the supernatant in all conditions (17 and 24 hours; neutralized and neutralized; 1% and 4%), education of bacteriocin any type, or other factor of great nature (Fig.12) and in this case is not excluded.

For the supernatant of the bacteria L. rhamnosus seen significant growth inhibition in all three groups (E coli ETEC, E. coli EPEC and Salmonella enteiica), mainly in 4% (Fig.13). This effect is observed mainly when using a non-neutralized supernatant, Reaffirming that it is due to the acidic products of fermentation, AVL�to give effect to the growth of the pathogen.

Although in the case of E. coli EPEC (CECT 742) and Salmonella typhimuhum (CECT 4594) the inhibition observed with the use of neutralized 24-hour supernatant, this effect can be explained, as in previous cases, the presence of a factor or bacteriocin any type of nutrient medium of probiotic bacteria (Fig.13).

The results of activity assays in liquid medium against viral pathogens that infect the digestive system: human rotaviruses Ito, Wa and Va70

Optimized protocols of infection, the detection of infectious foci and quantitative assessment of the protective effect against human rotaviruses Ito, Wa and Va70. To obtain as representative as possible of the results of the analyses of infection and protection were carried out on human cell line HT-29.

After amplification of the virus in cells MA-104 spent their titration on line HT-29. Obtained credits in blakebrough units (COMBAT) was 2,h6The FIGHT/ml for the virus Ito, of 6.80×104The FIGHT/ml for the virus Wa and 2.33×105The FIGHT/ml for the virus Va70. On the basis of the results of the titration the concentration of viruses adjusted so that they were suitable for infection. To ensure the correctness of the analyses of infection was performed using three consecutive serial decimal dilution and pastorality tests three times. Fig.14 shows the results of reducing the formation of infectious foci obtained with the use of supernatants without pre-concentrating the neutralized supernatant fluids from 24-hour cultures. These results show that the strains of the present invention reduce the formation of infectious foci all investigated viruses (Wa, Ito and Va70).

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1. Probiotic microorganism strain isolated from the feces of children fed exclusively on mother's milk, characterized by the fact that it consists of Lactobacillus rhamnosus HERO 22 A (CNCM I-4036).

2. Probiotic microorganism strain isolated from the feces of children fed exclusively on mother's milk, characterized by the fact that it consists of Lactobacillus paracasei HERO 7 (CNCM I-4034).

3. Probiotic microorganism strain isolated from the feces of children fed exclusively on mother's milk, characterized by the fact that it consists of Bifidobacterium breve HERO IN 15 (CNCM I-4035).

4. The microorganism strain according to any of claims. 1-3, characterized in that it presents in the form of pure biological culture.

5. The microorganism strain according to any of claims. 1-3, characterized in that it is selected.

6. The strain of probiotic microorganism according to any one of p�. 1-3, characterized in that it is presented in the form of viable cells.

7. The strain of probiotic microorganism according to any one of claims. 1-3, characterized in that it is presented in the form of non-viable cells.

8. The strain of probiotic microorganism according to any one of claims. 1-3 for use in food.

9. The strain according to claim 8, characterized in that it is used in pediatric and/or adult and/or special diet.

10. The strain according to claim 9 for use in the manufacture of dairy mixes for baby food.

11. The strain according to claim 10, characterized in that the formula consists of ready-to-drink milk for baby food and/or cereal for baby food, and/or products for baby food.

12. The strain according to claim 8 for use in the manufacture of dietary supplements.

13. The strain according to claim 8 for use in the production of special mixtures for oral and/or enteral nutrition.

14. The strain of probiotic microorganism according to any one of claims. 1-3 for use in the manufacture of a pharmaceutical product.

15. The strain of probiotic microorganism according to any one of claims. 1-3, suitable for stimulating the immune system, and/or prevention/treatment of asthma, and/or prevention/treatment of gastrointestinal disorders, and/or correcting/modulation main pathogens that infect the digestive system, and/or prevent/treat Aire�Oia and related diseases including metabolic syndrome and diabetes, and/or when the typical diseases associated with aging.

16. The strain according to claim 15, characterized in that the specified gastrointestinal disorders include changes of the passage of contents through the intestines, such as constipation, and changes in bioavailability of minerals, infections and malabsorption syndromes.

17. The strain according to claim 16, characterized in that these infections include gastric infection and gastrointestinal infection with acute or chronic diarrhea.

