KR100855846B1 - Method for improved breast milk feeding to reduce the risk of allergy - Google Patents

Abstract

In order to improve selected strains of Lactobacillus and breast milk for infant feeding, more precisely, the level of anti-inflammatory cytokine IL-10 in breast milk is increased; To reduce the risk of developing allergies in feeding babies and at the same time reduce the cause and amount of TGF-beta-2 in breast milk to reduce the risk of mastitis in the feeding mother. The product containing the cells of the selected strain is shown.

Lactobacillus

Description

METHOD FOR IMPROVED BREAST MILK FEEDING TO REDUCE THE RISK OF ALLERGY

The present invention relates to the selection and use of lactic acid bacteria for improved breast milk for infant feeding purposes.

Hygiene hypothesis of allergic disease suggests that environmental changes in advanced regions reduce microbial contact in childhood, leading to a rapid prevalence of allergic diseases such as atopic eczema, allergic rhinoconjunctivitis, and asthma. do. One such environmental change under discussion is the altered uptake of various types of natural microorganisms due to improved hygiene, living standards, and eating habits. This induces not only changes in the composition of the natural intestinal flora (Journal of Allergy and Clinical Immunology, 2001; 108: 516-520), but also changes in the milk composition of the lactating mother. These changes are associated with the prevalence of various types of allergies. In addition, infants who are later members of large families have a lower risk of allergies than early-born siblings (first of the litters). This suggests that maternal breast milk is "improved" as the number of pregnancy increases.

Breast milk contains various components important in the immune system, such as macrophages, immunoglobulins, or antibacterial proteins, which are thought to inhibit infection and inflammation in the gastrointestinal and respiratory tracts of lactating offspring. In addition, the presence of other potential immunomodulatory factors (eg, complex oligosaccharides, growth factors, enzymes, hormones or cytokines) are discussed. Due to these beneficial properties, along with the high availability of nutrients and low antigen content in human breast milk, breast milk is recommended by pediatricians as the best physiological food for infants, especially infants born to allergic families.

For this reason, breast milk contains a series of cytokines and chemokines that can potentially affect the development of allergies in infants. Components that modulate allergic reactions, such as cytokines, chemokines, and adhesion molecules, have already been reported to be secreted into breast milk at various lactation stages (S Rudloff et al, Allergy 1999, 54, 206-211). Cytokines or chemokines can be beneficial or detrimental to lactating infants.

In addition, cytokines delivered by animal milk are known to possess the ability to withstand migration through the GI tract of the offspring. For example, homozygous TGF-beta-1-knockout mice die of weaning inflammatory disease after weaning, which appears to survive the transfer of TGF-beta from breast milk until weaning (Kulkarni AB et al., Am J Pathology 1993; 143, 3-9). TGF-beta withstands migration to the colon. IL-10, too, probably survives in this way, so delivery from breast milk allows IL-10 to reach the GI tract and potentially induce a beneficial anti-inflammatory effect.

Furthermore, Hawkes JS et. al., Lipids 2001 Oct 36: 1179-81 report that long-chain polyunsaturated fatty acids are associated with aspects of immune regulation, including cytokine production. The aim of this study was to enrich docosahexaenoic acid (DHA) -rich tuna oil for the concentration of transforming growth factor beta 1 (TGF-beta-1) and TGF-beta-2 in breast milk. To investigate the effects of parental dietary supplements. In a random dietary intervention trial, the mother of a term infant was a 2000 mg oil containing placebo (n = 40), 300 mg DHA (n = 40), or 600 mg DHA (n = 40). Daily supplements were taken. DHA increases in breast milk and plasma were proportional to dietary DHA. There was no correlation between breast milk DHA status and TGF-beta-1 and TGF-beta-2 levels.

IL-10 is a widely reported and accepted anti-inflammatory cytokine. This means that IL-10 will induce an anti-inflammatory effect in the infant's GI tract. This is beneficial for infants-breast milk is generally considered to be anti-inflammatory for infants in that the infant does not overreact to pathogens / infections present in the intestine at an early age. In addition, animal data suggest a potential benefit for the progeny of IL-10 in breast milk. An Australian group examined tick allergies in animal models. Animals negative for the skin prick test (SPT) against ticks produced IL-10 and IL-4, which attenuated IgE (allergic reactions) and these animals did not show allergies. Allergic SPT + ve (histamine) animals produced only IL-4 and no IL-10 production. For this reason, IgE was not weakened and allergies were prevalent. Thus, IL-10 can attenuate the allergy-inducing effect of IgE and prevent the development of allergies.

