HK1166670A - A stabilized oil product and its method of preparation and method of increasing survival of a lactic acid bacteria strain - Google Patents

Description

Stable oil product, method for preparing same and method for improving survival of lactobacillus strains

Background

Technical Field

The present invention relates to the selection and use of lactic acid bacteria to improve breast milk for nursing infants.

Description of the Related Art

The hygiene of allergic diseases is believed that environmental changes in the industrialized world will lead to reduced exposure to microorganisms in the young leading to the development of allergic diseases in the growing epidermis, such as atopic eczema, allergic rhinitis, and asthma. One such environmental change is considered to be a change in the species of various natural microorganisms taken in due to improvements in sanitary conditions, living standards, eating habits, and the like. This leads to a change in the composition of our natural gut flora (journal of allergy and clinical Immunity, 2001; 108: 516-520) and also changes the composition of breast milk of the nursing mother. Such changes are associated with the common various types of allergic reactions. It has also been reported that infants born later in the family are less at risk of developing allergic reactions than their siblings (first in the family) born earlier. This also suggests that the mother's breast milk "improves" with the number of pregnancies.

Human milk contains various components important to the immune system, such as macrophages, immunoglobulins, or antibiotic proteins, which are thought to protect the gastrointestinal and respiratory tracts of breast-fed offspring from infection and inflammation. In addition, the presence of other potential immunomodulatory factors (e.g., oligosaccharide complexes, growth factors, enzymes, hormones, or cytokines) has been discussed. These beneficial properties in human milk, coupled with high nutritional availability and low antigen concentrations, are the physiological basis for the current paediatric specialist to consider breast milk as the best food for infants, especially for those with a history of allergic reactions.

Thus, breast milk is known to contain a range of cytokines and chemokines that can potentially affect the development of allergic reactions in infants. It has been previously reported that components that regulate allergic reactions, such as cytokines, chemokines, and adhesion molecules, are sequestered in the milk at various stages of lactation (S Rudloff et al, allergy 1999, 54, 206-211). Cytokines or chemokines may be beneficial or disadvantageous for breast-fed infants.

Cytokines delivered by the milk of animals are also known to have the ability to survive passage through the GI tract of the offspring. For example, assuming that pre-weaning survival is a result of TGF-beta-1 transmission through breast milk, homozygous TGF-beta-1-knockout mice die of a generalized inflammatory disease after weaning (Kulkarni AB et al, Am J Pathology 1993; 143, 3-9). TGF-beta passes through the intestine in various ways and remains active. IL-10 may also remain active in such a way that IL-10 delivered by breast milk reaches the GI pathway and potentially produces beneficial anti-inflammatory effects.

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 purpose of this study was to investigate the effect of maternal diet supplementation with tuna oil enriched with docosahexaenoic acid (DHA), which at certain concentrations in breast milk can be converted into growth factors beta 1(TGF-beta-1) and TGF-beta-2. In this randomized dietary intervention trial, the infant's mother was routinely supplemented with 2000mg of oil, which contained either a placebo (placebo) (n-40), or 300mg DHA (n-40), or 600mg DHA (n-40). The increase in DHA in breast milk and lymph is directly proportional to the dietary DHA. There was no correlation between the DHA status of the emulsion, the level of TGF-beta-1, and the level of TGF-beta-2.

IL-10 is an anti-inflammatory cytokine that has been widely discussed and accepted. This suggests that it has an anti-inflammatory effect in the GI tract of infants. This is beneficial for breast-fed infants, who are generally believed to help infants with some anti-inflammatory properties, and who should not over-react early in life to pathogens/infections in the gut. Furthermore, animal data indicate that IL-10 may be beneficial to offspring in emulsion. An animal model was observed by an australian panel in which an allergic reaction to ticks was observed. Animals negative for the cutaneous sting test (SPT) of ticks had IL-10 and IL-4 products, had IL-10 attenuated IgE (allergic response) and the animals did not develop an allergic reaction. In allergic SPT + ve (histidine) animals, only IL-4 was produced and no IL-10 product. Thus IgE is not reduced and allergic reactions occur. IL-10 therefore attenuates the allergic reaction induced by IgE action and can prevent the allergic reaction from occurring.

