MXPA06013955A - Method for improved breast milk feeding to reduce the risk of allergy - Google Patents

Abstract

Selected strains of Lactobacillus and products containing cells of the selected strains to improve breast milk for feeding to babies, more precisely to increase the levels of the anti-inflammatory cytokine IL10 in the milk and reduce the risk that the feeding baby will develop allergies and simultaneously reduce the cause and thereby the amount of TGF-beta-2 in the milk, thus resulting in reduced risk for the lactating mother to develop mastitis.

Description

METHOD OF FOOD FOR IMPROVED LACTATION IN ORDER TO REDUCE THE RISK OF ALLERGY BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the selection and use of lactic acid bacteria for improved lactation for infant feeding.

Description of the Related Art The hygiene hypothesis of allergic diseases suggests that environmental changes in the industrialized world have led to reduced microbial contact at an early age and have therefore resulted in the epidemic growth of allergic disease, such as atopic eczema. , allergic rhinoconjunctivitis and asthma. One of such environmental changes that has been discussed is our changed intake of natural microbes of various kinds due to improved hygiene, quality of life, eating habits, etc. This has led to shifts in the composition of our natural intestinal flora (Journal of Allergy and Clinical Immunology, 2001; 108: 516-520) and also to changes in the composition of breast milk of a breastfeeding mother. Such changes have been linked to the presence of allergies of various types. It has also been reported that infants who are the latest additions to larger families have a reduced risk of allergy compared to their siblings who were previously born (the first of the progeny). This can imply that a mother's mother's milk "improves" with the increasing number of pregnancies. Human milk contains a variety of important components in the immune system, such as macrophages, immunoglobulins or antimicrobial proteins, which are thought to protect against infection and inflammation in the gastrointestinal and respiratory tract of milk-fed offspring. In addition, the presence of other potentially immunomodulatory factors (eg, complex oligosaccharides, growth factors, enzymes, hormones or cytosines) has been discussed. These beneficial properties together with the high availability of nutrients and the low antigen content of human milk are the physiological basis of the current recommendation by pediatric experts that breast milk is the best food for infants, especially those with a history family allergy Therefore, it is known that maternal milk contains a series of cytosines and chymosins that potentially affect the development of allergy in the infant. It has been previously reported that components that modulate allergic reactions, such as cytosines, chymosins and adhesion molecules, are secreted in milk at various stages of lactation (S Rudloff et al., Allergy 1 999, 54, 206-21 1 ) . The cytosines or chymosins could be either beneficial or disadvantageous for the infant breastfed. It is also known that cytosines supplied by the breast milk of animals have the ability to survive passage through G l tract of the offspring. For example, homozygous knockout mice TGF-beta-1 died from diffuse inflammatory disease after weaning, presumably being saved until weaning by the transfer of TGF-beta supplied by breast milk (Kulkarni AB et al., Am J Pathology 1 993; 143, 3-9). TGF-beta survives the passage all the way to the colon. I L-10 probably also survives in this way and thus the supply from breast milk allows the I L-1 0 to reach the Gl tract and potentially induces beneficial anti-inflammatory effects. In addition, Hawkes JS et al. (Lipids 2001 Oct 36: 1 179-81) reported that long-chain polyunsaturated fatty acids have been associated with aspects of immune regulation that include the production of cytosine. The purpose of this study was to investigate the effect of maternal dietary supplementation with tuna oil, rich in docosahexenoic acid (DHA), on the concentration of transforming growth of beta factor 1 (TGF-beta-1) and TGF-beta-2 in breast milk In this trial of dietary intervention, randomized, mothers of term infants consumed a dietary supplement of 2000 mg of oil containing either placebo (n = 40), 300 mg DHA (n = 40), or 600 mg DHA (n = 40). The increase in DHA in the milk and plasma was proportional to the DHA in the diet. There was no relationship between the DHA status of the milk and the levels of TGF-beta-1 and TGF-beta-2. IL-10 is a well-documented and anti-inflammatory cytosine accepted. The implication is that it will have anti-inflammatory effects in the infant's Gl tract. It is good for the infant that breast milk is consider generally anti-inflammatory in the sense that the infant should not over-react to pathogens / infections that appear in the intestine early in life. In addition, animal data point to possible benefits for weaning I L-10 in milk. An Australian group observed an animal model where mite allergy was investigated. The animals that were negative to a skin bite test (SPT) for