TGF-beta-2 (converted growth factor) is also a widely reported growth factor / cytokine. Its source is, for example, platelets yielding an amount of TGF-beta / kg in a few mg amount. The factor and its isoforms can also be isolated from other tissues (microgram TGF / kg) and are predominantly found in the spleen and bone tissues. Human milk also contains this factor, for example macrophages (TGF-beta-1), lymphocytes (TGF-beta-1), endothelial cells (TGF-beta-1), keratinocytes (TGF-beta-2) It is also synthesized by granular layer cells (TGF-beta-2), chondrocytes (TGF-beta-1), glioblastoma cells (TGF-beta-2) and leukemia cells (TGF-beta-1).

Depending on cell type and condition, secretion of TGF-beta can be induced by a number of different stimuli including steroids, retinoids, EGF (epithelial growth factor), NGF, lymphocyte activator, vitamin D3, IL-1 . Synthesis of TGF-beta can be inhibited by EGF, FGF (fibroblast growth factor), dexamethasone, calcium, retinoids, follicle stimulating hormone. TGF-beta also affects the expression of its own genes, which may be important for wound healing. TGF-beta exists as at least five isotypes independent of TGF-alpha, that is, TGF-beta-1, TGF-beta-2, TGF-beta-3, TGF-beta-4, and TGF-beta-5 do. These isotypes of amino acid sequences show 70-80% homology. TGF-beta-1 is a dominant form, found almost everywhere, while other isotypes are expressed in a more limited spectrum of cells and tissues. Homotypes isolated from different species are evolutionarily closely conserved and have 98% sequence homology. Mature human, pig, ape, bovine TGF-beta-1 is identical and different from murine TGF-beta-1 at the single amino acid position. Human and chicken TGF-beta-1 are also identical.

In addition, members of the transforming growth factor beta (TGF-beta) population have been reported to be pleiotropic cytokines that play a key role in tissue morphogenesis and growth (Ingman WV, Bioessays 2002 Oct 24: 904-14). TGF-beta-1, TGF-beta-2, and TGF-beta-3 are abundant in mammalian regenerative tissue where development and circulatory remodeling persist in infancy and adulthood. Potential roles for TGF-beta have been identified in gonadal and secondary reproductive organ development, spermatogenesis and ovarian function, immunoregulation of pregnancy, embryo implantation, and placental development.

In Rautava et.al., Journal of Pediatric Gastroenterology and Nutrition 38: 378-388, April 2004, TGF-beta-2 and IL-10 are synergistic ways in which TGF-beta-2 encourages production of IL-1O. It has been reported to function.

Mastitis is an inflammation of the breast, often characterized by tenderness and erythema and sometimes fever and associated with TGF-beta-2. During mastitis, the tight junctions of the mammary alveolar cells open, accompanied by an increase in sodium, inflammatory cells, inflammation and immune mediators in breast milk. Mastitis is usually limited to one side and develops maximally in the first few weeks after lactation. In developed countries, mastitis is generally regarded as a low morbidity problem, in which affected women are treated primarily by midwife and nurse practitioners. Mastitis is 20-33% of women in large longitudinal longitudinal studies (Semba R., Annals of the New York Academy of Sciences. November 2000; 918: 156-62) who track lactating women in the United States, Finland and Australia. As clinically apparent mastitis develops in, it appears to be much more frequent than previously thought. For this reason, the number of lactating women with sub-clinical mastitis is logically greater than this.

Mastitis has recently been associated with higher human immunodeficiency virus (HIV) loads in breast milk and a higher risk of mother-to-child transmission of HIV (Semba, RD, N. Kumwenda, TE Taha, et al. 1999. Mastitis and immunological factors in breast milk of human immunodeficiency virus-infected women.J. Hum.Lact. 15 (4): 301-306).

In breast milk, TGF-beta-2 is synthesized by many other cells, including B- and T-cells, but originates mostly from the epithelium. For this reason, increased levels of TGF-beta-2 may be mediators or outcomes of subclinical breast inflammation. The sodium to potassium ratio (Na / K ratio) in breast milk is referred to as a well known predictor of infection and subclinical breast inflammation.