TGF-beta-2 (transfer growth factor) is also a widely discussed growth factor/cytokine. Sources include, for example, platelets, which produce TGF-beta/kg in milligram quantities. The factor and its homologues (see below) can also be isolated from other tissues (microgram TGF/kg) and predominate in spleen and bone tissues. Human milk also contains this factor and its synthesis is also via, for example, macrophages (TGF-beta-1), lymphocytes (TGF-beta-1), endothelial cells (TGF-beta-1), keratinocytes (TGF-beta-2), granulosa cells (TGF-beta-2), cartilage (TGF-beta-1), glioblastoma cells (TGF-beta-2), leukemia cells (TGF-beta-1).

Depending on the cell type and conditions, TGF-beta secretion can be induced by a range of different stimulatory factors including steroids, retinoids, EGF (epidermal growth factor), NGF, lymphocyte activators, vitamin D3, and IL 1. TGF-beta synthesis can be inhibited by EGF, FGF (fibroblast growth factor), dexamethasone, calcium, retinoids and follicle stimulating hormone. TGF-beta also affects the expression of its own genes and this may be important in wound healing. There are at least five homologs of TGF-beta known as TGF-beta-1, TGF-beta-2, TGF-beta-3, TGF-beta-4, TGF-beta-5, regardless of TGF-alpha. The amino acids of these homologues show 70-80% homology. TGF-beta-1 is a ubiquitous form and is almost ubiquitous, while other homologues are expressed only in a more localized spectrum of cells and tissues. Homologs isolated from different species are evolutionarily close and have 98% identical sequences. Adult, porcine, simian and bovine TGF-beta-1 are identical and differ from murine TGF-beta-1 at one amino acid position. TGF-beta-1 is also the same for humans and chickens.

It has also been reported that members of the transforming growth factor beta (TGF-beta) family are 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 reproductive tissues, with developmental and circulatory remodeling continuing to postnatal and adult stages. The potential roles of TGF-beta are identified in gonadal and secondary organ development, spermatogenesis and ovarian function, immune regulation of pregnancy, embryo transplantation and placental development.

Rautava et al in Journal of petri Gastroenterology and Nutrition 38: 378-388, April 2004 suggested that TGF-beta-2 and IL-10 show synergy with TGF-beta-2 in IL-10 production.

Mastitis is an inflammation of the udder, which is often characterized by tenderness and erythema and sometimes fever, and is associated with TGF-beta-2. During mastitis, the tight junctions of the acinar cells are opened, a process which is accompanied by an increase in sodium, inflammatory cells, and inflammatory and immunoregulatory agents in breast milk. Mastitis is usually unilateral and occurs most frequently in the first few weeks of breast feeding. Mastitis is generally considered a low incidence problem in industrialized countries, and affected women are often treated by midwives or nurses. Mastitis has been found to be more prevalent than previously thought through extensive and long-term investigations of women nursing in the United states, Finland and Australia (Semba R., Annals of the New York Academy of sciences. November 2000; 918: 156-62), suggesting that approximately 20-33% of women may develop clinically significant mastitis. And even more in lactating women.

Furthermore, Mastitis has recently been found to be associated with higher amounts of Human Immunodeficiency Virus (HIV) in breast milk and higher maternal-fetal risk of transmission of HIV (Semba, R.D., N.Kumwenda, T.E.Taha, et al 1999 Mastitis and immunological factors in Breast milk of human Immunodeficiency virus-induced genome.J.Hum.Lact.15 (4): 301-306).

TGF-beta-2 in breast milk is mostly of epithelial origin, even though it is synthesized by many other cells, including B-cells and T-cells. Thus an increase in TGF-beta-2 levels may be a regulator or a consequence of sub-clinical mammary inflammation. The ratio of sodium to potassium in breast milk (Na/K ratio) is said to be a well-known predictor of infection and subclinical mammary inflammation.

Furthermore, Kalliomaki et al, J Allergy Clin Immunol.1999 Dec; 104(6): 1251-7 it is believed that TGF-beta in colostrum can prevent the development of allergic diseases and increase the production of specific IgA in humans throughout the period of breast-feeding.

