LACTIC ACID BACTERIAL STRAINS

Abstract

A first aspect of the invention relates to a porcine lactic acid bacterial strain, wherein said bacterial strain is characterised by one or more of the following characteristics: (i) the ability to exhibit antimicrobial activity against E. coli; (ii) the ability to exhibit antimicrobial activity against S. enteritidis; (iii) the ability to suppress inflammation in IPEC cells induced by 12-O-tetradecaboylphorbol-13-acetate (PMA); (iv) the ability to block the attachment or invasion of IPEC cells by S. enteritidis; (v) the ability to block the attachment or invasion of IPEC cells by E. coli; (vi) the absence of antibiotic resistance to one or more antibiotics selected from the following: ampicillin; cefotaxime; chloramphenicol; erythromycin; gentamicin; tetracycline; vancomycin; metronizadole; nalidixic acid; and kanamycin; and (vii) the ability to exhibit heat stability when subjected to three cycles of heating, each cycle comprising heating at a temperature of 70° C. for a period of 15 minutes. Further aspects of the invention relate to compositions comprising said bacterial strains, and therapeutic uses of said bacterial strains.

Claims

1.-31. (canceled)

32. A pharmaceutical composition comprising at least 10.sup.3 colony forming units (CFU) of at least one Lactobacillus bacteria strain, and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the at least one Lactobacillus bacteria strain comprises a 16S rRNA gene sequence with at least 95% sequence identity to a 16S rRNA gene sequence selected from the group consisting of SEQ ID NOs 10, 11, 13, and 14, as determined by a sequence alignment performed using BLAST, and wherein the pharmaceutical composition is a solid formulation.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is lyophilized.

34. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is encapsulated in one or more capsules.

35. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition further comprises a preservative or stabilizer.

36. The pharmaceutical composition of claim 35, wherein the preservative comprises sodium benzoate, sorbic acid, or esters of p-hydroxybenzoic acid.

37. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition comprises from about 10.sup.3 to about 10.sup.11 colony forming units (CFU) of the at least one Lactobacillus bacteria strain per gram of the pharmaceutical composition.

38. The pharmaceutical composition of claim 32, wherein the at least one Lactobacillus bacteria strain exhibits at least one characteristic selected from the group consisting of: (i) antimicrobial activity against E. coli; (ii) antimicrobial activity against S. enteritidis; (iii) suppression of inflammation in intestinal pig epithelial cells (IPEC) induced by 12-O-tetradecaboylphorbol-13-acetate (PMA); (iv) an ability to block the adherence or invasion of the IPEC by S. enteritidis; (v) an ability to block the adherence or invasion of the IPEC by E. coli; and (vi) absence of antibiotic resistance to an antibiotic selected from the group consisting of ampicillin, cefotaxime, chloramphenicol, erythromycin, gentamicin, tetracycline, vancomycin, metronizadole, nalidixic acid, and kanamycin.

39. The pharmaceutical composition of claim 38, wherein the at least one Lactobacillus bacteria strain exhibits at least two characteristics selected from the group of claim 38.

40. The pharmaceutical composition of claim 38, wherein the at least one Lactobacillus bacteria strain exhibits at least three characteristics selected from the group of claim 38.

41. The pharmaceutical composition of claim 32, wherein the at least one Lactobacillus bacteria strain is of a species selected from the group consisting of Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus gasseri, Lactobacillus pentosus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus mucosae, and any combinations thereof.

42. A method of treating an intestinal disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising at least at least 10.sup.3 colony forming units (CFU) of at least one Lactobacillus bacteria strain, and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the at least one Lactobacillus bacteria strain comprises a 16S rRNA gene sequence with at least 93% sequence identity to a 16S rRNA gene sequence selected from the group consisting of SEQ ID NOs 10, 11, 13, and 14, as determined by sequence alignment performed using BLAST, and wherein the pharmaceutical composition is a solid formulation in unit dose form.

43. The method of claim 42, wherein the intestinal disorder is selected from the group consisting of salmonellosis, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), functional dyspepsia, functional constipation, functional diarrhea, functional abdominal pain, functional bloating, Epigastric Pain Syndrome, Postprandial Distress Syndrome, Crohn's disease, ulcerative colitis, gastroesophageal reflux disease (GERD), necrotizing enterocolitis, and any combination thereof.

44. The method of claim 42, wherein the administering is oral.

45. The method of claim 42, wherein the subject is a human.

46. The method of claim 42, wherein the at least one Lactobacillus bacteria strain is of a species selected from the group consisting of Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus gasseri, Lactobacillus pentosus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus mucosae, and any combinations thereof.

47. A method of improving intestinal microbiota in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising at least at least 10.sup.3 colony forming units (CFU) of at least one Lactobacillus bacteria strain, and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the at least one Lactobacillus bacteria strain comprises a 16S rRNA gene sequence with at least 95% sequence identity to a 16S rRNA gene sequence selected from the group consisting of SEQ ID NOs 10, 11, 13, and 14, as determined by sequence alignment performed using BLAST, and wherein the pharmaceutical composition is a solid formulation.

48. A method of improving growth in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising at least at least 10.sup.3 colony forming units (CFU) of at least one Lactobacillus bacteria strain, and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the at least one Lactobacillus bacteria strain comprises a 16S rRNA gene sequence with at least 95% sequence identity to a 16S rRNA gene sequence selected from the group consisting of SEQ ID NO 41, as determined by sequence alignment performed using BLAST, and wherein the pharmaceutical composition is a solid formulation.

49. The method of claim 48, wherein the subject comprises an early-weaned animal.

50. The method of claim 48, wherein the subject comprises a sheep, a goat, a cow, a pig, a horse, a cat, a dog, a turkey, a duck, a chicken, a fish, or a crustacean.

51. The method of claim 48, wherein the improving growth comprises gaining weight.

Description

[0234] The present invention is further described by way of non-limiting example, and with reference to the following non-limiting figures, wherein:

[0235] FIG. 1 shows an assay of antibacterial activity of conditioned media from Lactic Acid Bacteria.

[0236] FIGS. 2A and 2B show inhibitory activity against S. enteritidis S1400 (expressed as area of inhibition in a well diffusion assay) of conditioned media of all individual LAB cultured from faeces of organically-reared pigs.

[0237] FIGS. 3A and 3B show inhibitory activity against E. coli K88 (expressed as area of inhibition in a well diffusion assay) of conditioned media of all individual LAB cultured from faeces of organically-reared pigs.

[0238] FIGS. 3C and 3D show inhibitory activity (expressed as area of inhibition in a well diffusion assay) of conditioned media of all individual LAB cultured from faeces of organically-reared pigs.

[0239] FIGS. 4A, 4B, and 4C shown inhibition of adherence by (FIG. 4A) S. enteritidis S1400; and (FIG. 4B) E. coli K88 to IPEC cells in culture by LAB cultured from faeces of organically-reared pigs; (FIG. 4C) comparison between inhibition of S. enteritidis S1400 and E. coli K88.

[0240] FIG. 5 shows an assay of the antibiotic susceptibility of Lactic Acid Bacteria using discs impregnated with a defined amount of antibiotic.

[0241] FIG. 6 shows an evaluation of substrate profile of LAB using an API CH 50 kit [49 substrates, pale colour indicated positive reaction, except 25 where positive reaction is black, dark colour indicates no reaction].

[0242] FIGS. 7A-7C show the ΔCt (FIG. 7A), ratio (FIG. 7B) and fold-change (FIG. 7C) for IL-8 gene expression in IPEC cells treated with PMA and pig LAB.

[0243] FIGS. 8A and 8B show the stability of L. reuteri (FIG. 8A) and L. johnsonii (FIG. 8B) after freeze-drying in skimmed milk powder (SKP, (100 g/1), SKP+lactose (both 100 g/1), SKP+sucrose (both 100 g/1) or SKP (200 g/1).

[0244] FIGS. 9A-9D show the stability of isolated LAB to heat-treatment (FIG. 9A), the ratio (FIG. 9B) and fold-change (FIG. 9C) for IL-8 gene expression in IPEC cells treated with PMA and naive or heat-treated pig LAB; (FIG. 9D) Antibiotic susceptibility of native and heat-treated RINH vial 31.

[0245] FIG. 10 shows a protocol for the C3H/HeN mouse study to evaluate efficacy of vial 323 (L. mucosae) to counteract salmonella infection in vivo.

[0246] FIGS. 11A-11C show the distribution of S. enteritidis S1400 in tissues at 10 days post-infection in C3H/HeN mice that had or had not been co-treated with 323 (L. mucosae, LM).

[0247] FIGS. 12A-12B show spleen weight (mg/100 g BW) and intestinal (ileal) myeloperoxidase (μg) at 10 days post-infection in C3H/HeN mice that had or had not been co-treated with vial 323 (L. mucosae).

[0248] FIG. 13 shows a protocol for the C57 BI/6 mouse study to evaluate efficacy of vial 323 (L. mucosae) to counteract acute salmonella infection in vivo.

[0249] FIGS. 14A-14C shows the distribution of S. enteritidis S1400 in tissues at 6 days post-infection in C57BI/6 mice that had or had not been co-treated with RINH vial 323.

[0250] FIG. 15 shows spleen weight (mg/100 g BW) at 6 days post-infection in C57BI/6 mice that had or had not been co-treated with vial 323 (L. mucosae).

[0251] FIG. 16 shows a protocol for the C3H/HeN mouse study to evaluate efficacy of selected LAB from faeces of organically reared pigs to counteract salmonella infection in vivo.

[0252] FIGS. 17A & 17B show excretion of S. enteritidis in faeces at 7-8 days post-infection by C3H/HeN mice that had or had not been co-treated with selected LAB.

[0253] FIGS. 18A-18B show the distribution of S. enteritidis (Log 10 CFU/g) in caecum (18A[[b]]) and colon (18B) at 10 days post-infection of C3H/HeN mice that had or had not been co-treated with selected LAB.

[0254] FIGS. 19A-19C show the distribution of S. enteritidis (Log 10 CFU/g) in mesenteric lymph node (19A), liver (19B) and spleen (19C) at 10 days post-infection of C3H/HeN mice that had or had not been co-treated with selected LAB.

[0255] FIG. 20 shows the performance of pigs fed GGDK266 and GGDK31 versus a control (daily weight gain, DWG, in g/day) for days 0-7, 7-14 and 0-14.

[0256] FIG. 21 shows microbial diversity analysis using denaturing gel gradient electrophoresis (DGGE; Trial 1). DGGE using universal primers revealed no differences in overall microbial diversity between the treatments and placebo. Bands on the gel are visualised by silver staining.

[0257] FIG. 22 shows microbial diversity analysis using DGGE. DGGE using lactic acid bacteria (LAB) specific primers revealed significant differences in LAB diversity between treatment with GGDK266 and placebo in both caecal and ileal samples. Bands on the gel are visualised by silver staining.

[0258] FIG. 23 shows microbial diversity analysis using DGGE. DGGE using lactic acid bacteria (LAB) specific primers revealed significant differences in LAB diversity between treatment with GGDK266 and placebo in ileal samples. Bands on the gel are visualised by silver staining.

[0259] FIG. 24 shows microbial diversity analysis using DGGE. DGGE using lactic acid bacteria (LAB) specific primers revealed significant differences in LAB diversity between treatment with GGDK266 and placebo in caecal samples. Bands on the gel are visualised by silver staining.

[0260] FIG. 25 shows the gene ontology biological processes significantly down-regulated by oral administration of GGDK266.

[0261] FIG. 26 shows changes in immune response and response to stimuli in animals treated with GGDK266 versus animals treated with placebo (percent of genes versus a range of different GO annotations).

[0262] FIG. 27 shows the gene ontology biological processes significantly enriched by oral administration of GGDK266.

EXAMPLES

Materials and Methods

[0263] Materials: Pig faeces samples collected during the course of the study of outdoor- and indoor-reared pigs (Mulder et al, 2009) were used in these studies. The culture collection was based primarily on LAB collected from frozen samples 411, 412 and 416, which were from outdoor-reared pigs with particularly high levels of LAB in their faeces. MRS broth premix, agar and vancomycin, anaerobe gas packs and indicator and antibiotic discs were purchased from Oxoid, anaerobe catalyst from Fisher Scientific and cysteine-HCL, bromocresol green and skimmed milk powder from Sigma-Aldrich. Pig colostrum carbohydrate fractions were prepared as part of the SMART 163 programme of D. Kelly. DNA extraction kits were purchased from MP Biomedicals and PCR reagents and clean-up kits from Promega. API CH 50 kits were purchased from Biomerieux UK Ltd.

[0264] Standard media: MRS broth and MRS agar were prepared according to the manufacturer's instructions. LAMVAB agar was prepared according to the method of Jackson et al. (2002). Agar plates were prepared immediately before use. MRS broth was decanted (10 ml per tube) into sterile Hungate tubes under anaerobic conditions and stored at room temperature.

[0265] Carbohydrate-supplemented media: SMART 163 ammonium sulphate precipitate of pig colostrum: precipitated at 0, 20, 25, 30, 35, 45, 50, 55 or 65% saturation or soluble at 65% saturation were weighed out in proportion to the amounts recovered from 15 ml or 50 ml of colostrum. Carbohydrate fractions were each dispersed in 15 ml of MRS or LAMVAB agar, held at 45° C., and then individual plates were poured for each fraction. They were also dispersed in MRS broth (50 ml) and the supplemented broth decanted to eight (6 ml/tube) sterile Hungate tubes under anaerobic conditions.

