Probiotics, secretory IgA and inflammation

Abstract

Probiotics and ways to increase their effectiveness are provided. One embodiment of the present invention relates to a combination of probiotics with SIgA and possible uses of this combination. For example a use of a composition comprising SIgA and at least one probiotic for the preparation of a product to treat or prevent inflammation is disclosed.

Claims

1. A method for reducing the severity of an inflammatory condition selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, necrotizing enterocolitis, eczema, allergy, and atopy, the method comprising: administering a therapeutically-effective amount of a composition comprising isolated secretory immunoglobulin A (SIgA) and a probiotic micro-organism to a subject, wherein the isolated SIgA and the probiotic micro-organism are present in the form of a complex with each other, the isolated SIgA and the probiotic micro-organism are present in the composition in a stoichiometric ratio of a least 10:1, and the composition comprises between 10.sup.2 and 210.sup.7 cells of the probiotic micro-organism per daily dose.

2. The method of claim 1, wherein the subject is selected from the group consisting of newborns, infants, juveniles, adults, elderly, and mothers during pregnancy or lactation.

3. The method of claim 1, wherein the composition comprises milk obtained from an animal or plant source.

4. The method of claim 3, wherein the milk is selected from the group consisting of cows' milk, human milk, sheep milk, goat milk, horse milk, camel milk, rice milk, and soy milk.

5. The method of claim 1, wherein the composition comprises milk protein fractions or colostrum.

6. The method of claim 1, wherein the composition contains a prebiotic.

7. The method of claim 6, wherein the prebiotic is selected from the group consisting of oligosaccharides, dietary fibers, and mixtures thereof.

8. The method of claim 6, wherein the prebiotic is selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides, xylo-oligosaccharides, oligosaccharides of soy, glycosyl sucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), maltooligosaccharides, pectins, hydrolysates thereof, and mixtures thereof.

9. The method of claim 1, wherein the composition comprises between 10.sup.2 and 10.sup.10 cells of the probiotic micro-organism per daily dose.

10. The method of claim 1, wherein the composition comprises between 0.0001 mg SIgA and 10 mg SIgA per daily dose.

11. The method of claim 1, wherein the probiotic micro-organism is selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus, Saccharomyces, and mixtures thereof.

12. The method of claim 1, wherein the probiotic micro-organism is selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Enterococcus faecium, Saccharomyces boulardii and Lactobacillus reuteri, and mixtures thereof.

13. The method of claim 1, wherein the probiotic micro-organism is selected from the group consisting of Lactobacillus johnsonii (NCC533; CNCM I-1225), Bifidobacterium longum (NCC490; CNCM I-2170), Bifidobacterium longum (NCC2705; CNCM I-2618), Bifidobacterium lactis (2818; CNCM I-3446), Lactobacillus paracasei (NCC2461: CNCM 1-2116), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus (NCC4007; CGMCC 1.3724), Enterococcus faecium SF 68 (NCIMB10415), and mixtures thereof.

14. The method of claim 1, wherein at least 90% of the probiotic micro-organism present in the composition is associated with at least one isolated SIgA molecule as immune complexes.

15. The method of claim 1, wherein the isolated SIgA and the probiotic micro-organism are present in the composition in a stoichiometric ratio of at least 100:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows schematically how SIgA is believed to improve the effects of commensal bacteria, when associated with them by increasing the interaction with the intestinal mucosa of the host. Depicted are possible and documented routes of interaction of SIgA associated with commensal bacteria with the host intestinal mucosa.

(2) FIG. 2 shows the result of experiments testing the binding properties of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria to epithelial cells. Data are expressed as means CFU per 100 Caco-2 cellsSEM.

(3) FIG. 3 shows the result of experiments testing the binding properties of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria to epithelial cells and the influence of secretory IgA (SIgA) or secretory component (SC). Data are expressed as means CFU per 100 Caco-2 cellsSEM.