Moreover, various parts of the human and other mammalian bodies are inhabited by many different species of bacteria, including many different species of lactobacilli. Such bacteria are frequently symbiotic with their hosts, giving the host various beneficial effects, which are currently known to be different and dependent on the actual strain. For example, a different lactic acid bacterial strain, i.e. lactobacillus reuteri SD2112, has a specific antigen, either on its surface or is released by the bacteria in the mother's gastrointestinal tract. For example, the data from Valeur et al, AEM, 70, 1176-1181(2004) show that feeding Lactobacillus reuteri SD2112 can affect the levels of CD4+ T-chaperone cells in the ileum of healthy humans. Such observations have also been made in mammalian species and avians, which show that this may be a functional signaling system between the intestinal flora and the host. Through the so-called gut-mammary association, antigens from active strains are kept actively transported to the lymphatic region, Peyer's patches, below the epithelium of the GI tract. The epithelial antigen-specific B-cells are subsequently activated after they circulate from the GI tract to move other mucous membranes in the body, including saliva and mammary glands. Expression of specific molecules on these cells is thought to direct their adhesion to these tissues. Once in the mammary gland, these immune cells then initiate other processes to determine the levels of cytokines produced locally. This type of signal through the gut-mammary gland link has been shown to promote IgA secretion in breast milk and is also very likely to be applied to cytokine production.

It was earlier suggested (Laiho et al, Pediatric Research 53: 642-647, 2003) that the observed relationship between nutrients and inflammatory factors in breast milk suggests that the immunological properties of breast milk may be affected by intervention in the mother's diet. The same group noted that mothers with allergic disease had lower concentrations of TGF-beta-2 in the emulsion than mothers without disease. IL-10 was detected in their hands only at low levels and with natural frequency, and not differently in diseased and non-diseased maternal milk. This suggests that resistance to allergic disease is primarily through induction of oral tolerance to TGF-beta-2 and IL-10, and particularly TGF-beta-2 in breast milk may play a key role in the prevention and treatment of allergic disease. However, Weiner h, reported in Microbes and Infection, Volume 3, Issue 11, September2001, pages 947-954, states that since regulatory T cells generated by oral administration of antigens are triggered in a specific antigen but inhibited in a non-specific antigen, they modulate bypass inhibition when they encounter ingested autoantigens in the target organ. Thus, mucosal tolerance can be used to treat inflammation that occurs in nature due to lack of autoimmunity.

Different lactobacillus species, including lactobacillus reuteri, have been used in the formation of so-called biologies, meaning to provide viable and beneficial microorganisms to an animal, including humans. Lactobacillus reuteri is one of the most common inhabitants of the gastrointestinal tract of animals, and is commonly found in the intestine, and occasionally in the reproductive tract including humans, breast milk, and the oral cavity of healthy animals. It is known to have antibacterial activity. See, for example, U.S. patent nos.5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When lactobacillus reuteri cells live in anaerobic conditions in the presence of glycerol, the antibiotic substance they produce is called reuterin (B-hydroxy-propionaldehyde).

Lactic acid bacteria have earlier been reported to be used for preventing and treating various types of allergic reactions, for example see the following patents or patent applications, EP 1239032 to Stadler et al discloses novel recombinant strains, WO01/37865 to Clancy et al discloses the reduction of IgE levels by lactobacilli.

It is therefore an object of the present invention to provide a selected lactic acid bacterium and components thereof for improving breast milk for nursing infants, and a method for performing such selection. More specifically, it is an object of the present invention to increase the level of anti-inflammatory cytokine IL-10 in breast milk to reduce the risk of allergic reactions in nursing infants, while reducing the amount of TGF-beta-2 in breast milk to reduce the risk of mastitis in the nursing mother.

It is an object of the present invention to compensate for undesirable changes in the microbial flora by administering a selected, specific lactic acid bacterial strain to the mother before or during breast feeding.

It is another object of the present invention to employ the methods described herein or similar methods to select for specific cytokine effects that differentiate the tested strains on the relevant cell types and to use such specific lactic acid bacterial strains as maternal dietary components to stimulate increased production of IL-10 in breast milk while reducing the risk of mastitis as a result of reduced TGF-beta-2 levels, indicating reduced levels of subclinical inflammation in breast milk breasts and other tissues. Mastitis and subclinical mastitis are considered to interfere with breast feeding and to be detrimental to infants, and providing selected lactobacilli by the method of the present invention may improve the health of mothers and enable them to feed for longer periods of time.

It is a further object of the present invention to provide products, including formulations, comprising said strains, mutants, metabolites or components thereof, for administration to animals, including humans.

Other objects and advantages will become apparent from the following description and claims.

Summary of The Invention

The present invention comprises lactobacilli, and components thereof, the properties of which are selected to improve breast milk for feeding infants, more precisely to increase the level of the anti-inflammatory cytokine IL-10 in breast milk to reduce the risk of allergic reactions in the fed infant, while reducing the predisposition and thus the amount of TGF-beta-2 in breast milk, which means that the risk of mastitis in the feeding mother is reduced, thereby increasing the ability to feed and providing optimal protection and growth for young children. Here, the present invention also includes a method of selecting such lactobacillus strains. The present invention also includes a novel method of providing an even distribution of active ingredients in lactobacilli that are viable in formulated oil products.