[0266] Animals: Female C3H/HeN and C57BI/6 mice (5-6 weeks old) were purchased from Harlan UK. They were housed as groups or pairs in standard caging within HEPA-filtered flexifilm isolators situated in a class 2 containment facility. They had free access to a high quality rodent chow and sterile deionised water at all times and were allowed to acclimatise for 7 to 10 days prior to commencement of experiments. The Rowett Institute of Nutrition and Health (RINH) is licensed under the UK Animals (Scientific Procedures) Act 1986. Studies herein were carried out under the auspices of an approved Home Office Project Licence by staff holding the requisite Home Office Personal Licence (as defined and set out in the UK Animals (Scientific Procedures) Act 1986), and were reviewed and approved by the RINH Ethical Review Committee.

Methods

[0267] Culture of LAB: In initial studies, a small amount of frozen faeces (100 mg) was dispersed in 1 ml of maximum recovery diluent (MRD). Two further sequential ten-fold dilutions were made. All three suspensions were streaked out on MRS or LAMVAB agar plates. In later studies, the faeces sample was dispersed in 5 ml of MRD, further diluted (1:40) in MRD and 0.5 ml of this dilution spread over the surface of MRS or LAMVAB agar plates with or without supplemental pig colostrum carbohydrates. In all cases, the plates were incubated in an anaerobic jar for 72 hours at 37° C. Distinct colonies (at least 8 per plate) were picked off the agar plates and seeded into Hungate tubes containing MRS broth or where appropriate MRS broth containing pig colostral carbohydrates. The tubes were incubated for 48 hours at 37° C.

[0268] Frozen stock: An aliquot (0.7 ml) of each culture was drawn off with a sterile syringe and needle and dispensed into a plastic tube that was flushed with CO.sub.2 and contained 0.3 ml glycerol and 2 mg L-cysteine. The tube was sealed with a plastic stopper, labelled, the contents mixed, frozen and stored at −80° C.

[0269] Conditioned medium: The remaining culture was transferred to a Corning 15 ml centrifuge tube, centrifuged at 1000 g×5 min at room temperature, the supernatant decanted, aliquoted and frozen. The pellets were either extracted immediately for 16S rRNA gene analysis or frozen.

[0270] 16S rRNA gene analysis (Clarridge, 2004): Bacterial DNA was extracted using a FastDNA® Spin kit for Soil in conjunction with a Fastprep 120 bead beater system, according to the protocol supplied with the kit. PCR was carried out (reaction mix: buffer, 104 dNTPs (2 mM), 5 μl. 27F Primer (20 pmol/2 μl. 1492R Primer (20 pmol/μl)). 2 μl Go Taq Flexi Polymerase, 0.5 μl). MgCl2, 5 μl. H2O, 23.5 μl and 2 μl of extracted DNA) using MJ Research PTC-200 Peltier Thermal Cycler run through 35 cycles of 95° C. for 3 minutes, 95° C. for 30 seconds, 57° C. for 30 seconds and 72° C. for two minutes. Primer: 27F (F01) AGAGTTTGATCCTGGCTCAG; 1492R (RP2) ACGGCTACCTTGTTACGACTT. PCR product cleanup was done with a Wizard® SV Gel and PCR Clean-up kit (Promega), used according to the manufacturer's instructions. 16S PCR products were sequenced using fully automated genetic analysers based on capillary electrophoresis technology (Genomics Section, RINH, UoA) using the reverse and forward primers 519R and 926F. Bacterial strains were identified by comparison of sequences with known bacterial DNA sequences using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

[0271] Antibacterial activity: XLD agar was prepared as per manufacturer's instructions and cooled to 45° C. Salmonella enteritidis S1400 was added to the XLD agar [1 ml of a 1:1000 dilution of an overnight culture of salmonella in 200 ml XLD agar to give the equivalent of 106 CFU/ml]. The agar was poured into petri dishes and allowed to set. The plates were marked off into 4 quadrants and an approximately 5 mm well cut out in each quadrant. An aliquot (60 μl) of conditioned media or MRS broth was added to the wells. The plates were covered and incubated for 16 hours at 37° C. They were photographed using a digital camera. Images transferred to Photoshop, and the diameter of the well and zone of inhibition were determined using the measure tool. Values were calculated and stored on an Excel spreadsheet. The same procedure was used with Escherichia coli K88, except that MacConkey No 3 agar was used.

[0272] Antibiotic susceptibility: Pig LAB [0.5 ml of a 1:100 dilution of an overnight culture] was spread onto the surface of an MRS agar [90 mm] plate and dried off. The plates were marked off into 4 quadrants and in each quadrant was placed an antibiotic-containing disc [Ampicillin, 10 μg. Cefotaxime, 30 μg. Chloramphenicol, 10 μg. Erythromycin, 15 Gentamicin, 10 μg Kanamycin, 30 μg. Metronizadole, 50 μg. Nalidixic acid, 30 μg. Tetracycline, 30 μg. Vancomycin, 30 μg]. The plates were covered, placed in an anaerobic jar and incubated for 24 hours at 37° C. They were photographed using a digital camera. Images transferred to Photoshop, and the diameter of the zone of inhibition was determined using the measure tool. Values were calculated and stored on an Excel spreadsheet.

[0273] Prevention of adherence/invasion by salmonella in vitro: Monolayers of IPEC-J2 cells were grown to 3 days post-confluence in 24-well plates and synchronised by the addition of DTS media 24 hrs prior to use. Overnight cultures of pig LAB (10 ml) were centrifuged [1000 g×5 min at room temperature] and the bacteria re-suspended in 1 ml of phosphate buffered saline [PBS]. An aliquot (50 μl) of LAB was added to the wells. The plates were incubated for 2 hours at 37° C., 5% CO.sub.2, 95% humidity. An overnight culture of Salmonella enterica serovar Enteritidis S1400 [S. enteritidis S1400] was sub-cultured (0.5 ml in 10 ml) into Luria Bertani (LB) media and incubated aerobically for 2-3 hours at 37° C. until it reached an optical density (560 nm) of 0.8. This gave a concentration equivalent to 1×108 CFU/ml. The culture was centrifuged [1000 g×5 min at room temperature], the bacteria re-suspended in 10 ml of PBS. An aliquot (50 μl) was added to the wells of IPEC-J2 cells. Wells treated with PBS were used as controls. The plates were incubated for a further 2 hours at 37° C., 5% CO.sub.2, 95% humidity. The IPEC-J2 cell monolayers were washed 5 times with HBSS. A solution (0.5 ml) of PBS containing Triton-X100 (10 ml/litre) was added to each well, the monolayer scraped off and dispersed. Viable salmonella were estimated on XLD agar plates [incubated for 24 hours at 37° C.] by the Miles and Misra method [Robertson et al, 2003]. LAB were determined by the same procedure [incubated anaerobically for 48 hours at 37° C.].

[0274] Inhibition of inflammatory responses: Monolayers of IPEC-J2 cells were grown to 3 days post-confluence in 24-well plates and synchronised by the addition of DTS media 24 hrs prior to use. Overnight cultures of pig LAB (10 ml) were centrifuged [1000 g×5 min at room temperature] and the bacteria re-suspended in 1 ml of PBS. An aliquot (50 μl) of LAB was added to each well [3 wells for each sample] along with 220 ng 12-O-Tetradecaboylphorbol-13-acetate [PMA] per well. PMA or PBS alone served as controls. The plates were incubated for 2 hours at 37° C., 5% CO.sub.2, 95% humidity. Culture media was removed from the dishes and the cells washed twice with PBS. RLT buffer (0.5 ml) containing mercaptoethanol was added to each well, the cells scraped off and transferred to an eppendorf tube [for each sample scrapings from 3 wells were combined]. RNA extraction was done using RNeasy® Mini kit in accordance with the manufacturer's protocols and reverse transcription with a high capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real Time PCR was done on a 7500 Fast Real-time PCR system operating with 7500 Fast System v 1.4.0 Sequence Detection Software version 1.4 (Applied Biosystem). Primers for porcine IL-8 and TNF-α [IPEC-J2, SY100604186-096 IL-8-2 Reverse, SY100604186-090 TNF1 a Reverse, SY100604186-095 IL-8 2 Forward, SY100604186-089 TN Fa 1 Forward, and SY100604186-093] were prepared by Sigma Aldrich. The reaction mix was: 10 μl Power Sybergreen Master mix, 2.5 μl of forward primer, 2.5 μl of reverse primer and 5 μl of cDNA, The Real Time PCR was then run according to the Standard 7500 protocol [95° C., 10 min, 1 cycle. 95° C., 15 sec, 40 cycles. 60° C., 1 min, 40 cycles. 95° C., 15 sec, 1 cycle. 60° C., 1 min, 1 cycle. 95° C., 15 sec, 1 cycle. 60° C., 15 sec, 1 cycle]. Expression of IL-8 and TNF-α genes were analysed and compared to that of the ‘house-keeping’ gene β-actin. For comparison, values were given as the ratio of IL-8 and TNF-α per β-actin or fold-change.

[0275] For example:

[0276] a. Calculate ΔCt (2h) for IL-8 [Ct IL-8 minus Ct β-actin]

[0277] b. Calculate ΔCt (2h) for PMA [Ct PMA minus Ct β-actin]

[0278] c. Divide ΔCt (IL-8) with ΔCt (PMA)

[0279] d. Round up value to whole number

[0280] Substrate reactivity: The carbohydrate reactivity of individual LAB was determined using an API CH 50 kit (Biomerieux UK Ltd). Assays were done according to the manufacturer's instructions and reactions were recorded after incubation for 24 and 48 hours at 37° C. There are 50 capules on an API CH 50 plate. These contain various potential substrates and negative controls. The range of substrates is as follows: Monosaccharides 16, Monosaccharides/alcohols 4, Disaccharides 8, Trisaccharides 2, Polysaccharides 3, Alcohols 6, Others 7. For each substrate group the number of positive reactions is counted. This is divided by the maximum possible to give the rank for that substrate group. The sum of all the substrate scores gives the overall ranking for the bacterium. High Ranking indicates broad spectrum of substrate reactivity

[0281] Heat-treatment of LAB: A small amount of frozen faeces (100 mg) was dispersed in 5 ml of maximum recovery diluent (MRD). Sediment was allowed to settle out and the upper layer was decanted into eppendorf tubes (1 ml/tube). The tubes were heated at 50° C., 60° C. or 70° C. for 10 min. An aliquot (0.4 ml) of each was plated out on MRS agar and incubated in an anaerobic jar for 72 hours at 37° C. A small number of colonies were detected after heating at 70° C. Distinct colonies were picked off, seeded into Hungate tubes containing MRS broth and incubated for 48 hours at 37° C.

[0282] In a second study, a small amount of frozen faeces (100 mg) was dispersed in 5 ml of maximum recovery diluent (MRD). Sediment was allowed to settle out and the upper layer was decanted into eppendorf tubes (1 ml/tube). The tubes were heated at 50° C. for 20 min, 50° C. for 20 min plus 60° C. for 20 min or 50° C. for 20 min plus 60° C. for 20 min plus 70° C. for 20 min. An aliquot (0.5 ml) of each was plated out on MRS agar and incubated in an anaerobic jar for 48 hours at 37° C. A small number of colonies were detected, picked off, seeded into Hungate tubes containing MRS broth and incubated for 48 hours at 37° C.

[0283] In the third study, an overnight culture (10 ml) of isolated pig LAB was centrifuged (1000 g×5 min at room temperature), the pellet re-suspended in fresh MRS broth (10 ml). An aliquot (1 ml) was heated at 70° C. for 15 min and then plated out (0.5 ml) out on MRS agar and incubated in an anaerobic jar for 48 hours at 37° C. A small number of colonies were detected, picked off, seeded into Hungate tubes containing MRS broth and incubated for 48 hours at 37° C. This culture was centrifuged, re-suspended in MRS broth, heated again at 70° C. for 15 min, plated out on MRS agar, incubated in an anaerobic jar for 48 hours at 37° C., picked off, seeded into Hungate tubes containing MRS broth and incubated for 48 hours at 37° C. As before, this culture was centrifuged, re-suspended in MRS broth, re-heated at 70° C. for 15 min, plated out (0.5 ml) out on MRS agar, incubated in an anaerobic jar for 48 hours at 37° C., picked off, seeded into Hungate tubes containing MRS broth and incubated for 48 hours at 37° C.

[0284] Stability of freeze dried bacteria: Overnight cultures of LAB were centrifuged (1000 g×5 min at room temperature. Pellets were re-suspended in 2 ml sterile PBS and re-centrifuged. The subsequent pellets were then re-suspended in 5 ml of freezing solution [defatted skimmed milk powder (SKP), 100 g/1; SKP+lactose, both 100 g/1; SKP+sucrose, both 100 g/1; or SKP, 200 g/1]. The samples were frozen at −20° C. (2-3 hours) and then stored at −80° C. overnight. They were freeze-dried for 48 hours and dried material stored at room temperature. Viable bacteria in the samples were determined at 0 and approximately 40 and 80 days after completion of freeze drying. They were plated out on MRS agar and incubated anaerobically for 48 hours at 37° C.