In addition, Kalliomaki et.al., J Allergy Clin Immunol. 1999 Dec; 104 (6): 1251-7 suggested that TGF-beta in colostrum prevents the development of allergic diseases during exclusive lactation and promotes specific IgA production in human subjects.

Furthermore, many different bacterial species, including many different Lactobacillus species, live in various parts of the body of humans and other mammals. These bacteria coexist with the host for a long time and provide a variety of synergistically beneficial effects that are known today to depend on the actual strain of bacteria. Different lactic acid strains, such as L. reuteri SD2112, possess specific antigens that are released on their surface or by the bacteria in the parent's gastrointestinal tract. Valeur et al., AEM, 70 , 1 176-1181 (2004) data is provided to affect the levels of CD4 + T- cells in the secondary of a healthy human meeting the intake ruteri Lactobacillus (L. reuteri) as one kinds of examples SD2112 Show that you can give These results have also been observed in other mammalian species and birds, indicating that this process is the fundamental signaling system between the flora and the host. Through the so-called entero-mammary link, antigens from the active strain are actively transported to the lymph area below the epithelium of the GI tract, in other words the Peyer's patch. Then, antigen-specific B-cells are activated, which then travel through the circulation from the GI vascular epithelium to other mucous membranes in the body, including salivary glands and mammary glands. Expression of certain molecules in these cells is thought to induce their adhesion to the tissue. In the mammary gland, these immune cells induce other processes that determine the level of locally produced cytokines. This type of signaling through intestinal-wired links has been demonstrated in the production of secreted IgA in breast milk and is likely to be applied to cytokine production.

In previous literature (Laiho et al, Pediatric Research 53: 642-647, 2003), the observed association between nutritional and inflammatory factors in breast milk may affect the maternal immunological characteristics of dietary intervention. Proposed to prove that. In addition, these authors argue that mothers with allergic diseases have lower levels of TGF-beta-2 in breast milk compared to mothers without allergic diseases. In their study, IL-10 was detected only at low levels and frequencies naturally observed in breast milk, and there was no difference between mothers with and without allergic disease. Protection from allergic diseases is achieved primarily through the induction of oral tolerance to TGF-beta-2 and IL-10, especially where breastmilk TGF-beta-2 plays an important role in the prevention of allergic diseases. It was suggested that. However, Weiner H., in Microbes and Infection, Volume 3, Issue 11, September 2001, pages 947-954, found that regulatory T cells generated by oral antigens are induced in an antigen-specific manner although they are induced in an antigen-specific manner. Reported that they mediate bystander suppression when faced with self-antigens in target organs. Thus, mucosal tolerance can be used to treat inflammatory processes that are not autoimmune in nature.

Different Lactobacillus species , including Lactobacillus reuteri , are used in so-called probiotic compositions, which aim to provide living beneficial microorganisms to animals, including humans. Lactobacillus reuteri is one of the naturally occurring habitats of the gastrointestinal tract of animals and is commonly found in the gut of animals, including humans, sometimes in birth channels, breast milk, and mouth. It is known to possess antimicrobial activity (see US Patent No. 5,439,678, 5,458,875, 5,534,253, 5,837,238, 5,849,289). When L. reuteri cells are grown in the presence of glycerol under anaerobic conditions, they produce an antibiotic known as lutein (β-hydroxy-propionaldehyde).

Lactic acid bacteria have already been reported to be able to prevent and treat various types of allergies (eg EP 1239032 (Stadler et al.) With respect to new recombinant strains; with regard to reducing the amount of IgE with Lactobacillus ). WO 01/37865).

For this reason, the present invention aims to provide a screened lactic acid bacterium and its components and methods for screening for improved breast milk for infant feeding purposes. More precisely, the present invention reduces the risk of developing allergies in feeding babies, while at the same time reducing the cause and amount of TGF-beta-2 in breast milk, thereby reducing the risk of mastitis in the feeding mother. It is aimed at increasing the level of anti-inflammatory cytokine IL-10 in breast milk to reduce the risk of development.

For this reason, the present invention aims to compensate for negative changes in microbial flora by providing the parent with a specially selected lactic acid bacteria strain before and during lactation.

In addition, the present invention is directed to screening for specific lactic acid bacteria strains using the methods described herein or similar methods that distinguish specific cytokine effects of test strains on related cell types; Promoting increased production of IL-10 and a decrease in TGF-beta-2 levels in breast milk, indicating a lower level of subclinical inflammation in breast milk gland and other tissues and thus a lower risk of mastitis. In order to use these lactic acid bacteria strains. Mastitis and subclinical mastitis interfere with lactation and thus can be considered to block the benefits breast milk provides to infants- Lactobacillus screened by the method of the present invention improves maternal health so that Allow breast milk to be produced during the period.

