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Definitions

• the present invention refers to new strains of Bifidobacteru having the ability to produce glutamine and optionally arginine in vivo . 5 Background of the invention
• Glutamine is the most abundant amino acid in the body. It is a "nitrogen shuttle" between tissues and a fuel for enterocytes, colonocytes, lymphocytes and proliferating cells. The function of the gut is impaired in
• Glutamine depletion occurs in critically ill and injured patients, and may contribute to
• glutamine is easily conver- ted to glutamic acid (glutamate), i.e. glutamine is a relatively unstable compound that is difficult to incorporate into formulas intended for oral administration. Furthermore, the orally administered glutamine will in the sour environment of the stomach to a high degree be converted into glutamic acid and never reach the intestine and be absorbed as glutamine.
• Arginine enhances the immune function and promotes wound healing.
• Administration of arginine has been used in postoperative patients and patients under intensive care. In most clinical studies arginine has been administered together with other substances such as RNA and fish oil . There are indications that administration of arginine modulates post-operative immune response. Daly, John E., et al . , Surgery 112:55-67 , 1992 show that en- teral nutrition with supplemental arginine, RNA and omega-3-fatty acids in patients after operation improves the immune defence through different mechanisms. Arginine reduces complications in patients undergoing chemoradia- tion and surgery (Tepaske et al . , 2001; -Lancet 358: 696-
• Bifidobacterium spp. that is bifido- bacteria
• Bacteria from the Bifidobacterium spp. are regarded as probiotics, that is live bacteria that upon ingestion provide health beneficial effects to the host.
• High numbers of Bifidobacterium spp. in colon have been claimed to have health beneficial effects.
• the course of action of said beneficial effects is largely unknown.
• WO 01/83700 discloses a composition and method for treating and preventing gastro-intestinal injury, neonatal necrotising entero- colitis (NEC) and bacterial sepsis.
• the composition includes a combination of Gram (+) bacteria, in particular Lactobacillus and Bifidobacterium, and glutamine, and should be orally or naso-orally administrated.
• the com- position is said to block translocation of bacterial agents such as Gram (-) bacteria.
• Glutamine administered intravenously is very efficient but expensive and complicated.
• a better way to administer glutamine would of course be to use a bacterial strain having the ability to produce substantial amounts of glutamine in the intestines. Until now, however, no such strains have been described. Summary of the invention
• strains of Bifidobacterium are capable of producing glutamine in a growth medium mimicking the environment in the human colon. Said strains can thus be used for producing glutamine in vivo after oral or enteral administration to a mammal, especially a human. Some of the strains are also capable of producing arginine.
• Figure 1 is a schematic representation of the elec- trophoretic patterns obtained with Restriction Fragment Length Polymorphism (RFLP) for the strains CURE 19, CURE 21, CURE 26, CURE 28, and CURE 29, obtained by cleaving chromosomal DNA with EcoRI and Hind III, respectively, followed by hybridisation with a DIG-labelled 420 bp fragment probe (position 506 to 926, E. coli numbering) of the 16S rRNA gene of L . casei subsp . pseudopl ant arum
• RFLP Restriction Fragment Length Polymorphism
• FIG. 1 shows a photograph of separated DNA fragments obtained by cleaving chromosomal DNA of strains CURE 19 (lane 1) , CURE 29 (lane 2) and CURE 28 (lane 3) with the restriction enzyme EcoRI (Restriction Endonuc- lease Analysis, REA) .
• High Molecular Weight DNA marker (BRL) and DNA molecular weight marker VI (Roche) were used as standard (lane 4) .
• Figure 3 shows a photograph of separated DNA fragments obtained by cleaving chromosomal DNA of strains CURE 19 (lane 2), CURE 28 (lane 3) and CURE 29 (lane 4) with the restriction enzyme Hind III. High Molecular
• Figure 4 shows a photograph of separated DNA fragments obtained by cleaving chromosomal DNA of strains CURE 21 (lane 1) and CURE 26 (lane 2) with the restriction enzyme EcoRI. High Molecular Weight DNA marker (MD, USA) and DNA molecular weight marker VI (Roche) were used as standard (lane 3) .