(4) FIG. 4 shows the result of an experiment testing the effect of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria, alone or in combination with SIgA or SC, on transepithelial electrical resistance (TER) measuring epithelial permeability. Data are expressed as means ohms per cm.sup.2SEM.

(5) FIG. 5 shows the result of an experiment testing the effect of LPR, combined or not with SIgA or SC, on NF-B activation in Caco-2 cell monolayer. Decrease in NF-kB binding activity is indicative of attenuated inflammatory pathway(s) within the Caco-2 cell.

(6) FIG. 6 shows the result of an experiment testing the effect of LPR, combined or not with SIgA or SC, on S. flexneri invasion of Caco-2 cell. Two monoclonal SIgA molecules were used: a non-specific SIgA (SIgAnon-specific) recognizing a Salmonella epitope, and a specific anti-S. flexneri SIgA (SIgAC5). Data are expressed as means CFU per transwell filterSEM.

(7) FIG. 7 shows the result of an experiment testing the effect of probiotics on polymeric Ig receptor (pIgR) expression in a Caco-2 monolayer. (A) Different treatments were tested at 16 h, including combination of probiotics with non-specific SIgA and combination of S. flexeneri with specific anti-S. flexneri SIgA (western blot). (B) Semi-quantitative analysis of pIgR expression levels normalized to -actin by densitometric analysis of the bands identified in the gels in A. (C) Kinetic of pIgR expression over 24 h incubation of Caco-2 cells with various preparations (ELISA).

DETAILED DESCRIPTION

(8) Probiotic means microbial cell preparations or components of microbial cells with a beneficial effect on the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. et al Probiotics: how should they be defined Trends Food Sci. Technol. 1999:10 107-10).

(9) All probiotic micro-organisms may be used in accordance with the present invention. Preferably, they are selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus and Saccharomyces or mixtures thereof, in particular selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis. Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Enterococcus faecium, Saccharomyces boulardii and Lactobacillus reuteri or mixtures thereof, preferably selected from the group consisting of Lactobacillus johnsonii (NCC533; CNCM 1-1225), Bifidobacterium longum (NCC490; CNCM 1-2170), Bifidobacterium longum (NCC2705; CNCM 1-2618), Bifidobacterium lactis (2818, CNCM 1-3446), Lactobacillus paracasei (NCC2461; CNCM 1-2116), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus (NCC4007; CGMCC 1.3724), Enterococcus faecium SF 68 (NCIMB10415), and mixtures thereof.

(10) The composition of the present invention may also contain prebiotics. The addition of prebiotics is beneficial as it can, when combined with probiotics, delivers synergistic effects in terms of the health benefits. A composition comprising a combination of prebiotics and probiotics is commonly known as a symbiotic composition.

(11) Prebiotic means food substances that promote the growth of beneficial bacteria such as bifidobacteria or lactobacilli, and/or probiotics in the intestines. They are not broken down in the stomach or absorbed in the GI tract of the person ingesting them, but they are fermented by the gastrointestinal microflora and/or by probiotics.

(12) The prebiotics that may be used in accordance with the present inventions are not particularly limited and include all food substances that promote the growth of probiotics in the intestine. Preferably, they may be selected from the group consisting of oligosaccharides, optionally containing fructose, galactose, mannose; dietary fibers, in particular soluble fibers, soy fibers; inulin; or mixtures thereof. Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides, xylo-oligosaccharides, oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, pectins and/or hydrolysates thereof.

(13) Typical inflammatory conditions that may be treated or prevented by the use of the present invention include but are not limited to acute inflammations such as sepsis, infections, burns and chronic inflammations such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, necrotizing enterocolitis, skin inflammation, such as UV or chemical-induced skin inflammation, eczema, reactive skin, psoriasis, vitiligo, acne, liver inflammation, alcoholic cirrhosis, allergy, atopy, bone inflammation, rheumatoid arthritis, systemic lupus, Gougerot-Sjgren's syndrome, Reiter's syndrome, poliomyelitis, dermato-myositis, thyroiditis, Basedow, Hashimoto, type I diabetes, Addison's disease, auto-immunes hepatitis, celiac disease, Biermer's disease, multiple sclerosis, myasthenia, encephalomyelitis, eye inflammation, obesity-associated inflammation, age-related low-grade inflammation, Blau's syndrome, Alzheimer's disease, cardiovascular diseases, atherosclerosis, metabolic syndrome, gingivitis, paronditis and combinations thereof.

