Nutritional composition containing strains bifidobacterium longum and relieving symptoms of food allergy, especially in infants and children

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to microbiology. What is presented is using the strain Bifidobacterium longum NCC 2705 (CNCM-I2618) for preparing a complete nutrient composition used for relieving symptoms of allergy to food in the patients suffering allergies caused by ingestant allergens.

EFFECT: composition provides the secondary prevention of allergic reactions caused by the above ingestant allergens.

16 cl, 7 dwg, 1 ex

 

The technical field

The present invention relates to the field of Microbiology, in particular to the use of probiotics, especially strains of Bifidobacterium longum, in the manufacture of a nutritional composition designed for attenuation of allergic symptoms in allergic patients when they are exposed to allergens.

The level of technology

Allergies are the most common health problems affecting the lives of patients of all ages. Allergic diseases currently recognized by the world health organization (WHO, who) as an epidemic. It is shown that the prevalence of allergies over the past decade have increased. The modern way of life, especially in cities, is associated with a higher prevalence and greater severity of allergic manifestations.

It is shown that allergic sensitization in childhood, especially early childhood and especially to food allergens is critical and calls the keen interest from the point of view of the development of the allergic phenotype", or "atopy", contributing to subsequent sensitization to other allergens. Consequently, allergies in childhood may be the initial stage of the allergic cascade, leading to multiple allergies in older against whom they the process, which is usually referred to as the "atopic March". For example, in human populations has been demonstrated that children with sustainable food allergies in the early stages of life has dramatically increased the risk of allergic rhinitis (hay fever) or asthma in later childhood (Ostblom and others 2008). Children with milder forms of food allergies are also prone to increased risk of developing respiratory allergies, but to a lesser extent than children with sustainable food allergies. Therefore, the attenuation of the severity of food allergies can have a critical role in the containment of the "allergic March".

In this context, the control of allergic asthma and prevention of allergies in childhood and infancy are of the highest importance.

The immune system of infants is actively developing throughout the first few years of life. Through prevention, prevention, control, mitigate or modulation of allergic reactions in young patients, it is possible to influence not only their short-term allergic profile, but also on more distant in older age.

Prevention of allergies may be provided at different levels.

"Primary prevention" is an action to prevent or mitigate the risk of sensitization is allergens patients characterized by the absence or reduced levels of allergen-specific IgE antibodies. Prevention or mitigation of sensitization lead to the absence or weakening of allergic symptoms upon exposure to exposure to the same allergen. By modulating the ways in which the patient is sensitive in relation to a single allergen or group of allergens (primary prevention), can also be provided with the modulation and subsequent allergic reactions.

"Secondary prevention" is the action on the modulation of allergic symptoms, that is, manifestations or intensity of allergic reactions in patients already become sensitive to one or more allergens, when the patient is re-exposed to the specified allergen (s). By modulating the manifestations or the intensity of allergic symptoms (secondary prevention), are minimized associated with allergies inconvenience.

Taking into account these various concepts prevent allergies, it can be assumed that, based on their inherent mechanisms of action, some compounds could only act on one or both of these certain level of prevention. Some may, for example, only weaken sensitization to a specific allergen (primary profile is prevention), while other compounds may have an effect only on secondary prevention and to reduce the severity of allergic reactions. Other compounds may be able to influence the sensitization and symptoms, and thus, to be effective in the implementation of primary and secondary prevention.

Food allergens belong to the first allergens that babies face in the very early stages of their life: for babies who do not receive exclusive breastfeeding, is fairly typical meeting with proteins of cow's milk. Milk proteins actually are among the most frequent causes of food Allergy in infancy, followed by egg whites and wheat proteins. In General, food allergies in infants and young children may have cutaneous manifestations (rash, eczema, and other) and gastrointestinal symptoms (abdominal cramps, pain, especially in the abdomen, vomiting). Further sensitization and allergic attacks can also occur when a baby/small child is exposed to a new food product, such as cereals, vegetables, fruit, nuts or fish.

Strains of Bifidobacterium longum (B. longum) were described as playing an important role in the intestinal microbiota and associated with positive effects on health. Their presence in balticconnector in the intestinal microbiota is typical for healthy breastfed infants. They can promote effective digestion, strengthen the immune system and produce lactic and acetic acid, which regulate the pH level in the intestines. These bacteria can also inhibit the growth of Candida albicans, E. coli and other with pathogenic bacteria. C. longum in General or by certain strains of B. longum can be according to the who definition attributed to probiotics.

Have indication that certain strains of B. longum to some extent possess anti-allergic action. For example, patients suffering from allergic rhinitis due to hay fever Japanese cedar, showed a decrease in allergic symptoms after ingestion of B. longum W in the weeks before and during the pollen season (Xiao JZ, Kondo S, Yanagisawa N, Takahashi N, Odamaki T, Iwabuchi N, Miyaji K, Iwatsuki K, Togashi H, Enomoto K, Enomoto T. Probiotics in the treatment of Japanese cedar pollinosis: a double-blind placebo-controlled trial ("Probiotics in the treatment of pollinosis in Japanese cedar: double-blind, placebo-controlled trial"). Clin Exp Allergy. 2006 Nov; 36(11): 1425-35. Xiao JZ, Kondo S, Yanagisawa N, Takahashi N, Odamaki T, Iwabuchi N, Iwatsuki K, Kokubo S, Togashi H, Enomoto K, Enomoto T. Effect of probiotic Bifidobacterium longum BB536 [corrected] in relieving clinical symptoms and modulating plasma cytokine levels of Japanese cedar pollinosis during the pollen season. A randomized double-blind, placebo-controlled trial (the"Action of the probiotic Bifidobacterium longum BB536 in alleviating clinical symptoms and modulating the plasma levels of cytokines in pollinosis in Japanese Cervo the pollen seasons. A randomized, double-blind, placebo-controlled trial"). J Investig Allergol Clin Immunol. 2006; 16(2):86-93.)