• Figure 5 shows a photograph of separated DNA fragments obtained by cleaving chromosomal DNA of strains CURE 21 (lane 2) and CURE 26 (lane 3) with the restriction enzyme Hind III .
• High Molecular Weight DNA marker (MD, USA) and DNA molecular weight marker VI (Roche) were used as standards (lanes 1 and 4) .
• the invention refers to a strain of Bifidobacterium having the ability to survive in the intestinal tract and to produce glutamine in vivo . Especially glutamine is produced in the human colon.
• the bifidobacterial strains of the invention can grow on nutrient media having a pH below 7, especially 5.5-6.5. Rogosa agar is one example of such a medium, another is MRS.
• the new strains also have the ability to assimilate ammonia.
• the invention especially refers to strains belonging to the species Bifidobacterium infantis .
• the invention also refers to a strain of Bifidobacterium which has the 16S rRNA genes located on single DNA fragments with molecular sizes of about 2840 kb, obtained by cleaving chromosomal DNA with Hind III, followed by separation of fragments by agarose gel electrophoresis, and by hybridisation with a DIG-labelled 420 bp fragment probe (position 506 to 926, E. coli numbering) of the 16S rRNA gene of L . casei subsp. pseudoplantarum DSM 20008 using Southern blot hybridisation.
• a strain of Bifidobacterium which has the 16S rRNA genes located on single DNA fragments with molecular sizes of about 2840 kb, obtained by cleaving chromosomal DNA with Hind III, followed by separation of fragments by agarose gel electrophoresis, and by hybridisation with a DIG-labelled 420 bp fragment probe (position 506 to 926, E. coli numbering)
• Such strains are, for example, the strains, deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 23, 2002, Bifidobacterium infantis CURE 19 (DSM 15158) ; Bifidobacterium infantis CURE 21 (DSM 15159) ; Bifidobacterium infantis CURE 26 (DSM 15160) ; Bifidobacterium infantis CURE 28 (DSM 15161) ; Bifidobacterium infantis CURE 29 (DSM 15162) .
• the invention also refers to a strain of Bifidobacterium which has the 16S rRNA genes located on single DNA fragments with molecular sizes of about 895 kb, obtained by cleaving chromosomal DNA with EcoRI, followed by separation of fragments by agarose gel electro-phoresis, and by hybridisation with a DIG-labelled 420 bp fragment probe (position 506 to 926, E. coli numbering) of the 16S rRNA gene of L . casei subsp. pseudoplantarum DSM 20008 using Southern blot hyb- ridisation.
• said strain is able to produce glutamine without reducing glutamic acid.
• the invention especially refers to the strains Bifi dobacterium infantis CURE 21 and Bifidobacterium infantis CURE 26, which were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 23, 2002 and were given the accession numbers DSM 15159 and DSM 15160, respectively, or variants thereof. Said strains are able to produce glutamine without reducing glutamic acid.
• the invention also refers to a strain of Bifidobacterium which has the 16S rRNA genes located on single DNA fragments with molecular sizes of about 3420 kb, obtained by cleaving chromosomal DNA with EcoRI, followed by separation of fragments by agarose gel electro-phoresis, and by hybridisation with a DIG-labelled 420 bp fragment probe (position 506 to 926, E. coli numbering) of the 16S rRNA gene of L . casei subsp. pseudoplantarum DSM 20008 using Southern blot hyb- ridisation.
• said strain has the ability to produce arginine.
• the invention also especially refers to the following strains, which have all been deposited at the Deut- sche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 23, 2002, and been given a deposition number, that is Bifidobacterium infantis CURE 19, DSM 15158, Bifidobacterium infantis CURE 28, DSM 15161, and Bifi - o-acterium infantis CURE 29, DSM 15162, and to variants thereof. Said strains are able to produce in addition to glutamine also arginine.
