as) United States
`a2) Patent Application Publication 10) Pub. No.: US 2007/0258953 Al
`
` Duncanetal. (43) Pub. Date: Nov.8, 2007
`
`
`US 20070258953A1
`
`(54) LACTIC ACID UTILISING BACTERIA AND
`THEIR THERAPEUTIC USE
`
`(30)
`
`Foreign Application Priority Data
`
`(76)
`
`Inventors: Sylvia Helen Duncan, Aberdeen (GB);
`Harry James Flint, Aberdeenshire
`(GB)
`
`Correspondence Address:
`DRINKER BIDDLE & REATH
`ATTN: INTELLECTUAL PROPERTY GROUP
`ONE LOGAN SQUARE
`18TH AND CHERRY STREETS
`PHILADELPHIA, PA 19103-6996 (US)
`(21) Appl. No.:
`10/550,662
`(22) PCT Filed:
`Mar. 29, 2004
`
`(86) PCT No::
`
`PCT/GB04/01398
`
`§ 371(€)(),
`(2), (4) Date: Nov. 9, 2005
`
`Mar. 27, 2003
`
`(GB) wes ceeesssesssesssesseensceneeenee 0307026.5
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`(2006.01)
`AG6LK 35/74
`(2006.01)
`CI2N 1/20
`(52) US. Che
`cicceccccceccsssstescsscssssssneeeeee 424/93.4; 435/252.1
`
`ABSTRACT
`67)
`There is provided a method of isolating novel lactic acid
`utilizing bacteria from human faeces, as well as novelstrains
`so obtained. The use ofthe novellactic acid utilising bacteria
`in therapy, including prophylactic therapy, is described and
`is of particular relevance for lactic-acidosis, short bowel
`syndrome and inflammatory bowel disorders
`such as
`Crohn’s disease and ulcerative colitis. A probiotic compris-
`ing the live lactic acid utilising bacteria is also described.
`
`GenomeEx. 1018
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`

`Patent Application Publication Nov. 8, 2007 Sheet 1 of 13
`
`US 2007/0258953 Al
`
`Figure 1
`
`Sequence information for five of the lactate utilising
`
`strains.
`
`S D6 1L/1
`
`GATGAACGCTGGCGGCGTGCCTAACACTGCAAGTCGAACGAAGCACCTTACCTGATTCTTCGGATGAA
`
`GGTCTGGTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCTGTACAGGGGGATAACA
`
`GTTGGAAACGGCTGCTAATACCGCATAAGCGCACGAGAGGACATCCTCTTGTGTGAAAAACTCCGGTG
`
`GTACAGGATGGGCCCGCGTCTGATTAGCTGGTTGGCAGGGTAACGGCCTACCAAGGCGACGATCAGTA
`
`GCCGGTCTGAGAGGATGAACGGCCACATTGGAACTGAGACACGGTCCAACTCATACGGGAGGCAGCAG
`
`TGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCGTGAGTGAAGAAGTATTTCGGT
`
`ATGTAAAGCTCTATCAGCAGGGAAGATAATGACGGTACCTGACTAAGARAGCT CCGGCTAAATACGTGC
`
`CAGCAGCCGCGGTAATACGTATGGAGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGTIGCGTAGGT
`
`GGCAGTGCAAGTCAGATGTGAAAGGCCGGGGCTCAACCCCGGAGCTGCATTTGAAACTGCATAGCTAG
`
`AGTACAGGAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAG
`
`TGGCGAAGGCEGCCTGCTGGACTGTTACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTA
`
`GATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGCCGTATAGGCTTCGGTGCCG
`TCGCAAACGCAGTAAGTATTCCACCTGGG
`
`GAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATG
`TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTCTGACCACTCCGTA
`ATGGGAGTCTTCCTTCGGGACAGBAGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTG
`
`AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCAGTAGCCAGCAGGTAAGGCTG
`
`GGCACTCTGGAGAGACTGCCAGGGATAACCTGGAGGAAGGTGGGGACGACGTCAAATCATCATG
`
`CCCCTTATGATCTGGGCGACACACGTGCTACAATGGCGGTCACAAAGTGAGGCGAACCTGCGAG
`
`GGGGAGCAAACCACAAARAGGCCETCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAG
`
`CTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACA
`
`CCECCCGTCACACCATGGGAGTCGGAAATGCCCGAAGCCAGTGACCCAACCATATGGAGGGAGC
`TGTCGAAGGTGGAGCCGGTAACTGGGGTG
`
`SM 6/1
`
`GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCACCTTACGAGATTCTTCGGATGA
`
`TCGTTTGGTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCTGTACAGGGGGATAAC
`
`AGCTGGAAACGGCTGCTAATACCGCATAAGCGCACGAGGAGACATCTCCTAGTGTGAAAAACTCCGGT
`
`GGTACAGGATGGGCCCGCGTCTGATTAGCTGGTTGGCAGGGTAACGGCCTACCAAGGCAACGATCAGT
`
`AGCCGGTCTGAGAGGATGAACGGCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGAGGCAGC
`AGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCETGAGTGAAGAAGTATTICG
`GTATGTAAAGCTCTATCAGCAGGGAAGATAATGACGGTACCTGACTAAGAAGCTCCGGCTAAATACGT
`
`1
`
`2 3
`
`4
`
`5 6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`19
`
`20
`21
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`29
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`37
`38
`
`GenomeEx. 1018
`Page 2 of 28
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`