(14) The composition of the present invention may also be used to control and/or alleviate an inflammatory reaction of the body.

(15) The composition of the present invention can further be used to generate, improve or reinforce homeostasis and oral tolerance.

(16) The product prepared by the use of the present invention may be a food product, an animal food product or a pharmaceutical composition. For example, the product may be a nutritional composition, a nutraceutical, a drink, a food additive or a medicament.

(17) A food additive or a medicament may be in the form of tablets, capsules, pastilles or a liquid for example. Food additives or medicaments are preferably provided as sustained release formulations, allowing a constant supply of SIgA and probiotics for prolonged times.

(18) The product is preferably selected from the group consisting of milk powder based products; instant drinks; ready-to-drink formulations; nutritional powders; nutritional liquids; milk-based products, in particular yoghurts or ice cream; cereal products; beverages; water; coffee; cappuccino; malt drinks; chocolate flavoured drinks; culinary products; soups; tablets; and/or syrups.

(19) Milk may be any milk obtainable from animal or plant sources and is preferably cows milk, human milk, sheep milk, goat milk, horse milk, camel milk, rice milk or soy milk.

(20) Instead of milk, also milk derived protein fractions or colostrum may be used.

(21) The composition may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobials. They may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. Further, they may contain an organic or inorganic carrier material suitable for oral or enteral administration as well as vitamins, minerals trace elements and other micronutrients in accordance with the recommendations of Government bodies such as the USRDA.

(22) The composition of the present invention may contain a protein source, a carbohydrate source and/or a lipid source.

(23) Any suitable dietary protein may be used, for example animal proteins (such as milk proteins, meat proteins and egg proteins); vegetable proteins (such as soy protein, wheat protein, rice protein, and pea protein); mixtures of free amino acids; or combinations thereof. Milk proteins such as casein and whey, and soy proteins are particularly preferred.

(24) If the composition includes a fat source, the fat source more preferably provides 5% to 40% of the energy of the formula; for example 20% to 30% of the energy. DHA may be added. A suitable fat profile may be obtained using a blend of canola oil, corn oil and high-oleic acid sunflower oil.

(25) A source of carbohydrates may more preferably provide between 40% to 80% of the energy of the composition. Any suitable carbohydrate may be used, for example sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrins, and mixtures thereof.

(26) The product prepared by the present invention may be administered to humans or animals, in particular companion animals, pets or livestock. It has beneficial effects for any age group. Preferably, the product is intended in for infants, juveniles, adults or elderly. It may however also be administered to mothers during pregnancy and lactation to treat the infant.

(27) The composition of the present invention will be effective as long as probiotics and SIgA are administered simultaneously, briefly one after the other, for example in a maximal timeframe of less than 60 minutes, preferably less than 30 minutes, more preferred less than 15 minutes and most preferred less than 5 minutes, and/or are combined prior to administration as already present in the food product.

(28) However, it was found that the combination of probiotics and SIgA is in particular effective, if SIgA and probiotics are combined in complexes prior to administration. This has the advantage that the beneficial complexes do not need to form after consumption of the product, but that they are already present in the food product.

(29) Consequently, one embodiment of the present invention relates to the use of a composition comprising SIgA and probiotics, wherein the SIgA and at least one probiotic are at least partially associated in the composition.

(30) SIgA and at least one probiotic are preferably present as immune complexes, for example in a way that at least 90%, more preferably at least 95%, even more preferred all probiotic bacteria are present as immune complex in association with at least 1 SIgA molecule, for example with at least 5 SIgA molecules.