• the new strains have all been isolated from faeces from young children and selected by culturing on agar at a pH below 7.
• the strains have subsequently been characterised by ribotyping and REA.
• Another object of the invention is a composition comprising one or more strains of Bifidobacterium of the invention in combination with a carrier.
• carriers are oatmeal gruel, lactic acid fermented foods, inulin, lactulose, fructo-oligosaccharides, resistent starch, ⁇ -glucans and guar gum.
• Dietary fibres are for instance inulin, fructo-oligo-saccharides, maltodext- rins, ⁇ -glucans and guar gum.
• the invention thus also refers to a composition as described comprising in addition dietary fibres.
• compositions of the invention such as suspensions, tablets, capsulas, powders, can be administrated orally. They can also be administrated as an enema.
• the composition of the invention can be a food com- position wherein the carrier is a food product.
• the glutamine producing BifidoJbacterium-strains can be given to young children, elderly people, athletics and ordinary consumers that wish to keep-fit to improve muscle func- tion and avoid immune depression following exercise.
• the arginine producing Bifidobacterium-strains can be given to ordinary consumers that want to keep-fit and avoid negative influence on immune function.
• the composition of the invention can be a pharmaceutical composition, wherein the carrier is a therapeu- tically acceptable carrier.
• the glutamine producing strains, as well as the arginine producing strains can in addition be used in formulas for enteral feeding.
• the ammonia assimilating Bifidobacterium strains of the invention can be given to patients with temporary kidney failure such as seen in patients under intensive care following surgery and complications or following other diseases, such as severe infections, intoxications. In such cases the kidney function can be expected to return and a treatment aimed at reducing the nitrogen load from the gut may avoid the need for dialysis.
• the ammonia assimilating strains can be administered to patients with liver failure and encephalopathy, e.g. in hepatitis or intoxication or following alcohol abuse. A reduced absorption of nitrogenous substances from the gut will in those situations improve encephalopathy and liver function.
• the ammonia assimilating Bifidobacterium strains can be given to young children, elderly people or con- sumers with underlying diseases that hamper their liver capacity for the conversion of ammonia to urea or having an increased absorption of nitrogen from the gut, e.g. patients with chronic renal or liver failure of small to moderate degree not yet in need of transplantation or dialysis.
• composition of the invention can also comprise one or more Lactobacillus strains .
• said bacteria can protect the bifidobacteria from the harmful influence of oxygen.
• the invention refers to one or more strains belonging to the species Bifidobacterium infantis for use in therapy.
• the invention refers to the use of one or more of the strains Bifidobacterium infantis CURE 19, DSM 15158; Bifidobacterium infantis CURE 21, DSM 15159; Bifidobac- terium infantis CURE 26, DSM 15160; Bifidobacterium infantis CURE 28, DSM 15161; Bifidobacterium infantis CURE 29, DSM 15162; or a variant thereof for the preparation of a medicament for treatment of intensive care patients with multiple organ dysfunction and intestinal failure, for prophylaxis in chemotherapy patients and patients with inflammatory diseases and postoperative administration after major surgery.
• Experimental Isolation of Strains All strains were isolated from faeces of young children, age one week to one year.
• the faeces samples were serial diluted in a dilution solution (0.9% [w/v] NaCl, 0.1% [w/v] peptone, 0.1% [w/v] Tween 80, 0.02% [w/v] cystein-HCl) and spread on Rogosa agar plates. Isolates were selected with respect to their ability to grow on Rogosa agar, pH 5.4, and repeated isolation from one individual. The isolates were picked from the Rogosa agar plates after incubation at 37°C for 72 hours. They were identified to genus level by genus specific PCR (Roy et al .
• strains could be isolated at least twice from the same individual with one to four weeks between the samplings, which strongly indicate that the strains had a certain ability to colonise the GI-tract.