`

`Patent Application Publication Nov. 8, 2007 Sheet 2 of 13
`
`US 2007/0258953 Al
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`GCCAGCAGCCGCGGTAATAGATATGGAGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGTGCGTAG
`
`GTGGCAGTGCAAGTCAGATGTGAAAGGCCGGGGCTCAACCCCGGAGCTGCATTTGAAACTGCWYRGCT
`
`AGAGTACAGGAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACC
`
`AGTGGCGAAGGCGGCCTGCTGGACTGTTACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGAT
`
`TAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGCCGTATAGGCTCCGGTGC
`
`CGCCGCTAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCABRAGGAATTGAC
`
`GGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTT
`
`GACATCCTTCTGACCGCACCTTAATCGGTGCTTTCCTTCGGGACAGAAGAGACAGGTGGTGCATGGTT
`
`GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCAGTAGC
`
`CAGCAGGTAAGGCTGGGCACTCTGGAGAGACTGCCAGGGATAACCTGGAGGAAGGTGGGGACGACGTC
`
`AAATCATCATGCCCCTTATGATCTGGGCGACACACGTGCTACAATGGCGGTCACAGAGTGAGGCGAAC
`
`CCGCGAGGGGGAGCAAACCACAAAAAGGCCGTCCCAGTnCGGACTGTAGTCTGCAACCCGACTACACA
`
`GAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACA
`
`CCGCCCGTCACACCATGGGAGTCGGAAATGCCCGAAGCCAGTGACCCAACCTTTATGAAGGAAGCCnG
`
`TCCAAGGTTGAACCCGTTAACTGGGGnnTT
`
`Ss3/4
`
`GAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAGGT
`
`ATATTGAATTGAAGTTTTCGGATGGATTTCAATGATACCGAGTGGCGGACGGGTGAGTAACGCGTGGG
`
`TAACCTGCCTCATACAGGGGGATAACGGTTAGAAATGACTGCTAATACCGCATAAGCGCACA
`
`GTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAG
`
`TTGGTGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACA
`
`TTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAG
`
`GAAACTCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGC
`
`AGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTA
`
`ATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCGACGCAA
`
`GTCTGAAGTGAAATACCCGGGCTCAACCTGGGAACTGCTTTGGAAACTGTGTTGCTAGAGTGCT
`
`GGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTG
`
`GCGAAGGCGGCTTACTGGACAGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGAT
`
`TAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGTGAGCAAAGCTCATCG
`
`GTGCCGCCGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAG
`
`GAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAC
`
`CTTACCAAATCTTGACATCCCTCTGAAAARYCCYTTAATCGGRTITCCTCCTTCGGGACAGAGGT
`
`GACAGGTGGTGCATGGTTIGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG
`
`CGCAACCCCTATTGTCAGTAGCCAGCAGGTGAAGCTGGGCACTCTGATGAGACTGCCAGGGATA
`
`ACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTIATGATTTGGGCTACACACGTG
`
`CTACAATGGCGTAAACAAAGAGAAGCGAGCCTGCGAGGGGGAGCAAATCTCAAAAATAACGTCT
`
`CAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCAGATCAG
`
`GenomeEx. 1018
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`