mites had production of I L-1 0 and I L-4, obstructing the I L-10 IgE (allergic response) and showing the animals null allergy . In the allergic animals SPT + ve (histamine), only I L-4 was produced and there was no production of I L-10. Therefore, IgE was not decreased and allergy prevailed. In this way, IL-10 obstructs the allergy-inducing effects of IgE and can prevent the development of allergy. TGF-beta-2 (Transforming Growth Factor) is also a well documented growth factor / cytosine. Their sources include, for example, platelets that produce quantities in milligrams of TGF-beta / kilogram. The factor and its isoforms (see below) can also be isolated from other subjects (microgram TGF / kg) and found predominantly in spleen and bone tissues. Human milk also contains this factor and is also synthesized, for example, by macrophages (TGF-beta-1), lymphocytes (TGF-beta-1), endothelial cells (TGF-beta-1), keratinocytes (TGF-beta-). 2), granulosa cells (TGF-beta-2), condorcytes (TGF-beta-1), glioblastoma cells (TGF-beta-2), leukemia cells (TGF-beta-1). Depending on the type of cell and conditions, the secretion of TGF-beta can be induced by a number of different stimuli, including spheroids, retinoids, EGF (Epidermal Growth Factor), NGF, lymphocyte activators, vitamin D3 and IL1. The synthesis of TGF-beta can be inhibited by EGF, FGF (Fibroblast Growth Factor), dexamethasone, calcium, retinoids and follicle stimulating hormone. TGF-beta also influences the expression of its own gene and this may be important in wound healing. TGF-beta exists in at least five isoforms, known as TGF-beta-1, TGF-beta-2, TGF-beta-3, TGF-beta-4, TGF-beta-5, which are not related to TGF-alpha . The amino acid sequences of these isoforms have homologies of the order of 70-80 percent. TGF-beta-1 is the prevailing form and is found almost ubiquitously while the other isoforms are expressed in a narrower spectrum of cells and tissues. The isolated isoforms of different species are evolutionarily accurate and have sequence identities of the order of 98 percent. Mature, human, porcine, simian and bovine TGF-beta-1 are identical and differ from murine TGF-beta-1 in a single amino acid position. Human and chicken TGF-beta-1 are also identical. It has also been reported that members of the transforming growth factor beta family (TG F-beta) are pleiotropic cytosines with key functions in morphogenesis and tissue growth (Ingman WV, Bioassays 2002 Oct 24: 904-14). TGF-beta-1, TGF-beta-2 and TGF-beta-3 are abundant in mammalian reproductive tissues, where development and cyclic remodeling continue after birth and adult life. The potential functions of TGF-beta have been identified in the development of gonads and secondary sex organs, spermatogenesis and ovarian function, immunoregulation of pregnancy, embryo implantation and development of the placenta. Rautava er al. , in Journal of Pediatric Gastroenterology and Nutrition 38: 378-388, April 2004, declares that TGF-beta-2 and I L-1 0 appear to function in a synergistic manner with TGF-beta-2 favoring the production of IL-10 . Mastitis is an inflammation of the breast that is often characterized by sensitivity and erythema and sometimes fever and is associated with TGF-beta-2. During mastitis, the tight junctions of the alveolar mammary cells open and this process is accompanied by an increase in sodium, inflammatory cells and inflammatory and immunological mediators in breast milk. Mastitis is usually unilateral and the highest incidence is in the first weeks of lactation. In industrialized countries, mastitis has generally been considered a problem of low morbidity, since affected women are usually treated by nurse practitioners and midwives. Mastitis seems to be more common than previously believed, since the large longitudinal studies that have followed lactating women in the UA, Finland and Australasia (Semba R., Annals of the New York Academy of Sciences, November 2000; 91 8: 156-62) suggest that 20-33% of women develop clinically apparent mastitis. The number of women breastfeeding with sub-clinical mastitis is consequently logically even greater. Mastitis has also recently been linked to an increased burden of human immunodeficiency virus (HIV) in breast milk and an increased risk of mother-to-child transmission of HVI. (Semba, RD, N. Kumwenda, TE Taha, et al., 1 999. Mastitis and immunological factors in breast milk of women infected with human immunodeficiency virus J. Hum. Lact. 1 5 (4): 301 -306 ). TGF-beta-2 in maternal milk is mainly of epithelial origin even if it is synthesized by many other cells, including B and T cells. Consequently, the increased level of TGF-beta-2 could be a mediator of or an effect of a sub-clinical breast inflammation. The proportion of sodium and potassium in the mother's milk (proportion of Na / K) is said to be a good predictor of infection and sub-clinical breast inflammation. Also, Kalliomaki ef al. , in J Allergy Clin I mmunol. 1 999 Dec; 1 04 (6): 1 251 -7, has suggested that TGF-beta in colostrum can prevent the development of allergic disease during exclusive breastfeeding and promote the specific production of IgA in human subjects.