(31) The composition may also contain at least one other kind of other food grade bacteria, preferably selected from the group consisting of lactic acid bacteria, bifidobacteria, enterococci or mixtures thereof. These other food grade bacteria may contribute to obtain a healthy gut microflora and will hence contribute to achieve the object of the present invention even more effectively.

(32) The present invention also relates to a food composition comprising SIgA and at least one probiotic micro-organism. The SIgA and the probiotic micro-organism may preferably be combined in the food composition. SIgA and the probiotic micro-organism may preferably be present in a stoichiometric ratio of at least 10:1, preferably at least 100:1, most preferably at least 2000:1 to 100000:1. Obviously, the more SIgA molecules are attached to the surface of the probiotic micro-organism the more effective this combination will be. The upper limit of SIgA saturation is determined by the surface of the probiotic micro-organisms and by the number of available binding sites for SIgA.

(33) Typically, the probiotics will be effective in a large range amount. In general, it is preferred if the product comprises between 10.sup.2 and 10.sup.10 cells of probiotics per daily dose.

(34) The amount of SIgA required to achieve an effect, is equally not limited. It is generally preferred if the product comprises between 0.0001 mg SIgA and 100 mg SIgA per daily dose.

EXAMPLE 1

(35) Binding to Epithelial Cells

(36) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37 C. with different doses of bacteria, indicated in the figure legend in absence of antibiotic or FCS. Fresh overnight cultures of LPR, BL818 and E. coli TG-1 bacteria were used. Cells were then washed prior to enumeration. Bound bacteria were counted by plating on MRS or LB plates. For each experiment, triplicate tests were performed. Data were expressed as means of bound bacteria per 100 Caco-2 cellsSEM. Triplicates were performed for each experiment. In a subsequent experiment, cells were incubated with 210.sup.7 bacteria for 16 hours at 37 C., in the presence of increasing doses of either SIgA or SC as indicated in the legend to FIG. 3. Cells were then washed prior to enumeration. Bound bacteria were counted by plating on MRS or LB plates. For each experiment, triplicate tests were performed. Data were expressed as means of bound bacteria per 100 Caco-2 cellsSEM. Triplicates were performed for each experiment.

(37) A preferential binding to polarized Caco-2 cells of LPR and BL818 is observed in comparison to E. coli TG-1 (FIG. 2). There is a dose-dependent binding capacity of probiotics to intestinal epithelial cells. It can be observed that binding properties could be differentiated between the two strains.

(38) For subsequent experiments 210.sup.7 CFU of probiotics were used, as this amount did not lead to any pH change in the medium on one hand, and showed an efficient binding ratio on the other hand.

(39) Increasing the dose of monoclonal SIgA potentiated the capacity of both LPR and BL818 to bind to polarized Caco-2 cell monolayers. Secretory component by itself did not exhibit such properties (FIG. 3). The 1 g dose of SIgA that confers a significant improvement in probiotic binding capacity was selected for subsequent experiments. This dose enables a final complex constituted of 50,000 to 100,000 units of SIgA for 1 bacterium.

(40) Results are shown in FIGS. 2 and 3.

EXAMPLE 2

(41) Barrier Function in Polarized Caco-2 Cell Monolayer

(42) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 24 h at 37 C. with 210.sup.7 CFU of bacteria in absence of antibiotic or FCS. Bacteria were tested either alone or in combination with SIgA or SC at concentrations indicated in the legend to FIG. 4. Transepithelial electrical resistance (TER) was measured at 3, 6, 9, 15 and 24 h. Controls include incubation with SIgA and SC alone. Triplicates were performed for each experiment.

(43) A 20-25% increase in transepithelial electrical resistance (TER) resulted from the incubation of polarized Caco-2 cell monolayer with LPR or BL818 alone, suggesting that probiotics potentiated epithelial barrier function. This remained true when the bacteria were combined with SIgA or SC (FIG. 4). SIgA or SC by themselves did not lead to any TER change.

(44) Results are shown in FIG. 4.