In addition, we discussed the effect on the immune system Allergy different cultures of probiotics or mixtures of probiotics, for example, EP 1858336 (WO 2006697949), describes a mixture of probiotics, which can reduce the risk of developing allergies to wheat albumin flour and globulins. JP 2006028050 describes suppressive properties in relation to skin allergies compositions containing such probiotic bacteria such as Lactobacillus and Streptococcus. In the publication WO 2009072889 (Jan knoll and others) describes Bifidobacterium, which can be used to improve pulmonary function in subjects suffering from allergies to dust mites. In JP 10309178 indicated, as selected human Bifidobacterium can help to cure food allergies or promote oral immune tolerance.

However, there is still a need to be able to in a certain way to ease allergic reactions and symptoms in populations of young children and infants. This is particularly important in relation to maturation as intestinal and immune systems in young children, with many new allergens, which are small children, especially during weaning baby from the breast.

The problem of allergic re the capabilities of foodborne at a young age even more complicated by the special nutritional needs of infants and young children. The caloric density of calories, variety of desirable nutrients, protein content and protein quality - all these factors are among play an important role in ensuring the most appropriate for infants and young children nutrition. Also important is the availability of micronutrients such as vitamins and minerals, especially when their concentration is limited to certain recommended ranges, age-appropriate target patients. In this regard, matrix, delivering food to young children and infants, by their very nature are complex, but their diversity is low: for example, infants, even potentially prone to allergies, usually require a certain protein balance in the matrix proteins derived from milk. The presence of such numerous nutrients may potentiate the action of food allergens. In such complex matrices weak food allergens themselves unable to cause allergic reactions, then you can become more powerful in triggering allergic reactions. Similarly, one can observe the decrease or disappearance of actions in such complex food matrices compounds that can neutralize allergens or exhibit prophylactic against allergies effect. In the lastnosti, it is impossible to predict whether the connection is usually recognized as having a preventive effect against allergies, to keep such activity and to what extent, in such complex matrices, as a nutritional composition for infants or small children.

There is a need to reduce the symptoms of food allergies in the group of infants and young children who have a history of allergic episodes and/or are available.

There is a need for complete nutrient composition, which not only ensures the supply of various nutrients, but also would reduce the severity of allergic reactions.

There is a need to provide nutritional compositions for modulation of allergic reactions in young patients suffering from allergies from mild to moderate severity, because such patients have special needs, dictated incomplete maturity of their intestinal and immune systems.

There is also a need to modulate allergic reactions in young children who do not have the tolerance to potent pharmaceutical drugs, for example, with the help of nutritional interventions, such as inclusion in the diet on a regular basis immunomodulators.

There is a need to provide nutritious whom is osili, modulating allergic reaction in young patients during and shortly after weaning baby from the breast, when his intestine undergoes a substantial transformation occurs when the flow of new solid foods potentially containing new allergenic proteins, and therefore, the patient is especially vulnerable to sensitization to food allergens.

There is a need in the relief of allergic symptoms by providing effective compositions may reduce exposure to intact allergens, even though this song may not reduce sensitization to allergens directly.

There is a need for compositions that would be a positive effect in secondary prevention of allergies, not necessarily showing the effect on the same Allergy in primary prevention.

Finally, there is a need for compositions most suitable for young patients, which by weakening symptoms could help minimize allergic phenotype and, thus, could reduce sensitization to new allergens in older age. There is a need to curb the atopic March.

The essence invented the I

In the first object of the present invention provides a complete nutritional composition designed to alleviate symptoms of allergies triggered by food products for patients suffering from allergies caused by food allergens, especially in young patients, young children and infants.

The second object of the present invention provides a composition, which greatly facilitates secondary prevention of allergic reactions caused by food allergens may not impact on the primary prevention against the same allergen.

The third object of the present invention provides a composition that contains probiotics, in particular belonging to the genus Bifidobacterium.

Another object of the invention, the composition of the invention is particularly effective for babies/small children during weaning baby from the breast.

The invention also extends to reduction sensitization to other allergens at an older age.

Brief description of drawings

Figure 1/Table 1. Th2 cytokine profile is offset peripheral blood mononuclear (RVMS) after co-culture with B. longum NCC 2705, registered Nestec SA as CNCM-I2618, or with other strains.

Figure 2/Table 2. The mRNA profile of Th2-semenichenko in human peripheral blood after co-culture with B. longum or with other strains.

Figure 3. Schematic description of food Allergy to egg white albumin (OVA) model in mice.

Figure 4. The view expressed in points clinical indicator showing the attenuation of the symptoms of food Allergy in mice treated with 5×108CFU/ml of B. longum NCC 2705 with drinking water.

Figure 5. Serum levels of protease 1 fat cells mouse (MSR-1).

Figure 6/Table 3. Cytokine production the mesenteric LN lymphocytes and splenocytes re-stimulated ex vivo.

Figure 7/Table 4. The levels of gene expression in the ileum

Disclosure of inventions

Definition.