In addition, various body parts of humans and other mammals are inhabited by many different species of bacteria, including a number of different species of Lactobacillus. Such bacteria often coexist with their host, giving beneficial synergistic effects of various kinds, nowadays it is known that they are also diverse and depend on the current strain of bacteria. The different strains of lactic acid bacteria, for example, L. reuteri SD21 12, have specific antigens, either on their surface or released by bacteria in the gastro-intestinal tract of the mother. The data in Valeur et al. , AEM, 70, 1 1 76-1 1 81 (2004) show that L. reuteri SD21 1 2 ingested can affect the levels of CD4 + T helper cells in the ileum of a healthy human as an example. Such observations have also been made in other mammalian and avian species indicating that this may be a fundamental signaling system between the intestinal flora and the host. Through the so-called whole-mammary bond, the antigens of the active strains are actively transported to the lymphoid region, i.e., the Peyer patches, below the Gl tract epithelium. The antigen-specific B cells are then activated, after which they migrate from the ep tract of the Gl tract through the circulation to other mucosal membranes in the body, including the salivary and mammary glands. The expression of specific molecules in these cells is through directing their adhesion to these tissues. Once in the mammary gland, these immune cells are then directed to other processes to determine the levels of locally produced cytosines. This type of signaling through the entero-mammary link has been demonstrated in the generation of secretory IgA in maternal milk and it is highly probable that it is also applied to the production of cytosine. It has been suggested previously (Laiho et al., Pediatric Research 53: 642-647, 2003) that the associations observed between the nutritional and inflammatory factors in breast milk show that it may be possible to influence the immunological properties of breast milk through dietary intervention of the mother. The same group observed that mothers with an allergic response have a lower concentration of TGF-beta-2 in breast milk compared to those without allergy. In his hands, I L-1 0 was detected, only at low levels and frequency in a natural way in breast milk, without difference between mothers with allergic disease or without it. It was suggested that the protection of allergic disease was mainly through the induction of oral tolerance for TGF-beta-2 and I L-1 0 and that particularly the TGF-beta-2 of breast milk can play a key role with respect to to the prevention of allergic disease. Nevertheless, Weiner H. Reported in Microbes and Infection, Volume 3, Topic 1 1, September 2001, pages 947-954, which due to regulatory T cells, generated by oral antigens, are activated in a specific manner of antigen, but are suppressed in a manner nonspecific antigen, mediate the surrounding suppression when they find the self-antigen fed into the target organ. Thus, mucosal tolerance can be used to treat inflammatory processes that are not autoimmune in nature. Different species of Lactobacillus, including Lactobacillus reuteri, have been used in so-called probiotic formulations, which means they supply an animal, including humans, with live and beneficial microorganisms. Lactobacillus reuteri is one of the naturally occurring inhabitants of the gastrointestinal tract of animals and is normally found in the intestines and occasionally in the birth canal, breast milk and mouth of healthy animals, including humans. They are known to have antibacterial activity. See, for example, Patents of E. U. Nos. 5,439,678, 5,458, 875, 5, 534, 253, 5,837, 238 and 5,849, 289. When L. reuteri cells grow under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (ß-hydroxy). propionaldeh ído). The lactic acid bacterium has also been reported previously that is used to prevent and treat allergies of various kinds, for example, the following patents / patent applications EP 1 239032 by Stadler et al. , with respect to new recombinant strains, WO 01/37685 by Clancy et al. , with respect to the decrease in the amount of IgE by lactobacilli. Accordingly, an object of the invention is to provide selected lactic acid bacteria and components thereof for improved breast milk for feeding babies, and a method for such selection. More precisely, an object of the invention is to increase the levels of anti-inflammatory cytosine I L-1 0 to reduce the risk that the baby will develop allergy at the same time as the cause and therefore the amount is reduced. of TGF-beta-2 in milk and the risk that the breastfeeding mother develops mastitis. Accordingly, an object of the invention is to compensate for the negative changes in the microbial flora by giving specifically selected strains of lactic acid bacteria to mothers before and during lactation. Another object of the invention is to select, by using the method described herein or similarly distinguish the influence of specific cytosine from the strains tested on relevant cell types and the use of such certain strains of lactic acid bacteria dietary components for mothers, which stimulates the increased production of IL-1. in breast milk at the same time that the level of TGF-beta-2 is reduced, indicating a lower level of sub-clinical inflammation in the milk glands of the breast and other tissue and therefore reducing the risk of mastitis. Mastitis and sub-clinical mastitis can be considered to interfere with breastfeeding and thereby avoid the benefit to the infant providing breast milk - the lactobacilli selected by the method of the present invention can then improve the health of the mothers and enable them Give milk for a longer time. A further object of the invention is to provide products containing said strains, mutants, metabolites or components thereof, including agents for administration to animals, including humans. Other objects and advantages will be more fully apparent from the following statement and appended claims.