EXAMPLE 3

(45) NF-kB Activation in Polarized Caco-2 Cell Monolayer

(46) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm2 Transwell filter. Cells were incubated for 16 h at 37 C. with 210.sup.7 CFU of LPR in absence of antibiotic or FCS. Bacteria were tested either alone or in combination with SIgA or SC at concentrations indicated in the legend to FIG. 5. S. flexneri, S. typhi and H. pylori (210.sup.7 CFU) were used as pathogenic controls. Nuclear and cytoplasmic extracts were prepared and analysed by electrophoretic mobility shift assay (EMSA) and Western blot using anti-IB-specific monoclonal antibody. Triplicates were performed for each experiment.

(47) Exposure to pathogenic bacteria led to much more pronounced activation of nuclear NF-B compared to non-pathogenic bacteria (FIG. 5).

(48) Disappearance of IB (lower panel) reflects activation of the pathway leading to nuclear translocation of NF-B. In that respect, while LPR alone has a mild effect on NF-B activation, combination of LPR with SIgA or SC reduced NF-B activation in Caco-2 cells (BL818 not tested). Incubation of epithelial cells with pathogenic S. flexneri led to total disappearance of IB expression.

(49) Results are shown in FIG. 5.

EXAMPLE 4

(50) Anti-Pathogenic Activity

(51) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37 C. with 210.sup.7 CFU of LPR in absence of antibiotic or FCS. LPR was tested alone or in combination with with either 0.2 g of SC, 1 g of polyclonal SIgA or 1 g of specific anti-S. flexneri LPS SIgAC5. After incubation with LPR cells were washed and then incubated with 10.sup.7 S. flexneri for 6 hours, washed again and incubated with 50 mg/ml gentamycin for 45 min. Finally, cells were lysed and intracellular S. flexneri were enumerated on LB agar plates. Triplicates were performed for each experiment.

(52) Addition of LPR reduced infection of polarized Caco-2 cell monolayer by S. flexneri in a dose dependent manner. The effect was highly enhanced upon combination with SIgA. Full prevention of infection was achieved when S. flexneri LPS-specific SIgAC5 antibody was used (FIG. 6).

(53) Results are shown in FIG. 6.

EXAMPLE 5

(54) Expression of Polymeric Ig Receptor in Polarized Caco-2 Cell Monlayer

(55) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37 C. with 210.sup.7 CFU of LPR or BL818 in absence of antibiotic or FCS. Probiotics were tested alone or in combination with with either 0.2 g of SC, 1 g of polyclonal SIgA. Control S. flexneri was tested alone or in combination with 1 g of specific anti-S. flexneri LPS SIgAC5. After washing, Caco-2 cells were directly recovered from the Transwell filters and lysed. Nuclei were removed and cell debris as well as cytoplasms were analysed by Western blot using anti-pIgR antibody and antisera to human SC and -actin as controls. Triplicates were performed for each experiment.

(56) In a subsequent experiment cells were incubated following the same procedure and then recovered from the transwell filter at 8, 16 and 24 h of incubation. Quantitative analysis of pIgR was performed by ELISA on cell debris/cytoplasm fractions. Total proteins were determined by BCA protein assay. Values were normalized to protein content and data expressed as means of ng pIgR/mg of total proteinSEM.

(57) pIgR expression in epithelial cells was normalized to -actin expression. As revealed by Western blot (upper panel) and densitometric analysis of the respective bands (lower panel) there was an increase of pIgR level following overnight exposure of polarized Caco-2 monolayers to combinations of LPR or BL818 with either SIgA or SC compared to probiotics alone (FIG. 7a). Specific anti-S. flexneri LPS SIgAC5 prevented interaction of the pathogen with the Caco-2 cell polarized monolayer, thus explaining the decrease in pIgR expression when compared to S. flexneri treatment alone.

(58) The results further showed a time-dependent increase of polymeric Ig receptor (pIgR) level following exposure of polarized Caco-2 cell monolayers to probiotic combinations with SIgA or SC (FIG. 7b).

(59) Results are shown in FIG. 7.

(60) It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.