It is also an object of the present invention to provide a product containing the above strains, variants, metabolites or components thereof, including drugs for administration of animals, including humans.

Other objects and advantages are more apparent from the following detailed description and the appended claims.

Summary of the invention

The present invention increases the level of anti-inflammatory cytokine IL-10 in breast milk, which improves breast milk for infant lactation; It relates to selected Lactobacillus strains and their components in view of their ability to reduce the risk of allergy in feeding babies and at the same time reduce the cause and amount of TGF-beta-2 in breast milk. This means reduced risk of developing mastitis in the feeding mother and improvement in breastfeeding capacity, overall protection, and optimal growth that breast milk provides to infants. The present invention also relates to a method for screening such Lactobacillus strains. In addition, the present invention provides a delivery means of the active component, relates to a novel method of protecting a living Lactobacillus bacteria (Lactobacillus) in the five days to prepare the product.

Other objects and features of the present invention are more apparent from the following detailed description and the appended claims.

1 is a flow chart of a manufacturing process that may be used to prepare the products of the present invention.

The present invention improves the level of anti-inflammatory cytokine IL-10 in breast milk, in order to improve breast milk for infant feeding; Feeding mothers reduce the risk of allergies in feeding babies and at the same time reduce the amount of TGF-beta-2 and the causative factors that induce the production of TGF in breast milk. Presents products for reducing the risk of developing mastitis. The reduction in TGF-beta-2 associated with the present invention is considered to be an anti-inflammatory effect of selected lactic acid bacteria consumption, which means that the incidence of subclinical mastitis in the lactating group, which recently ingested these bacteria daily, will be lowered. An example of a strain selected by the present invention is L. reuteri SD2112 (ATCC 55730).

In the findings that led to the present invention during clinical trials, there were three factors related to the level of IL-10 in maternal colostrum: a) whether to be treated with selected lactic acid bacteria; b) number of infants previously born to the same mother; c) the number of previous pregnancy. Thus, the higher the number of previously born infants or the number of previous pregnancies in the mother, the more IL-10 is present in colostrum. At 4 weeks before birth, even if a specially selected lactic acid bacterium is given to a premature mother, the ability to increase the level of IL-10 in colostrum, whether or not he or she is pregnant, is improved.

In addition, because in some situations increased systemic expression of IL-10 is known to prevent type I diabetes, strains selected according to the invention may also be useful for this purpose.

In the selection method used in the present invention, optimal strains that induce IL-10 in breast milk and show reduced levels of TGF-beta-2 are selected by using established mouse models and traditional assay methods. Other similar methods of detecting cytokine production in breast milk may also be used. The details thereof will be more clearly understood from the examples. Suitably, the product of the present invention contains viable cells of the selected strains; However, if isolated metabolites or portions of these cells are responsible for the activity of the viable cells of these strains, the products of the invention may contain these metabolites or portions in addition to or on behalf of the viable cells. The product of the present invention can be any product for female consumption, for example oil drop products, foods, tablets, capsules, powder-flavored powders and the like. Particularly useful products of the present invention are oil residues (Example 3) which help to keep the active ingredient stable for extended periods of time. Lactobacillus bacteria (Lactobacillus) cells oil for improved stability of the bacteria - have been used in the composition (see: US patent 4518696, Gehrman et al .). However, any existing lactic acid bacterium composition contained in oil or fat does not include the key step of drying the oil by vacuum means prior to preparation to increase the stability of the bacterial culture, as listed in the Examples of the present invention.

The concentration of selected Lactobacillus cells required for the efficacy of the product of the invention depends on the food type and the amount of food ingested (or the time of use of the non-food dental care product in the mouth), but in general the product Approximately 10 ⁵ -10 ⁸ CFU (colony-forming unit) or more per day intake. A maximum amount of 10 ¹⁰ -10 ¹¹ CFU is possible and can be used to increase efficacy without negatively affecting the sensory stimulatory properties (flavor or odor) of the product. If the product is a yoghurt or other lactic acid bacteria fermentation product, the lactic acid bacteria fermentation strains used to produce such products are preferably standard fermentation broths (eg, S. thermophilus and L. bulgaricus in yogurt). . Importantly, the selected active strains possessing the desired cytokine effect according to the invention are compatible with any standard culture used for such products, and the important properties of each strain used should not be negated by the use of other strains. . This can be readily determined by screening assays known in the art. As described above, the strain used in the present invention may be added at a level equivalent to approximately 10 ⁶ -10 ⁸ CFU per daily serving of yogurt, before and after fermentation of the product.