Brief Description of the Invention The invention herein comprises strains of Lactobacillus and components thereof, which have been selected for their ability to improve breast milk to feed babies, more precisely to increase the levels of anti-inflammatory cytosine IL10. in the milk for reduced risk that the feeding baby will develop allergy at the same time the cause is reduced and therefore the amount of TGF-beta-2 in the milk, meaning a reduced risk that the breastfeeding mother will develop mastitis and thus increasing the ability to give breast milk and the overall protection and optimal growth that gives the child. The invention herein also includes the method of selecting such strains of Lactobacillus. The invention also includes a novel method for protecting viable Lactobacillus in a formulated oil product, as a means of delivering the active component. Other objects and features of the inventions will be more apparent from the following disclosure and appended claims.

Brief Description of the Drawings Figure 1 is a flowchart of a manufacturing process that can be used to make the product of the invention.

Detailed Description of the Invention and Preferred Modalities of the Same The present invention provides a product to improve breast milk for infant feeding, more precisely to increase the levels of anti-inflammatory cytosine I L1 0 in milk and reduce the risk that the feeding baby will develop allergies and simultaneously reduce the factors that trigger the production of the cellular signaling substance (TFR) and therefore the amount of TGF-beta-2 in milk, thus resulting in a reduced risk that the breastfeeding mother will develop mastitis. The decrease in TGF-beta-2 associated with the invention is considered to be an anti-inflammatory effect of the consumption of selected lactic acid bacteria, which means that the incidence of sub-clinical mastitis is decreased in the lactating group that has recently ingested daily the bacteria. An example of a strain selected by the invention herein is L. reuteri SD21 1 2 (ATCC 55730). In a clinical trial in the research leading to this invention, there were only three factors in the analysis that were related to the level of I L-1 0 in the colostrum of the mothers: a) treatment or not with lactic acid bacteria selected, b) the number of children previously born to the mother and c) the number of previous pregnancies. In this way, the greater the number of infants or pregnancies that a mother has, the greater the I L 1 0 that occurs in her colostrum. Giving the waiting mother specifically selected lactic acid bacteria four weeks prior to delivery also has the ability to increase the level of I L-1 0 in colostrum without requiring prior pregnancies. Increased systemic expression of IL-10 is also known in some situations that avoids type 1 diabetes, therefore, the selects selected in accordance with this invention could also be useful for this purpose. In the selection method used here, the best strains to induce I L1 0 in breast milk and demonstrate a level reduced TGF-beta-2, are selected by using an established mouse model and traditional analytical methods. Other similar methods can also be used for the detection of cytosine production in milk. The details of this will be understood more clearly in the Examples. The product of the invention preferably contains living cells of the selected strain (s); however, if the isolated metabolites or parts of such cells are responsible for the activity of the living cells of the strain (s), the products of the invention may include such metabolites or parts in addition to or in place of the cells. living The product of the invention can be any product for consumption by women, such as a product of oil drops, food products, tablets, capsules, powder bags and the like. The products that lend themselves particularly for use in the invention include a drop of oil (as in Example 3) which will help to keep the active ingredient stable for a long time. Lactobacillus cells have been used in oil formulations for improved stability of bacteria, see, for example, U.S. Patent. No. 451 8696 by Gehrman er al. But none of the existing formulations of lactic acid bacteria in oils and fats that we have found contain the important step of drying the oil by vacuum prior to formulation for increased stability of the bacterial cultures, as set forth in the examples in the present. The concentration of selected Lactobacillus cells, necessary for the efficacy of a product of the invention depends on the type of food and the amount of food to be ingested (or the time of use in the mouth of a non-food dental treatment product), but it is usually preferable that it has an equivalence of approximately 1 05 - 1 08 CFU (colony forming units) or more per daily intake of a product. Amounts of up to about 1010 - 101 1 CFU are possible and can be used to increase efficiency without adversely affecting the organoleptic characteristics of the product (its taste or odor). When the product is a yogurt or other lactic acid fermentation product, the lactic acid fermentation strain (s) used to produce the product would be (preferably) standard cultures (eg, in yogurt, S. thermophilus and L. bulgaricus). It is important that the selected active strain have the desired cytosine effects according to the invention herein to be compatible with any standard culture used in the product, so that the important properties of each strain used are not negated by the use of the other strain (s). Of course, this can be easily determined by selection examinations known in the art. The strains used for the invention herein may be added either before or after fermentation of the product at an equivalent level of about 1 06 - 108 CFU per day, serving yogurt or more as discussed above. Preferably, the product of the invention does not contain other antibacterial components, at least none that inhibit or eliminate the selected strain (s) of Lactobacillus, or metabolites or components thereof, or that interfere with the activity. The Lactobacillus strain (s), or metabolites or components thereof, can be an additive mixture in the ingredients by means known in the art for formulation of such products. When cells are used and if the preparation of the selected food or other product of the invention requires a heating step, the Lactobacillus strain (s) should be watered after heating. Once selected cells of Lactobacillus are found in the product, it is preferred not to heat the product to 60-70 degrees C or above for a prolonged period of time. The features of the invention will be understood more clearly by reference to the following examples, which should not be considered as limiting the invention.