In this description, the following terms have the following meanings

"Full nutritional composition". For the purposes of this document full nutritional composition is a composition which contains a significant amount, usually 20% or more of the basic food nutrients recommended for this age. These essential nutrients are usually provided in such quantities and proportions to match 20% or more is recommended for this age dosage of specific nutrients when used in quantities adequate to provide recommended for this age of consumption of calories. Full nutritional composition usually is about contains a source of protein, the source of lipids, a source of carbohydrates in a balanced proportion, which meets the General recommendation for this age. Usually it also includes micronutrients such as vitamins and minerals, as well as a source of essential amino acids and a source of essential fatty acids. However, it is understood that full nutritional composition may not contain all the specific nutrients or all of the recommended quantities that meet all the nutritional needs of an infant or small child. Full nutritional composition excludes compositions containing only Bifidobacterium, or those in which Bifidobacterium are the dominating share content.

"The symptoms of allergies" mainly include the symptoms caused by allergens. Such symptoms include skin (redness, rash, itching, dermatitis, eczema), eye (itching and redness), gastrointestinal (it's congestion, abdominal pain, cramps, vomiting, diarrhea), respiratory (itchy nose, nasal congestion, rhinitis, asthma), and in severe cases, the system (dizziness, confusion, anaphylaxis) symptoms.

"Primary prevention of allergies" means all measures aimed at the prevention or mitigation of allergic (immunologic) sensitization, such as the prevention or reduction of the JV is Titicaca IgE antibodies.

"Secondary prevention of allergies" means the prevention or attenuation of allergic disease/allergic symptoms in sensitized individuals.

"The period of weaning is a period during which infants are adapted from eating clean liquid to solid or semi-solid food and adapt to the transition from food quasi-individual (usually milk or mixture for infants) to a variety of food.

"Sensitization" means the induction/development of allergen-specific IgE antibodies.

"Probiotic" refers to the preparation of microbial cells or components of microbial cells with a beneficial effect on the health or well-being of the host body (C Salminen, Ouwehand A. Benno Y., etc. "Probiotics: how should they be defined" ("Probiotics: how to determine") Trends Food Sci. Technol. 1999:10 107-10). This definition of probiotics is widely accepted and is in accordance with the who definition. Probiotic may contain a single strain of microorganism, a mixture of different strains and/or a mixture of bacteria of different species and genera. However, in the case of mixtures can be used the term in the singular "probiotic" is used to denote a probiotic mixture or preparation. For the purpose of this is part II of the invention as probiotics are considered microorganisms of the genus Bifidobacterium.

"Prebiotic" usually means unassimilated component of the food product, which has good effects on the body of the recipient, by selectively stimulating the growth and/or activity of microorganisms present in the intestine of the host body, and thereby contributes to the improvement of the health status of the organism.

Strain Bifidobacterium longum (B. longum) NCC 2705 (room 2705 in meeting Nestle) is a strain of B. longum with the international security identification number CNCM-I2618 (Collection Nationale de Cultures de Microorganismes at the Institut Pasteur, Paris, France).

The inventors have demonstrated that allergic reaction and the symptoms can be alleviated when sensitized young mammals provided complete nutritional composition, which contains the B. longum NCC 2705. This defines a positive effect in secondary prevention of allergies.

This effect is accompanied by a small but significant reduction sensitization of young mammals to allergens (i.e. observed primary prevention of allergies).

The effect of the composition.

The invention relates to the use of microorganisms of the genus Bifidobacterium, more specifically Bifidobacterium longum (B. longum), and more specifically, strain B. longum NCC 2705 to get the full nutritional composition designed to alleviate symptoms in patients suffering from allergies, vyzyvae the diversified food allergens. The inventors have shown that the use of Bifidobacterium longum in General and B. longum NCC 2705, in particular, led to the weakening of the symptoms of food allergies in the group of mice treated with a nutrient composition containing a specified strain of Bifidobacterium. It's been proven in the induction of allergic reactions (provocation) after sensitization. The model simulates food allergies in humans, when people (usually infants/young children) naturally become sensitive to food allergens, and further exposed to these allergens again. When this bacterial strain B. longum NCC 2705 showed a protective effect.

In one embodiment of the present invention the effect of the composition, more specifically is the effect in secondary prevention of allergies. In the model in mice, the symptoms of the allergies were actually significantly reduced, as illustrated by the lower rates on the clinical scale of severity of allergic manifestations. As a rule, can include all the symptoms or selected recognized the symptoms of allergies.

In one embodiment of the invention symptoms include diarrhea, skin irritation or respiratory symptoms, or their combination.

In one embodiment of the invention symptoms may be accompanied by the release of biochemical mediators, such as tryptase chymase, histamine, leukotrienes.

In one embodiment of the invention the composition also has an effect on the sensitization of patients to allergens. In practice, using containing B. longum NCC 2705 compositions, apparently, achieved a small but significant in their effect primary prevention of allergies. Animals showed a lower sensitization to food allergens. The effect of this composition is of great interest in that it manifests itself in reduced symptoms, and reduction sensitization, that is, reducing the risk of subsequent symptoms with repeated exposure to exposure to allergens.

In one embodiment of this composition reduces sensitization to other allergens at an older age. It is assumed that the composition stimulates (or at least not blocking) the natural processes of the immune maturation and, therefore, capable of a long-term effect of reducing sensitization to the same allergens or other allergens (reduction sensitization at an older age). It is assumed that by reducing Allergy symptoms (secondary prevention) and short-term sensitization (as part of primary prevention), the composition of the invention may allow the passage of natural immune when Sravana, that provides such long-term beneficial effect.

The composition according to the invention or intended for use in the invention.