• the strains were identified by ribotyping, that is Restriction Frag- ment Length Polymorphism, RFLP, of the 16S rRNA gene, and by REA, that is Restriction Endonuclease Analysis.
• Bifidobacterium infantis CURE 19 able to grow on Rogosa agar and to ferment oatmeal gruel to some degree, "sour" smell after fermentation.
• Bifidobacterium CURE 20 able to grow on Rogosa agar and to ferment oatmeal gruel to some degree, "sour" smell after fermentation.
• Bifidobacterium CURE 22 able to grow on Rogosa agar and to ferment oatmeal gruel to some degree, "nice" smell after fermentation.
• Bifidobacterium CURE 23 able to grow on Rogosa agar and to ferment oatmeal gruel to some degree, "nice" smell after fermentation.
• Bifidobacterium infantis CURE 24 able to grow on Rogosa agar and to ferment oatmeal gruel to some degree, "nice" smell after fermentation.
• Bifidobacterium CURE 25 able to grow on Rogosa agar and to ferment oatmeal gruel .
• Bifidobacterium infanti s CURE 26 ; able to grow on Rogosa agar and to ferment oatmeal gruel .
• Bifidobacterium dentium CURE 27 ; able to grow on Rogosa agar and to ferment oatmeal gruel .
• Bifidobacterium infantis CURE 28 ; able to grow on Rogosa agar and to ferment oatmeal gruel .
• Bifidobacterium infantis CURE 29 ; able to grow on
• the isolated strains 1-12 were tested for production of glutamine in broth by the following procedure.
• test strains were cultured at 37 °C for 4 days in a growth medium (broth) modified from the medium descri- bed by Matteuzzi et al . (Ann. Microbil. (Inst. Pasteur, 1978, 129 B: 175-181).
• the broth was composed of: sodium acetate, 10 g/1; ascorbic acid, 10 g/1; ammonium sulphate ((NH ) 2 S0 4 ), 5 g/1; dipotassium hydrogen phosphate (K 2 HP0 4 ) , 3 g/1; potassium di-hydrogen phosphate (KH 2 P0 4 ) , 3 g/1; MgS0 4 x 7H 2 0, 0.32 g/1; FeS0 4 x 7H 2 0, 0.01 g/1;
• MnS0 4 H 2 0, 0.007 g/1; NaCl, 0.01 g/1; yeast extract, 0.5 g/1; glucose, 20 g/1; Tween 80, 1 ml/1. pH was adjusted with 1 M NaOH to 6.18-6.24 prior to autoclavation.
• the glutamine concentration in the broth was mea- sured before inoculation with bacteria and after growth of the test strain. After growth, the culture was centrifuged and sterile filtered, and the cell-free supernatant subsequently frozen at -80 °C.
• the amino acids were analysed in an automated analyser (Biochrom 20, Pharmacia Biotech) after addition of sulpho-salicylic acid and pH adjustment with lithiumhydroxide.
• the amino acid production of each test strain was measured after growth of the test strain at 37 °C for 4 days in a growth medium (broth) modified from the medium described by Matteuzzi et al . (1978) .
• the broth was composed of: sodium acetate, 10 g/1; ascorbic acid, 10 g/1; ammonium sulphate ((NH 4 ) 2 S0 4 ), 5 g/1; dipotassium hydrogen phosphate (K 2 HP0 4 ) , 3 g/1; potassium dihydrogen phosphate (KH 2 P0 4 ) , 3 g/1; MgS0 4 x 7H 2 0, 0.32 g/1; FeS0 4 x 7H 2 0, 0.01 g/1; MnS0 4 H 2 0, 0.007 g/1; NaCl, 0.01 g/1; yeast extract, 0.5 g/1; glucose, 20 g/1; Tween 80, 1 ml/1.
• CURE 21 did not produce any citrulline or arginine but significant amounts of asparagine acid and tyrosine.
• CURE 26 did not produce any citrulline but very small amounts of arginine and a wide spectrum of different amino acids .