Example 1 . Strain Selection Method The selection of the Lactobacillus strains to be used according to this invention can be carried out in the following manner in stages: Evaluation of stimulation of I L 1 0 production and reduction of TGF-beta-2 in human breast milk by cells of Lactobacillus strains for selection of strains This is an example of the selection method; certain variations and the alteration of this method can be done by someone expert in the matter without departing from the invention herein.

Materials and Methods Animals: fifty-one BALB / c germ-free mice (males and females) are purchased from Wisconsin University, USA. The mice are transported in sterile plastic film transporters. Fifteen mice are transferred to each of 3 isolators (isolators # 2, 3 and 4). Two cages each containing 2 males and 4 females are placed in insulator # 1 (feeder).

Animal Handling Mice are maintained in flexible, sterile film insulators (Class Biolog ically Clean, Ltd, Madison, WI, USA). The isolators are sterilized with 2% acetic acid solution (FMC Corporation, Philadelphia, PA, USA) containing 0. 1% Naconal (Stepan Co., Rocksport, I L, USA). Five to six mice are housed in polystyrene cages sterilized with peracetic acid with stainless steel wire lids. Mice of the same genus are placed in each cage. Mice are fed autoclavable Agway 3500 rodent food (Agway, Granville, Creedmoore, NC, USA). The food, water and bed are placed in an autoclave. The feed and bed are autoclaved inside stainless steel cylinders and then transferred aseptically to insulators. The bottles with running water are autoclaved and then sterilized with peracetic acid when placed in insulators. All mice They received food and water to taste. Water and food levels are checked daily. The bed changes once a week. The animals were kept under a light / dark cycle for 1 2 hours. The ambient temperature and relative humidity are checked daily.

Bacteria The lactic acid bacteria to be examined are identified and characterized by biochemical and molecular biology techniques. Lactobacilli are first grown in 10 ml of Man-Rogosa-Sharp medium (MRS) (BBL, Cockeysville, MD), incubated for 1 h at 37 ° C and then transferred to 90 ml of MRS, incubated at 37 ° C. C for 18 h and transferred to 1 000 ml of MRS. After 1 8 h of incubation at 37 ° C, the cultures are rotated at 3000 rpm for 10 minutes in a refrigerated centrifuge (SORVALL RC2-B, SORVALL, Norwalk, CN USA), the supernatant is discarded and the granule is rinsed twice with sterile phosphate buffered saline (PBS) at 3000 rpm for 10 min. The granule of each strain is re-suspended in 30 ml of PBS. The cultures are placed in 1.2-ml cryo-flasks and stored at -70 ° C until used. Before offering the bacteria to the mice, the purity and concentration of the cultures are verified. Serial 1/10 dilutions of the lactobacillus suspensions were grown on M RS medium plates with 1.5% agar and incubated in anaerobic jars (GasPak: BBL, Cockeysville, MD, E AU) containing anaerobic generators ( Anaeroid Gen: Oxoid Ltd., Wake Road, Basig nstoke, Hampshire, England, GB) for 48 h at 37 ° C. The Crops were checked for colony morphology and reuterin production.

Examination Treatments (in this example) Control. Strain: Lactobacillus reuteri SD21 12, ATCC 55730. Strain: Lactobacillus 4000 Strain: Lactobacillus 4020 Determination of colonization of Lactobacillus One day after the mice were placed in the isolators, fecal samples of mice were taken in each cage to examine the absence of microorganisms. The mice are denied water from 1 1:00 a.m. to 7:00 p.m. After this period of time, 1.2 ml of suspension of lactic acid bacteria are added to each bottle containing 200 ml of water and offered to the mice. Mice in isolator # 2 receive L. reuteri SD21 12. The suspension added to water contains 2.0 x 1010 cfu / ml of L. reuteri SD21 1 2. Mice in isolator # 4 receive strain of Lactobacillus 4000 and mice in isolator # 5 they receive strain of Lactobacillus 4020. The suspension of lactic acid bacteria added to each bottle of water contains 3.0 x 1010 cfu / ml. The mice in insulator # 3 receive only water (control). Fecal samples (1 0 granules) of the mice (cage group) in each isolator are taken per week to examine the colonization of lactic acid bacteria and possible contamination with other microorganisms.