Other objects and advantages of the present invention will become apparent from the following description and claims.

Brief description of the drawings

FIG. 1 is a flow diagram of a manufacturing process that may be used to manufacture the products of the present invention.

Detailed description and preferred embodiments of the invention

The present invention provides a product for improving the breast milk of a nursing baby, more precisely a product for increasing the level of the anti-inflammatory cytokine IL-10 in breast milk and for reducing the risk of allergic reactions in the nursing baby, while reducing the amount of the causative factor triggering the production of the cell signaling substance (TGF), i.e. TGF-beta-2, in the milk, thereby reducing the risk of mastitis in the nursing mother. The reduction of TGF-beta-2 associated with the present invention is believed to be an anti-inflammatory effect produced by consumption of the selected lactic acid bacteria, meaning a reduction in subclinical mastitis in the group of lactic acid bacteria that have recently been ingested daily. An example of the selected strain of the present invention herein is Lactobacillus reuteri SD2112(ATCC 55730).

In clinical trials, in the studies leading to the present invention, only three factors were correlated in the analysis with the level of IL-10 in the mother's colostrum: a) treatment with or without selected lactic acid bacteria; b) the number of children previously born by the mother; and c) the number of previous pregnancies. Thus, the greater the number of pregnancies or the number of babies, the greater the IL-10 in her colostrum. The specific selection of lactic acid bacteria administered to the pregnant mother for 4 weeks prior to delivery also increases IL-10 levels in colostrum without requiring prior pregnancy.

It is known that in certain cases increasing the systemic expression of IL-10 also prevents type I diabetes, and therefore the strains selected according to the invention can also be used for this purpose.

Among the selection methods used herein, the best strains for inducing IL-10 in breast milk and showing TGF-beta-2 reduction were selected using an established murine model and conventional analytical methods. Other methods similar to the detection of cytokine products in an emulsion may also be used. The details of which will be more clearly understood by way of example. The product of the invention preferably comprises viable cells of the selected strain; however, if the activity of the isolated metabolite or fraction of such cells on the viable cells of the strain is reliable, the product of the invention may include such metabolite or fraction in addition to or instead of the viable cells. The product of the invention may be any product for women, such as oil drip products, food products, tablets, capsules, sachets, and the like. Products particularly suitable for use in the present invention include an oil droplet (as in example 3) which helps to keep the active ingredient stable for an extended period of time. Lactobacillus cells can be used in the oil composition to improve the stability of the bacteria, as seen in U.S. patent 4518696 to Gehrman et al. However, in the present forms of lactic acid bacteria present in oils and fats we have not found that the inclusion of the important step as mentioned in the examples herein, i.e. drying the oil by means of vacuum before shaping, to improve the stability of the bacterial culture medium, is not present.

The concentration of the selected lactic acid bacteria cells required to achieve the effect in the product of the invention depends on the type of food and the amount of food to be ingested (or the time of use of a non-food chewing therapy product in the oral cavity), but the daily intake of the product is generally preferably equal to about 10⁵-10⁸CFU (colony forming unit) or more. Up to about 10 in number¹⁰-10¹¹CFU is possible and can be used to improve the effect without affecting the organoleptic properties of the product (its taste or smell). When the product is a yogurt or other lactic acid fermentation product, the lactic acid fermenting strains producing the product are preferably standard media (e.g., s. thermophilus and l. bulgaricus in yogurt). It is important that the strains selected according to the invention have the desired cytokine effect, compatible with any standard medium used in the product, so that the important properties of each strain employed are not affected by the use of the other strains. This can be determined by screening assays known in the art. The strains used in the present invention may be added before or after fermentation of the product at a level of about 10 days in the form of yoghurt⁶-10⁸CFU or more as discussed above.

Preferably, the product of the invention does not comprise other antibacterial ingredients, at least no substances inhibiting or killing the selected lactobacillus strain, or metabolite or component thereof, or substances affecting its activity.

The lactobacillus strain or metabolite, or component thereof, may be added to the raw materials in a manner well known in the art to formulate a particular type of product. In case cells are used and if a heating step is required for preparing the selected food or other products of the invention, the lactobacillus strain should be added after heating. Once the selected Lactobacillus cells are located in the product, it is preferred not to heat the product to 60-70℃ or above for a longer period of time.