[0285] Bulk preparation of GGDK31 and GGDK266: Two 500 ml batches of MRS broth were prepared in 500 ml glass screw-top bottles, autoclaved and allowed to cool to room temperature (in proximity to gas flame) whilst being flushed with CO.sub.2. Four ml of a 24 hour culture of GGDK31 or GGDK266 was added to each bottles of MRS and the lids lightly closed. The bottles were placed in an anaerobic jar and incubated at 37° C. for 24 hours. The culture was centrifuged [1000 g×5 min at room temperature] in 6 sterile 50 ml centrifuge tubes. The supernatant was discarded, tubes refilled with culture and re-centrifuged until all the bacteria had been recovered. Each of the 6 tubes contained almost equal amounts of bacteria. The bacteria in each tube were re-suspended in 40 ml of sterile PBS, re-centrifuged and the supernatant discarded. The bacteria in each tube was re-suspended in 20 ml of SKM (100 g/1), frozen at −20° C. (2-3 hours) and then overnight at −80° C., freeze-dried for 48-72 hours and stored at 4° C. To evaluate viable bacteria in the sample, one tube of freeze dried material was re-suspended in 20 ml of MRS broth, incubated at room temperature for 2 hours, diluted, plated out on MRS agar and incubated anaerobically for 48 hours at 37° C.

[0286] L. mucosae in vivo Study 1: Sixteen (6 week) old female C3H/HeN mice were dosed with an overnight culture of vial 323 (L. mucosae; 50 μl; >109 CFU) at day −7, −4, −2 and 0 and daily thereafter up to day +9. A further 16 mice (control) were given media. On day 0, eight mice (L. mucosae-treated) and eight control mice were given, by gavage, a single dose of Salmonella enteritidis S1400 (50 μl;≥108 CFU). In addition, eight mice (L. mucosae-treated) and eight control mice were given a single dose of culture medium. Body weight and health score were monitored twice daily post-salmonella infection. The mice were euthanased (isoflurane overdose and exsanguination) and dissected at 10 days post-salmonella infection. Stomach, representative portions of jejunum and ileum, caecum plus contents, colon plus contents, spleen and liver and one kidney and the mesenteric lymph node were collected under near aseptic conditions for microbiology. Representative portions of upper jejunum, mid jejunum, ileum, caecum and ascending and descending colon were placed in neutral buffered formalin or RNA-later and stored for future analysis.

[0287] L. mucosae in vivo Study 2: Five (6 week) old female C57BI/6 mice were dosed with an overnight culture of vial 323 (L. mucosae; 50 μl; >109 CFU) at day −7, −4, −2 and 0 and daily thereafter up to day+5. A further 5 mice were given media. On day 0, all ten mice were given, by gavage, a single dose of Salmonella enteritidis S1400 (50 μl; >107CFU). The mice were euthanased and dissected on day 6, according to the procedure for study 1.

[0288] Novel pig LAB in vivo: Four (6 week) old female C3H/HeN mice were dosed with an overnight culture of RINH vial 31 (L. reuteri; 50 μl; >109 CFU), four with RINH vial 32 (L. reuteri). Four with vial 323 (L. mucosae), four with RINH vial 46 (L. reuteri), four with RINH vial 47 (L. reuteri) and eight with MRS. This was done at day −6, −4, −2 and 0 and daily thereafter up to day+9. On day 0, all lactobacilli-treated mice and four control mice were given, by gavage, a single dose of Salmonella enteritidis S1400 (50 μl;≥108 CFU). In addition, the remaining four control mice were given a single dose of culture medium. The mice were euthanased and dissected on day 10, according to the procedure for study 1.

[0289] Microbiology: Tissues were homogenised [1:100 w/v] in MRD using a Janke-Kunkel Ultra-Turrax T25 tissue homogeniser at 20,000 rpm for 30 seconds, as were jejunal and ileal contents. Up to eight sequential dilutions (1: 10 v/v) of the primary homogenates were made, plated out onto XLD agar and MacConkey No. 3 agar and incubated overnight at 37° C. Viable counts were estimated as before [Robertson et al, 2003].

[0290] Statistical analysis: Where appropriate data were initially assessed by one-way analysis of variance (ANOVA) regarding treatment outcome. If ANOVA indicated that there were significant differences (p<0.05) amongst all groups, the data was then analysed by the Tukey-Kramer Multiple Comparisons Test or the Kruskal-Wallis Multiple Comparisons Test as appropriate. This was done using the Instat Statistical Package (GraphPad Software Inc., San Diego, USA).

[0291] Based on the outputs from the multiple comparison tests, means in tables or graphs were marked with superscript letters. Means that differed significantly from each other (p<0.05) were allocated distinct superscript letters. Means that did not differ significantly from each other were allocated common superscript letters.

Results

1. Isolation of LAB

[0292] Faeces from organically-reared piglets were plated out on selective agars and were incubated under anaerobic conditions. From all studies, a total of 436 individual colonies of Lactic Acid Bacteria [LAB] were picked off, seeded into MRS broth and incubated under anaerobic conditions. Each culture was given a unique RINH vial number and an aliquot was frozen down in MRS media containing 30% glycerol and L-cysteine (˜2 mg/ml) and stored at −80° C. 16S rRNA gene analysis was done and bacterial strains were identified by comparison of sequences with known bacterial DNA sequences (Table 1).

[0293] The majority of the cultured LAB colonies were L. johnsonii and L. johnsonii-related strains [L. johnsonii, L. johnsonii/gasseri, L. johnsonii/taiwanensis] (240/436) and L. reuteri or L. reuteri-related [L. reuteri, L. reuteri/pontis, L. reuteri/vaginalis, L. reuteri/acidophilus (169/436)]. There were 7 L. plantarum/pentosus colonies, 19 other species and 5 uncultured strains.

2. Anti-Salmonella Activity In Vitro

[0294] Conditioned media from isolated LAB were screened for anti-bacterial activity against Salmonella enteritidis S1400 using a well-diffusion assay (FIG. 1).

[0295] Conditioned media from individual colonies of LAB varied greatly in their activity against S. enteritidis (FIG. 2A). This was not strain dependent. The range of anti-salmonella activities amongst L. johnsonii was similar to that amongst L. reuteri. On an arbitrary basis, the cultures were separated into groupings on the basis of their capacity to inhibit salmonella in vitro (FIG. 2B [[2b]]). Group 1 had <20000 units of inhibition, Group 2 20000-40000 units of inhibition, Group 3 40000-60000 units of inhibition, Group 4 60000-80000 units of inhibition, Group 5 80000-100000 units of inhibition and Group 6>>100000 units of inhibition (FIG. 2B Group 1 comprised of 14 strains (3.4% of total), Group 2 of 95 strains (22.8%), Group 3 of 99 strains (23.7%), Group 4 of 99 strains (23.7%), Group 5 of 86 strains (20.6%) and Group 6 of 24 strains (5.8%). The latter group comprised of seventeen L. johnsonii and L. johnsonii-related, six L. reuteri or L. reuteri-related strains and one uncultured strain.

3. Anti-E. coli K88 Activity In Vitro

[0296] Conditioned media from LAB were also screened for anti-Escherichia coli K88 activity by the well diffusion assay. Activity against E. coli K88, as with salmonella, varied greatly between individual colonies of LAB (FIG. 3A). The range and variation in the activity was similar amongst the L. johnsonii and L. reuteri strains. In general, there was no direct correlation between the anti-salmonella and anti E. coli K88 activities for any individual LAB (FIG. 3C, 3D). However of the ten strains in E. coli K88 group 5 (FIG. 3B), seven had relatively high activities against both pathogens, two had high activity against E. coli K88 but moderate activity against salmonella and one was active primarily against E. coli K88.

4. Initial Selection of Candidate LAB

[0297] Thirty-three strains were identified for further testing in vitro (Table 2).

[0298] These comprised 18 L. johnsonii and L. johnsonii-related strains, 11 L. reuteri or L. reuteri-related and 4 L. plantarum and L. plantarum-related strains (Table 2a).

5. Attachment I invasion of Pig Intestinal Epithelial [IPEC-J2] Cells

[0299] The capacity of LAB to block adhesion/invasion of IPEC cells by S. enteritidis and E. coli K88 was evaluated (FIG. 4A, 4B,). The candidate LAB all greatly reduced attachment and invasion of IPEC cells by salmonella. Most of them were also very effective against E. coli K88. However, 3 of the strains had only limited effects on adhesion/invasion of IPEC cells by E. coli K88.

6. Susceptibility of LAB to Antibiotics.

[0300] The susceptibility of the candidate LAB to a range of antibiotics was evaluated (Table 4, FIG. 5). All but one strain (RINH vial 266) exhibited some degree of resistance to individual antibiotics. All were susceptible to ampicillin (10 μg), cefotaxime (30 μg) and chloramphenicol (10 μg). The majority were susceptible to erythromycin (15 gentamicin (10 μg), tetracycline (30 μg) and vancomycin (30 μg). Most strains were resistant to metronizadole (50 μg) and nalidixic acid (30 μg) and to a lesser extent kanamycin (30 μg). 23

7. Refined Selection of Candidate LAB

[0301] Twenty-three high ranking strains were identified for further testing in vitro.

8. Substrate Specificity of LAB

[0302] The candidate LAB were screened for substrate reactivity using an API CH 50 kit (Table 5, 6, FIG. 6). L. johnsonii, L. reuteri and L. plantarum each exhibited strain-specific general substrate reaction profiles. In addition, most strains of each genotype exhibited fine differences in their substrate reactivity, indicative that they were unique individual strains.

9. Suppression of Inflammation in Pig Intestinal Epithelial [IPEC-J2] Cells

[0303] The ability of candidate LAB to block or suppress inflammatory responses triggered in IPEC cells by 12-O-Tetradecaboylphorbol-13-acetate [PMA] was tested (FIGS. 7A-7C; Table 7). The candidate strains varied greatly in their capacity to block interleukin-8 (IL-8) gene-expression triggered by PMA. Five strains (RINH vial 29, 30, 31 86 and 266) had potent anti-inflammatory effects.

10. Final Selection of Candidate LAB

[0304] Fourteen strains were identified having killing and blocking activities against salmonella and E. coli K88, susceptibility to antibiotics carbohydrate reactivity and capacity to suppress inflammation in vitro. Seven of these were particularly preferred. The latter set comprised 4 L. plantarum-related, 3 L. johnsonii-related and one L. reuteri. Two of these LAB strains [GGDK266 and GGDK31] were prepared in bulk for evaluation in a trial with newly-weaned piglets (Table 8).

11. Freeze Drying and Storage of LAB

[0305] The survival and viability of LAB after freeze drying in skimmed milk powder [SKP], SKP plus lactose or SKP plus sucrose was evaluated (FIGS. 8A and 8B). FIG. 8A depicts the stability of L. reuteri and FIG. 8B depicts the stability of L. johnsonii. Small losses in viability were evident on storage for 42 and 84 days at room temperature of samples dried in SKP. This was less marked when skimmed milk powder and sugars were used in combination. However, the 24 latter preparations tended to be hygroscopic and difficult to maintain. Bulk preparations of GGDK266 and GGDK31 were therefore prepared by drying the bacteria in skimmed milk powder [100 g/1] (Table 8).

12. Heat-Treatment Studies

[0306] Suspensions of faeces from organically reared pigs were heat treated for varying periods of time at 50-70° C., plated out on MRS agar, colonies picked off and cultured in MRS broth [RINH vial 417-506]. The strain types recovered were variable and clostridium species formed a high proportion, the isolated strains remained sensitive to heat.

[0307] Isolated cultures of LAB were subject to heating three times for 15 minutes at 70° C. (FIGS. 9A-9C). Viable bacteria decreased by 3-4 log orders after heat-treatment for the first time. However, the surviving bacteria had a degree of heat-resistance. With one exception, losses of viable bacteria were low when the bacteria were re-cultured and re-heated a further two times.

[0308] Heat-treatment three times at 70° C. altered the biological activities of the strains (FIGS. 9A-9C). RINH vial 521 (vial 255 heat-treated) was not able to block attachment of pathogens to IPEC cells and the capacity of RINH vial 520 (vial 230 heat-treated) to prevent attachment was reduced. The ability of RINH vial 517 (vial 31 heat-treated) to abolish inflammatory responses triggered in IPEC cells was abolished. In contrast, the biological properties of RINH vial 518 (vial 85 heat-treated) and RINH vial 519 (vial 86 heat-treated) were similar to those of the native strains.

13. Mouse Infection Studies

[0309] 13.1 L. mucosae (RINH Vial 323)

[0310] C3H/HeN mice develop a persistent but non-lethal, intestinal and systemic infection, which has many characteristics of the major form of human salmonellosis, when challenged with high levels of Salmonella enteritidis S1400. In contrast, C57BI/6 mice develop a severe primarily systemic, infection, reminiscent of acute infection in humans, when challenged with the same pathogen. To evaluate the capacity of L. mucosae (vial 323) to ameliorate salmonellosis, C3H/HeN and C57BI/6 mice were treated with L. mucosae prior to and post-challenge with Salmonella enteritidis (FIGS. 10, 13). The mice were euthanased and dissected 6 (C57BI/6) or 10 (C3H/HeN) days post-infection.