18. The strain according to claim 16, characterized in that said malabsorption syndromes include disorders affecting the anatomy of the intestine, such as the syndrome shortened small intestine, and disorders affecting the physiology of the intestine, such as cystic fibrosis pancreas, malabsorption of sugars, in particular lactose, changes in the absorption of lipids, food Allergy and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

19. Probiotic composition comprising the microorganism strain according to any of claims. 1-7 and a carrier suitable for oral administration.

20. Probiotic composition comprising a mixture of microorganisms according to any of claims. 1-7 and a carrier suitable for oral administration.

21. Composition according to claims. 19 or 20, characterized in that it further comprises �another probiotic material.

22. Composition according to claims. 19 or 20, characterized in that it further comprises a prebiotic material.

23. Composition according to claims. 19 or 20, characterized in that the carrier is a pharmaceutically acceptable carrier, such as capsules, tablets or powder.

24. Composition according to claims. 19 or 20, characterized in that the carrier is a food product.

25. A composition according to claim 24, characterized in that the said food product is selected from the group consisting of milk and products obtained from milk, in particular dairy products and cheeses; cereals and their derivatives, including the dough for baking bread; soups and other similar products in dehydrated form; fermented meat products; processed products of fruits, juices and non-alcoholic beverages; foodstuffs for use in special diet.

26. Composition according to claims. 19 or 20 for use in food.

27. A composition according to claim 26, characterized in that it is used in pediatric and/or adult and/or special diet.

28. The composition of claim 27 for use in the manufacture of dairy mixes for baby food.

29. A composition according to claim 28, characterized in that the formula consists of ready-to-drink milk for baby food and/or cereal for baby food, and/or products for baby �warning.

30. The composition of claim 26 for use in the manufacture of dietary supplements.

31. The composition of claim 26 for use in the production of special mixtures for oral and/or enteral nutrition.

32. Composition according to claims. 19 or 20 for use in the manufacture of a pharmaceutical product.

33. Composition according to claims. 19 or 20, suitable for stimulating the immune system, and/or prevention/treatment of asthma, and/or prevention/treatment of gastrointestinal disorders, and/or correcting/modulation main pathogens that infect the digestive system, and/or prevention/treatment of obesity and related diseases, including metabolic syndrome and diabetes, and/or when the typical diseases associated with aging.

34. A composition according to claim 33, characterized in that the specified gastrointestinal disorders include changes of the passage of contents through the intestines, such as constipation, and changes in bioavailability of minerals, infections and malabsorption syndromes.

35. A composition according to claim 34, characterized in that these infections include gastric infection and gastrointestinal infection with acute or chronic diarrhea.

36. A composition according to claim 34, characterized in that said malabsorption syndromes include disorders affecting the anatomy of the intestine, such as the syndrome shortened small intestine, and dist�STS, affecting the physiology of the intestine, such as cystic fibrosis pancreas, malabsorption of sugars, in particular lactose, changes in the absorption of lipids, food Allergy and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.



 

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SUBSTANCE: invention relates to novel strains of Bacillus thuringiensis B-1272 and Bacillus thuringiensis B-1273 deposited in the collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution of Science "State Research Centre of Virology and Biotechnology "Vector". The index of neutralisation of the infectious activity of virus A/H3N2 while using the preparations based on the culture fluid of any of the proposed strains is 0.5-3.2 lg.

EFFECT: strains have the ability to neutralise the infectious activity of the human influenza virus.

2 cl, 1 tbl, 8 ex

FIELD: chemistry.

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17 cl, 21 dwg, 7 tbl, 18 ex

FIELD: biotechnology.

SUBSTANCE: method of preparing the nutrient medium for cultivation of lactobacilli comprises preliminary preparing of acid hydrolysate of blood of slaughtered animals, yeast autolysate and milk whey with known methods. The acid hydrolyzate of blood of slaughtered animals is deoxidised to pH of 6.7-6.9 and is added to yeast autolysate, milk whey, glucose and cabbage broth in a predetermined ratio of components. Then the mixture is heated and boiled for 5-7 minutes. The pH of the culture medium is determined and adjusted to 6.7-6.9. The resulting nutrient medium is filtered and packaged in vials, followed by sterilisation by autoclaving at 0.5 atm for 25-30 minutes.