• CURE 26 was the only test strain that increased the concentration of proline in the broth. All test strains produced threonine .
• CURE 29 35 28 0 0 2 0 0 Thr -threonine; Tyr-tyrosine; Cys-cysteine; Asp-asparagine acid; Ala-alanine; Gly-glycine; Ile-isoleucine
• the values represent mean values of three or two samples derived from three or two separate cultures .
• the strains were examined as to the cleavage pattern of the chromosomal DNA, through restriction- endonuclease analysis - REA - method according to Stahl M, Molin G, Persson A, Ahrne S & Stahl S, International
• Schematically REA can be described as follows: Chromo- somal DNA from the strains involved in the study were prepared and cleaved by restriction endonucleases . 0.75 ⁇ g of each DNA was separately digested at 37°C for 4 h with 10 units of EcoRI and Hind III; each endonuclease was used separately. The cleaved DNA fragments are separated as to size by gel electrophoresis using submerged horizontal agarose slab gels.
• the gels consisted of 150 ml of 0.9 % agarose (ultrapure DNA grade; low electro- endo osmosis; BioRad Laboratories, Richmond, USA) and were cast as slab gels (150 by 235 mm) .
• 0.2 ⁇ g of the High Molecular Weight DNA marker (Bethesda Research Laboratories, MD, USA) together with 0.5 ⁇ g of a DNA molecular weight marker VI (Roche, Germany) were used as standards.
• Minimal band distortion and maximal sharpness were achieved by applying the sample DNA in Ficoll loading buffer (2g of Ficoll, 8 ml of water, 0.25% brom- phenol) .
• the probe was a 420 bp fragment (position 506 to 926, E. coli numbering) of the 16S rRNA gene of L. casei ssp. pseudoplantarum DSM 20008, obtained by PCR and labelled by DIG DNA labelling technique according to the instructions supplied by the manufacturer (Boehringer Mannheim, Bromma, Sweden) .
• the amount of probe used in the reactions was 50 ng.
• the test resulted in one single band with a molecular weight of about 2840 kb in for all strains (CURE 19, 21, 26, 28 and 29) when chromosomal DNA was cleaved by Hind III.
• chromosomal DNA was cleaved by EcoRI, the genes for 16S rRNA ended up on a single fragment with the molecular weight of about 895 kb in strains CURE 21 and 26, while another single band with a molecular weight of about 3420 kb was obtained for strains CURE 19, 28 and 29.
• Test 1 Effects of Lactobacillus and Bifidobacterium strains on DPS induced colitis in rat
• DDS Extran Sodium Sulfate
• Sprague Dawley rats were divided into six groups, one control group (colitis without administration of bacteria) , and five groups to which different bacterial strains ⁇ Lactobacillus plantarum 299v, Lactobacillus paracasei 8700:2, Lactobacillus gasseri LG1 , Bifidobacterium 3B1 and Bifidobacterium infantis CURE 19, respectively) were administered.
• the bacterial strains were administered orally for 7 days prior to the induction of colitis (day 0) and were continuously adminis- tered for 7 days in combination with DDS (5% w/v dissolved in water) .
• the degree of colitis were determined daily with DAI (Disease Activity Index) . Sampling was performed day 14 and bacterial translocation and quantity in the intestine determined.
• DAI Disease Activity Index
• infantis CURE 19 compared to the groups receiving Lactobacillus paracasei 8700:2 and Lactobacillus gasseri LG1 after 6 and 7 days.
• the bacterial translocation to the mesenteric lymph nodes decreased significantly in all groups as well as the translocation of Enterobacte- riaceae to the liver.
• Test 2 Survival of Lactobacillus and/or Bifidobacterium in the GI -tract after oral administration
• Lactobacillus or Bifidobacterium provides different positive effects, e.g. pre- venting antibiotic associated diarrhoea (D'Souza et al . , 2002, BMJ 324:1361)

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