`

`Patent Application Publication Nov. 8,2007 Sheet 3 of 13
`
`US 2007/0258953 Al
`
`AATGCTGCGGTGAATACGTTCCCGGETCTTGTACACACCGCCCGTCACACCATGGGAGTCGGAA
`
`ATGCCCGAAGCCAGTGAACCCAATGCGAAAGCAGGGAGCTGTCGAAGGCAGGTCTGATAACTGGGGTG
`
`Ss2/1 and Ssc/2
`
`AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGABA
`
`CACCTTATTTGATTTTCTTCGGAACTGAAGATTTGGTGATTGAGTGGCGGACGGGTGAGTAACG
`
`CGTGGGTAACCTGCCCTGTACAGGGGGATAACAGTCAGAAATGACTGCTAATACCGCATAAGAC
`
`CACAGCACCGCATGGTGCAGGGGTAAAAACTCCGGTGGTACAGGATGGACCCGCGTCTGATTAG
`
`CTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGTGAACGGC
`
`CACATTGGGACTGAGACACGGCCCARACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAAT
`
`GGGGGAAACCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATCTCGGTATGTAAAGCTCTAT
`
`CAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGC
`
`GGTAATACGTAGGGGGCAAGCGTTATCCGGAATTACTGGGTGTAAAGGGTGCGTAGGTGGTATG
`
`GCAAGTCAGAAGTGAAAACCCAGGGCTTAACTCTGGGACTGCTTTTGAAACTGTCAGACTGGAG
`
`TGCAGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACATC
`
`AGTGGCGAAGGCGGCTTACTGGACTGAAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACA
`
`GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGCCGTAGAGGC
`
`TTCGGTGCCGCAGCCAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAACTCA
`
`AAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGA
`
`ACCTTACCTGGTCTTGACATCCTTCTGACCGGTCCTTAACCGGACCTTTCCTTCGGGACAGGAG
`
`TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA
`
`GCGCAACCCCTATCTTTAGTAGCCAGCATATAAGGTGGGCACTCTAGAGAGACTGCCAGGGATA
`ACCTGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCAGGGCTACACACGTG
`
`CTACAATGGCGTAAACAGAGGGAAGCAGCCTCGTGAGAGTGAGCAAATCCCAAAAATAACGTCT
`
`CAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCGAATCAG
`
`AATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTA
`
`ACGCCCGAAGTCAGTGACCCAACCGTAAGGAGGAGCTGCCGAAGCGGGACCGATAACTGGGGTG
`
`AAGTCGTAACCAGGTAGCCGT
`
`W-=Aor?t
`YeTorec
`R=GoraA
`
`N = Unknown
`
`1
`
`2
`
`3 4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`121
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`30
`31
`32
`
`33
`
`34
`
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`

`Patent Application Publication Nov. 8,2007 Sheet 4 of 13
`
`US 2007/0258953 Al
`
`acetate/10
`
`butyrate
`
`L-lactate
`
`o~~
`
`o&; 2 i
`
`plusL2- 32
`L2-7
`
`SM6/‘1
`
`
`
`plusL2- 32 Co-cultureonstarch
`
`
`
`FSISESSNTISTIS
`
`
`L1-92plusL2-
`12-32
`
`oOo or Oo
`
`YW
`
`FT ON TO
`
`INW) UOd}Je1}UBDUOD
`
`32
`
`Fig.2
`
`GenomeEx. 1018
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`

`

`Patent Application Publication Nov. 8,2007 Sheet 5 of 13
`
`US 2007/0258953 A1
`
`oO_
`
`@D-lactate
`gformate
`f]acetate
`
`
`©85—
`
`!
`
`DLlactate
`
`RSG
`
`
`
`
`
`
`
`
`
`
`
`
`FEEeer stat
`
`WINE
`
`
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`
`SM6/1L2-7L4-92P2SR1/1$$2/1S$SC/2S$S3/4
`
`Strain
`
`SL6/1/1
`
`Fig.3a
`
`GenomeEx. 1018
`Page 6 of 28
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`Genome Ex. 1018
`Page 6 of 28
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`

`

`Patent Application Publication Nov. 8,2007 Sheet 6 of 13
`
`US 2007/0258953 Al
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`GenomeEx. 1018
`Page 7 of 28
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`Genome Ex. 1018
`Page 7 of 28
`
`

`

`Patent Application Publication Nov. 8, 2007 Sheet 7 of 13
`
`US 2007/0258953 A1
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`GenomeEx. 1018
`Page 8 of 28
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`Genome Ex. 1018
`Page 8 of 28
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`

`

`Patent Application Publication Nov. 8,2007 Sheet 8 of 13
`
`US 2007/0258953 A1
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`GenomeEx. 1018
`Page 9 of 28
`
`Genome Ex. 1018
`Page 9 of 28
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`

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`Patent Application Publication Nov. 8,2007 Sheet 9 of 13
`
`US 2007/0258953 Al
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`Page 10 of 28
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`Genome Ex. 1018
`Page 10 of 28
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`