[0311] Systemic tissues: Oral treatment with L. mucosae limited the capacity of S. enteritidis to cause systemic infection both in C3H/HeN and C57BI/6 mice (FIG. 11A-11C; 14A-14C[[11a-c; 14a-c]]). High numbers of viable salmonella were detected in the mesenteric lymph node, liver and spleen of mice. In contrast, the numbers present in these tissues were greatly reduced if the mice had been co-treated with RINH vial 323 (L. mucosae). Salmonella infection caused enlargement of the spleen (FIG. 12A; 15). This tissue response was significantly reduced in mice treated with both RINH vial 323 (L. mucosae) and salmonella.

[0312] Intestine: Intestinal myeloperoxidase [MPO], a marker for neutrophils, was determined in C3H/HeN mice treated with salmonella or salmonella plus RINH vial 323 (L. mucosae). MPO in the intestine was greatly increased by salmonella infection, due to recruitment of neutrophils to the intestine part of the host response to infection (FIG. 12B[[12b]]), Co-treatment with RINH vial 323 (L. mucosae) reduced MPO activity in the intestine of salmonella-infected mice, indicating that the intestinal inflammatory responses to infection were lowered in these animals.

13.2 Novel pig LAB

[0313] Four LAB were selected: RINH vial 31, RINH vial 32, RINH vial 46 and RINH vial 47 (All L. reuteri; LR31, LR 32, LR 36 and LR47 respectively). To assess their efficacy to ameliorate a pathogen infection, C3H/HeN mice were treated with these LAB or RINH vial 323 (L. mucosae, LM] prior to and post-challenge with Salmonella enteritidis (FIG. 16). The mice were euthanased and dissected 10 days post-infection. Faecal excretion of S. enteritidis was reduced, if the mice had been co-treated with LAB (FIG. 17A, 17B). LR31 and LR32 tended to have the greatest effects on faecal salmonella outputs.

[0314] Intestine: Treatment with LR31, LR32, LM, LR46 or LR47 significantly reduced the numbers of salmonella in the caecum (FIG. 18A). Furthermore, LR31, LR32, LR46 and LR47 but not LM lowered salmonella numbers in the colon (FIG. 18B). The reductions tended to be greater with LR31 and LR32. In contrast to the large intestine, the LAB had no significant effects on numbers of salmonella in the small intestine.

[0315] Systemic tissues: Treatment with LR31, LR32, LM, LR46 or LR47 greatly reduced the numbers of salmonella detected in the spleen and liver (FIGS. 19A-19C). The reductions were more marked with LR31 and LR32 than with LM, LR46, or LR47. Salmonella numbers in the mesenteric lymph node were lowered following treatment with LR31, LR32 and LR46 but not with LM or LR47.

Discussion

[0316] The LAB strains isolated (total of 436 individual colony picks) from faeces of organically-reared pigs were predominantly L. reuteri, L. johnsonii, L. gasseri, L. pentosus, strains with a small number of L. plantarum, L. acidophilus, L. vaginalis, a single L. mucosae and several uncultured strains. Most of the LAB produced substances that could inhibit the growth of S. enteritidis and/or E. coli K88 in vitro. The potency of these anti-pathogen effects varied greatly between the individual bacterial strains. A proportion of LAB had high activity against S. enteritidis but low activity against E. coli K88 and vice-versa, but the majority had similar activities against both pathogens.

[0317] Thirty-three strains were selected on the basis of anti-microbial potency as determined in vitro. These bacteria were further screened for their ability to block adherence/invasion of intestinal pig epithelial cells (IPEC) by pathogens in vitro and their susceptibility to antibiotics.

[0318] Twenty-three strains were assayed for substrate range and specificity and their capacity to suppress inflammation in IPEC cells in vitro. From these, fourteen LAB (5 L. johnsonii, 6 L. reuteri and 3 L. plantarum) with particularly favourable properties were identified.

[0319] Two LAB strains [GGDK266 and GGDK31] were prepared in bulk for in vivo evaluation in newly-weaned piglets. Other potentially important candidate strains were present in this set of 14 LAB.

[0320] The survival and viability of LAB after freeze drying in various solutions was also evaluated. Small losses in viability were evident on prolonged storage of samples dried with skimmed milk powder. This was less marked when skimmed milk powder and sugars were used. However, the latter preparations were hygroscopic and were difficult to maintain. It was therefore decided to use a skimmed milk powder suspension for freeze drying and storage of LAB. The bulk preparations of GGDK266 and GGDK31 were freeze-dried in this medium.

[0321] Heat stability is a useful feature for LAB to be used in pelleted animal foods. Five heat-conditioned viable strains of isolated pig LAB were obtained. However, the biological properties in vitro and probiotic potential of three of the strains were adversely affected by heat-treatment. Nonetheless, two of the bacteria retained the biological properties of their native non-heat-treated forms.

[0322] Five pig LAB (L. reuteri [4] or L. mucosae [1]) were tested for ability to ameliorate salmonellosis in vivo. Treatment of mice with these LAB greatly reduced the pathogenicity of S. enteritidis.

14. Evaluation of Oral Administration of Organic Lactobacilli Probiotic Strains on the modulation of the Gut Microbiota and Performance of early Weaned Pigs

[0323] In vivo trials were carried out on early weaned piglets to test the effect of two probiotic strains according to the invention, Lactobacilli strains GGDK266 and GGDK31.

Trial Design

Animals:

[0324] 24 Large—White×Redon piglets [0325] Early weaned (21 days old, ≈7-8 kg), born in a local farm [0326] Weighted then distributed equally between the different group [0327] 3 experimental treatments (n=8): [0328] A—Basal diet+Placebo [0329] B—Basal diet+probiotic GDDK 266−dose 10×10.sup.12 [0330] C—Basal diet+probiotic GDDK 31—dose 10×10.sup.12 [0331] Observation period: 14 days

Diet:

[0332] Diets based on barley, wheat & soybean meal [0333] Feed composition

TABLE-US-00001 Barley 36.5 Wheat 21 SBM 48 19 Corn 10 Soy oil 4 Sugar 4 Potato protein 2 Premix 3.5 [0334] feed ad libitum in pelleted form

Tissue Sampling and Measurements

[0335] Sampling: Day 0 Slaughter of 6 “naive” piglets for collection of the caecum Individual collection of faeces (if possible) [0336] Day 7 individual collection of faeces during weight measurement [0337] Day 14 Slaughter of 24 piglets for collection of:

TABLE-US-00002 Content (5 g): Tissus (10 cm): Gastric Jejunum Jejunum Ileum Ileum Caecum Caecum Lymphatic nodes (distal ileum level) [0338] Storage: All samples were weighed, frozen in liquid nitrogen and stored at −80° C. [0339] Performance: Daily Weight gain (DWG), Feed Intake (FI) and Feed Conversion Ratio (FCR)
(1.sup.st step) [0340] Analysis: Determination of the microbiota profile in the different gut content samples by [0341] (2nd step) the molecular microbiology technique Denaturing gradient gel electrophoresis (DGGE). [0342] Molecular analysis of gene expression data using pig affymetrix gene expression arrays to determine gene modulation patterns. [0343] Determination of immunity markers in intestinal tissues
Microbial analysis using Denaturing Gel Gradient Electrophoresis DGGE (Trial 1)
DGGE methodology

[0344] DNA is extracted from faecal or tissue samples utilizing the MP Bio FastDNA™ spin kit for soil sample—116560000. The DNA is then amplified using Muyzer primers, as it is essential to use primers with a GC Clamp to be run on the gel. For samples of lactobacillus, specialised lactobacillus primers with a GC clamp were used.

TABLE-US-00003 Amplicon Annealing DGGE Target Primer Sequence Size temperature gradient Group Primer (5′-3′) (bp) (° C.) (%) All MF ATTACCGCGGCTGCTGG 233 55 35-70 Bacteria MR-GC.sup.a GC-Clamp- CCTACGGGAGGCAGCAG LABs Lac1 AGCAGTAGGGAATCTTCCA 327 55 30-50 Lac2-GC.sup.a GC-Clamp- ATTYCACCGCTACACATG.sup.c

Annotations:

[0345] .sup.aThe GC clam is as follows:

CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG

[0346] .sup.cY=C or T

PCR Program:

[0347]

TABLE-US-00004 Time Temperature Cycles 5 minutes 94° C. 1 30 seconds 94° C. 35 30 seconds 55° C. 2 minutes 72° C. 10 minutes 73° C. 1

[0348] DGGE is a genetic analysis technique in which amplified PCR products are separated by the denaturants formamide and urea within the gel, based on the genetic sequence by as little as a single base difference. DGGE can be utilised to visualise the differences in microbial diversity between samples. DNA obtained from a range of samples can be used in DGGE e.g. tissue and faecal samples. Bands on the gel were visualised using silver staining.

Molecular analysis and gene expression profiles of pig tissues
RNA extraction and Affymetrix Microarray analysis

[0349] RNA was isolated from both animal tissue and cultured cells for use on Affymetrix GeneChips. For animal tissue, approximately 200 mg tissue sample was removed from RNAlater (Ambion) and lyzed in Trizol (Invitrogen) using a polytron homogenizer. The tissue was further homogenized by passing the lysate through a syringe fitted with a 19G needle 3-5 times. The samples were incubated for 5 min at RT to permit the complete dissociation of nucleoprotein complexes. Then, chloroform, isopropanol and ethanol steps were performed according to the manufacturer's instructions. Briefly, 0.2 mL of chloroform was added per 1 mL of Trizol, vortexed and incubated at RT for 5 min. The samples were centrifuged at 12,000×g for 15 min at 4° C. The resultant aqueous phase was transferred to a fresh tube, and the RNA was precipitated by the addition of 0.5 mL of isopropanol per 1 mL of Trizol. The tubes were shaken vigorously by hand for 10s, incubated at 4° C. for 10 min and centrifuged at 12,000×g for 10 min at 4° C.

[0350] The RNA precipitate was washed with ice-cold 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of Trizol. The samples were vortexed and centrifuged at 7,400×g for 5 min at 4° C. After air-drying the resultant RNA pellet, the RNA was resuspended in up to 100 μL RNase-free water. Total RNA was further extracted with the RNeasy kit (Qiagen) according to the manufacturer's instructions, including an RNase-free DNase I (Qiagen) digestion step.

[0351] Cultured cells were homogenized by adding 350 μL Buffer RLT+1% β-mercaptoethanol. The cells were scraped off culture dishes with a filter tip and further homogenized by passing the lysate through a syringe fitted with a 19G needle 3-5 times. The cell lysate was then further processed using the RNeasy kit (Qiagen) according to the manufacturer's instructions, including an RNase-free DNase I (Qiagen) digestion step.

[0352] RNA concentration and integrity was ascertained using a Nanodrop instrument and/or Agilent Bioanalyzer, and purified RNA was stored at −70° C.

[0353] 250 ng RNA was processed for Affymetrix GeneChips using the GeneChip 3′ IVT Express Kit (Affymetrix) according to the manufacturer's instructions. aRNA quality was determined by Agilent 2100 Bioanalyzer. Hybridization to the GeneChip Mouse Genome 430 2.0 and GeneChip Human Genome U133 Plus 2.0 (Affymetrix) on a GeneChip Fluidics Station 450 (Affymetrix) was performed at the Institute of Medical Sciences Microarray Core Facility (University of Aberdeen, UK). Chips were scanned with an Affymetrix GeneChip Scanner 3000 (Affymetrix). Image quality analysis was performed using Gene Chip Operating Software (GCOS) (Affymetrix). Further quality analysis, normalization (gcRMA), statistical analysis and heatmap generation was performed with the freely available software packages R (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org). Microarray data were submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo).

Results

Performance of Pigs Fed Probiotics GGDK266 and GGDK31

[0354] The results for pigs fed probiotics GGDK266 and GGDK31 are shown in FIG. 20. DWG (Daily weight gain), FI (food intake) and FCR (feed conversion ratio) are shown below:

TABLE-US-00005 GGDK266 DWG FI FCR D 0-d 7  +++ (*) + + D 7-d 14 = + + D 0-d 14 + + +

[0355] Piglets fed GGDK266 exhibited significantly improved daily weight gain (DWG) during the first week post-weaning relative to GGDK31 and placebo fed piglets.

Microbial diversity analysis using DGGE (Trial 1)

[0356] DGGE using universal primers revealed no differences in overall microbial diversity between the treatments and placebo (see FIG. 21).

[0357] DGGE using lactic acid bacteria (LAB) specific primers revealed significant differences in LAB diversity between treatment with GGDK 266 and placebo in both caecal and ileal samples (see FIG. 22).

[0358] DGGE using LAB specific primers revealed significant differences in LAB diversity between the treatment with GGDK266 and placebo in ileal samples (see FIG. 23).

[0359] DGGE using LAB specific primers revealed significant differences in LAB diversity between the treatment with 266 and placebo in caecal samples (see FIG. 24).

