COMPOSITION AND USES THEREOF

Abstract

A colostrum-enriched composition comprising at least 0.1 to 12 wt % immunoglobulin (at least 0.1 to 12g per 100 g composition dry weight) isolated from whey.

Claims

1. An enriched whey fraction composition comprising lactose, oligosaccharides and immunoglobulin G (IgG), which when in a dry form is substantially free of caseins and comprises 65-75% (w/w) total protein (including 20-30% (w/w) BSA, 0.1-12% IgG (w/w), 1-5% (w/w) -lac, 1-15% (w/w) -Ig)), 0.1-1.0% (w/w) fat, 3-13% (w/w) inorganic constituents and 16-25% (w/w) carbohydrate content, wherein the dry form components in combination amount to 100% (w/w).

2. An enriched whey fraction composition comprising lactose, oligosaccharides and immunoglobulin G (IgG), which is substantially free of caseins, and wherein the IgG is at a concentration of about 0.1-12 wt % of the composition.

3. An enriched whey fraction composition of claim 1 or claim 2, further comprising at least about 1 wt % to 3.5 wt % -lactalbumin or at least about 1.5 wt % to 9.1 wt % -lactoglobulin, or a combination thereof.

4. An enriched whey fraction composition of any one of claims 1 to 3, wherein the IgG is glycosylated.

5. An enriched whey fraction composition of any one the preceding claims, wherein the whey fraction is defatted.

6. An enriched whey fraction composition according to any one of the preceding claims, wherein the whey fraction is derived from a human, or domestic animals selected from cow, sheep, goat, camel, donkey.

7. An enriched whey composition according to any one of the preceding claims, in which the composition is converted to a wet form by adding a wetting agent so that the composition is diluted by a factor 1:50 to 1:100.

8. An enriched whey fraction composition of claim 2, which when in a dry form comprises 65-75% (w/w) total protein (including 20-30% (w/w) BSA, 0.1-12% IgG (w/w), 1-5% (w/w) -lac, 1-15% (w/w) -Ig)), 0.1-1.0% (w/w) fat, 3-13% (w/w) inorganic constituents and 16-25% (w/w) carbohydrate content, wherein the dry form components in combination amount to 100% (w/w).

9. An enriched whey fraction composition of claim 7 or claim 8, wherein the composition in dry form comprises: 71.04% (w/w) total protein, including 26.96% (w/w) BSA, 9% IgG (w/w), 3% (w/w) -lac, 9.1% (w/w) 3-Ig), and 0.6% (w/w) fat, 8% (w/w) inorganic constituents and 19.7% (w/w) carbohydrate content.

10. An enriched whey fraction composition of any one of the preceding claims, wherein the whey fraction is prepared from a colostrum fraction of milk, from a transitional milk stream or from a mature milk stream.

11. An enriched whey fraction composition according to any one of the preceding claims, for use in a method of treating or preventing diseases in a subject, wherein the disease is associated with lower counts of commensal bacteria such as observed in formula fed infants, elderly or immune-compromised individuals or individuals on antibiotics.

12. An enriched whey fraction composition of claim 11, wherein the subject is suffering from inflammatory bowel diseases (such as Crohn's disease, irritable bowel disease, ulcerative colitis), periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multi-organ failure, asthma, and allergic diseases (such as allergic rhinitis (hay fever), food allergy, and atopic dermatitis (eczema)).

13. An enriched whey fraction composition according to any one of claims 1 to 10 for use in increasing adherence of commensal bacteria in the gut.

14. A food product comprising the enriched whey fraction composition of any one of claims 1 to 10.

15. A food product according to claim 13, wherein the food product is a dairy product, a beverage, infant food, a cereal, a biscuit, confectionary, a cake, a food supplement, a dietary supplement.

16. A food product according to claim 14, wherein the dairy product is a yoghurt, a milk drink, a flavoured milk drink, a probiotic drink, ice cream, frozen yoghurt.