The composition of the invention can be a full nutritional composition, such as a mixture for infants, or it can represent a significant part of the total diet. Preferably the composition is a complete nutritional composition, which provides all or almost all of the nutritional needs of the target organism with its use as the sole source of nutrients. In one embodiment the composition comprises intended for babies porridge. In another embodiment, such as baby food, the composition is one part of a complete diet, preferably 50% or more, or 20% or more (quantitatively and qualitatively). In one embodiment the composition comprises intended for babies porridge. In one embodiment the composition may be a liquid composition for children, containing cereal or baby food.

Used probiotic refers to the genus Bifidobacterium. Although the inventors cannot exclude that other probiotics would be similar or close to the action, experimental data, is able to prove that other kind or strains of probiotics are equally effective, are not available, for IP the connection observed for Lactococcus lactis NCC 2287 (SL131) CNCM 1-4154 and Lactococcus lactis NCC 2180 (SL60) CNCM 1-4199, described in another, simultaneously pending application of the same authors.

In one embodiment of the invention, the probiotic is a strain of B. longum NCC 2705 CNCM-I2618.

In one embodiment of the invention the composition comprises between 104and 1011colony forming units (CFU) in 1 g of dry composition. When the composition is a mixture for infants, the number of probiotic mixture for infants may be between 105and 108CFU per 1 g of the mixture for feeding babies. In one embodiment the composition comprises between 105and 5×107CFU/g, which is the dose that demonstrates the presence of physiological actions. In one embodiment, it was determined that probiotics, low-dose can have beneficial effects, in particular, when the composition also contains prebiotics and proteins in a limited quantity. In this embodiment of the probiotics present in the composition in amounts of between 103and 105CFU/g Prebiotics can be the oligosaccharides and/or proteins that can be present in an amount of not more than 4 g/100 kcal or less than 2 g/100 kcal, or less than 1.8 g/100 kcal, or less than 1.5 g/100 kcal of the composition.

The probiotic may be mixed with dry or wet composition of the invention. Can be applied in specific ways or modes of processing in the order in which Ucrania stability or viability of probiotics in the composition. The probiotic can be used in dry or in wet form. After mixing the probiotic composition of the mixture can be processed in a way that has no significant effect on the viability of probiotics. In yet another embodiment of probiotics during or after mixing partially or completely inactivated. In one embodiment of probiotics to use in compositions of the invention have been translated in inaktivirovannoj and/or incapable of replication status. This can be achieved, for example, heat treatment or other described methods.

In one embodiment of the invention the composition contains prebiotics. It is known that probiotics contain carbohydrates and, more specifically, the oligosaccharides. In addition, it is known that they are widely used as ingredients of functional foods. They resist hydrolysis by enzymes of the digestive tract of man and can reach the colon in an undigested state, providing a carbohydrate material, especially suitable for the growth of bifidobacteria or other probiotics. Oligosaccharides can be obtained from, for example, glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, or mixtures thereof. Peeled offer for sale prebiotic foods such as fructooligo the sugars, contain more than about 95% of dry substance in the form of oligosaccharides.

Preferably one embodiment the composition is a nutritional composition that contains at least one prebiotic.

Preferably one embodiment contains prebiotic oligosaccharides derived from glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, or mixtures thereof. More preferably the oligosaccharide contains fructooligosaccharide. Most preferably, the prebiotic comprises a mixture of fructooligosaccharide and inulin. Preferably this mixture contains PREBIO1® or a mixture of commercially available RAFTILOSE® and RAFTILINE®.

Preferably one embodiment the prebiotic component of the composition contains from about 50% to about 90% of fructooligosaccharide. More preferably, it contains from about 60% to about 80% of fructooligosaccharide. The most preferable content of about 70% of fructooligosaccharide. Preferably one embodiment a prebiotic contains from about 10% to about 50% of inulin. More preferably, it contains from about 20% to about 40% inulin. Most preferably it contains about 30% inulin. In one embodiment the prebiotic component may be present between about 0.1% and 10% of the composition.

In one embodiment, the composition of the invention contains, in addition, Apple extract containing polyphenols. Apple extract can help to reduce symptoms of allergies to food products for patients suffering from allergies caused by food allergens. As such, Apple extract acts in a synergistic manner with the probiotic Bifidobacterium, modulating at the same time, reducing or attenuating allergic reactions in patients suffering from food allergies. In one embodiment the composition is used for baby food and/or intended for babies porridge, which naturally represent a suitable carrier for the composition. In the following embodiment of the baby food or intended for infant cereals contain Apple extracts or derived from apples material. In one embodiment of the invention the Apple extract is a protein extract, industrial output Val de Vire Bioactives (Conde sur Vire, France) under the name "Pomactiv HFV". In one embodiment Apple extract is similar to or received from the specified Pomactiv HFV and has a similar effect.

The proposed mechanism of action.

In recent decades, constantly increasing prevalence of allergic diseases and currently they are considered by the who as epidemic. In General terms, an Allergy is believed to be a consequence of imbalance between the immune responses Th1 Th2, leading to a strong shift towards the production of Th2 mediators. Therefore, without claiming to theoretical depth, it is assumed that allergies can be reduced, inhibited or prevented by restoring proper alignment between the Th1 and Th2 components of the immune system. This implies the need to reduce Th2 responses or increased, at least temporarily, the Th1 response. The first of these may differ decreased production of Th2 cytokines, such as IL-5, the latter can vary the increased production of these Th1 cytokines like IFNγ. Alternatively, in sensitised individuals most desirable may be a General anti-inflammatory effect, for example, through induction of T-regulatory (Treg) cells, capable of downward regulation of the actions of both Th1 and Th2 cells. This can be shown through the ability to stimulate the secretion of IL-10.