[0360] Overall the microbial diversity analysis revealed significant clustering of the LAB population in piglets fed GGDK266 indicating that the populations in individual animals on this treatment has a similar and stable microbiota.

Molecular analysis of ileal tissue samples: Affymetrix pig arrays
Downregulated in GDK266 versus placebo

[0361] Gene ontology analysis of differentially expressed gene revealed that a significant reduction in immune system processes and pro-inflammatory activation in response to feeding young piglets probiotic GGDK266 relative to placebo (see FIG. 25).

[0362] Results reveal that GGDK266 had a very specific and targeted effect on the immune system and the functional groups associated with response to stimuli (see FIG. 26).

Upregulated in GGDK266 versus Placebo

[0363] In contrast to the effects on the immune system, GGDK266 promoted metabolic processes particularly in relation to nitrogen (see FIG. 27). Without wishing to be bound by theory, it is believed that these effects may explain the improved DWG in animals fed GGDK266.

Top differentially expressed genes between GGDK266 and Placebo

TABLE-US-00006 affy. id Gene Name Product FC p-value Ssc.645.1.S1_at CSTA Cystatin A 44.06 0.00000 Ssc.11608.1.A1_at TIP_HUMAN T-cell immunomodulatory protein precursor 28.92 0.00030 Ssc.10837.1.A1_at ROBO1 Roundabout homolog 1 precursor 13.35 0.00178 Ssc.8960.1.A1_at BPI Bactericidal permeability-increasing protein 11.65 0.00476 precursor Ssc.16234.1.S1_at TCN1 Transcobalamin 1 precursor 11.48 0.00023 Ssc.1411.1.S1_at THBS4 Thrombospondin 4 precursor 8.92 0.00198 Ssc.837.1.A1_at BPI Bactericidal permeability-increasing protein 4.55 0.00573 precursor Ssc.30008.1.A1_at ESR1 Estrogen receptor 4.48 0.00053 Ssc.13539.1.A1_at PLAGL1 Zinc finger protein PLAGL1 4.42 0.00881 Ssc.26324.1.S1_at NP_981932 Iodotyrosine dehalogenase 1 protein 4.26 0.00200 Ssc.29413.1.A1_at B3GALT2 UDP-Gal:betaGlcNAc beta 1,3- 4.00 0.00046 galactosyltransferase 2 Ssc.27410.1.S1_at MYCN N-myc proto-oncogene protein 3.80 0.00261 Ssc.25176.1.A1_at GOLPH4 Golgi phosphoprotein 4 3.80 0.00009 Ssc.15890.1.S1_at VNN1 Pantetheinase precursor 3.61 0.00271 Ssc.23427.1.A1_at CYB561 Cytochrome b561 3.29 0.01512 Ssc.16186.1.S1_at CD3E T-cell surface glycoprotein CD3 epsilon chain −2.62 0.00764 precursor Ssc.22676.1.S1_at CXCR6 C-X-C chemokine receptor type 6 −2.63 0.01652 Ssc.15565.1.S1_at LCP2 Lymphocyte cytosolic protein 2 −2.76 0.00024 Ssc.18652.1.S1_at IL16 Interleukin-16 precursor −2.97 0.01132 Ssc.181.1.S1_at TRGV9 T-cell receptor gamma chainV region PT- −3.04 0.01615 gamma-1/2 precursor Ssc.23489.1.S1_at CD8A T-cell surface glycoprotein CD8 alpha chain −3.08 0.00071 precursor Ssc.428.6.S1_a_at TCA_HUMAN T-cell receptor alpha chain C region −3.15 0.00027 Ssc.10357.1.A1_at FMN2 Formin 2 −3.46 0.00308 Ssc.27354.1.S1_at STXBP5 Tomosyn −3.88 0.02438 Ssc.28909.3.A1_at TPH2 Tryptophan 5-hydoxylase 2 −4.36 0.00717 Ssc.25976.1.S1_at GZMH Granzyme H precursor −5.46 0.00179 Ssc.11070.1.S1_at IGHM Ig alpha-1 chain C region −9.07 0.00115 Ssc.16566.1.S1_at LCT Lactase phlorizin hydrolase precursor −11.31 0.00328 Ssc.13273.1.A1_at GCNT3 glucosaminyl (N-acetyl) transferase 3, mucin type −19.75 0.00016 Ssc.11098.1.S1_at IFITM3 Interferon-induced transmembrane protein 3 −51.36 0.00044

[0364] Gene expression data revealed that a number of genes were significantly increased including antimicrobial peptides (eg. CSTA, BP1) and immune-regulatory genes (TIP). In contrast GGDK266 reduced the expression of a diverse panel of genes involved in pro-inflammatory immunity (IFITM3, IL-16).

CONCLUSIONS

[0365] Cellular and metabolic processes, particularly in relation to nitrogen, are increased in animals treated with GGDK266 relative to placebo. [0366] Immune system processes are downregulated in animals treated with GGDK266 relative to placebo. Examples include T-cell markers CD3 and CD8, T cell receptor chains, chemokines/cytokines and IFN-related genes. [0367] Animals administered with GGDK266 exhibited a stable population of lactic acid bacteria revealed by clustering of the bacterial profile of the individual induced by the administration of probiotic GGDK266. [0368] FCR and performance were significantly improved during the first weeks of post-weaning life. [0369] This improvement in growth performance correlated with the reduction in inflammatory immune responses and the increase in specific metabolic processing.

[0370] Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

TABLE-US-00007 TABLE 1 Summary of bacteria colonies selected from cultures of faeces from organically-reared pigs. Total number of cultured colony picks 443 Media: LAMVAB agar 55 LAMVAB agar + pig colostral carbohydrate 88 MRS agar 29 MRS agar + pig colostrum carbohydrate 176 Glucose-free MRS agar + carbohydrate 57 MRS agar after heat-treatment at up to 70° C. 38 Main strains identified: Lactobacillus reuteri Lactobacillus johnsonii Lactobacillus plantarum Five isolated LAB were heated once, twice or three times at 70° C. for 15 min. Surviving bacteria were re-grown. In stock 5 LAB heated once at 70° C. 5 LAB heated twice at 70° C. 5 LAB heated three times at 70° C.

TABLE-US-00008 TABLE 2 Candidate LAB strains for further study selected on the basis of killing activity in well diffusion assays (note 266 and 161 contain LR) Pathogen killing (units) RINH Well diffusion assay Vial no. anti-SE anti-KSS 85 LR 129886 60168 255 LJ 101477 64390 266 LJ 101335 60168 436 LJ 81656 85010 161 LP 77894 103346 12 LJ 162709 42977 16 LJ 117621 41365 29 LR 174471 45720 31 LR 116867 46907 86 LR 98520 75147 230 LJ 95705 64340 256 LJ 94012 77459 314 LJ 103497 48936 361 LJ 100770 40254 17 LJ 144765 23072 30 LR 125463 36050 32 LR 168892 32572 258 LP 70724 68612 260 LP 78197 68562 320 LJ 66350 78044 364 LJ 99137 55123 433 LJ 95083 51461 15 LP 77459 58669 218 LJ 62329 50416 220 LJ 68612 53834 356 LJ 72986 55302 363 LJ 79125 45555 131 LR 42223 44108 434 LR 10000 81656 166 LJ 17064 79621 431 LR 48657 31674 47 LR 20722 34633 46 LR 19867 34633 LJ. L. johnsonii, LR. L. reuteri, LP. L. Plantarum

TABLE-US-00009 TABLE 2a Identification of candidate LAB strains (by 16S rRNA gene sequence) selected on the basis of killing activity in well diffusion assays (note 266 and 161 contain LR) RINH forward reverse Vial no. sequence sequence 85 Lactobacillus reuteri Lactobacillus reuteri 255 Lactobacillus johnsonii, Lactobacillus johnsonii, taiwanensis, acidophilus gasseri 266 Lactobacillus johnsonii Lactobacillus johnsonii 436 lactobacillus johnsonii Lactobacillus johnsonii str. 466 F19785 161 Lactobacillus plantarum, Lactobacillus plantarum, pentosus, paraplantarum pentosus 12 Lactobacillus johnsonii, Lactobacillus johnsonii, gasseri, taiwanensis gasseri 16 Lactobacillus johnsonii, Lactobacillus johnsonii gasseri, taiwanensis 29 Lactobacillus reuteri, Lactobacillus reuteri pontis, vaginalis, frumenti 31 Lactobacillus reuteri Lactobacillus reuteri 86 Lactobacillus reuteri Lactobacillus reuteri 230 Lactobacillus johnsonii, Lactobacillus johnsonii taiwanensis, acidophilus 256 Lactobacillus johnsonii, Lactobacillus johnsonii taiwanensis, acidophilus 314 lactobacillus johnsonii uncultered bacterium BR0315 361 lactobacillus johnsonii lactobacillus johnsonii str. NCC2822 F19785 17 Lactobacillus johnsonii, Lactobacillus johnsonii gasseri, taiwanensis 30 Lactobacillus reuteri, Lactobacillus reuteri pontis 32 Lactobacillus reuteri Lactobacillus reuteri 258 Lactobacillus plantarum, Lactobacillus plantarum, pentosus, helveticus pentosus, paraplantarum 260 Lactobacillus plantarum, Lactobacillus pentosus, pentosus, paraplantarum plantarum, paraplantarum 320 lactobacillus johnsonii Lactobacillus johnsonii NCC2822 F19785 364 lactobacillus johnsonii lactobacillus johnsonii 466 F10785 433 lactobacillus johnsonii lactobacillus johnsonii str. CECT 289 F19785 15 Lactobacillus plantarum, Lactobacillus plantarum, pentosus pentosus 218 Lactobacillus johnsonii, uncultured Firmicutes, taiwanensis Lactobacillus johnsonii 220 Lactobacillus johnsonii, uncultured Firmicutes, taiwanensis Lactobacillus johnsonii 356 lactobacillus johnsonii lactobacillus johnsonii NCC2822 F19785 363 lactobacillus johnsonii lactobacillus johnsonii 466 F10785 131 Lactobacillus reuteri Lactobacillus reuteri 434 Lactobacillus reuteri lactobacillus reuteri NM99-1 166 Lactobacillus johnsonii, Lactobacillus johnsonii taiwanensis, acidophilus 431 lactobacillus reuteri lactobacillus reuteri str. Probio-16 JCM 1112 47 Lactobacillus reuteri Lactobacillus reuteri 46 Lactobacillus reuteri Lactobacillus reuteri

TABLE-US-00010 TABLE 3 Candidate LAB strains for further study selected on the basis of killing activity in well diffusion assays and capacity to block adherence of pathogen to IPEC cells RINH Inhibition of adherence (%) Vial no. SE KSS 85 88.31 87.93 255 82.37 99.93 266 88.03 98.09 161 98.32 96.94 12 96.89 99.92 29 93.7 99.91 31 98.64 99.75 86 81 99.98 256 82.47 99.92 361 85.07 99.44 17 84.56 99.66 30 96.44 99.91 32 87.74 99.86 230 78.89 82.45 258 96.37 86.5 260 90.22 88.79 314 79.68 94.2 433 99.99 96.23 16 87.68 45.38 218 91.53 86.49 363 85.61 99.93 364 82.13 78.12 15 79.19 99.52 131 95.5 96.03 220 91.04 78.6 320 92.7 44.17 356 82.15 78.4 434 94.78 98.85 436 99.97 1 166 91.45 95.97 431 96.35 86.47 47 90.47 99.47 46 83.51 99.7

TABLE-US-00011 TABLE 4 Area of inhibition of LAB by defined amounts of antibitiotic (arbitrary units) ampicillin cefotaxime chloramphenicol erythromycin gentamicin kanamycin metronidazole nal. acid tetracycline vancomycin 12 244011 340402 186699 13151 0 0 0 0 37668 22581 15 277117 311725 204282 214008 0 0 0 0 0 16 266033 294166 187805 64681 17000 7157 0 0 0 105209 17 387224 400570 235430 277145 9193 0 0 0 50328 117741 29 410335 444193 190293 114511 0 0 0 0 252497 11483 30 292728 335927 77133 208117 31261 0 0 0 187805 31402 31 334789 410966 165904 262226 38221 0 0 0 214037 24901 32 404496 402291 247436 350238 71608 23786 0 0 261979 10691 46 359232 402588 210421 251461 29550 0 0 0 21382 25069 47 328283 410579 185515 270105 30342 0 0 0 211556 22231 85 356114 369916 204992 309439 0 0 0 0 276800 3971 86 250812 381270 183399 250805 41858 0 31264 0 16643 13355 131 349955 473065 248521 123562 82466 14932 0 0 19354 7479 161 338497 412977 258724 261133 51991 4536 29126 0 20435 5542 166 268783 417393 185508 251607 61136 17671 0 0 24606 0 218 209117 271547 148617 0 0 0 0 0 88668 122870 220 209371 319970 165815 34230 58814 32572 0 0 34636 111666 230 254614 335143 164405 51078 65717 45705 0 0 36644 41991 255 330364 392169 217758 59224 56563 8486 0 0 29872 0 256 456892 502325 228531 71258 93058 0 0 0 20955 42203 258 401257 271932 195909 233326 28608 0 0 0 223143 0 260 286400 364573 203796 33393 78821 78364 0 0 21757 62792 266 287070 322869 198614 247085 54008 3079 6437 2737 48286 107882 314 297057 332853 154830 44115 0 0 0 0 90259 356 291920 339895 203692 62656 10472 5890 0 0 24194 8202 361 320695 323713 201886 234140 0 0 0 0 91863 363 275304 308159 193271 44491 86683 0 0 0 28212 18146 364 288514 341651 194320 143978 45880 0 0 0 18322 103995 431 339016 380459 226484 311725 74991 0 0 0 0 26302 433 241710 203588 174124 63381 19139 0 0 0 19965 79034 434 198112 261065 172223 68052 6049 0 0 0 60344 45863 436 290458 287331 185812 142842 0 0 0 0 52279 61810 Nal. Acid, naladixie acid.