17. An infant formula comprising the enriched whey fraction composition of any one of claims 1 to 10.

18. A method for producing an enriched whey fraction composition comprising lactose, oligosaccharides and at least 0.1-12 wt % immunoglobulin G (IgG) of the composition, and which is substantially free of caseins, the method comprising the steps of: (a) defatting and de-caseinating a whey colostrum fraction; (b) providing a colostrum, a transitional milk stream or a mature milk stream that is substantially free of casein and is substantially fat-free, while retaining lactose, glycoproteins and oligosaccharides; and (c) freeze-drying the defatted and de-caseinated whey fraction.

19. A composition for use in increasing adherence of commensal bacteria in the gut, the composition comprising at least one of glycosylated IgG, glycans, monosaccharides or a combination thereof.

20. A composition according to claim 18, wherein the glycan comprises sialic acid, N-Acetylglucosamine, fucose, mannose, galactose, fructose, glucose, or a combination thereof.

21. A composition according to any one of claim 18 or 19, wherein the IgG is purified from a whey fraction obtained from a colostrum fraction of milk, from a transitional milk stream, or from a mature milk stream.

22. A composition according to claim 20, wherein the purified IgG is active at a concentration of between 2 to 24 mg/ml.

23. An enriched whey fraction composition as claimed in any one of claims 1 to 10, or a food product as claimed in any one of claims 13 to 15, or an infant formula claim 16, for use in a method of increasing the levels of commensal bacteria in the gastrointestinal system of a mammal.

24. An enriched whey fraction composition of claim 22, wherein the mammal is an infant human, an adolescent human or an adult human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:

[0082] FIG. 1 is a bar chart illustrating a 1.5 to 6.1-fold increase in Bifidobacterium longum NC/MB 8809 adherence to HT29 cells after exposure to various dilutions of the BWCF

[0083] FIG. 2 is a bar chart illustrating a 1.4 to 1.5-fold increase in Bifidobacterium longum subsp. infantis ATCC 15697 adherence to HT29 cells after exposure to various dilutions of the BWCF

[0084] FIG. 3 is a bar chart illustrating 0.5 to 1.7-fold increase in Bifidobacterium breve 2258 adherence to HT29 cells after exposure to various dilutions of the BWCF

[0085] FIG. 4 is a bar chart illustrating a 0.1 to 1.6-fold increase in Bifidobacterium breve UCC2003 adherence to HT29 cells after exposure to various dilutions of the BWCF

[0086] FIG. 5 is a bar chart illustrating a 5.8 to 6.4-fold increase in Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to individual components of the whey colostrum; -lactalbumin (-lac), -lactoglobulin (-Ig), IgG, as well as mixtures thereof. There was a 5.8 to 6.4-fold increase in Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after exposure to IgG and a combination of IgG, Ig and -lac.

[0087] FIG. 6 is a bar chart illustrating Bifidobacterium longum subsp. infantis 15697 adherence to HT-29 cells after prior exposure of HT-29 cells to individual components; -lactalbumin, -lactoglobulin, IgG and mixtures thereof. There was a 0.4-fold increase and a 14.7-fold increase in Bifidobacterium longum subsp. infantis 15697 adherence to HT-29 cells after prior exposure of HT-29 cells to a combination of IgG, -Ig and -lac, and IgG alone, respectively.

[0088] FIG. 7 is a bar chart illustrating Bifidobacterium longum subsp. infantis 2258 adherence to HT-29 cells after prior exposure of HT-29 cells to individual components; -lactalbumin, -lactoglobulin, IgG and mixtures thereof. There was an 11.3-fold increase in Bifidobacterium longum subsp. infantis 2258 adherence to HT-29 cells after prior exposure of HT-29 cells to IgG alone.