Suitably, the product of the invention does not contain other antibiotic components, in particular components which inhibit or kill selected Lactobacillus strains or their metabolites or components or interfere with their activity.

Lactobacillus bacteria (Lactobacillus) strain or a metabolite or components thereof may be additives that are mixed with these components to the means known in the art for the preparation of products of this type. If cells are used and a heating step is required for the production of selected foods or other products of the invention, Lactobacillus strains should be added after heating. If selected Lactobacillus cells are present in the product, it is desirable not to heat the product to 60-70 ° C. or higher for an extended period of time.

Features of the invention will be more clearly understood with reference to the following examples which are not limiting of the invention.

Example 1 Screening Method of Strains

The selection of Lactobacillus strains that can be used in the present invention can be performed in the following stepwise manner:

Lactobacillus for strain selection ( Lactobacillus Evaluation of Stimulation of IL-10 Production and TGF-beta-2 Reduction in Human Breast Milk by Strain Cells

This is an example of a selection method; Certain modifications to this method can be made by those skilled in the art without departing from the scope of the present invention.

Materials and methods

Animals: 51 sterile BALB / c mice (male and female) are purchased from Wisconsin University (USA). These mice are transported in sterile plastic film transporters. Fifteen mice are transferred to three isolation devices (isolators # 2, 3, and 4), respectively. Two cages, each containing two males and four females, are placed in containment # 1.

Animal handling

Mice are maintained in sterile flexible film isolates (Class Biologically Clean, Ltd, Madison, Wis., USA). The containment is sterilized with a 2% peracetic acid (FMC Corporation, Philadelphia, PA, USA) solution containing 0.1% Naconal (Stepan Co., Rocksport, IL. USA). Five to six animals are housed in a peracetic acid sterilized polystyrene cage with a stainless steel wire grid. Position identical mice in each cage. Mice are fed autoclaved Agway rodent chow 3500 (Agway, Granville, Creedmoore, NC, USA). Feed, water and litter are autoclaved. Feed and litter are autoclaved in stainless steel cylinders and then transferred to the separator under aseptic conditions. The bottle containing the tap water is autoclaved and then sterilized with peracetic acid when placed in the separator. All mice are given free feed and water. Feed and water levels are checked daily. The litter is replaced once a week. These animals are maintained in a 12 hour light cycle. Room temperature and relative humidity are checked daily.

bacteria

The lactic acid bacteria tested are identified and characterized by biochemical and molecular biological techniques. These Lactobacillus strains were first grown in 10 ml of Man-Rogosa-Sharp (MRS) medium (BBL, Cockeysville, MD) and incubated at 37 ° C. for 18 hours, then transferred to 90 ml of MRS and 37 Incubate at 18 ° C. for 18 h and transfer to 1000 ml MRS. After 18 hours of incubation at 37 ° C., the culture was spun at 3000 rpm for 10 minutes in a chilled centrifuge (SORVALL RC2-B, SORVALL, Norwalk, CN, USA), the supernatant was discarded and the pellet was 3000 for 10 minutes. Wash twice with sterile phosphate buffer (PBS) at rpm. Pellets of each strain are resuspended in 30 ml of PBS. Cultures are placed in 1.2-ml cryovials and stored at -70 ° C until use. Prior to providing these bacteria to mice, the purity and concentration of the cultures are checked. Serial 1/10 dilutions of Lactobacillus suspensions were cultured on MRS medium plates containing 1.5% agar and containing an anaerobic generator (Anaero Gen: Oxoid Ltd., Wake Road, Basingstoke, Hampshire, England, GB). Incubate for 48 hours at 37 ° C. in anaerobic disease (GasPaK: BBL, Cockeysville, MD, USA). The culture is tested for colony form and reuterin production.

Inspection Processing (in this embodiment)

contrast.

Strains: Lactobacillus reuteri SD2112, ATCC 55730.