"Conventionalization" with altered Schaedler flora Sixty days after colonization with test strains, the initiated mice are "conventionalized" with altered Schaedler flora. C3H mice containing altered Schaedler flora were purchased from Taconic Farms, Inc. (Germantown, NY, USA). Two mice were placed in each isolator and the stool samples were taken immediately to examine the presence of the test strain. Control mice and mice colonized with test strains are deprived of water overnight. The next day, 10 stool granules of C3H mice are placed in each bottle of water to drink, suspended in water and offered to mice. This procedure is followed by 3 consecutive days. The feces of BALB / c and C3H mice are obtained once a week for 1 month to verify the colonization of altered Schaedler flora and the presence of test strains.

Evaluations Forty-five days after monocolonization with test strains, and 30 days after "conventionalization", five mice from each treatment group are sacrificed and samples taken from animal spleens for T-lymphocyte isolation and determination. of cytosine. Preparation of spleen cells: Spleens are aseptically removed and placed in cold PBS + 0.5% bovine serum albumin (BSA) (Sigma Chem. Co., St. Louis, MO, USA) + 0. 1% sodium azide (NaN3) (Sigma). A single cell suspension of each spleen is prepared by perfusing it with 5 ml of medium RPM I-1640 (Sigma) + 0. 1 mg / ml gentamicin (Sigma). The cell suspension is transferred in sterile conical tubes of 15 ml and centrifuged at 2000 rpm for 10 min in a refrigerated centrifuge. The supernatant was decanted and the red blood cells of each tube underwent lysis by re-suspending the cells in 0.5 ml PBS 1 X, then adding 9 ml of distilled water, mixing well and finally adding rapidly 1 ml of PBS 1 0X and good mixing. The suspension is centrifuged as above, then the supernatant is discarded and the cells are re-suspended in 5 ml of RPMI-1640 of complete medium. A sample of the suspension is verified under the microscope and if different particles of the cells are present, the suspension is filtered through a sterile nylon cloth and centrifuged again. The granule is re-suspended in 5 ml of complete RPMI-1640 medium, rinsed twice and finally re-suspended in 5 ml of complete medium. Cell viability is determined by trypan blue exclusion (Sigma). 20 μl of cell suspension is diluted in 380 μl of trypan blue solution to 0. 1% (dilution 1: 20) and quantified in a hematocytometer. The concentration of the cell suspension is adjusted to 1.0 x 106 cells / ml in full RPM I-1640. Cell culture: Cells are grown in 96-well flat bottom tissue culture plates (Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA) with RPMI-1 640 complete medium in the absence (no stimulated) and in the presence of inducing agents such as Concavalin A (5 μg / ml), LPS (1 μg / ml) of Salmonella typhimurium, phorbol 12-myristate-1 3-acetate (PMA, 10ng / ml), ionomycin (I 0.5 μg / ml) and L. reuteri inactivated by heat (4.5 mg protein / ml). To each cavity were added 100 μl of cell suspension containing 1.0 x 105 cells and fifty μl of heat-inactivated L. reuteri (4.5 mg protein / ml). Each set is passed with 5 repetitions. The cell cultures were incubated in a 5% CO2 atmosphere for 48 h at 37 ° C with the mitogen and for 96 h with L. reuteri. The supernatants of the 5 cavities were collected, deposited and stored at -70 ° C until they were used for cytosine assays. Quantification of cytosine: Cytokines (I L-10, TGF-beta-2) are measured in supernatants by ELI SA by using Quantikine ™ M equipment (R &D Systems, Minneapolis, MN, USA) following the procedure recommended by the maker.