In one embodiment of the invention the composition contains hydrolyzed or partially hydrolyzed proteins. Nutrient composition based on (partially) hydrolyzed proteins, particularly suitable for the immune system and the gastrointestinal tract of infants/young children, as hydrolyzed proteins are easier to digest and have reduced allergenicity as compared with intact proteins. In addition to this is, not pretending to theoretical depth, we can assume that hydrolyzed proteins may be the preferred substrate for probiotic and intestinal bacteria (especially demonstrating the diversity of peptidase activity) compared to intact proteins, leading to increased activity of the strain of the probiotic and, thus, the synergism between gidrolizovannykh protein and probiotics. This is the most suitable composition for the immune system and the gastrointestinal tract of the infant/young child, prone to food allergies. In this case, the combination of B. longum NCC 2705 with gidrolizovannykh protein has the highest relevance for the invention.

In one embodiment of the hydrolyzed proteins are gidrolizovannykh proteins whey and/or casein. In one embodiment of the hydrolysed proteins derived as a result of actions on proteins (e.g. whey proteins) trypsin and/or chymotrypsin. In one embodiment of the hydrolyzed proteins contain soy protein or egg whites. In one embodiment of the hydrolysed proteins derived as a result of the action of proteases, such as Protamex® and/or Flavourzyme® (Novozyme, Denmark). In one embodiment, the proteins of the composition, preferably hydrolyzed proteins, proteins contain eggs or cereal. In one embodiment of the hydrolyzed proteins obtained actually is alcalase. The composition of the invention may contain a mixture of two or more of these sources of protein.

Target group.

The composition of the invention is most appropriately focused on the use of relatively young patients, although its effect on adults is not excluded or possible. Preferably, these patients actually young enough to have passed the phase of maturation of the immune system and gastrointestinal tract. In these patients, the effect of the composition may be more intense or faster. In one embodiment the composition is a mixture for infants intended for infant cereal and/or baby food. Preferably the composition is addressed to young people aged less than 6 years of age between birth and 3 years or between birth and weaning baby from the breast. In one embodiment the composition is a starting mixture or mixture for feeding babies. Preferably, the nutritional composition comprises the vast majority of the nutrients needed to supply young people.

In one embodiment the nutritional composition is a composition cereals intended for babies/young children aged 1-4 years. The composition may more specifically be intended for introduction in the period stun the value of breastfeeding and/or up to 12 months thereafter. The period of weaning from breastfeeding is really important for the invention, because infants during weaning are exposed to a variety of foods, while still undergoing maturation and reorganization of their immune system and gastrointestinal tract. Effective management of allergic reactions is, therefore, in this period of time is of particular importance.

In respect of the application of the compositions of the invention babies, small children or babies can be allergic children. Allergic children are children, small children or infants that suffered at least one attack, allergic reactions (light, moderate or heavy) to the food allergen. In one embodiment of the invention the children, small children or infants are recognized as having severe allergies to food allergens and/or experienced more than one moderate or severe bout of food allergies. Symptoms of allergies can include various well-known symptoms such as skin irritation or redness, gastrointestinal symptoms or respiratory symptoms.

Food allergens covered by the present invention may include all types of naturally occurring allergens or commonly found in food, especially in food products for young people (e.g. the R, infants, small children, children).

The experimental results.

Probiotics, mainly belonging to the genera Lactobacillus and Bifidobacterium, have been tested in trials on humans and animals for their ability to prevent allergic sensitization and development of allergic symptoms in sensitized individuals. Here it is analyzed whether a particular strain of B. longum, namely B. longum NCC 2705, potential to mitigate the development of allergic sensitization or symptoms.

At the initial stage was developed and applied in vitro system based on human blood lymphocytes to determine cytokine profiles induced by different strains of B. longum. It was assumed that the observed profiles are predictive for the production of cytokines induced in vivo by the same strains, and thus, the biological effects reported in these strains.

Cells of the allergic individual or of the individual, prone to the development of allergies differ in their tendency to the production of Th2 cytokines. To partially simulate this condition in vitro model was developed cell culture Th2-biased mononuclear cells of peripheral blood (RVMS) of a person as an alternative to applying RVMS allergic donors. Th2-shift was caused by what antivirovym RVMS with interleukin (IL)-4+anti-CD40-antibodies (for details, see "Methods"); after 3 days of cultivation, to induce Th2-shift, for an additional 48 hours were added bifidobacteria, leading to a total duration of cultivation in 5 days. Main-defined indicators were the cytokines IFNg, IL-10 and IL-5 measured ELISA (enzyme-linked immunosorbent assay) in the supernatants of cells stimulated B. longum.

Figure 1/table 1 shows that B. longum NCC 2705 induced similar amounts of IFNg and IL-10, resulting in the ratio of IFNg/IL-10, amounting to 1.3. Other strains of C. longum studied in parallel, such as B. longum NCC 3001 (W; claimed Morinaga as ATSS BAA-999 and B. longum NCC 435 (BL23, ATSS 15707), had the ratio of IFNg/IL-10, largely shifted to IFNg. In addition, the B. longum NCC 2705 proved to be an effective inhibitor of the secretion of IL-5. These data indicate that B. longum NCC 2705 in terms of Th2-shift is a powerful producer of IL-10 and therefore may possess antiallergic activity through anti-inflammatory activity.

Figure 2/table 2 demonstrate the proof of these results on the levels of mRNA for B. longum NCC 2705 compared with B. longum W and (from E. coli). The levels of expression of different genes was analyzed after 10 and 24 hours after the start of culturing Th2-biased human RUMS with different stimuli.