The features of the present invention will be more clearly understood in conjunction with the following examples, which are not intended to limit the invention.

Example 1: method for selecting strains

The selection method of the lactobacillus strain employed according to the present invention may be carried out by the following steps:

evaluation of Lactobacillus cells for selection of strains production of IL-10 and reduction of TGF-beta-2 stimulated in human milk

This is an example of a selection method; certain modifications and substitutions may be made by those skilled in the art without departing from the scope of the invention.

Materials and methods

Animals: 51 sterile BALB/c mice (male and female) were purchased from Wisconsin University, USA. The mice were loaded into a sterile plastic film carrying case. 15 mice were transferred into 3 isolation boxes (isolation boxes #2, 3, and 4). Two cages each containing 2 males and 4 females were placed in isolation box #1 (breeding).

Animal treatment

Mice were placed in a sterile, flexible, thin film isolation box (Class biologicalcalin Clean, Ltd, Madison, WI, USA). The isolation box was sterilized with a 2% peracetic acid (FMC Corporation, philiadelphia, PA, USA) solution containing 0.1% Naconal (Stepan co., Rocksport, il.usa). 5 to 6 mice were placed in peracetic acid sterilized polystyrene cages with stainless steel wire lids. Mice of the same sex were placed in each cage. Mice were fed with autoclaved rodent chow 3500(Agway, Granville, Creedmoore, NC, USA). The food, water and mattress are all sterilized by high heat and high pressure. The food and bedding are autoclaved in a stainless steel cylinder and then aseptically transferred into an isolation box. Bottled, triggerable water is autoclaved and then sterilized with peracetic acid when placed in an isolation box. All mice received food and water ad libitum. Food and water levels were checked daily. The mattress is replaced once a week. Animals were maintained on a 12 hour day/night cycle. The indoor temperature and relative humidity were checked daily.

Bacteria

The detection of lactic acid bacteria is characterized and characterized by biochemical and molecular biological techniques. Lactobacillus was first grown in 10ml Man-Rogosa-Sharp (MRS) medium (BBL, Cockeysville, Md.), incubated at 37 ℃ for 18 hours and then transferred to 90ml MRS, incubated at 37 ℃ for 18 hours and then transferred to 1000ml MRS. After incubation at 37 ℃ for 18 hours, the medium was spun in a refrigerated centrifuge (SORVALL RC2-B, SORVALL, Norwalk, CN, USA) at 3000rpm for 10 minutes, the supernatant was discarded, the pellet was washed twice with sterile Phosphate Buffered Saline (PBS) and centrifuged at 3000rpm for 10 minutes. The pellet of each strain was resuspended in 30ml of PBS. The medium was placed in 1.2-ml cryovials and stored at-70 ℃ until use. The purity and concentration of the medium was checked before the bacteria were provided to the mice. Continuous 1/10 dilutions of Lactobacillus suspension were cultured on MRS media plates with 1.5% agar and incubated in anaerobic jars (GasPaK: BBL, Cockeysville, Md., USA) containing anaerobic products (Anaero Gen: Oxoid Ltd., Wake Road, Basingstoke, Hampshire, England, GB) at 37 ℃ for 48 hours. The culture medium was checked for clonal morphology and reuterin production.

Detection processing (in this embodiment)

Control

The strain is as follows: lactobacillus reuteri SD2112, ATCC 55730.

The strain is as follows: lactobacillus 4000

The strain is as follows: lactobacillus 4020

Example 2: determination of Lactobacillus clones

One day after the mice were placed in the isolation chamber, fecal samples from the mice in each cage were tested for the presence of microorganisms. Mice were deprived of water from 11:00am to 7:00 pm. After this period, 1.2ml of the lactic acid bacteria suspension was added to a bottle containing 200ml of water and provided to the mice. Mice in isolation box #2 received lactobacillus reuteri SD 2112. The suspension added to the water contained 2.0x10 oil¹⁰cfu/ml of Lactobacillus reuteri SD 2112. Mice in isolator #4 received lactobacillus 4000 strain, while mice in isolator #5 received lactobacillus 4020 strain. The lactic acid bacteria suspension added to each bottle of water contained 3.0x 10¹⁰cfu/ml. Mice in isolation box #3 received only water (control). Fecal samples (10 pellets) from mice (cage group) in each isolation box were taken once a week to detect lactic acid bacteria colonies and possible other microbial contamination.