`

`Patent Application Publication Nov. 8, 2007 Sheet 10 of 13
`
`US 2007/0258953 A1
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`Page 11 of 28
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`Genome Ex. 1018
`Page 11 of 28
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`

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`Patent Application Publication Nov. 8, 2007 Sheet 11 of 13
`
`US 2007/0258953 Al
`
`2s sis
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`
`GenomeEx. 1018
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`Genome Ex. 1018
`Page 12 of 28
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`

`

`Patent Application Publication Nov. 8, 2007 Sheet 12 of 13
`
`US 2007/0258953 A1
`
`jw9}e}0e7-7]——
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`GenomeEx. 1018
`Page 13 of 28
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`Genome Ex. 1018
`Page 13 of 28
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`

`

`Patent Application Publication Nov. 8,2007 Sheet 13 of 13
`
`US 2007/0258953 Al
`
`Es5382s
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`Fig.5continued
`
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`US 2007/0258953 Al
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`Nov. 8, 2007
`
`LACTIC ACID UTILISING BACTERIA AND THEIR
`THERAPEUTIC USE
`
`[0001] This invention relates to improvements in health
`and nutrition for both animals and humans following the
`ingestion of specific bacteria capable of utilising lactic acid.
`
`[0002] Under normal conditions the concentration of lac-
`tic acid (lactate) in the mammalian gut is very low despite
`the fact that many bacterial species, such as /actobacilli,
`streptococci, enterococci and bifidobacteria that reside in
`the intestine produce this acid in large quantities as a
`fermentation end product. Lactic acid is also produced by
`host tissues.
`
`It has been hypothesised that the accumulation of
`[0003]
`lactic acid is normally prevented by the ability of certain
`other bacteria that inhabit the gut to consumelactic acid and
`to use it as a source of energy. The identity of the micro-
`organisms that are postulated to conduct
`this metabolic
`process in the mammalian large intestine has largely not
`previously been elucidated, Bourriaud et al (2002). Kanau-
`chi et al (1999) revealed that a strain of Bifidobacterium
`longum was co-incubated with a strain of Eubacterium
`limosum on germinated barley feedstuff for three days there
`was a marked increase in acetate formed and a small
`increase (less than 3 mM) in butyrate formed when com-
`pared to the incubations with £. limosum alone.
`
`In the rumen ofcattle and sheep the species Sele-
`[0004]
`nomonas ruminantium, Veillonella parvula and Megas-
`phaera elsdenii are regarded as the most numerousutilisers
`of lactate (Gilmour et al., 1994; Wiryawan and Brooker,
`1995). The contribution of Megasphaera elsdenii appears to
`be particularly significant in the rumen, based on the high
`proportion of carbon flow from lactic acid to propionic acid
`and this species employs the acrylate pathway for this
`purpose (Counotte et al., 1981). Megasphaera elsdenii pro-
`duces a variety of end products including propionate,
`butyrate, caproate and branched chain fatty acids from
`lactate—see Ushidaet al (2002), Kung and Hession, (1995).
`This probably reflects the ability of this species to use lactate
`despite the presence of other carbon sources such as sugars,
`whereas Selenomonasuseslactic acid only in the absence of
`other energy sources. This has led to interest in the use of
`Megasphaera as a probiotic organism that might be added to
`animal (Kung and Hession, 1995; Ouwerkerk et al., 2002),
`or even humandiets to prevent the harmful accumulation of
`lactic acid. In ruminant animals (cattle and sheep) accumu-
`lation of lactic acid occurs when a large amountof readily
`fermentable substrate (such as starch and sugars) enters the
`rumen. Rapid fermentation, particularly by organisms such
`as Streptococcus bovis, drives down the pH, creating more
`favourable conditions for the proliferation of lactic acid
`producing bacteria such as /actobacilli, and S. bovis itself.
`Normalpopulations of bacteria capable of utilising lactate
`(lactate utilisers) are unable to cope with the greatly
`increased production oflactic acid. Unaided, lactic acid may