Strains: Lactobacillus 4000

Strains: Lactobacillus 4020

Lactobacillus ( Lactobacillus ) Determination of colonization

The next day after the mice are placed in the separator, fecal samples are taken from the mice in each cage to check for the absence of microorganisms. Mice stop supplying water from 11:00 am to 7:00 pm. Thereafter, 1.2 ml of lactic acid bacteria suspension is added to each bottle containing 200 ml of water and given to mice. Mice of Separator # 2 take L. reuteri SD2112. The suspension added to water contains 2.0 × 10 ¹⁰ cfu / ml of L. reuteri SD2112. Mice of Separator # 4 ingest Lactobacillus 4000 strains, and Mice of Separator # 5 ingest Lactobacillus 4020 strains. The lactic acid bacteria suspension added to each water bottle contains 3.0 x 10 ¹⁰ cfu / ml. Mice in Separator # 3 only consume water (control). Fecal samples (10 pellets) from mice (cage group) in each separator are taken weekly to check for lactic acid colonization and possible contamination with other microorganisms.

“Conventionalization” with the changed Schaedler flora

At 60 days after colonization with the test strain, the mice are “cultured” with the altered Sedler flora. C3H mice carrying a modified Sedler fungal flora were purchased from Taconic Farms, Inc. (Germantown, NY, USA). Two mice are placed in each separator and fecal samples are taken immediately to check for the presence of the test strain. Control mice and mice colonized with the test strain stop the water supply overnight. The next day, 10 fecal pellets from C3H mice were placed in each drinking water bottle and suspended in water and fed to the mice. This procedure is repeated for three consecutive days. Fecals from BALB / c and C3H mice are collected once a week for the presence of altered cowler colonization and test strains.

evaluation

At 45 days after monocolonization and 30 days after culture, 5 mice were euthanized from each treatment group and spleen samples were taken from these animals for T-lymphocyte isolation and cytokine determination with the test strain.

Preparation of Spleen Cells: Spleens are removed aseptically and cold PBS + 0.5% bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, MO, USA) + 0.1% sodium azide (NaN3) (Sigma). A single cell suspension from each spleen is prepared by sparging it with 5 ml RPMI-1640 medium (Sigma) + 0.1 mg / ml gentamicin (Sigma). The cell suspension is transferred to a 15 ml sterile conical tube and centrifuged for 10 minutes at 2000 rpm in a cooled centrifuge. The supernatant is discarded and erythrocyte cells from each tube are resuspended in 0.5 ml PBS 1X and mixed with sufficient addition of 9 ml of distilled water, finally lysing by quickly adding 1 ml of PBS 1OX and mixing well. do. The suspension is centrifuged as described above, the supernatant is discarded and these cells are then resuspended in 5 ml RPMI-1640 complete medium. Suspension samples are examined under a microscope, and if particles other than cells are present, the suspension is filtered through a sterile nylon cloth and centrifuged once again. The pellet is resuspended in 5 ml complete RPMI-1640 medium and washed twice, and finally resuspended in 5 ml complete medium. Cell viability is determined by exclusion of trypan blue (Sigma). 20 μl of cell suspension is diluted in 380 μl of 0.1% trypan blue solution (1:20 dilution) and counted on a hematocytometer. The concentration of cell suspension is adjusted to 1.0 × 10 ⁶ cells / ml in complete RPMI-1640.

Cell culture: Cells were derived from inducing agents such as Concavalin A (5 μg / ml), Salmonella typhimurium , LPS (1 μg / ml), phorbol 12 -myristate-13-acetate (PMA; 10 ng / ㎖) , Ino azithromycin (I; 0.5 ㎍ / ㎖) , heat inactivation of Lactobacillus member (stimulation of ruteri (L. reuteri) (4.5 ㎎ protein / ㎖) And sterile 96 well flat-bottom tissue culture plates (Fisherbrand, Fisher Scientific, Pittsburgh, Pa., USA) containing RPMI-1640 complete medium in the presence of the same. 100 μl of cell suspension with 1.0 × 10 ⁵ cells and 50 μl of heat inactivated Lactobacilli L. reuteri (4.5 mg protein / ml) are added to each well. Each set is repeated five times. Cell cultures are incubated with L. reuteri for 48 hours at 37 ° C. with mitogen under 5% CO ₂ atmosphere for 96 hours. Supernatants from five wells are collected and collected and stored at -70 ° C until used for cytokine analysis.