By using an altered schaedler flora "normalization

The mice starting with the test strain were "normalized" with the altered schaedler flora 6 days after colony formation. C3H mice receiving the altered Schaedler's flora were purchased from Taconic Farms, Inc (Germantown, NY, USA). Two mice were placed in each isolation box and immediately a stool sample was taken to detect the presence of the test strain. Control mice and mice with clones of the test strains were deprived of water overnight. The next day 10 fecal pellets of C3H mice were placed into each bottle of drinking water, suspended in water, and provided to the mice. The procedure was continued for 3 days. Feces from BALB/C and C3H mice were taken once a week for one month to check for altered Schaedler flora clones and for the presence of the test strains.

Evaluation of

45 days after the test strains had formed monoclonals, and 30 days after "normalization", 5 mice in each treatment group were euthanized, and animal samples were isolated for spleen and T-lymphocytes, and cytokines were measured.

Preparation of spleen cells: the spleens were sterilized and placed in cold PBS + 0.5% Bovine Serum Albumin (BSA) (Sigma Chemical co., St Louis, MO, USA) + 0.1% sodium azide (NaN3) (Sigma). Single cell suspension from spleen was obtained by perfusing it with 5ml of RPMI-1640 medium (Sigma) +0.1mg/ml of gentamicin (Sigma). The cell suspension was transferred into a 15ml sterile conical tube and centrifuged in a refrigerated centrifuge at 2000rpm for 10 minutes. The supernatant was decanted and the red blood cells in each tube were lysed by resuspending the cells in 0.5ml PBS 1X, then adding 9ml distilled water, mixing well, and finally adding 1ml PBS 10X quickly and mixing well. The suspension was centrifuged as above and the supernatant was removed to resuspend the cells in 5ml of RPMI-1640 complete medium. A sample of the suspension is examined under a microscope and if particles other than cells are present, the suspension is filtered through a sterile nylon cloth and centrifuged again. The pellet was resuspended in 5ml of RPMI-1640 complete medium, washed twice and finally resuspended in 5ml of complete medium. Cell viability was determined by the talofulen blue (Sigma) exclusion method. Mu.l of the cell suspension were diluted in 380. mu.l of a 0.1% solution of dolol blue (1: 20 dilution) and counted using a hemocytometer. The concentration of the cell suspension was adjusted to 1.0X 10 in complete RPMI-1640⁶cells/ml。

Cell culture: cells were cultured in sterile 96-well flat-bottomed tissue culture plates (Fisherbrand, Fisher Scientific, Pittsburgh, Pa., USA) in RPMI-1640 complete medium with or without (non-stimulating) induction agents, such as Concavalin A (5. mu.g/ml), LPS from Salmonella typhimurium (1g/ml), phorbol 12-myristate 13-acetateAcid (PMA; 10ng/ml), ionomycin (I; 0.5. mu.g/ml), and heat-inactivated Lactobacillus reuteri (4.5mg protein/ml). Containing 1.0x 10⁵Mu.l of cell suspension and 50. mu.l of heat-inactivated Lactobacillus reuteri (4.5mg protein/ml) of cells were added to each well. Each set was repeated 5 times. Cell culture media at 5% CO₂The incubation with mitogen for 48 hours at 37 ℃ in the atmosphere and with Lactobacillus reuteri for 96 hours. Supernatants were collected from 5 wells, pooled and stored at-70 ℃ until used for cytokine detection.

And (3) cytokine quantification: cytokines (IL-10, TGF-beta-2) in the supernatant were used as murine Quantikine^TMM kit (R)&D Systems, Minneapolis, MN, USA) were tested by ELISA following the manufacturer's recommended protocol.

Results

Cytokines are immune system proteins, which are modulators of biological responses. They cooperate with antibodies to interact with the T cell immune system and amplify the immune response. Cytokines include monokines synthesized by macrophages and lymphokines produced by activated T lymphocytes and natural killer cells (NK).

The test strain of Lactobacillus reuteri SD2112 showed a higher (P < 0.05) IL-10 concentration than cells from strain 4000, 4020 or control mice. Moreover, the level of TGF-beta-2 is higher than that of the SD2112 strain. This strain was selected according to the invention.

b. Determination of IL-10 production and TGF-beta-2 reduction stimulated in human milk by Lactobacillus strains

This example demonstrates that the selected strain gives the expected effect in vivo. Lactobacillus reuteri, ATCC55730 (available from The American Type Culture Collection, Manassas, Va., USA) was detected. The tested lactobacillus strains were grown in MRS broth (Difco) and harvested in the exponential growth phase by centrifugation at 1000x g, washed twice with phosphate buffered saline (PBS; pH 6.8) and resuspended in the same buffer. The culture was then added to the oil droplet product, following the procedure of example 3 below.