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3 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: method of determining sensitivity of fluorescence spectra comprises cultivation of P. aeruginosa strains on nutrient media, stimulating the synthesis of pioverdine, centrifugation and filtration of cultures for obtaining samples, sample irradiation with spontaneous ultraviolet radiation in the wavelength range of 200-300 nm, obtaining the fluorescence spectra, the resistance marker of the strain to antibiotics is the presence in a band of fluorescence of the peak in the wavelength range of 435-445 nm. The sensitivity marker of the strain to antibiotics is the presence in a band of fluorescence of the peak in the wavelength range of 455-470 nm.

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2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

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9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a method for preparing a recombinant protein of type III interferon-like factor (ILF III) of the producing strain E. coli. The inclusion bodies E. coli are washed and dissolved with using 2% aqueous γ-cyclodextrin. That is followed by the sequential Ni-Sepharose, Q-Sepharose and SP-Sepharose chromatographic procedures. Refolding of a target protein is performed with using a mixture of cysteamine and cystamine at pH 10.5. The Amberchrome Profile XT20, Amberchrome Profile HPR10 and Kromasil 300-5C18 chromatographic procedures are sequentially performed.

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3 dwg, 4 ex

FIELD: biotechnology.

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4 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: strain of micromycete Aspergillus foetidus 379-K-5-1 is proposed, producing a complex of pectinases, β-glucanase, xylanase, cellulase, chitinase, mannanase and protease for the destruction of polysaccharides of a plant and microbial material. The strain is deposited in the Departmental collection of useful microorganisms for agricultural purposes of the Russian Academy of Agricultural Science (RCAM) of the State Scientific Institution of the Russian National Research Institute for Agricultural Microbiology under the registration number of RCAM 01136. The advantage of the new strain is to obtain the more active complex of enzymes catalyzing the hydrolysis of polysaccharides, as well as endopolygalacturonase and pectinesterase. The strain Aspergillus foetidus RCAM 01136 is produced using efficient methods of selection and mutagenesis using UV irradiation and nitrosoguanidine from the known strain.

EFFECT: invention enables to expand the range of the enzymes obtained, used for the destruction of polysaccharides.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to the strain Lactobacillus rhamnosus CNCM I-3690 and to a dairy food product containing the above strain. The presented strain possesses the mannose-specific adhesive properties. The strain possesses the antimicrobial properties in relation to, e.g. Escherichia coli, Salmonella enteritidis and Lysteria monocytogenes.

EFFECT: strain possesses the immunomodulatory properties, particularly possesses an ability to inhibit an inflammatory reaction of HT-29 epithelial cells.

2 cl, 2 dwg, 5 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: inventions relates to the field of biotechnology. A composition and a method for controlling the number of molluscs of the class Gastropoda and Bivalvia are claimed. The composition contains piliferolide A or 11-hydroxy-12-ene-octadecanoic acid, originating from Pseudomonas fluorescens ATCC 55799 of a suspension of its cells in the amount, effective for the regulation of the number of molluscs, and clayey mineral. The clayey mineral represents kaolinite, smectite, attapulgite or any their combination. The method of regulating the number of molluscs includes the introduction of one or more than one clayey mineral and piliferolide A or 11-hydroxy-12-ene-octadecanoic acid in the place, where control over the number of molluscs is desirable. The clayey mineral is taken in the quantity, effective for increasing the effectiveness of piliferolide A or 11-hydroxy-12-ene-octadecanoic acid by at least 20%. The presence of the clayey mineral enhances the molluscocidal effect of piliferolide A or 11-hydroxy-12-ene-octadecanoic acid.

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4 cl, 5 dwg, 6 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to novel strains of Bacillus thuringiensis B-1272 and Bacillus thuringiensis B-1273 deposited in the collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution of Science "State Research Centre of Virology and Biotechnology "Vector". The index of neutralisation of the infectious activity of virus A/H3N2 while using the preparations based on the culture fluid of any of the proposed strains is 0.5-3.2 lg.

EFFECT: strains have the ability to neutralise the infectious activity of the human influenza virus.

2 cl, 1 tbl, 8 ex

FIELD: medicine, pharmaceutics.

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41 cl, 16 dwg, 13 tbl

FIELD: chemistry.