[0089] FIG. 8 is a bar chart illustrating Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to purified IgG (extracted and purified from day 1 colostrum) in the concentration range 2-24 mg/mL. There was a 2.5 to 43-fold increase of Bifidobacterium longum NCIMB 8809 after prior exposure to the different range of IgG concentrations.

[0090] FIG. 9 is a bar chart illustrating Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to the milk oligosaccharide 3-sialyllactose at 4 mg/mL. There was no increase in adhesion of Bifidobacterium longum NCIMB 8809 after prior exposure to 3-sialyllactose.

[0091] FIG. 10 is a bar chart illustrating Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to two commercial prebiotics oligofructose (Orafti p95) (Beneo Orafti, Dublin) and an oligofructose-enriched inulin (Raftilose) at 4 mg/mL. There was no increase in adhesion of Bifidobacterium longum NCIMB 8809.

[0092] FIG. 11 is a bar chart illustrating Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to IgG and sodium metaperiodate treated IgG at 24 mg/mL. There was no increase in adhesion of Bifidobacterium longum NCIMB 8809 when exposed to the sodium metaperiodate-treated IgG unlike non-treated IgG which still gave a significant increase (3-fold) in adhesion of Bifidobacterium longum NCIMB 8809. Although not being bound by theory, this data supports the hypothesis that the glycan terminal moieties of IgG are responsible for its observed effect on HT-29 cells, which leads to HT-29 cells being better colonisers for commensal bacteria such as Bifidobacterium longum NCIMB 8809.

[0093] FIG. 12 is a bar chart illustrating Bifidobacterium longum NCIMB 8809 adherence to HT-29 cells after prior exposure of HT-29 cells to purified IgG (isolated from mature milk) at 16 mg/mL. There was a significant 3.9-fold increase of Bifidobacterium longum NCIMB 8809 adhering to HT-29 cells after prior exposure to purified IgG isolated from mature milk.

DETAILED DESCRIPTION OF THE DRAWINGS

[0094] Materials and Methods

[0095] Generation of Bovine Whey Colostrum fraction (BWCF)

[0096] A fraction was isolated from bovine colostrum (Day 1) as follows: [0097] The colostrum as collected from Holstein Friesian cattle on-site at the Teagasc Food Research Centre, Moorepark (Fermoy, Cork, Ireland) and was defatted and deproteinized through conventional methods as described by Kobata & Ginsburg (Oligosaccharides of human milk. IV. Isolation and characterization of a new hexasaccharide, lacto-N-neohexaose. Arch Biochem Biophys 150, 273-281 (1972)). [0098] In brief, aliquots were centrifuged at 5000 rpm for 20 min at 4 C. in a Sorvall RC6 plus to separate the fat. [0099] The aqueous phase was collected and 1 M-HCI was added until a pH of 4.6 was reached. [0100] The sample was then heated to 35 C. for 2hr and precipitated caseins were removed by centrifugation at 5000 rpm for 30 min at 25 C. [0101] The pH of the supernatant was then neutralised using 4M NaOH. [0102] The de-fatted, de-caseinated solution was collected, freeze-dried and stored at 80 C.

[0103] Generation of Bovine Whey Milk (Mature) [0104] A fraction was isolated from mature milk as follows: [0105] The mature milk as collected from Holstein Friesian cattle on-site at the Teagasc Food Research Centre, Moorepark (Fermoy, Cork, Ireland) and was defatted and deproteinized through conventional methods as described by Kobata & Ginsburg (Oligosaccharides of human milk. IV. Isolation and characterization of a new hexasaccharide, lacto-N-neohexaose. Arch Biochem Biophys 150, 273-281 (1972)). [0106] In brief, aliquots were centrifuged at 5000 rpm for 20 min at 4 C. in a Sorvall RC6 plus to separate the fat. [0107] The aqueous phase was collected and 1 M-HCl was added until a pH of 4.6 was reached. [0108] The sample was then heated to 35 C. for 2 hr and precipitated caseins were removed by centrifugation at 5000 rpm for 30 min at 25 C. [0109] The pH of the supernatant was then neutralised using 4M NaOH. [0110] The de-fatted, de-caseinated solution was collected, freeze-dried and stored at 80 C.