Cytokine Quantitation: Cytokines (IL-10, TGF-beta-2) are measured in supernatants by ELISA using the murine Quantikine ™ M kit (R & D Systems, Minneapolis, MN, USA), following the procedure recommended by the manufacturer. .

result

Cytokines are immune system proteins that function as biological response regulators. They mediate T cell immune system interactions with antibodies and amplify immune responsiveness. Cytokines include monookine synthesized by macrophages and lymphokine produced by activated T lymphocytes and natural killer (NK) cells.

The L. reuteri SD2112 test strain shows a higher IL-10 concentration than cells (P <O.05) from strain 4000, 4020 or control mice. TGF-beta-2 levels are also higher in the SD2112 strain. The strain is selected in the present invention.

b. Lactobacillus ( Lactobacillus ) Stimulation of IL-10 Production and TGF-beta-2 Reduction in Human Breast Milk by Strain Cells

This example confirms that the selected strains provide a beneficial effect in vivo. Lactobacillus reuteri , ATCC 55730 (The American Type Culture Collection, Manassas, VA, USA) is tested. The test Lactobacillus strains were grown in MRS liquid medium (Difco), harvested during exponential growth by centrifugation at 1000 xg, washed twice with phosphate buffer (PBS; pH 6.8) and resuspended in the same buffer. . The culture is then prepared as an oil drop product, according to the method described in Example 3 below.

This study is a double-blind, placebo-controlled study that identifies potential in selected allergic lactic acid bacteria and was performed in pediatrics at regional hospitals in Jonkoping, Motala, Norrkoping, and Linkoping University Hospital (Sweden).

Pregnant women from households with a history of allergic disease were randomly given oral doses of Lactobacillus reuteri SD2112, ATCC 55730 (daily dose 1 × ^{10 8} CFU), or placebo for 4 weeks prior to birth. The history of allergic disease was confirmed by telephone interview by one experienced allergy research nurse. A total of 232 households were included in the trial and evenly randomized between the experimental and placebo groups from January 2001 to April 2003.

Compliance was assessed by collecting used bottles of the study product and evaluating what remained there.

Way

This study uses colostrum obtained from 109 mothers within the first 3 days of childbirth and mature breast milk obtained one month after birth. Breast milk samples were collected into sterile plastic tubes using a manual breast pump and stored at −70 ° C. until analysis.

After thawing, breast milk samples were centrifuged to remove fat and cell compartments (Bottcher, 2000). The remaining whey is analyzed immediately with respect to the amount of IL-10, the remaining whey is stored at -70 ° C in portions and then TNF-α, IL-4, TGF-beta-1, TGF-beta-2 Soluble CD14 (sCD14), total IgA, secretory IgA (slgA), sodium and potassium were analyzed. Levels of TGF-beta-1, TGF-beta-2, sCD14 (soluble CD14) were analyzed with a commercial ELISA kit (R & D Systems, Abingdon, UK) according to the manufacturer's recommendations. Analysis of TGF-beta-1 and TGF-beta-2 was performed after acid treatment to preactivate latent TGF-beta, as described in the previous document (Bottcher 2000). Levels of IL-10 and TNF-α were measured with a commercial ELISA reagent kit (CLB PeliPair reagent set, Amsterdam, the Netherlands), according to the manufacturer's protocol. The lower limit of detection is 62.5 pg / ml for TGF-beta-1 and TGF-beta-2, 250 pg / ml for sCD14, 2.3 pg / ml for IL-10, 7.8 pg / ml for TNF-α It was.

Total IgA and slgA were analyzed by ELISA, as described in the previous document (Bottcher 2000). The lower limit of detection was 31.2 ng / ml for both tests. Sodium and potassium levels were measured in the Clinical Chemistry Department at Linkoping University Hospital, according to standard procedures (ion selective electrodes). Serum IgE levels for inhalation allergy in one panel were analyzed by UniCapPharmacia CAP System ™ Phadiatop (Pharmacia Diagnostics, Uppsala, Sweden).

Statistical analysis

The size of the study group was estimated by estimating 80% power to detect true differences in clinical manifestation in the L. reuteri group compared to the placebo group. This estimate is based on the assumption that clinical manifestations of allergy or eczema occur in at least 40% of individuals in the placebo group and can be reduced by half in the experimental group. The number of people required was 91 individuals, which could be evaluated in groups, and the dropout frequency was estimated to be 25%. The randomized list was created by an external company and sorted by center in a block size of four. Based on the above estimates, including expected dropout frequency and block size, the total number of individuals requiring registration was determined to be 116 women per group and their offspring.