SUBSTANCE: present group of inventions relates to biotechnology. The group of inventions discloses the pseudomonade type Pseudomonas azotoformans, strain F30A, which is deposited under registration number DSM 22077, supernatant fluid, an enzymatic product and an agricultural composition based on the Pseudomonas azotoformans F30A strain, use of the Pseudomonas azotoformans F30A strain or supernatant fluid thereof to improve seed germination, plant germination and/or plant growth, a method of improving seed germination and a method of producing an agricultural composition. The method of producing an agricultural composition includes mixing the Pseudomonas azotoformans F30A strain or supernatant fluid thereof with one or more liquid and/or solid carriers, as well as with one or more microorganisms which stimulate plant growth, biological control microorganisms, organic fertilisers and/or agrochemicals in an effective amount. The method of improving seed germination, plant germination and/or plant growth includes applying an enzymatic product or agricultural composition on seeds, plants and/or surroundings in an effective amount.

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17 cl, 21 dwg, 7 tbl, 18 ex

FIELD: biotechnology.

SUBSTANCE: method of preparing the nutrient medium for cultivation of lactobacilli comprises preliminary preparing of acid hydrolysate of blood of slaughtered animals, yeast autolysate and milk whey with known methods. The acid hydrolyzate of blood of slaughtered animals is deoxidised to pH of 6.7-6.9 and is added to yeast autolysate, milk whey, glucose and cabbage broth in a predetermined ratio of components. Then the mixture is heated and boiled for 5-7 minutes. The pH of the culture medium is determined and adjusted to 6.7-6.9. The resulting nutrient medium is filtered and packaged in vials, followed by sterilisation by autoclaving at 0.5 atm for 25-30 minutes.

EFFECT: invention enables to increase the yield of biomass and to increase the intensity of its growth.

3 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises a solid and a liquid phase. The solid phase of the nutrient medium comprises a hydrolyzate of pancreatic fish flour, the growth stimulator of hemophilic microorganisms, sodium chloride, yeast extract, glucose, agar, potassium tellurite - 2% solution and distilled water at a given ratio of the components. The liquid phase comprises the hydrolyzate of pancreatic fish flour, enzymatic peptone, yeast extract, glucose, agar, sodium chloride, potassium tellurite - 2% solution, blood serum of cattle and distilled water at a given ratio of the components.

EFFECT: invention enables to increase the inhibitory properties of the nutrient medium relating to microbes-associants of staphylococci.

4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to the strain Lactobacillus rhamnosus CNCM I-3690 and to a dairy food product containing the above strain. The presented strain possesses the mannose-specific adhesive properties. The strain possesses the antimicrobial properties in relation to, e.g. Escherichia coli, Salmonella enteritidis and Lysteria monocytogenes.

EFFECT: strain possesses the immunomodulatory properties, particularly possesses an ability to inhibit an inflammatory reaction of HT-29 epithelial cells.

2 cl, 2 dwg, 5 tbl, 3 ex

FIELD: chemistry.

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EFFECT: invention enables to obtain L-lysine with high efficiency.

2 cl, 1 tbl, 8 ex

FIELD: food industry.

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EFFECT: invention allows to increase accuracy of lipase-producing microorganisms detection.

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Rhodococcus wratislaviensis KT112-7 has destructive activity against a group of stable toxic chlor-aromatic compounds - polychlorinated biphenyls (PCBs). A strain of bacteria Rhodococcus wratislaviensis is deposited in the Russian Collection of Microorganisms IBPM n.a. GK Scriabin RAS under the number VKM Ac-2623D and can be used for decomposition of mono-, di- and trichlorinated congeners, ortho-, meta- and para-substituted biphenyls, as well as a commercial mixture of PCBs.

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4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine and concerns a pharmaceutical composition. The presented composition contains a recombinant lysozyme of FMV P. aeruginosa bacteriophage, ethylene diamine tetraacetate and TrisHCl in the following proportions, wt %: Lysozyme 0.001; TrisHCl 0.68; ethylene diamine tetraacetate 0.049; water - the rest.

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4 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to novel strains of Bacillus thuringiensis B-1272 and Bacillus thuringiensis B-1273 deposited in the collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution of Science "State Research Centre of Virology and Biotechnology "Vector". The index of neutralisation of the infectious activity of virus A/H3N2 while using the preparations based on the culture fluid of any of the proposed strains is 0.5-3.2 lg.

EFFECT: strains have the ability to neutralise the infectious activity of the human influenza virus.

2 cl, 1 tbl, 8 ex

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