[0111] Generation of Bovine Whey Milk (Transitional) [0112] A fraction was isolated from transitional milk as follows: [0113] The transitional milk as collected from Holstein Friesian cattle on-site at the Teagasc Food Research Centre, Moorepark (Fermoy, Cork, Ireland) and was defatted and deproteinized through conventional methods as described by Kobata & Ginsburg (Oligosaccharides of human milk. IV. Isolation and characterization of a new hexasaccharide, lacto-N-neohexaose. Arch Biochem Biophys 150, 273-281 (1972)). [0114] In brief, aliquots were centrifuged at 5000 rpm for 20 min at 4 C. in a Sorvall RC6 plus to separate the fat. [0115] The aqueous phase was collected and 1 M-HCl was added until a pH of 4.6 was reached. [0116] The sample was then heated to 35 C. for 2hr and precipitated caseins were removed by centrifugation at 5000 rpm for 30 min at 25 C. [0117] The pH of the supernatant was then neutralised using 4M NaOH. [0118] The de-fatted, de-caseinated solution was collected, freeze-dried and stored at 80 C.

[0119] Bacterial Strains Conditions

[0120] Bifidobacterium longum subsp. infantis ATCC 15697 and Bifidobacterium adolescentis ATCC 15703 was purchased from DSMZ (Germany). Bifidobacterium longum NCIMB 8809, Bifidobacterium breve UCC2003, Bifidobacterium breve NCFB 2258, and Lactobacillus rhamnosus GG were obtained from Teagasc Food Research Centre culture collection. The strains were stored in deMan Rogosa Sharpe (MRS) [Difco, Sparks, Md., USA] broth containing 50% glycerol at 80 C. until they were re-cultured according to the supplier instructions. The strains were cultured twice in MRS media supplemented with L-cysteine (0.05%w/v) prior to use with the exception of Lactobacillus rhamnosus GG which was cultured twice in un-supplemented MRS. The strains were cultured overnight at 37 C. under anaerobic conditions generated using a Anaerocult A system (Merck, Dannstadt, Germany). The strains were grown overnight and used at an optical density (OD600) of 0.45-0.5. To prepare mid-exponential phase cells, overnight cultures were adjusted to an optical density of 0.3 and the cultures were then grown for a further 2 hours and then adjusted to an optical density (OD600 nm) of 0.45-0.5 (corresponding to 5107 CFU/mL).

[0121] Cell culture and exposure of HT-29 cells to milk components

[0122] The human colon adenocarcinoma cell line HT29 was purchased from the American Type Culture Collection (ATCC). HT29 cells were routinely grown in McCoy's 5A modified medium (Sigma). All cells were routinely maintained in 75 cm.sup.2 tissue culture flasks and incubated at 37 C. in a humidified atmosphere (5% CO2). Cells were passaged when the confluency of the flasks was approximately 80%. HT29 cells were seeded into 12 well plates (Corning) at a concentration of 110.sup.5 cells/well and incubated at 37 C. in 5% CO.sub.2 in a humidified atmosphere. The cells were fed every second day with McCoy's media (10% FBS) until 100% confluency was achieved (typically 3-4 days). Prior to sample exposure, the cells were washed and placed in 2% FBS McCoy's media. After 24 hours, BWCF, purified IgG, -lac and -Ig, were re-suspended in pre-heated McCoy's 5A (0% FBS) each at given concentrations as indicated in the figures (see FIGS. 1-8). The samples were then filter sterilized with a 0.2 pm (individually packed sterile) filter. Non-supplemented McCoy's 5A medium was used as a non-treated control (NT). The cells were then washed with phosphate buffered saline and the samples were applied. The plates were then incubated at 37 C. incubator for 24 hours in a in a humidified atmosphere (5% CO.sub.2) prior to bacterial exposure.