`accumulate to levels that can cause acute toxicity, laminitis
`and death (Nocek, 1997; Russell and Rychlik, 2001).
`
`[0005] Similar events occurring in the large intestine can
`also cause severe digestive and health problems in other
`animals, for example in the horse where high lactate levels
`and colic can result from feeding certain diets.
`
`In humanslactic acid accumulation is associated
`[0006]
`with surgical removal of portions of the small and large
`intestine, and with gut disorders such as ulcerative colitis
`and short bowel syndrome (Day and Abbott, 1999). High
`
`concentrations of lactic acid in the bloodstream can cause
`toxicity (Hove et al., 1994), including neurological symp-
`toms (Chanet al., 1994). Muchof this lactic acid is assumed
`to derive from bacterial fermentation, particularly by bifi-
`dobacteria and by lactobacilli and enterococci. Lactic acid
`can also be produced by host
`tissues, but
`the relative
`contributions of bacterial and host sources are at present
`unclear.
`
`[0007] Conversely, the formation of other acid products, in
`particular butyric acid (butyrate), is considered to be ben-
`eficial as butyric acid provides a preferred energy source for
`the cells lining the large intestine and has anti-inflammatory
`effects (Inan et al., 2001, Pryde et al., 2002). Butyrate also
`helps to protect against colorectal cancer and colitis (Archer
`et al., 1998; Csordas, 1996).
`
`[0008] We have now established a method of isolating
`novelbacteria that are remarkably active in consuminglactic
`acid. The bacteria have been isolated from human faeces.
`Preferably the method allows isolation of bacteria which
`convert the lactic acid to butyric acid. According to this
`method several new bacteria that are remarkably active in
`converting lactic acid to butyric acid have beenisolated.
`
`[0009] One group of these bacteria is from the newly
`described genus Anaerostipes caccae (Schwiertz et al.,
`2002). Although some main characteristics of A. caccae are
`described in this publication, its ability to use lactate was not
`reported and has only recently been recognised as described
`herein.
`
`[0010] The invention relates to a method for selecting a
`strain of lactic acid-utilising bacteria, which method com-
`prises the steps of:
`
`a) providing (for example isolating) a bacterial
`[0011]
`culture from a human faecal sample;
`
`[0012]
`
`b) selecting a single colony of bacteria;
`
`c) growing said colony in a suitable medium
`[0013]
`containing lactic acid; and
`
`4d) selecting a strain of bacteria consuming rela-
`[0014]
`tively large amountsoflactic acid, all of the above steps
`being conducted under anaerobic conditions.
`
`In the above method, the reference to “relatively
`[0015]
`large amounts of lactic acid” is defined as meaning the
`bacteria used at least 10 mM of D, L or DLlactic acid during
`growth into stationary phase, per 24 hours at 37° C.
`in
`YCEFALG or YCFAL medium.
`
`[0016] Preferably thestrain of lactic acid utilising bacteria
`also produces high level of butyric acid and the method of
`the invention may therefore comprise an additionalstep of:
`
`e) selecting a strain of bacteria producing rela-
`[0017]
`tively large quantities of butyric acid.
`
`In the abovestep the reference to “relatively large
`[0018]
`quantities of butyric acid” is defined as meaning the bacteria
`produces at least 10 mM ofbutyric acid during growth into
`stationary phase, per 24 hours at 37° C. in YCFALG or
`YCFAL medium.
`
`[0019] Preferably the strain of lactic acid utilising bacte-
`rium must be capable of converting lactate produced by
`another gut bactertum from dietary components such as
`resistant starch.
`
`[0020] Preferably the lactic acid used in step c) is both D-
`and L-isomers of lactic acid.
`
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`US 2007/0258953 Al
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`
`[0021] Preferably the suitable medium to grow bacteria is
`nutritionally rich medium in anaerobic Hungate tubes.
`
`[0022] Preferably the selected strain of bacteria is re-
`purified using nutritionally rich medium in anaerobic roll
`tubes.
`
`[0023] A further aspect of the invention is a bacterial strain
`that produces butyric acid as its sole or predominant fer-
`mentation product from lactate and which has been isolated