Cultivation of Th2-biased RVMS with B. longum NCC 2705 or NCC 3001 (W) leads to a moderate induction IFNg comparison with LPS, known for a strong inducing factor IFNg. Levels of mRNA expression of IL-10 induced Blongum NCC 2705, were higher than induced Blongum W. The mRNA levels of IL-5 was not changed, whereas GATA-3 was decreased after incubation with any of the strains. In General, these discoveries confirm the results obtained for the levels of protein content (Table.1.)

The effect Blongum NCC 2705 in vivo was investigated in a model of food Allergy in mice (OVA Food Allergy Mouse Model, illustrated in Fig.3.

Mouse strain BALB/c mice were sensitized at weekly intervals egg albumin (OVA)+cholera toxin, administered by mouth for 7 weeks. In this model, oral provocation high dose of OVA at the end of the period of sensitization leads to clinical symptoms such as diarrhea, combing, ruffled coat, cyanosis and loss of mobility.

NCC 2705 was given to mice in drinking water (5×108OE/ml; ad libitum) or during the sensitization phase from day 1 to 43 (primary prevention), during the last week of the experiment (days 43 - 50; secondary prevention), or throughout the entire test period (days 1 - 50 all the time).

Figure 4 shows the clinical symptoms observed in two independent experiments.

In mice, tucked therapy with Blongum NCC 2705, after provocation developed significantly less severe clinical symptoms, which sensitized animals not subjected to this treatment (positive control). The most pronounced effect was in secondary prevention, but it also remained significant and primary prevention, and when Blongum NCC 2705 was given throughout time (experiment 1, upper chart). In the second experiment confirmed the protective effect of secondary prophylaxis with trends that have been observed to effect in primary prevention or during the whole observation time (experiment 2, bottom chart).

As shown in Figure 5, in addition to the scale clinical indicators as criteria for activation putting in fat cells after allergen provocation were identified serological levels of protease-1 fat cells mouse (MSR-1). The OVA provocation led to a significant increase in serum levels MSR-1 in the positive control group compared with the negative control group. In mice subjected to processing NCC 2705, compared with positive control group there was a tendency for modulating the serum levels MSR-1 (though insignificant).

Figure 6/table 3 shows the production of cytokines re-stimulated ex vivo lymphocytes. With this purpose, after performing provocations were selected lymphocytes from mesenteric lymph nodes (MLN), again what was stimulirovalis 1 mg/ml OVA and were cultured for 72 hours. Using multiplex analysis (Mesoscale®) were measured levels of IL-1, IL-2, IL-4, KS, TNFa, IFN-γ, IL-5 and IL-10. MLN lymphocytes of mice treated NCC 2705 in primary prevention, has produced less IL-2, IL-4, TNFa, IFNg, IL-5 and IL-10 in comparison with the positive control.

The levels of expression in the intestine of various associated with allergic manifestations genes was determined by quantitative polymerase chain reaction (PCR) in real time (qRT-PCR - see method below). Evaluation of gene expression was performed on the tissue of the ileum for a group of positive control, negative control group and the treated group Blongum NCC 2705 during sensitization (primary prevention) and in the last weeks before provocation (secondary prevention). Figure 7/table 4 presents the results: sensitization led to increasing regulation of transcription, mRNA for typical Th2 cytokines, such as IL-4, IL-5, IL-9 and IL-13 (negative control against positive control) and IL-10. Treatment with NCC 2705 (primary or secondary prevention) led to a pronounced down-regulation of these markers.

Thus, the experiments showed lower regulation of immune responses through the attenuation of the expression of relevant genes, when animals received Blongum NCC 2705 (primary and secondary prevention is). It is believed, contributes to the anti-allergic effects of the studied strain of Bifidobacterium with the weakening of food allergies in this model.

Conclusions.

These data show that the use of Blongum NCC 2705 sensitized animals leads to a weakening of allergic symptoms during its exposure to the impact of the sensitizing allergen. Similarly, though less clearly, the consumption of C. longum NCC 2705 provided a protective effect during the sensitization phase (primary prevention). This discovery is consistent with reduced secretion of cytokines by cells of mesenteric lymph nodes, as well as reduced expression of the key associated with allergies genes in the intestine, suggesting that the modulation of components of the immune system contributes to the protective effect.

Analytical determination.

Reagents and bacterial biomass.

Bacterial biomass was obtained by culturing each strain under optimal conditions in a liquid nutrient medium. Was determined the kinetics of growth for each strain and accordingly biomass were taken after 3 hours after reaching the phase of maturity. In a moment of culture were washed twice in cold PBS (saline phosphate buffer) and frozen in PBS with 20% glycerol at -80°C in 50 μl of the aliquot is. LPS (lipopolysaccharide from E. coli) was purchased from Sigma (Buchs, Switzerland).

Isolation and cultivation Th2-biased human RUMS.

Mononuclear cells from peripheral blood (RVMS) people were selected from the filters obtained from the "Centre de Transfusion of the CHUV". Captured by the filters, the cells were washed back into the bag for blood collection 90 ml of balanced salt solution Hanks (HBSS) (Sigma). Cells were diluted 1:2 HBSS and RVMS were separated by centrifugation on density gradient on Histopaque 1077 (Sigma). Cells were collected at the interphase were washed twice with HBSS. RWMS was resuspendable in the environment Dulbecco modified by the method of Claims (cIMDM, Sigma), supplemented with 10% fetal bovine serum (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1% penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). The cells were cultured in 48-hole tablet (Milian, Meyrin, Switzerland) with 1.5×106cells/ml in the presence of 50 ng/ml IL-4 (Bioconcept) and 1 μg/ml anti-SO antibodies (R&D Systems, Abington, UK) in cIMDM to induce cytokines of the Th2 phenotype. LPS was used at a concentration of 1 µg/ml After 3 days of cultivation were added probiotics in quantities of 107, 106and 105CFU/ml After adding ingredients cultivation RWMS was continued for an additional 48 hours, resulting in a total duration of culturing in 5 days.