The study was double-blind, and placebo-controlled survival determined the potential performance of selected lactic acid bacteria in allergic reactions, performed onMotala andpediatrics in county Hospital, and University Hospital inSweden。

Pregnant women from a family with a history of allergic disease received randomly oral Lactobacillus reuteri SD2112, ATCC55730 at a daily dose of 1x10 for 4 weeks prior to the study⁸CFU or placebo. The history of allergic disease was confirmed by a skilled nurse by telephone investigation. In summary, 232 families were included in the experiment from 1 month 2001 to 4 months 2003, and equally randomized into experimental and placebo groups.

The fit was assessed by collecting bottles of all used study products and then assessing their adherence.

Method of producing a composite material

The study included colostrum, taken from the first 3 days post partum, and mature milk, taken one month post partum, from 109 mothers. A manual breast pump was used to collect breast milk samples into sterile plastic tubes and stored at-70 ℃ until analysis.

After thawing, breast milk samples were centrifuged to remove fat and cellular debris (Bottcher, 2000). A portion of the remainder was used immediately for analysis of IL-10 concentration and the remainder was stored at-70 ℃ and subsequently used for analysis of TNF- α, IL4, TGF-beta-1, TGF-beta-2, soluble CD14(sCD14), total IgA, secretory IgA (sIgA), and sodium and potassium.

Levels of TGF-beta-1, TGF-beta-2 and CD14 (soluble CD14) were analyzed using 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 of the potential TGF-beta precursors, as described earlier (Bottcher 2000). The levels of IL-10 and TNF-. alpha.were determined using a commercial ELISA kit (CLB PeliPair reagent set, Amsterdam, the Netherlands) according to the manufacturer's recommendations. The detection lower limit is 62.5pg/mL for TGF-beta-1 and TGF-beta-2, 250 pg/mL for CD14, 2.3pg/mL for IL-10, and 7.8pg/mL for TNF- α.

The total amount of IgA and IgA was measured using ELISA described earlier (Bottcher 2002). The lower limit of detection was 31.2ng/mL for both assays. Sodium and potassium levels inThe clinical chemistry of University Hospital was tested according to standard protocols (ion selective electrodes). IgE levels in serum corresponding to inhaled allergensPharmacia CAP System^TM(Pharmacia Diagnostics, Uppsala, Sweden).

Statistical analysis

The study group was calculated to be of a size that assumed that a 80% ratio of the lactobacillus reuteri group compared to the placebo group was sufficient to show clinically true differences. The basis of the calculations is to assume that the incidence of clinical development of allergic reactions or eczema is 40% in the placebo group and can be reduced by half in the experimental group. The frequency of 91 objects required per group and half the way spent can be calculated to be estimated at 25%. The random list is completed by the outer companies and each center is hierarchically sampled with 4 as the word size. The total number of subjects to be enrolled is based on the above calculations, including expected half-way waste rates and word size, resulting in 116 women per group and their offspring.

Consideration of ethics

According to the Helsinki code, the participants received a written message and signed to agree. The lactobacillus reuteri SD2112 test strain in oil is considered a well-discussed and safe food for both young children and adults and therefore does not raise ethical concerns.

A small problem is also seen with research procedures with different tests, i.e. a large proportion of this at risk population will develop allergies and pass allergy screening procedures. The study was conducted by the University Hospital ofAgreed to by the ethical review committee of (c).

Results

In the study, we examined the effect on breast milk when pregnant women routinely orally administered lactobacillus reuteri strain SD2112 prior to delivery, four weeks.

Maternal distribution was similar for both groups compared. The number of weeks when the mother daily ingested the study product did not differ between the two groups, as for all breastfeeding within one month. The discontinuity between the two groups, up to as much as a month, is mainly due to the treatment schedule for infant protection (one of the exclusion criteria). No difference between the two groups was seen.

The cytokine distribution in breast milk in the Lactobacillus reuteri test strain panel was varied by increasing levels of the anti-inflammatory factor IL-10 in colostrum (mean 6.61pg/mL [ range 1.15-150]) over the mother in the placebo panel (4.78pg/mL [1.15-150 ]); p is 0.046. It was also seen that the level of TGF-beta-2 was reduced in the Lactobacillus reuteri SD2112 group. The level of TGF-beta-2 in the Lactobacillus reuteri group (mean 674pg/mL [102.5-2800] vs 965pg/mL [211.7-2800]) was significantly lower than in the placebo group; p is 0.020. While the levels of the other parameters are similar in both groups.