`according to the method of the invention described above.
`Such novel bacterial strains include:
`
`the bacteria Anaerostipes caccae strain L1-92
`[0024]
`deposited at NCIMB (National Collections of Industrial,
`Marine and Food Bacteria in Aberdeen, United Kingdom)
`under No 138017 on 4 Nov. 2002 and at DSM under No
`14662 on 4 Nov. 2002.
`
`strain Ss2/1
`the Clostridium indolis bacterial
`[0025]
`deposited at NCIMB under No 41156 on 13 Feb. 2003;
`
`the bacteria strain SM 6/1 of Eubacterium hallii
`[0026]
`deposited at NCIMB under No. 41155 on 13 Feb. 2003.
`
`[0027] Another aspect of the invention is a strain of
`bacteria having a 16S rRNA gene sequence which has at
`least 95% homology to one of the sequences shown in FIG.
`1, preferably 97% homology (ie. differs at less than 3% of
`residues out of approximately 1400 from one of the
`sequences shownin FIG.1).
`
`[0028] Another aspect of the invention is the use ofat least
`one of the above-mentioned bacterial strains in a medica-
`mentor foodstuff.
`
`[0029] Another aspect of the invention is a method to
`promote butyric acid formationin the intestine of a mammal,
`said method comprising the administration of a therapeuti-
`cally effective dose of at least one of the above described
`strains of live butyric acid producing bacteria. The bacterial
`strain may be administered by means of a foodstuff or
`suppository or any other suitable method.
`
`[0030] Another aspect of the invention is a method for
`treating diseases associated with a high dosageoflactic acid
`such as lactic-acidosis, short bowel syndrome and inflam-
`matory bowel disease,
`including ulcerative colitis and
`Crohn’s disease, which method comprises the administra-
`tion of a therapeutically effective dose of Anaerostipes
`caccaeor at least one above-mentionedstrains of live lactic
`acid utilising bacteria. Advantageously the strain selected
`mayalso produce a high level of butyric acid.
`
`[0031] Further, another aspect of the invention is a pro-
`phylactic method to reduce the incidence or severity of
`colorectal cancer or colitis in mammals caused in part by
`high lactic acid and low butyric acid concentrations, which
`method comprises the administration of a therapeutically
`effective dose of at least one above identified strains oflive
`lactic acid utilising bacteria and/or butyric acid producing
`bacteria mentioned above or of Anaerostipes caccae.
`
`[0032] Another aspect of the invention is the use of live
`Anaerostipes caccae or at least one of the above mentioned
`lactic acid utilising bacteria as a medicament. Advanta-
`geously the strain chosen may produce butyric acid as its
`sole or predominant fermentation product from lactate.
`Preferably the bacteria are used in the treatment of diseases
`associated with high levels of lactic acid such as lactic
`acidosis, short bowel syndrome and inflammatory bowel
`disease including ulcerative colitis and Crohn’s disease.
`
`[0033] According to another aspect of the invention at
`least one lactate-utilising strain of bacteria as mentioned
`above or Anaerostipes caccae are used in combination with
`lactic acid producing bacteria including those such as Lac-
`tobacillus spp. and Bifidobacterium spp. or other additives
`or growth enhancing supplement currently used as probiot-
`ics.
`
`[0034] The combination of strains would potentially
`enhance the health-promoting benefits of the lactic acid
`bacterium by converting its fermentation products (lactic
`acid alone or lactic acid plus acetic acid) into butyrate.
`Indeedit is possible that certain health-promoting properties
`currently ascribed to lactic acid bacteria might actually be
`due to stimulation of other species such as lactate-consumers
`in vivo, particularly where probiotic approaches (see below)
`are used to boost native populations in the gut. Furthermore
`the presence of the lactic acid producing bacteria in a
`combined inoculum could help to protect the lactate con-
`sumer against oxygen prior to ingestion.
`
`[0035] The growth andactivity of the novel bacteria may
`be promoted by means of providing certain growth require-
`ments, required for optimal growth and enzymeexpression
`to the bacteria, present in the animal or human gastrointes-
`tinal tract. These bacterial growth enhancing nutrients are
`often referred to as prebiotics or synbiotics.
`
`[0036] Thus the invention provides methods to promote