Ethical Considerations

Under the Helsinki Declaration on Medical Research in Human Subjects, participants were provided with written information and signed a informed consent. The L. reuteri SD2112 test strain in oil has been fully reported and considered a safe product for both infants and adults, and consequently, registering pregnant women and their offspring was not considered an ethical issue.

In addition, in several different tests, this study procedure is considered a minor issue with regard to the fact that allergic diseases can develop in many of these risky populations and that allergic screening procedures should be performed. The protocol was approved by the Ethical Review Board of Linkoping University Hospital.

result

In this study, we investigated the effects of breast milk on pregnant women taking oral Lactobacillus reuteri strain SD2112 4 weeks before delivery.

The maternal profile comparing the two groups was similar. The total number of weeks the mother consumed the study product daily did not differ between the two groups in terms of one-month exclusive lactation. Differences in the two groups up to 1 month of age were mainly caused by referral to the neonatal ward (one of the exclusion criteria). No difference was observed between the two groups.

In the L. reuteri test strain group, the cytokine profile of breast milk was higher in colostrum (average 6.61 pg / mL [range 1.15-150] than in samples obtained from mothers in the placebo group (4.78 pg / ml [1.15-150]). ]) By increased levels of anti-inflammatory cytokine IL-10 (p = 0.046). Meanwhile, a decrease in the level of TGF-beta-2 was observed in the L. reuteri SD2112 group. TGF-beta-2 was much lower in the L. reuteri group (average 674 pg / ml [102.5-2800]) than in the placebo group (965 pg / ml [211.7-2800]) (p = 0.020). . The levels of the other parameters were similar in the two groups.

There was no difference in breast milk obtained at 4 weeks postpartum and after stopping daily intake of probiotics.

Example 3: Preparation of Products Containing Selected Strains

In this embodiment, a product called "Reuteri Drops" is produced. This product is an oil-based composition that has good stability and half-life and contains L. reuteri SD2112. A unique feature of the production process is the drying step of the oil, which removes most of the moisture from the oil.

The oils used in the present invention are pure edible vegetable oils, preferably sunflower oils. Although oils such as pure sunflower oil are not expected to contain moisture, the unexpected effect of the processing step of drying the oil under vacuum is the significantly increased stability of Lactobacillus in the composition. For this reason, the oil used in the present invention should be an oil from which moisture can be removed. It is already known that the stability of these cultures correlates closely with the water activity of the preparation, but it is not known to dry the oil under vacuum to stabilize Lactobacillus .

Description of Manufacturing Process

A flowchart of the preferred manufacturing process is shown in FIG. Details of one such possible process that can be used in the present invention are as follows.

1. Mixing of ingredients: in a Bolz mixing machine / tank (Alfred BOLZ Apparatebau GmbH, Wangen im Allgau, Germany), medium-chain triglycerides (eg Akomed R, Karlshamns AB, Karshamn Sweden) and sunflower oils (eg Akosun, Karlshamns) ) Is mixed with silicon dioxide (Cab-o-sil M5P, M5P, Cabot).

2. Homogenization: Connect a sine pump and a dispax (Sine Pump, Arvada, Colorado) to a Bolz mixer and homogenize the mixture.

3. Vacuum-drying: The mixture is dried in a Bolz tank under 10 mBar vacuum for 12 hours.

4. Add Lactobacillus reuteri : Transfer approximately 20 kg of dried oil mixture to a 50 L stainless steel container. L. reuteri powder (preferably freeze-dried; the amount of L. reuteri used ) varies depending on the amount required in the oil, but in one example 0.2 kg with 10 ¹¹ CFU / g Culture medium is added). Mix it slowly until it is homogeneous.

5. Mixing: Reduce the premix with L. reuteri to Bolz mixer.

6. Release. The suspension is discharged into a 200 L glass vessel and covered with nitrogen. The suspension is held in a container until the bottle is filled.

Although specific specific embodiments have been described herein, those skilled in the art will readily appreciate that modifications are possible without departing from the spirit or scope of the invention.

Claims (11)

A method for screening lactobacillus strains administered to women to improve the breast milk of women for infant feeding, wherein when administered to women, the level of IL-10 (Interleukin-10) is increased in breast milk and transforming growth factor (TGF)- Lactobacillus strains identified as lowering the level of beta-2 are selected using cell culture and cytokine assays. delete delete delete delete delete delete delete delete delete delete

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