And the And cytokines.

Using DuoSet kits from R&D systems in accordance with manufacturer's instructions was measured levels of the cytokines IFN-γ human IL-5 human IL-10 human IFN-γ mice, IL-13 and mouse IL-10A mouse.

Evaluation of ELISA results.

Size OD (optical density) was converted to PG/ml using a standard curve of translation. To agree on significant fluctuations from donor to donor, typically observed for RVMS received from various people-donors, the data were standardized according to the conventional internal standard. The magnitude of IFN-γ in PG/ml obtained for each donor upon stimulation with LPS, was equal to 100%. Normalizing the values of IL-10 for 100% taken number of IL-10 induced by strain B. lactis NCC 2818 (deposited Nestec SA as CNCM-I3446). Finally, 100% was made the number of IL-5a induced IL-4 and antibody anti-SB exclusively in pure culture medium (i.e. in the absence of probiotics).

Quantification of gene expression by PCR in real time.

RVMS (1,5×106cells/ml) were cultured in cIMDM with IL-4 (50 ng/ml) and anti-CD40 (1 μg/ml) for 3 days. Then to RWMS was added ingredients and after 10 h and 24 h were selected cells. Total RNA was extracted from subjected to stimulation RVMS kit "SV Total RNA Isolation System (Promega, Wallisellen, the Doorman is I), including processing of DNA Asai according to the manufacturer's instructions. The total RNA was determined quantitatively by using the dye ribogreen kit for the quantitative definitions RNA (Molecular Probes, Basel, Switzerland). Was performed by reverse transcription of 1 μg total RNA using a kit "Multiscribe Reverse Transcriptase kit (Applied Biosystems, Foster City, CA, USA). The total RNA was mixed with 50 µm random hexamers, 0.5 mmol dNTP, 20 E inhibitor RNA-ASE (Applied Biosystems), 62.5 E MultiScribe reverse transcriptase, IX RT buffer and 5.5 mmol MgCl2in the final volume of 100 μl. Human IFNg, IL-10, IL-5, Tbet, GAT A3, FoxP3 (Applied Biosystems) was quantitatively determined by PCR in real time (Applied Biosystems, ABI PRISM 7900HT) using a kit for assessing the level of gene expression Taqman. Quantitative indicators were subjected to normalization by three genes of the household": (β-actin, GAPDH and HPRT (Applied Biosystems).

Based on the obtained values of the threshold cycle (Ct) for each gene using the ACt were determined relative and normalized indicators of mRNA expression. The Ct value for each gene was adjusted by the average Ct three genes of the household". The results in expression relative expression was calculated by the formula: 2-ΔCt×K.

The model of food Allergy to OVA in mice.

All studies were approved by the internal Committee is ω ethics of the company Nestec and Veterinary service of the Canton of Vaud, Switzerland (resolution No. 1970). Six-week female mice of BALB/c mice (Harlan Laboratories, France) were sensitized by oral (gavage feeding) through weekly intervals to 20 mg of egg albumin (OVA) from Fluka (Buchs, Switzerland)+10 μg/mouse of cholera toxin (used as adjuvant; List Biologicals, purchased from LuBioscience, Lucerne, Switzerland), for 7 weeks. One week after the last sensitization was performed oral provocation 100 mg OVA. At different phases of the experiment was carried out nutritional intervention with B. longum NCC 2705 (5×108CFU/ml in drinking water); for primary prevention - during sensitization, for secondary prevention - since the late phase of sensitization, or during the entire time of the test (Fig.3). After a 30 minute run provocations mouse for 30 min was observed individually. Clinical symptoms were recorded and quantitatively determined in the following way (the assessment of the severity of allergies in points): 0: no symptoms, less than 4 episodes tangle free; 1: 4-10 episodes of scratching around the nose and head, diarrhea is absent; 2: more than 10 episodes of scratching or vzdelavani wool and lack of mobility or soft stool; 3: diarrhea, or difficulty breathing or cyanosis; 4: diarrhoea in combination with immobility after boost, vzdelavani SERS and, difficulty breathing or cyanosis; 5: anaphylaxis. Four hours later after provocation mouse wordplays (displacement of the cervical vertebrae), were selected blood and the last cm of the ileum and frozen in liquid nitrogen. Serum MSR-1.

Protease-1 fat cells of mice (MSR-1) was determined quantitatively in the mouse serum by means of ELISA method purchased from Moredun Scientific (Penicuik, Scotland) according to the manufacturer's instructions. Data on concentrations MSR-1 were obtained by the conversion of OD values in PG/ml using a polynomial translation.

Isolation and cultivation of cells in mesenteric lymph nodes.

Mesenteric lymph nodes (MLN) were gomogenizirovannykh the plunger of the syringe in the strainer for cells (BD Falcon, Milian, Meyrin, Switzerland). Cells were tsentrifugirovanie and were washed twice in RPMI medium (Sigma) supplemented with 10% fetal bovine serum FBS (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1% penicillin/streptomycin (Sigma), 0.1% gentamicin (Sigma), 0.1% of β-monothioglycerol (Sigma). The cells were cultured in 96-well flat-bottomed tablet (Corning, Milian) in the absence or presence of OVA (1 mg/ml) with 3×106cells/ml After 72 h culture plates were frozen.

Cytokines in the supernatant of cultures MLN.