`the growth and enzyme expression of the micro-organism
`and hence removalof lactate and production of butyrate in
`vivo, for example, via a prebiotic or symbiotic approach
`(Collins and Gibson, 1999).
`
`[0037] Another aspect of the invention is a method for
`treating acidosis and colic in animals, particularly in rumi-
`nants and horsesor other farm animals, by administration of
`a therapeutically effective dose of Anaerostipes caccae or at
`least one of the lactate utilising bacteria mentioned above.
`Advantageously the bacteria can be administrated as feed
`additives.
`
`[0038] For the use, prevention or treatment of conditions
`described herein, the bacteria or prebiotic(s) or symbiotic(s)
`are preferentially delivered to the site of action in the
`gastro-intestinal tract by oral or rectal administration in any
`appropriate formulae or carrier or excipient or diluent or
`stabiliser. Such modesof delivery may be of any formulation
`includedbutnot limited to solid formulations such as tablets
`or capsules; liquid solutions such as yoghurts or drinks or
`suspensions. Ideally, the delivery mechanism delivers the
`bacteria or prebiotic or symbiotic without harm through the
`acid environment of the stomach and through the rumen to
`the site of action within the gastro-intestinal tract.
`
`[0039] Another aspect of the invention is the useofat least
`one bacterial strain mentioned aboveor Anaerostipes caccae
`in a methodto produce butyric acid from lactate and acetate.
`The method includes
`the fermentation of the above
`described microorganism selected for both their lactic acid
`utilising and butyric acid producing abilities in a medium
`rich in lactate and acetate. The method can be used in
`industrial processes for the production of butyrate on a large
`scale.
`
`BRIEF DESCRIPTION OF THE FIGURE
`
`FIG. 1: Sequence information of 16S rRNAforfive
`[0040]
`lactic acid utilising strains.
`
`FIG. 2: Co-culture experiment. Concentration of
`[0041]
`SCFA are shown after 24 hours growth in YCFA medium
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`US 2007/0258953 Al
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`Nov. 8, 2007
`
`[0050] Bacterial strains were isolated by selection as
`with 0.2% starch as energy source (values for acetate,
`single colonies from the nutritionally rich medium in
`initially present in the medium, are shown on a 10 fold
`reducedscale). Butyrate production by 4. caccae 1-92, and_anaerobicroll tubes as described by Barcenilla et al. (2000).
`by E, hallii L2-7 and SM 6/ 1,is stimulated by co-culture
`The isolates were grown in M2GSCbroth and the fermen-
`with B. adoloscentis L2-32, while L-lactate disappears from
`tation end products determined. Butyrate producing bacteria
`the co-cultures.
`.
`.
`:
`:
`were re-purified using roll tubes as described above. Strains
`L1-92, S D8/3, S D7/11, A2-165, A2-181, A2-183, L2-50
`[0042] FIG. 3: SCFA formation and lactate utilisation for
`and L2-7 were all isolated using this medium. Omitting
`new and existing isolates. Acids produced or consumed
`rumenfluid and/or replacing the sugars with one additional
`guring aVCEAmodere shownfor aeuncured for
`:
`ws
`ours: a
`medium containing
`m
`actate
`carbon source such as DL lactate increased the selectivity of
`(YCFAL): b) YCFA medium containing 10 mM glucose and
`the roll tube medium and this medium wasusedto isolate
`35 mM DLlactate (YCFALG); c) YCEA medium with no
`strain S D6 1L/1. Strains G 2M/1 and SM 6/1 wereisolated
`:
`.
`,
`.
`addition. Carbon recoveries (%) for growth on lactate, and
`from medium where DL-lactate was replaced with mannitol
`lactate plus glucose, respectively, were as follows: SM 6/1
`(0.5%). Separately, non-rumenfluid based media routinely
`(94.6, 76.4); SL 6/1/1 (100.2, 78.7); L1-92 (96.2, 97.9);
`used for isolating Selenomonas sp., namely Ss and Sr
`§82/1 (92.1, 90.1); SSC/2 (104.4, 96.9); SR1/1 (103, 93.8).
`This suggests that there may be unidentified fermentation|medium (Atlas, 1997) was used to isolate other strains.
`products in the case of SM 6/1, SL 6/1/1 and SS3/4 when
`Inoculating Sr medium roll tubes with dilutions of faecal
`grown on glucosepluslactate.
`samples resulted in the isolation of strain Sr1/1 while the Ss
`medium resulted in the isolation of strains Ss2/1, Ss3/4 and
`Ssc/?
`[0043] FIG. 4: Time course of SCFA formation and growth