Results Cytosines are immune system proteins that are biological response modifiers. They coordinate antibodies and T-cell immune system interactions and amplify immune reactivity. Cytosines include monoquinas synthesized by macrophages and lymphokines produced by activated T lymphocytes and natural eliminating cells (NK). The L. reuteri test strain shows a higher concentration (P <0.05) of I L-1 0 than the cells of strain 4000, 4020 or the mice of control. Also TGF-beta-2 levels are higher for strain SD21 1 2. This strain is selected according to the present invention. b. Confirmation of I L1 0 production stimulation and reduction of TGF-beta-2 in human breast milk by cells from Lactobacillus strains This example confirms that the selected strain gives the desired effect in vivo. Lactobacillus reuteri, ATCC55730 (available from The American Type Culture Collection, Manassas, VA, USA) is examined. The Lactobacillus test strain is grown in MRS broth (Difco) and harvested during the exponential growth phase by centrifugation at 1000 xg, rinsed twice with phosphate buffered saline (PBS, pH 6.8) and re-suspended in the same regulator. After this, the culture is formulated in an oil droplet product, following the methods described in example 3 below. This study is a double-blind, controlled-survival placebo that confirms the potential of the selected lactic acid bacteria in allergy, conducted in the departments of Pediatrics in the county hospitals of Jónkóping, Mótala and Norrkoping and the University Hospital in Linkóping, Sweden. Pregnant women from families with a history of allergic disease were randomized to receive orally lactobacillus reuteri SD21 1 2, ATCC 55730, daily dose 1 x1 08 CFU or placebo during the four weeks before reaching term. The History of allergic disease was confirmed by a telephone interview by an experienced allergy research nurse. In all, 232 families were included in the trial and were also randomly ordered between the experimental endpoint and the placebo, from January 2001 to April 2003. Compliance was determined by collecting all the used bottles of the study product and then an estimate of what remained in them.

Methods The present study includes colostrum, obtained within the first 3 days after delivery, and mature milk, obtained one month after delivery, from 109 of the mothers. Milk samples were collected by using a manual milk pump in sterile plastic tubes and stored at -70 ° C until analysis. After thawing, the milk samples were centrifuged to remove the fat and cell compartments (Bóttcher, 2000). The remaining serum was immediately analyzed with respect to the IL-10 content and the rest of the serum was stored in aliquots at -70 ° C and subsequently analyzed for TNF-a, I L4, TG F-beta-1, TGF- beta-2, soluble CD 14 (sCD 14), total IgA, secretory IgA (slgA) and sodium and potassium. The levels of TGF-beta-1, TG F-beta-2 and sCD 14 (soluble CD 14) were analyzed with commercial equipment from ELI SA (R & D Systems, Abingdon, United Kingdom) according to the manufacturer's recommendations. The analyzes of TGF-beta-1 and TGF-beta-2 were carried out after acid treatment to pre-activate latent TGF-beta, as described previously (Bóttcher, 2000). The levels of I L-1 0 and TNF-a were determined with commercial ELISA reagent equipment (CLB PeliPair reagent equipment, Amsterdam, The Netherlands) according to the manufacturer's protocol. The lower limit of detection was 62.5 pg / mL for TGF-beta-1 and TGF-beta-2, 250 pg / mL for sCD14, 2.3 pg / mL for IL-10 and 7.8 pg / mL for TNF-a. The total IgA and slgA were analyzed with ELISA as described previously (Bóttcher, 2002). The lower limit of detection was 31.2 ng / mL for both tests. Sodium and potassium levels were measured in the Department of Clinical Chemistry at the University Hospital of Linkóping in accordance with standard routines (selective ion electrodes). Serum IgE levels for a panel of inhalant allergens were analyzed with UniCap® Pharmacia CAP System ™ Phadiatop® (Pharmacia Diagnostics, Uppsala, Sweden).

Statistical Analysis The size of the study group was calculated assuming 80% energy to detect a true difference in clinical manifestations in the L. reuteri group compared to the placebo group. The calculation is based on the assumption that clinical manifestations of allergy or eczema occur in at least 40% of the subjects in the placebo group and can be reduced by half in the experimental group. The Necessary subjects were 91 subjects that could be evaluated by group and the frequency of attrition was assumed at 25%. A random ordering list was carried out by an outside company and stratified by scepter into block sizes of four. The total number of subjects needed to be included based on the previous calculation, including the expected dropout frequency or block size, was estimated at 16 women and their weaning by group.

Ethical Considerations According to the Declaration of Helsinki on medical research with human subjects, participants received written information and signed an informed consent. The test strain L. reuteri SD21 12 in oil was considered as a well-documented and safe product for both children and adults and therefore it was not observed as an ethical problem to include pregnant women and their children. Study procedures with different examinations were also seen as a minor problem with respect to the fact that a large proportion of this population at risk would develop allergic disease and undergo selection procedures. The protocol was approved by the Ethical Review Panel at the University Hospital of Linkóping.