Breast milk obtained 4 weeks after delivery and discontinued daily intake of biological product were not different compared to placebo panel.

Example 3 preparation of products containing selected strains

In this example, a product called "royy oil droplets" was made. The product contains Lactobacillus reuteri SD2112 with good stability and viability on the basis of oil. The unique process in the production of the product is a drying step which removes most of the water in the oil.

The oil used herein according to the invention is a pure edible vegetable oil, preferably sunflower oil. Although an oil, such as a pure sunflower oil, is not expected to contain water, the process step of drying the oil by placing it in a vacuum has the unexpected effect of significantly improving the stability of the lactobacilli in the finished product. Therefore, the oil used in the present invention should be as water-free as possible. Although the stability of such media has previously been known to be closely related to the activity of water in the finished product, it was not known that drying oil under vacuum was used to stabilize the lactobacilli.

Description of the manufacturing Process

A preferred manufacturing process flow diagram is shown in figure 1. Details of one such possible process may be used in the present invention as follows.

Mixing of raw materials

1. Medium chain triglycerides (e.g., Akomed R, (Karlshamns AB, Karshamn Sweden) and sunflower oil with silica (e.g., Akosun, Karlshamns), and fumed silica [ Cab-o-sil M5P, M5P, Carbott (Cabot) ], were mixed in a Bolz mixer/tank (Alfred BOLZ Apprartabau GmbH, Wangen im Allgauu, Germany).

2. And (4) homogenizing. A Sine Pump and disperser (Sine Pump, Arvada, Colorado) was connected to the Bolz mixer and the mixture was homogenized.

3. And (5) drying in vacuum. The mixture was dried under 10mBar vacuum in a Bolz pot for 12 hours.

4. Adding lactobacillus reuteri. Approximately 20kg of the dried oil mixture was transferred to a 50 liter stainless steel container. Adding Lactobacillus reuteri powder (preferably lyophilized, the amount of Lactobacillus reuteri used varies depending on the amount put in the oil, but one example is adding 0.2kg with 10¹¹CFU/g culture). Mix slowly until homogenous.

5. And (4) mixing. Premixing of the lactobacillus reuteri is carried out back in the Bolz mixer.

6. And (4) discharging. The suspension was discharged into a 200 liter stainless steel vessel and blanketed with nitrogen. The suspension was maintained in the container until the bottle was filled.

Although certain representative embodiments have been described herein, modifications may be made by one skilled in the art without departing from the spirit or scope of the invention.

Lactobacillus reuteri strain (abbreviated Lactobacillus reuteri strain) SD2112, deposited at ATCC55730 on 12.07.1995, and addressed to 20852 Rockville, Md.

Claims (11)

1. A stable oil product comprising a lyophilized Lactobacillus strain in an edible vegetable oil, wherein the edible vegetable oil is produced by vacuum drying prior to addition to the Lactobacillus strain.

2. The oil product of claim 1, wherein the edible vegetable oil is sunflower oil.

3. The oil product of claim 1, wherein the edible vegetable oil is a medium chain triglyceride.

4. The oil product of any one of claims 1 to 3, wherein the vacuum drying step of the edible vegetable oil enables survival of a freeze-dried culture of the Lactobacillus strain prior to addition to the Lactobacillus strain.

5. A method of improving the survival of a lactobacillus strain, the method comprising mixing a freeze-dried survival culture of the lactobacillus strain with an edible vegetable oil that has not been vacuum dried.

6. A process for preparing an oil product according to any one of claims 1 to 4, said process comprising the steps of:

a) mixing edible vegetable oil to obtain a mixture;

b) drying the mixture under vacuum to obtain a dried mixture; and

c) adding the dry mixture to a culture of lactobacillus reuteri to obtain a suspension.

7. The method of claim 6, wherein the edible vegetable oil comprises sunflower oil.

8. The method of claim 6, wherein the edible vegetable oil further comprises medium chain triglycerides.

9. The method of claim 6, wherein the culture is lyophilized.

10. The method of claim 6, wherein the mixture is dried under a vacuum of 10mBar for 12 hours.

11. The method of claim 6Method, wherein the amount of Lactobacillus reuteri used is 10¹¹CFU/g of 0.2kg of Lactobacillus reuteri culture.