TABLE-US-00012 TABLE 5 Substrates in capsules of API CH 50 Kit Substrates in capules of API CH 50 kit 1 glycerol polyol 2 erythritol polyol 3 D-arabinose monosaccharide 4 L-arabinose monosaccharide 5 D-ribose monosaccharide 6 D-xylose monosaccharide 7 L-xylose monosaccharide 8 D-adonotol alcohol 9 Methyl-βD-Xylopyranoside cyclic 10 D-galactose monosaccharide 11 D-glucose monosaccharide 12 D-fructose monosaccharide 13 D-mamose monosaccharide 14 L-sorbose monosaccharide 15 L-rhamose monosaccharide 16 dulcitol monosaccharide/alcohol 17 inositol polyol 18 D-mamitol polyol 19 D-sorbitol sugar/alcohol 20 Methyl-αD-Mannopyranoside cyclic 21 Methyl-αD-Glucopyranoside cyclic 22 N-acetylglucosamine monosaccharide 23 amygdalin glycoside 24 arbutin glycoside 25 esculin ferric citrate 26 salicin glycoside 27 D-cellobiose disaccharide 28 D-maltose disaccharide 29 D-lactose (bovine) disaccharide 30 D-Melibiose disaccharide 31 D-saccharose disaccharide 32 D-trehalose disaccharide 33 inulin polysaccharide 34 D-melezitose trisaccharide 35 D-rafinose trisaccharide 36 amidon (starch) polysaccharide 37 glycogen polysaccharide 38 xylitol monosaccharide/alcohol 39 gentobiose disaccharide 40 D-turanose disaccharide 41 D-lyxose monosaccharide 42 D-tagatose monosaccharide 43 D-fucose monosaccharide 44 L-fucose monosaccharide 45 D-arabitol monosaccharide/alcohol 46 L-arabitol monosaccharide/alcohol 47 potassium gluconate sequestrant 48 potassium 2-ketogluconate sequestrant 49 potassium 5-ketogluconate sequestrant

TABLE-US-00013 TABLE 6 Substrate profile of LAB using an API CH 50 kit alcohol/ monosaccharides monosaccharides disaccharides trisaccharides polysaccharides alcohols others 17 0.4 0.0 0.8 0.5 0.3 0.0 0.4 30 0.2 0.0 0.0 0.0 0.0 0.0 0.3 31 0.3 0.0 0.6 0.5 0.0 0.0 0.6 32 0.3 0.0 0.5 0.5 0.0 0.0 0.3 46 0.2 0.0 0.5 0.5 0.0 0.0 0.4 47 0.2 0.3 0.5 0.5 0.0 0.0 0.4 85 0.1 0.0 0.3 0.0 0.0 0.0 0.4 86 0.3 0.0 0.5 0.5 0.0 0.0 0.4 131 0.4 0.0 1.0 1.0 0.3 0.3 0.9 161 0.7 0.3 0.9 1.0 0.0 0.3 0.9 166 0.4 0.0 0.6 0.0 0.7 0.0 0.3 220 0.1 0.0 0.5 0.5 0.0 0.0 0.4 230 0.3 0.0 0.8 0.5 0.0 0.0 0.4 255 0.1 0.0 0.6 1.0 0.3 0.0 0.4 256 0.2 0.0 0.5 1.0 0.0 0.2 0.6 258 0.6 0.3 1.0 1.0 0.7 0.7 0.9 260 0.4 0.3 0.9 1.0 1.0 0.0 0.6 266 0.3 0.0 0.9 0.5 0.3 0.0 0.4 320 0.3 0.0 0.5 0.5 0.3 0.0 0.3 363 0.4 0.0 0.8 1.0 0.3 0.0 0.4 364 0.4 0.0 0.8 0.5 0.3 0.0 0.4 433 0.2 0.0 0.5 0.0 0.0 0.2 0.3

TABLE-US-00014 TABLE 7 Candidate LAB strains selected on the basis of killing activity, capacity to block adherence of pathogen to IPEC cells, antibiotic susceptibility, substrate reactivity and ability to suppress inflammation (note 266 and 161 contain LR) RINH forward reverse Vial no. sequence sequence 266 Lactobacillus johnsonii Lactobacillus johnsonii 31 Lactobacillus reuteri Lactobacillus reuteri 258 Lactobacillus plantarum, Lactobacillus plantarum, pentosus, helveticus pentosus, paraplantarum 260 Lactobacillus plantarum, Lactobacillus pentosus, pentosus, paraplantarum plantarum, paraplantarum 255 Lactobacillus johnsonii, Lactobacillus johnsonii, taiwanensis, acidophilus gasseri 161 Lactobacillus plantarum, Lactobacillus plantarum, pentosus, paraplantarum pentosus 256 Lactobacillus johnsonii, Lactobacillus johnsonii taiwanensis, acidophilus 86 Lactobacillus reuteri Lactobacillus reuteri 85 Lactobacillus reuteri Lactobacillus reuteri 32 Lactobacillus reuteri Lactobacillus reuteri 230 Lactobacillus johnsonii, Lactobacillus johnsonii taiwanensis, acidophilus 131 Lactobacillus reuteri Lactobacillus reuteri 30 Lactobacillus reuteri, Lactobacillus reuteri pontis 364 lactobacillus johnsonii 466 lactobacillus johnsonii F10785

TABLE-US-00015 TABLE 8 Identity for pig LAB strains selected for bulk preparation (note 266 and 161 contain LR) Bacteria Bacteria RINH Seq code identified Seq code identified vial no primer 926F by BLAST primer 519R by BLAST GGDK266 266 S10CM218 Lactobacillus S10CM171 Lactobacillus johnsonii johnsonii GGDK31 31 S10BL123 Lactobacillus S10BL141 Lactobacillus reuteri reuteri

TABLE-US-00016 31   123 with 926F SEQ ID NO: 1 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAG GCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAG GTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGCCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAA GCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAAT CGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTT TGTAACGCCCAAAGTCGGTGGCCTAACCATTATGGAGGGAGCCGCCTAAGTGCGGGACAGATGACTGGGGTGAAGTCGTA ACAAGGTAGCCTGTATTTTCTTGCGGTTGTTCCCCCCCCNGGCGGGACTGCCTTACTCCTTTCACCNCCCGCGCCCCTGG AGGGGGCCGGAACCCCCCTCCCAACCCCCCTAACCCACCTCCTTCCTTTTAACCNGCT 31   141 with 519R SEQ ID NO: 2 GACTTTCTAGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAACAACAGAGCTTTACGAGC CGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGCGGAAGATTCCCTAGTGCTGCCTCCCGT AGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCG TTACCTTACCAACTAGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAAACAAAAGCCATGTG GACTTTCTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCA CCCGTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAATCAGTTGGGCCAGTGCGTACGACTTGC ATGTATTAGGCACACCGCCGGCGTTCATCCTGAGCCATGATCAAACTCTANGCGTCAGTTTTACGGTCTCGGCTCGTTTC TCTGTTNTCTGACATCAACGTGCGTTACATTTGCGGTTTACGCATTGATTGTACTCCCTCCACATAGGTGGCGGCATACC CTTCGTGCTCCTCTACTCATCTCGTTCATTACAACTCGCTTTGTTACCTTCCCGGTGGGGTTCTCTACCTCCTTCGTTTT CTCTCACCTCATTCTCTCTCCCATCCTCTCNCTTTCCTCTTGCTC 161   282 WITH 926F SEQ ID NO: 3 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAG ACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTAGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAG GTGGGGATGACGTCAAATCATCATGCCCCTTGATGACCTGGGCTAGACACGTGCTACAATGGATGGTACAACGAGTTGCG AACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTACGGATGTGTAGGCTGCAACTCGCCATACATGAAGTCG GAATCGCTAGTAATCGCGGATACAGCATGCCGCGGTGAATACTGTTCCCGGGCCTATGTGACACACCGCCCGTCACACCA TGAGCAGTTTGTAATCACCCACACAGTCGGTGGGGTAACCTTTATAGGAACCAGCCGCCTACAGTGCGGGACCGATGATT ATGGGTGCACTCGTATCACTGTAACTTAAACCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATACGTTACCTGCAAC CCTGAAGGGCGGAATCCCTCCAACGATTATCAAT 161   300 WITH 519R SEQ ID NO: 4 GTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAG CCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCG TAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCC GTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGC GGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTC ACCAGTTCGCCACTCACTCAAATGTAAATCATGATGAAGCACCAATCAATACCAAGTTCGTTCGACTTGCATGTATTA GGCACGCCGCCAGCGTTCGTCGCTGAGCCATGATCAAACTACTAAAGGCCCCCNATGCCTCCCACCCGCTTTGTTGCCGG GGCCCCCCGTTCCCATACCCCTTTTGGACGTTTTCCAGCCCCTTGGCGGGCCCTGTACCTCCCCCCAGGGCGGGGAATGC CTTAATTGCGTTNACCTTGCACCCCCTGAAGGGGCGGAATCCCTCCAACGATTACCT 255   504 with 925F SEQ ID NO: 5 GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTCGCATAACCTAAGAGATT AGGTGTTCCCTTCGGGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTCACATGTTGGGTTAAGTCCC GCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGA AGGTGGGGATGACGTCAAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAGAATGGACGGTACAACGAGATA GCGAACCTGCGAAGAGCTAAGCGGATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGC TTGGAATCGCTAGTAATGGGGGATCAGCACTGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA TGAGAGTCTGTAACTCCCAAAGTCGGTGGGATAACCTTCTATAGCGAGTGAGTCCGTTCGATGGGTAGGGACAAGATGAA TGAGCGGTGAAAGGTCGTTAAACCAAGGGTAGCAAGTAAGGATCCCTTTGGGGGTTTTATCTCCACGGGGGGGGTGTTTC TTTTCTGTCTTTA 255   530 with 519R SEQ ID NO: 6 ACTTTCTAGAGTTAGATGATACCGTTCAACATGACAGATGGCCACGTTTACTTACTCTCACTGACTACTGTTCTTTCATC TCACACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGAGCTTTGCGTCCCATTGTG GAACATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGG CTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGCCGAA CCATCTTTCACAACTCTAAACATGCTTGTAGTGTTGTTATTCCGGTATTAACATTCTGTTTCCAGGTTGTTATTCCCAGC TGATCTCGGGGCAGGGTTTACCCCAACGTTGGTTTACCTTCACCCCCGGTTNCGGCCCGGCTTCGNCCTTGGGTTAGTAC TNACGATTCTGCTATTATATACGATGGGCTAGACGACCAGCCTAACACAATTTCAATTTCGTNAAGTGTCGAGAGGNCCT ACGGTCGTCCCGTTAACGTGTAGNCNATTTGGCTTATTTGTTAAGTTGTCCANCGGGCCACCGACCCCCAGGGCCCGGTT GGTCCGGGTTTCCCCCATTGCAACGTCGCCAAAGTGCGGAAATTTCGAAAATACCCTTAACCAATGAAAAAAACATA 258   414 with  F SEQ ID NO: 5 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAG ACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGGTTAAGTCCCT CAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAA GGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCG AACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTGGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAA TCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGT TTGTAACACCCAAAGTGGGTGGGGGTAACCTTTTTAGGAAACCAGCCCGCCCTAAAGGGTGGGGAACAAGAATGAATTAA GGGGGTTGAAAAGTTCCGTTAAACCAAAAGGGGTTAGCCCCNGNTNNGANNNNNNNNNGAC 258   438 with 519R SEQ ID NO: 8 GCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTGTCTTCTTTAACAACAGAGTTTTACGAGCC GAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGT TACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGG TCCAAGTTGTTATGGGGTATTAGCATCTGTTTCCAGGGTGTTATTCCCCCGCTTGGTGGGCAGGGTTTCCCACGTGTTAC TCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTTGCATGTAT TAGGCACGCCGCCAGCGTTCGTCCTGAGCCATGATCAAACTCNGA indicates data missing or illegible when filed