Using a kit for multiplex analysis of 9 analytes Th1/Th2 mouse (Meso Scale Discovery, Gaithersburg, MD, USA) according to the accordance with the manufacturer's instructions have been measured levels of murine IL-4, IL-5, IL-10, IFN-γ, IL-1β, IL-2, IL-8, TNF-α, IL-12T.

Analysis of gene expression in the intestine by using a low-density microarray.

Isolation and quantification of RNA.

Total ribonucleic acid (RNA) was isolated from the ileum according to the manufacturer's Protocol using the kit "SV Total RNA isolation System kit purchased from Promega (Dubendorf, Switzerland). RNA was quantified according to the manufacturer's Protocol using the kit quant-IT Ribogreen Reagent kit provided by Promega (Dubendorf, Switzerland).

Reverse transcription.

Reverse transcription was performed on 1 µg of total RNA using a kit "Multiscribe Reverse Transcriptase kit from Applied Biosystems (Foster City, CA, USA). The total RNA was mixed with 50 µm random hexamers, 0.5 mmol dNTP, 20 E, an inhibitor of RNA-ASE (Applied Biosystems), 62,5 E MultiScribe reverse transcriptase, IX RT buffer and 5.5 mmol MgCl2in the final volume of 50 µl. Reverse transcription was carried out on a T3 thermocycler (Biometra, Gottingen, Germany) according to the following program cycle: 10 min at 25°C, 30 min at 48°C, 5 min at 95°C with termination at 4°C.

Analysis on microchips low density (Low Density Array, LP (A).

Plan for LDA was developed in online on the website of the Applied Biosytems (http://www3.appliedbiosystems.com/index.htm). Loading, execution, and analysis were performed according to the Protocol of the manufacturer on the device to share the public dimension ABI Prism NT".

Quantitative results were subjected to normalization by three genes of the household": β-actin, GAPDH and HPRT. Based on the obtained values of the threshold cycle (Ct) for each gene using the ΔCt were determined relative and normalized indicators of mRNA expression. The Ct value for each gene was adjusted by the average Ct three genes of the household". The results were calculated as relative expression using the formula 2-ΔCt×K, where K is a multiplier to 106. The magnification of results expression was normalized to levels of expression in the negative control group.

Example 1

Below is an example of the composition of the mixture for infants according to the present invention. This song is for illustrative purposes only. The protein source is a conventional mixture of whey protein and casein.

Nutrient100 kcal1 liter
Energy value (kcal)100670
Protein (g)1,8312,3
Fat (g) 5,335,7
Linoleic acid (g)0,795,3
α-Linolenic acid (mg)101675
Lactose (g)11,274,7
Prebiotic (100% GOS), (g)0,644,3
Minerals (g)0,372,5
Na (mg)23150
K (mg)89590
Cl (mg)64430
Ca (mg)62410
P(mg)31210
Mg (mg)750
Mn (µg)850
Se (µg) 213
Vitamin A (mcg, retinology equivalent (RE))105700
Vitamin D (µg)1,510
Vitamin E (mg, tocopherole equivalent (TE))0,8of 5.4
Vitamin K1 (μg)854
Vitamin C (mg)1067
Vitamin B1 (mg)0,070,47

Vitamin B2 (mg)0,151,0
Niacin (mg)16,7
Vitamin B6 (mg)0,0750,50
Folic acid (µg)960
Pantothenic acid (mg)0,453
Vitamin B12 (µg)0,32
Biotin (µg)2,215
Choline (mg)1067
Fe (mg)1,28
I (g)15100
Cu (mg)0,060,4
Zn (mg)0,755
Bifidobacterium longum NCC 2705 (see experimental part)2·107CFU per 1 g powder

1. The use of Bifidobacterium longum NCC 2705 (CNCM-I2618) to get the full nutritional composition designed to alleviate symptoms of allergies to food products for patients suffering from allergies caused by food allergens, with this composition provides significant secondary prophylaxis against allergic reactions caused by these food allergens.

2. Application under item 1, where the specified composition has a str is the functioning to influence the sensitization of these patients to these allergens.

3. Application under item 1, where the specified strain Bifidobacterium longum is a probiotic.

4. Application under item 1, where the specified composition contains 105-108CFU in 1 g of dry composition.

5. Application under item 1, where the specified composition comprises at least one prebiotic, preferably containing fructooligosaccharide.

6. Application under item 1, where the specified composition contains Apple extract containing polyphenols, and where specified Apple extract helps to reduce the symptoms of allergies to food products for patients suffering from allergies caused by food allergens.

7. Application under item 1, where the specified composition contains hydrolysed proteins.

8. The use according to any one of the preceding paragraphs, where the specified composition reduces sensitization to other allergens at an older age.

9. Application under item 1, where these symptoms are gastrointestinal, skin, or respiratory, or their combination.

10. Application under item 1, where the specified composition contains cereal proteins.

11. Application under item 1, where the specified composition is a mixture for infants designed for babies porridge, a liquid composition for children, containing cereal or baby food.

12. Application under item 1, where the specified composition is a composition cereals, etc is naznachennoe for infants aged 1-4 years.

13. Application under item 1, where the specified composition is provided to infants during breastfeeding and/or up to 12 months thereafter.

14. Application under item 1, where these patients are young children aged less than six years, preferably aged between birth and 3 years.

15. Application under item 1, where these symptoms are accompanied by the release of biochemical mediators, such as tryptase, chymase, histamine, leukotrienes.

16. The use according to any one of paragraphs.1, 3, or 4, where the specified Bifidobacterium undergoes inactivation to make it non-replicated.



 

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