[0123] Adhesion Assays

[0124] On the day of the assay, as mentioned above, the primarily anaerobic strains were used at an optical density (OD600) of 0.45-0.5. The bacteria were then centrifuged at 5000 g for 10 minutes, after which the supernatant was removed and the pellet was washed with 0% FBS McCoy's media and centrifuged again. This wash step was repeated twice after which the bacteria was re-suspended in 0% FBS McCoy's media. Bacterial exposure to eukaryotic cells was for 2 hours at 37 C. under anaerobic conditions using an Anaerocult A system (Merck, Dannstadt, Germany). The cells were then washed four times with PBS to remove non-adherent bacteria. The cells were then lysed with 500 l per well of 0.1% Triton X100; (Sigma, Steinheim, Germany) for 10 minutes at 37 C. The lysates were serially diluted and enumerated by spread-plating on MRS plates. The bacteria cells are then spread plated onto De Man, Rogosa and Sharpe (MRS) agar plates. The plates were incubated anaerobically at 37 C. in a 5% CO.sub.2 incubator for 48 hrs. CFU were then counted. Adhesion was determined as the fold change difference between the control treated cells adherent bacteria and the treated cells adherent bacteria. Fold adhesion=[CFU/mL of recovered adherent bacteria of treated cells CFU/mL of recovered adherent bacteria of non-treated cells]. Adhesion assays were performed in triplicate.

[0125] Periodate Treatment of IgG IgG was treated with sodium metaperiodate (Na104) to produce IgG-P (IgG-periodate). IgG (24 mg/mL) was incubated with 10 mM Na104 at room temperature for 30 min. Excess Na104 was removed by centrifugal filtration using 3 kDa MWCO with three phosphate buffered saline, pH 7.4 (PBS) washes and the retentate containing GMP-P was re-suspended in 0% McCoy's media. This was then exposed to HT-29 cells for 24 hours as per adhesion assay protocol.

[0126] Statistical Analysis

[0127] Data are expressed as meanstandard deviation (SD) of the results of three independent assays conducted in triplicate. Graphs were generated using Microsoft Excel. Where student t-tests were used p0.05 was considered significant.

[0128] Experimental

[0129] The inventors investigated whether a bovine whey colostrum (or a bovine whey fraction from transitional or mature milk) containing IgG, -lactalbumin and -lactoglobulin could alter the cell surface of HT-29 cells and make it then more favourable for colonisation by commensal strains. A de-fatted, de-caseinated bovine colostrum fraction (Day 1 colostrum) was originally generated and exposed to HT-29 cells for 24 hours. After 24 hours, the sample was washed off and different Bifidobacterial strains were introduced to the HT-29 cells. Increases in the adherence of all 7 commensal strains tested were observed ranging from 0.6-6.1-fold (FIG. 1-8).

[0130] The increase in attachment of the commensal strains to the epithelial cell surface is hypothesised to be related to a change in the glycosylation pattern of the epithelial cell surface induced by BWCF exposure. It was also investigated what bioactive component was being retained by the whey colostrum and found that IgG acting independently and/or synergistically with both -lactalbumin and -lactoglobulin (in the case of one strain B. longum 8809) was the most likely candidate with the activity being concentration and strain dependent (FIG. 1-8).

[0131] The data presented here shows an increase in commensal strain adherence. When the colostrum fraction is added to the HT29 cells, the fraction appears to be able to induce or cause an alteration in the HT29 cell environment allowing for better commensal strain adherence. IgG with or without -lactalbumin and -lactoglobulin alters the cell surface of HT-29 cells to create a more favourable phenotype for commensal colonisation. Exposure of IgG isolated from day 1 colostrum, induced a change to the cell surface of intestinal cells. This change on the cell surface leads to a dramatic increase in the ability of infant commensal bacteria to attach to the cell, which may result in multiple health benefits to the infant.