TABLE-US-00017 S12KG200 GGDK 31-2 22F SEQ ID NO: 10 TGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCT TGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAAC ACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCT AATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTT TGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGT AACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGC CACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGG GAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGA AGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGA GAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAAC TACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATT TATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGC CTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAG AAGAGGACAGTGGAACTC S12KG201 GGDK 31-1 519F SEQ ID NO: 11 TCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATG TGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTG AGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGA TATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGAC GCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGT CCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAG TGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGG TTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCG CTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTG CATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTG AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCA TGCCCTTATGACCTGGGCTA S12K202 GGDK 31-1 92GF SEQ ID NO: 12 GAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA CATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGA CAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG TCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGC ACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTC AGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACG GTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTT CTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCT AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTAC ACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCT AACCATTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGAAG TCGTAACAAGGTAGCCGTA S12KG203 GGDK 31-1 926R SEQ ID NO: 13 CTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCTCCGGCACTGAAGGGCGG AAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGT ATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAG ACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTC CACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCG GTTTCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTA AGCAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGC CACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTG GTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCC AACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGC TCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGT AGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAAC TCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAA TGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAAACAA AAGCCATGTGGCTTTTGTTGTTATGC S12KG204 GGDK 31-1 519R SEQ ID NO: 14 TTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTC TTCTCCAACAACAGAGCTTTACGAGTCCGAAACCCTTCTTCACTCACGCG GTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGC CTCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTC TCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAAC TAGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTT CAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTT CCAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCACCC GTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAA TCAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCG TTCATCCTGAGCCATGATCAAAC S12KG205 KKDK 31-1 RP2 SEQ ID NO: 15 CCGCCTTAGGCGGCTCCCTCCATAATGGTTAGGCCACCGACTTTGGGCGT TACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA TTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTA GGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTAGC TTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAA CTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGA CACGAGCTGACGACGACCATGCACCAACTGTCATTGCGTCCCCGAAGGGA ACGCCTTATCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCT TCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCCTTTGAGTTTCCACCTTGCGGTCGTACTCCCCAGGCGGAGTG CTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAG CACTCATCGTTTACGGCATGGACTACCAGGG
NCIMB 41847 GGDK161—contains both Lactobacillus plantarum and Lactobacillus reuteri Lactobacillus plantarum

TABLE-US-00018 S12KG218 GGDK 161-1 27F SEQ ID NO: 16 GTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGC ATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGA AACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGC ATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATC ACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGAGGTAACGGCTC ACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGG GACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC CACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGT TTCAGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAAC TGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGC CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGG CGTAAAGCGAGCGCAGGCGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCT CAACCGAAGAAGTGCATCGGAAACTGGGAAGCTTGAGTGCAGAAGAGGAC AGTGGAACTCCATGTGTAGCGGTGAAATGCGT S12KG219 GGDK 161-1 S19F SEQ ID NO: 17 CGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGT GAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGA GTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGAT ATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACG CTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTC CATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGT GCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGC TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGC AAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGC ATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGA GACTGCCGGTGACAAACCGGA S12KG220 GGDK 161-1 926F SEQ ID NO: 18 TGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTT GACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGA TACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGG GCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA TGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCA TTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCG CTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGT ACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGG GTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGA AGTCGTAACAAGGTAGCCCGTA S12KG221 GGDK 161-1 926R SEQ ID NO: 19 ACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGA AACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTA TCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGA CCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTC ACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAG TTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAA AAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCC ACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGG TTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTA ACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCT CCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGT CGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAAT ACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCG GACCATGCGGTCCAAGTTGT S12KG222 GGDK 161-1 519B SEQ ID NO: 20 GCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGT TCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGC GGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTG CCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACC CTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCAT CTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTT TCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTT TCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACC AGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATAC CAAAGTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCT GAGCCAGATCAAACTCTAA S12KG223 GGDK 161-1 RP2 SEQ ID NO: 21 CCACCTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGT TACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA TTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTA GGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGC TTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAA CTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGA CACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGA ACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCT TCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATG CTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAG CATTCATCGTTTACGGTATGGACTACCAGGGTATCTA
NCIMB 41847 GGDK161—contains both Lactobacillus plantarum and Lactobacillus reuteri Lactobacillus reuteri

TABLE-US-00019 S12KG309 cGGDK 161-1 27F SEQ ID NO: 22 ATGCTAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGAT TGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAA CCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCAT AACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCAC TCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTAC CAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAA CTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCA CAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTT CGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGT TCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGT AAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTA ACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAG TGGAAC S12KG310 cDGGDK 161-1 591F SEQ ID NO: 23 TCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGAT GTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTT GAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAG ATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGA CGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAG TCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCA GTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGAGTACGACCGCAAGG TTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCG CTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTG CATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTG AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCA TGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGA GTCGCAAGCTCGCGAGAG S12KG311 cGGDK 161-1 926F SEQ ID NO: 24 GGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTG ACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATG ACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA GTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGG CACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGT CAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGAC GGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGT TCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGC TAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTA CACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCC TAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGAA GTCGTAACAAGGTAGCCGTA S12KG312 cGGDK 161-1 926A SEQ ID NO: 25 TCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAA CCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATC TAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACC AGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCAC CGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGGTT TCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGC AACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCAC CTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTT GGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAAC AACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCC ATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGG AGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAACTCG GCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGC ACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAAACAAAAG CCATGTGGCTTTT S12KG313 cGGDK 161-1 519R SEQ ID NO: 26 TTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTC TTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGG TGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCC TCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCT CTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACT AGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTC AAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTC CAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCACCCG TCCGCCACTCACTGGTAATCCATCGTCAATCAGGTGCAAGCACCATCAAT CAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCGT TCATCCTGAGCCATGATCAAAC S12KG314 cGGDK 161-1 RP2 SEQ ID NO: 27 GCGGCTCCCTCCATAAAGGTTAGCGCCACCGACTTTGGGCGTTACAAACT CCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGC GGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTT GCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTAGCTTACTCTC GCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAG GTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTG TCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAAC AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCT GACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTA TCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAG CTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTC CTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGC GTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATC GTTTACGGCAT
NCIMB 41848 GGDK255—Lactobacillus reuteri

TABLE-US-00020 S12KG237 GGDK 255-1 27F SEQ ID NO: 28 GTGTGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGT GCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGT AACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGAT GCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGG CTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAA GGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATC GGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGT AGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAG TGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCG TGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAA AGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTG CAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATA TGGAAGAACACCAGTG S12KG238 GGDK 225-1 519F SEQ ID NO: 29 TCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATG TGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTG AGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGA TATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGAC GCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGT CCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAG TGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGG TTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCG CTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTG CATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTG AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCA TGCCCCTTATGACCTGGGCTACACACGTGCTAC S12KG229 GGDK 225-1 926F SEQ ID NO: 30 TGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTT GACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAAT GACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGG GCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACG TCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCG TTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCG CTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGT ACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGC CTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGA AGTCGTAACAAGGTAGCCGTA S12KG240 GGDK 255-1 526R SEQ ID NO: 31 TACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGG AAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGT ATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAG ACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTC CACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCG GTTTCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTA AGCAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGC CACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTG GTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCC AACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGC TCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGT AGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAAC TCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAA TGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAAACAA AAGCCATGTGGCTTTTG S12KG241 GGDK 255-1 519R SEQ ID NO: 32 TTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTC TTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGG TGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCC TCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCT CTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACT AGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTC AAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTC CAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCACCCG TCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAAT CAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCGT CCATCCTGAGCCATGATCAAAC S12KG242 GGDK 255-1 RP2 SEQ ID NO: 33 CCGCCTTAGGCGGCTCCCTCCATAAAGGTTAGGCCACCGACTTTGGGCGT TACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA TTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTA GGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTAGC TTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAA CTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGA CACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGA ACGCCTTATCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCT TCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTG CTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAG CACTCATCGTT
NCIMB 41849 GGDK 258—Lactobacillus plantarum

TABLE-US-00021 S12KG267 GGDK 258-3 27F SEQ ID NO: 34 GTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGC ATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGA AACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGC ATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATC ACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGAGGTAACGGCTC ACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGG GACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC CACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGT TTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACT GTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCC AGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGC GTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCT CAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGAC AGTGGAACTC S12KG268 GGDK 256-3 591F SEQ ID NO: 35 GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTG AAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAG TGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATA TATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGC TGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCC ATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTG CTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCT GAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCA AATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCA TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAG ACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATG CCCCTTATGACCTGGGCTAC S12KG269 GGDK 258-8 926F SEQ ID NO: 36 GTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCT TGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGG ATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTG GGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC GTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG ATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCC ATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATC GCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTG TACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGG GGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTG AAGTCGTAACAAGGTAGCCCGTA S12KG270 GGDK 258-3 926R SEQ ID NO: 37 ACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGA AACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTA TCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGA CCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTC ACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAG TTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAA AAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCC ACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGG TTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTA ACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCT CCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGT CGGCTACGTATCATTGCCATGGTGAGCCGTTACCTCACCATCTAGCTAAT ACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCG GACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTC S12KG271 GGDK 258-3 519R SEQ ID NO: 38 TTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTC TTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGG CGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCC TCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCT CTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCTCACCATCT AGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTC AAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTC CAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAG TTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCA GAGTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGA GCCATGATCAAAC S12KG272 GGDK 258-3 RP2 SEQ ID NO: 39 CCACCTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGT TACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA TTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTA GGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGC TTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAA CTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGA CACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGA ACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCT TCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATG CTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAG CATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTA CCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCT
NCIMB 41850 GGDK 266—contains both Lactobacillus iohnsonii and Lactobacillus reuteri Lactobacillus iohnsonii

TABLE-US-00022 S12KG273 GGDK 266-1 27F-repeat SEQ ID NO: 40 GTGCCTAATACATGCAAGTCGAGCGAGCTTGCCTAGATGATTTTAGTGCT TGCACTAAATGAAACTAGATACAAGCGAGCGGCGGACGGGTGAGTAACAC GTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAACAGATGCTAA TACCGGATAACAACACTAGACGCATGTCTAGAGTTTGAAAGATGGTTCTG CTATCACTCTTGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAAC GGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCAC ATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAA TCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGA AGGGTTTCGGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAG TAACTGGCCTTTATTTGACGGTAATTACTTAGAAAGTCACGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTAT TGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTT CGGCTCAACCGGAGAAT S12KG274 GGDK 266-1 519F SEQ ID NO: 41 TCCGGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGAT GTGAAAGCCTTCGGCTCAACCGGAGAATTGCATCAGAAACTGTTGAACTT GAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAG ATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGA CGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAG TCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCA GTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAG GTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGAAGCAACGGGAAGAACCTTACCAGGTCTTGACATCCAGT GCAAACCTAAGAGATTAGGTGTTCCCTTCGGGGACGCTGAGACAGGTGGT GCATGGCTGTCGTCAGCTCGTGT S12KG275 GGDK 226-1 926F-repeat SEQ ID NO: 42 GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTG ACATCCAGTGCAAACCTAAGAGATTAGGTGTGTCCCTTCGGGGACGCTGA GACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGG GCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG TCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CGGTACAACGAGAAGCGAACCTGCGAAGGCAAGCGGATCTCTTAAAGCCG TTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAATCG CTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGT ACACACCGCCCGTCACACCATGAGAGTCTGTA S12KG276 GGDK 255-1 926R SEQ ID NO: 43 ACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGA AACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTA TCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGA CCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCC ACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTTCAACAG TTTCTGATGCAATTCTCCGGTTGAGCCGAAGGCTTTCACATCAGACTTAT TGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCC ACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTAA GTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTA CCAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCT CCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACT CGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAAT GCACCGCAGGTCCATCCAAGAGTGATAGCAGAACCATCTTTCAAACTCTA GCACATGCGTCTAGTGTTGT S12KG277 GGDK 266-1 519R SEQ ID NO: 44 ACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTT TCTTCACTACCAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGC GGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTG CCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGT CTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAA CTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGCAGAACCATCTTT CAAACTCTAGACATGCGTCTAGTGTTGTTATCCGGTATTAGCATCTGTTT CCAGGTGTTATCCCAGTCTCTTGGGCAGGTTACCCACGTGTTACTCACCC GTCCGCCGCTCGCTTGTATCTAGTTTCATTTAGTGCAAGCACTAAAATCA TCTAGGCAAGCTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTC GTCCTGAGCCATGATCAAACT S12KG278 GGDK 266-1 RP2 SEQ ID NO: 45 CTACCTTAGACGGCTGACTCCTATAAAGGTTATCCCACCGGCTTTGGGTG TTACAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT ATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGT AGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATCCG CTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATTGTAGCACGTGT GTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCC TCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCA ACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG ACACGAGCTGACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGG AACACCTAATCTCTTAGGTTTGCACTGGATGTCAAGACCTGGTAAGGTTC TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC CGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACACCCCAGGCGGAGT GCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTA GCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCT ACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCT
NCIMB 41850 GGDK 266—contains both Lactobacillus iohnsonii and Lactobacillus reuteri Lactobacillus reuteri