Results In the study, we examined the effect on breast milk when pregnant women were taking Lactobacillus reuteri strain SD21 1 2 orally 4 weeks before delivery. The maternal profiles that compare the two groups were similar. The numbers of weeks that the mothers had ingested daily the study product do not differ between the two groups, in terms of exclusive breastfeeding in a month. The interruptions in the two groups, up to one month of age, originated mainly by reference to neonatal protection, (one of the exclusion criteria). No differences were observed between the two groups. In the L. reuteri test strain group, the cytosine profile in breast milk was changed to the increased level of anti-inflammatory cytosine IL 10 in colostrum (median 6.61 pg / mL [range 1-1.5-150 [ ), in comparison with the samples of the mothers in the placebo group (4.78 pg / mL [1-1.5-50]); p = 0.046. Meanwhile, a decrease in the level of TGF-beta-2 was observed in the group L. reuteri SD21 1 2. TGF-beta-2 was significantly lower in the L. reuteri group (median 674 pg / mL [102.5-2800 ] against 965 pg / mL [21 1 .7-2800]) than in the placebo group; p = 0.020. The levels of the other parameters were similar in the two groups. The mother's milk obtained 4 weeks after the supply and the interruption of the daily intake of probiotics showed no difference compared to the placebo group.

Example 3 Manufacturing of products containing the selected strain In this example, a product called "Drops of Reuteri" was made. The product is an oil-based formulation that contains L. reuteri developed for good stability and shelf life. The unique characteristic of the production process is a drying stage of the oil to remove most of the water in the oil. The oil used in the invention herein is a pure edible vegetable oil, preferably sunflower oil. Although an oil such as pure sunflower oil would not be expected to contain some water, an unexpected effect of the oil drying processing step by placing it in vacuum is a significantly increased stability of the lactobacilli in the formulation. Accordingly, the oil used in the invention must be an oil from which it is possible to remove the water. Although it was previously known that the stability of such cultures correlates very closely with the water activity of the formulation, it was not known to dry the oil under vacuum for the stabilization of the lactobacilli.

Description of the manufacturing process. A flow chart of the preferred manufacturing process is shown in Figure 1. The details of such a possible process that can be used in the invention are presented hereinafter.

Mixture of ingredients. 1 . Medium chain triglyceride mixture (eg, Akomed R, (Karishamns) with silicon dioxide, Cab-o-sil M5P, Cabot) in a Bolz mixing machine / tank (Alfred BOLZ Apparatebau GmbH, Wangen im Allgau, Germany). 2. Homogenization. A Sine and dispax pump (Sine Pump, Arvada, Colorado) are connected to the Bolz mixer and the mixture is homogenized. 3. Vacuum drying. The mixture is dried under a vacuum of 10 mbar in the Bolz tank, for 12 hours. 4. Addition of Lactobacillus reuteri. Approximately 20 kg of dry oil mixture is moved to a 50 liter stainless steel vessel. L. reuteri powder is added (preferably lyophilized, the amount of L. reuteri used would vary depending on the amount desired in the oil, but an example would be to add 0.2 kg of culture having 1 01 1 CFU per g). Mix slowly until homogeneous.

. Mixture. The pre-mix with L. reuteri is conducted back to the Bolz mixer. 6. Download. The suspension is discharged into a 200 liter glass vessel and covered with nitrogen. The suspension is kept in the container until it is filled in bottles. Although certain representative embodiments have been established herein, those skilled in the art will readily appreciate that modifications can be made without departing from the spirit or scope of the invention.

Claims (1)

REVIVALITION IS 1. A method for selecting a bacterial strain of lactic acid for administration to a woman to improve a woman's breast milk for feeding a baby, characterized in that it comprises the selection of a strain of Lactobacillus, the administration of which strain of Lactobacillus has been found that it increases levels of I L-10 and decreases the level of TGF-beta-2 in breast milk when administered to women. 2. A method for increasing the anti-inflammatory activity in a breast milk, characterized in that it comprises administration to the female cells of the lactic acid bacteria strain selected by the method according to claim 1. 3. A method for increasing I L-1 0 in a breast milk at the same time that the levels of TGF-beta-2 are decreased, characterized in that it comprises the administration to the cells of the female of the strain of acid bacteria lactic selected by the method according to claim 1. The method according to claim 3, characterized in that the cells of the strain of lactic acid bacteria are administered to the woman before the birth of a child. The method according to claim 3, characterized in that the cells of the strain of lactic acid bacteria are administered to the woman after the birth of a child. 6. A product containing cells of a strain of lactic acid bacteria, selected by the method according to the claim

1 . The product according to claim 6, characterized in that it also comprises an edible vegetable oil. 8. The product according to claim 7, characterized in that the edible vegetable oil is sunflower oil. 9. The product according to claim 8, characterized in that the oil is prepared by a vacuum drying step. 10. An oil product that allows the increased survival of dead microbial cultures, characterized in that the product is prepared by a vacuum drying step of the oil.