[0132] Fermentation of inulin-type fructans selectively stimulate growth of Bifidobacteria, and thus increase the number of potentially health-promoting bacteria and reduce the number of potentially harmful species. Both oligofructose and inulin are prebiotic. Lactoferrin has shown to retain bifidogenic activity of certain strains of Bifidobacterium longum subsp. infantis, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium breve. Compounds which are known to elicit bifidogenic activity such as oligosaccharides, lactoferrin, an oligofructose-enriched inulin etc. do not induce the observed effect of enhanced adhesion (see FIGS. 9 and 10), hence demonstrating this activity occurs through a non-prebiotic mechanism. BWCF with minimal processing has shown to alter the HT-29 cell surface to increase counts of beneficial bacteria. Thus, the ability to alter the intestinal surface environment is unique to the colostrum fraction.

[0133] A sample of IgG isolated from the bovine whey colostrum fraction was treated with sodium metaperiodate (a reagent used to open saccharide rings, basically cleave all glycans away). The treated IgG was exposed to HT-29 cells and was found that the treated IgG did not induce the same effect on Bifidobacterium (i.e. no increased adhesion) when compared to non-treated IgG (see FIG. 11). This suggests that the glycans of IgG are responsible for the observed effect of increased adhesion.

[0134] It was also shown that there was a significant 3.9-fold increase of Bifidobacterium longum NCIMB 8809 adhering to HT-29 cells after prior exposure to purified IgG isolated from mature milk. The bioactivity of IgG is preserved in mature milk albeit at lower levels. Hence, the observed bioactivity of IgG is preserved across lactation. Both colostral and mature IgG may be used commercially to achieve the observed effect.

[0135] Bovine whey colostrum (and whey derived from transitional or mature milk) and its derived components IgG, -lactalbumin and -lactoglobulin have not been shown previously to have the ability to alter the intestinal surface, which subsequently leads to increased commensal colonisation. IgG has been mostly associated with vaccination and combatting pathogenic infection. This invention offers a different application for whey colostrum (and whey generated from a transitional or mature milk stream) and its components IgG, -lactalbumin and -lactoglobulin in increasing commensal colonisation in the infant and adult gastrointestinal tract.

[0136] Bovine milk is used as the main understudy for human milk and most infant formulae are based on bovine milk. IgG is found in large amounts in bovine colostrum (46.40 g/L) and it is the dominant immunoglobulin in bovine colostrum and milk. The whey colostrum has also been tested with two adult strains (Bifidobacterium adolescentis ATCC 15703 and Lactobacillus rhamnosus GG) also giving rise to increased adhesion in vitro, suggesting applications in foods other than infant formula, possibly as a supplement for adults suffering from gut dysbiosis.

[0137] In conclusion, IgG (preferably glycosylated) with or without -lactalbumin and -lactoglobulin, and free glycans, alters the cell surface of HT-29 cells to create a more favourable phenotype for commensal colonisation. Exposure of intestinal cells to IgG isolated from day 1 colostrum, or from transitional milk (between 3 and day 14) or from day 14-onwards mature milk, induced a change to the cell surface of intestinal cells.

[0138] This change on the cell surface leads to a dramatic increase in the ability of infant commensal bacteria to attach to the cell, which may result in multiple health benefits to the infant. This should increase commensal colonization in infants (first stage formula application) resulting in a microbiota platform closer to that of breast-fed infants. Also, gut dysbiosis can cause many chronic inflammatory diseases, such as inflammatory bowel disease, obesity, cancer, and autism. It could be associated with ailments which are associated with lower counts of commensal bacteria such as asthma, allergic disease and bowel problems.

[0139] In the specification, the terms comprise, comprises, comprised and comprising or any variation thereof and the terms include, includes, included and including or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

[0140] The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.