TABLE-US-00023 S12KG279 GGDK-266-2 27F SEQ ID NO: 46 GTGTGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGT GCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGT AACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGAT GCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGG CTTCGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAA GGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATC GGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGT AGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAG TGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCG TGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAA AGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTG CAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTA S12KG280 GGDK-266-2 519F-repeat SEQ ID NO: 47 CGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGT GAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGA GTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGAT ATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACG CTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTC CATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGT GCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGT TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCG S12KG281 GGDK-266-2 926F-repeat SEQ ID NO: 48 GAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA CATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGA CAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG TCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGC ACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTC AGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACG GTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTT CTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCT AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTAC ACACCGCCCGTCACACC S12KG282 GGCK-266-2 926R-repeat SEQ ID NO: 49 ACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGA AACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTA TCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGA CCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCC ACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGG TTTCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTAA GCAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCC ACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGG TTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCA ACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCT CCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAACT CGGCTATGCATCATCGCTTGGTAACCGTTCACCTTACCAACTAGCTAATG CACCGCAGGT S12KG283 GGDK-266-2 591R SEQ ID NO: 50 TTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTC TTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGG TGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCC TCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCT CTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACT AGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTC AAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTC CAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCACCCG TCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAAT CAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCGT TCATCCTGAGCCATGATCAAACTCT S12KG284 GGDK-266-2 RP2 SEQ ID NO: 51 TCCCGCCTTAGGCGGCTCCCTCCATAATGGTTAGGCCACCGACTTTGGGC GTTACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACG TATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTG TAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTA GCTTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTG TGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTC CTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGC AACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC GACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGG GAACGCCTTATCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTT CTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCC CCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAG TGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCT AGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGC TACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTT CGCCACTGGTG
NCIMB 41850 GGDK 266—contains both Lactobacillus iohnsonii and Lactobacillus reuteri Lactobacillus reuteri

TABLE-US-00024 S12KG381 27F SEQ ID NO: 52 GTGTCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACG GGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTT TTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGC ATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGG GCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACT GTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATT TATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTT GAGTGC S12KG382 519F SEQ ID NO: 53 TTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAA CCGGGCAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGG CTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGC TAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGG AATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACC TTAGAGATAAGGCGTCCCTACGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTC A S12KG383 926F SEQ ID NO: 54 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCG GGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAG TTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGAC CTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTG TAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCATTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGA AGTCGTAACAAGGTAGCCGTA S12KG384 926R SEQ ID NO: 55 TACTCCCCAGGCGGAGTGCTTAATGCGTGAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACT ACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCC ATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGGTTTCCGATGCACTTCTTCGGTTAAG CCGAAGGCTTTCACATCAGACCTAAGCAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCG GCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAACAACAGAG CTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAG GAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACT AGCTATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAAACAAAAGCC S12KG385 519R SEQ ID NO: 56 GTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTGCTTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTC TTCACTCACGCGGTGTTGCTCCATCAGGCTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGTT CCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCAT CCCAGAGTGATAGCCAAAGCCATCTTTCAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCC CGCTCCGGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAATCAGTTG GGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCGTTCATCCTGAGCCATGATCAAAC S12KG386 RP2 SEQ ID NO: 57 TCCCGCACTTAGGCGGCTCCCTCCATAATGGTTAGGCCACCGACTTTGGGCGTTACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGC CCGGGAAGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAA CGGCTTTAAGAGATTAGCTTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA TGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTG CGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTA TCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAACCACATGCTCCACCGCTTGTGCGGGCCCCCG TCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAA CACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCC NCIMB 42008 GGDK266a-L. johnsonii (sample 4a) S12KG399 27F SEQ ID NO: 58 GCGTGCCTAATACATGCAAGTCGAGCGAGCTTGCCTAGATGATTTTAGTGCTTGCACTAAATGAAACTAGATACAAGCGAGCGGCGGACGGG TGAGTAACACGTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAACAGATGCTAATACCGGATAACAACACTAGACGCATGTCTAGA GTTTGAAAGATGGTTCTGCTATCCACTCTTGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAATGATGCAT AGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGAC GAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAGTAACTGG CCTTTATTTGACGGTAATTACTTAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTA TTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTCGGCTCAACCGGAGAATTGCATCAGAAACTGTTGAACTTGA GTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTA S12KG400 519F SEQ ID NO: 59 TGTCCGGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTCGGCTCAACCGGAGAATTGCATCAGAAAC TGTTGAACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGC TCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCT AAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGA ATTGACGGGGGCCCGCACAAGCGGTGGGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTGCAAACCTA AGAGATTAGGTGTTCCCTTCGGGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT S12KG401 926F SEQ ID NO: 60 GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTGCAAACCTAAGAGATTAGGTGTTCCCTTCG GGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAG TTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCTTATGACC TGGGCTACACACGTGCTACAATGGACGGTACAACGAGAAGCGAACCTGCGAAGGCAAGCGGATCTCTTAAAGCCGTTCTCAGTTCGGACTGT AGGCTGCAACTCGCCTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCATGAGAGTCTGTAACACCCAAAGCCGGTGGGATAACCTTTATAGGAGTCAGCCGTCTAAGGTAGGACAGATGATTAGGGTGA AGTCGTAACAAGGTAG S12KG402 926R SEQ ID NO: 61 TACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACT ACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCC ATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTTCAACAGTTCTGATGCAATTCTCCGGTTGAGC CGAAGGCTTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGG CTGCTGGCACGTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCG CGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAACAG AGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCG TAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAA CTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGACAGAACCATCTTTCAAACTCTAGACATGCGTCTAGTG S12KG403 519R SEQ ID NO: 62 GTGACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAACAGAGCTTTACGAGCCGAAACCCTTC TTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGT CCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCA TCCAAGAGTGATAGCAGAACCATCTTTCAAACTCTAGACATGCGTCTAGTGTTGTTATCCGGTATTAGCATCTGTTTCCAGGTGTTATCCCA GTCTCTTGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTCGCTTGTATCTAGTTTCATTTAGTGCAAGCACTAAAATCATCTAGGC AAGCTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCA S12KS404 RP2 SEQ ID NO: 63 TCCTACACTTAGACGGCTGACTCCTATAAAGGTTATCCCACCGGCTTTGGGTGTTACAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGG CCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGA ACGGCTTTAAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATG ATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCAACTAATGACAAGGGTT GCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACACCTA ATCTCTTAGGTTTGCACTGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC CGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCC AACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGC MCIMB 42009 GGDK286h-L. reuteri (sample 6e) S12KG411 37F SEQ ID NO: 64 GTGTGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGAC GGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCT TTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATG CATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGAGGCAGCAGTAGGGAATCTTCCACAATGG GCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACT GTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGT S12KG412 519F SEQ ID NO: 65 TATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAAC CGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGG CTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGC TAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGG AATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACC TTAGAGATAAGGCGTTCCCTTCGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGA S12KG413 926F SEQ ID NO: 66 GTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGG GGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGT TGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACC TGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTTCAGTTCGGACTG TAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCATTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGA AGTCGT S12KG414 926R SEQ ID NO: 67 TACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACT ACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCC ATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGGTTTCCGATGCACTTCTTCGGTTAAG CCGAAGGCTTTCACATCAGACCTAAGCAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCG GCTGCTGGCACGTAGTTAGCCGTGACTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAACAACAGAGC TTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGG AGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCC S12KG415 519R SEQ ID NO: 68 GTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTC TTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCGTAGGAGTATGGACCGTGTCTCAGTT CCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCAT CCCAGAGTGATAGCCAAAGCCATCTTTCAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCC CGCTCCGGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCAATCAGTTG GCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCCGGCGTTCAT S12KG416 RP2 SEQ ID NO: 69 TCCCGCCTTAGGCGGCTCCCTCCATAATGGTTAGGCCACCGACTTTGGGCGTTACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCC CGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAA CGGCTTTAAGAGATTAGCTTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA TGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTG CGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTA TCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCTTTGAGTTTCAACCTTGCGGTCGTACTCC NCMIMB 42010 GGDK161a-L. plantarium (sample 7a) SEQ ID NO: 70 GTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAAC ACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAA GATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGAGGTAACGGCTCACCATGGCAATGATACGTAGCCGACC TGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCT GATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTAT TGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGT AAAGCGAGCGCAGGCGGTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAG AGGACAGTGGAACTCATGTGT S12KG418 519F SEQ ID NO: 71 TCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTG GGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTG TCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAA GTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAAT TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAA GAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA ATCATCATGCCCCTTATGACCTGGGCTACACAC S12KG419 926F SEQ ID NO: 72 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCG GGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAG TTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGAC CTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTG TAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCACCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTACGGTGA AGTCGTAACAAGGTAGCCCGTA S12KG420 526R SEQ ID NO: 73 GTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGAC TACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTC CATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGA GCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGC GGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGA GTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCTCACCATC TAGCTAATACGCCGCGGGACCATCCAAAAGTGATA S12KG421 519R SEQ ID NO: 74 TGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCT TCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTC CCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCTCACCATCTAGCTAATACGCCGCGGGACCAT CTAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCC CGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTC GTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCATGATCAAACTCTA S12KG422 RP2 SEQ ID NO: 75 ACTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGA ACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCT TTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGATTGCGCTC GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCT TAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT TCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCA
NCIMB 42011 GGDK161b—L.reuteri (sample 11a)

TABLE-US-00025 S12KG441 27F SEQ ID NO: 76 TAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCA CCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGT AGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATA CCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGC TATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACG GCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACA ATGGAACTGAGACACGGTCCATACTCCTACGGGAGGGCAGCAGTAGGGAA TCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGA AGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAG TAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTGATCCGGATTTAT TGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTT CGGCTTAACCGAAGAAGTGCATCGGAGACGGGCGACTTGAGTGCA S12KG442 519F SEQ ID NO: 77 TTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCT GATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGA CTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCG TAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAAC TGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGG TAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCT TCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGC AAGGTTGAAACGCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCT TGCGCTAACCTTANAAGGCGTCCCCTTCGGGGACTCAATGACAGGTGGTG CATGGTT S12KG443 926F SEQ ID NO: 78 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTC TTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCA ATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTT GGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGA CGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG GACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATYCTCTTAAAG CCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAA TCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCT TGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGT GGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGG TGAAGTCGTAACAAGGTAG S12KG444 926R SEQ ID NO: 79 No results S12KG445 519R SEQ ID NO: 80 GTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCA CGTTCTTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCA CGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTG CTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATC AGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTAC CAACTAGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCAT CTTTCAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCT GTTTCCAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTC ACCCGTCCGCCACTCACTGGTAATCCATCGTCAATCAGGTGCAAGCACCA TCAATCAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCC GGCGTTCATCCTGAGCCA S12KG446 RP2 SEQ ID NO: 81 CTCCCTCCATAAAGGTTAGGCCACCGACTTTGGGCGTTACAAACTCCCAT GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCAT GCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGC CTACAGTCCGAACTGAGAACGGCTTTAAGAGATTAGCTTACTCTCGCGAG CTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCAT AAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACC GGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTACTAACAAGGG TTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGA CGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTATCTCT AAGGTTAGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCG AATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTG AGTTTCAACCTTGGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTA GCTCCGGCACTGAAGGGCGGAA
NCIMB 42012 GGDKZ66c—L.reuteri (sample 1a)

TABLE-US-00026 512KG381 27F SEQ ID NO: 82 GTGTGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGT GCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGT AACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGAT GCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGG CTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAA GGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATC GGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGT AGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAG TGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCG TGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAA AGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTG C S12KG382 519F SEQ ID NO: 83 TTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCT GATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCAA CTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCG TAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAAC TGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGG TAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCT TCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGC AAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCT TGCGCTAACCTTAGAGATAAGGCGTCCCTTCGGGGACGCAATGACAGGTG GTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTA GTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGAGGGACGACGTCA S12KG383 926F SEQ ID NO: 84 GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTC TTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCA ATGACAGGTGGTGCATGGTCGTCGTCAGCTGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTG GGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGAC GTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG ACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCC GTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATC GCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTG TACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGG CCTAACCATTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTG AAGTCGTAACAAGGTAGCCGTA S12KG384 928R SEQ ID NO: 85 TACTCCCCAGGCGGAGTGCTTAATGCGTGAGCTCCGGCACTGAAGGGCGG AAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGT ATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAG ACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTC CACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCG GTTTCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTA AGCAACCGCCTGCCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTG CCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCT GGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTC CAACAACAGAGCTTTACTGAGCCGAAACCCTTCTTCACTCACGCGGTGTT GCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCC GTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTCT CAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAG CTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCAA ACAAAAGCC S12KG385 519R SEQ ID NO: 86 GTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCA CGTGCTTCTCCAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCA CGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTG CTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATC AGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTAC CAACTAGCTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCAT CTTTCAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCT GTTTCCAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTC ACCCGTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCA TCAATCAGTTGGGCCAGTGCGTACGACTTGCATGTATTAGGCACACCGCC GGCGTTCATCCTGAGCCATGATCAAAC S12KG386 RP2 SEQ ID NO: 87 TCCCGCACTTAGGCGGCTCCCTCCATAATGGTTAGGCCACCGACTTTGGG CGTTACAAACTCCCATGGTGTGACGGCGGTGTGTACAAGGCCCGGGAACG TATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTG TAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTA GCTTACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTG TGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTC CTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGC AACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC GACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGG GAACGCCTTATCTCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAAGG TTCTTCGCGTAACTTCGAATTAAACCACATGTCCACCGCTTGTGCGGGCC CCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGA GTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACC TAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCG CTACCCATGCTTTCGAGCC

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