`in batch culture of E. hallii L2-7 on media containing DL
`lactate, glucose, or DL lactate plus glucose.
`
`EXAMPLE 2
`
`[0044] FIG.5: Time course of SCFA formation and growth
`in batch culture of strain SS2/1 on media containing DL
`lactate, glucose, or DL lactate plus glucose.
`
`DETAILED DESCRIPTION
`[0045] The experimental work performed showsthe fol-
`lowing:
`1. Certain human colonic anaerobic bacteria,
`[0046]
`including A. caccae strains, are strong andefficient uti-
`lisers of lactic acid.
`[0047]
`2. Certain human colonic anaerobic bacteria,
`including A. caccaestrains, are strong and efficient pro-
`ducers of butyric acid.
`.
`.
`.
`.
`[0048] 3. Certain human colonic anaerobic bacteria,
`including A. caccae strains, convert lactic acid to butyric
`acid.
`
`EXAMPLE 1
`Isolation and Characterisation of Bacteria
`[0049] A faecal sample was obtained from a healthy adult
`female volunteer that had not received antibiotics in the
`previous 6 months. Whole stools were collected, and 1 g was
`mixed in 9 ml anaerobic M2 diluent. Decimalserial anaero-
`bic dilutions were prepared and 0.5ml inoculated into roll
`tubes by the Hungate technique, under 100% CO, (Byrant,
`1972).
`
`A. caccae and other Human Colonic Bacterial
`Isolates Consumes Lactic Acid and Acetic Acid and
`
`ui
`
`Produces Butyric Acie wien Grown in Rumen
`[0051] Table 1
`summarises the fermentation products
`formed by twelvestrains of anaerobic bacteria when grown
`under 100% CO, in a rumen fluid-containing medium con-
`taining 0.5% lactate (M2L) or 0.5% lactate, 0.2% starch,
`0.2% cellobiose and 0.2% glucose (M2GSCL)as the energy
`sources. Ten of these strains were isolated from human
`faeces as described above in Example 1. Strains 2221 and
`NCIMB8052 are stock collection isolates not from the
`human gut and are included for comparison. Table 1 dem-
`onstrates that three strains, L1-92 (4. caccae), SD6 1L/1 and
`SD 6M/1 (both E.hallii-related) all consumedlarge amounts
`of lactate (>20 mM) on both media examined, M2L and
`M2GSCL,and produced large quantities of butyric acid. A.
`caccae 1-92 in particular consumed large amounts of
`lactate and produced large amounts of butyrate. Acetate is
`also consumed byall three strains. The other 9 butyrate
`producing bacteria tested either consumedrelatively small
`amounts of lactate, or consumed no lactate, on this medium.
`L-lactate concentrations were determined enzymatically and
`glucose concentrations were determined by the glucose
`oxidase method (Trinder, 1969). Analyses were conducted in
`a robotic clinical analyser (Kone analyser, Konelab Corpo-
`ration, Finland).
`
`TABLE1
`
`Table 1. Comparison of human faecal isolates for the ability to utilise
`(negative values) or produce (positive values) lactate on a rumen fluid
`based medium (M2) supplemented with lactate (M2L) and lactate plus glucose,
`cellobiose and soluble starch (0.2% each)
`(M2GSC).
`
`Strain ID
`S D8/3
`S D8/3
`SL 6/1/1
`SL 6/1/1
`
`Closest relative
`Adhufec 406*%+
`
`E, haliti
`
`Medium
`M2L
`M2GSCL
`M2L
`M2GSCL
`
`Formate Acetate
`1.15
`0.97
`21.66
`0.77
`-19.74
`-9.78
`
`Butyrate
`
`10.88
`35.48
`22.58
`
`Lactate
`-3.94
`6.43
`-32.41
`-21.85
`
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`US 2007/0258953 Al
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`TABLE 1-continued
`
`Table 1. Comparison of human faecal isolates for the ability to utilise
`(negative values) or produce (positive values) lactate on a rumen fluid
`based medium (M2) supplemented with lactate (M2L) and lactate plus glucose,
`cellobiose_and soluble starch (0.2% each)
`(M2GSC).
`
`Closest relative
`E, hallii
`
`HucA19*
`
`ND*
`
`But. Fibrisolvens
`
`F. prausnitzti
`
`Roseburia sp.
`
`Roseburia sp.
`
`Coprococcus sp.
`
`Strain ID
`SM 6/1
`SM 6/1
`G 2M/1
`G 2M/1
`8 D7 11/1
`8 D7 11/1
`2221
`2221
`8052
`8052
`A2-165
`A2-165
`A2-183
`A2-183
`A2-181
`A2-181
`L2-50
`L2-50
`L1-92
`L1-92
`
`Medium
`M2L
`M2GSCL
`M2L
`M2GSCL
`M2L
`M2GSCL
`M2L
`M2GSCL
`Cl. acetobutylicum =M2L
`M2GSCL
`M2L
`M2GSCL
`M2L
`M2GSCL
`M2L
`M2GSCL
`M2L
`M2GSCL
`Anaerostipes caccae M2L
`M2GSCL
`
`1.85
`1.37
`19.4
`
`0.13
`1.98
`17.47
`
`Formate Acetate
`0.79
`-19,01
`1.31
`-5.06
`2.82
`0.01
`0.51
`0.43
`-3.61
`-5.57
`-12.42
`-1.79
`0.62
`-6.97
`0.86
` -12.70
`-0.26
`-11.05
`2.32
`4.47
`-29.42
`-27.03
`
`-0.15
`0.58
`0.33.
`1.06
`19.37
`
`0.63
`
`Lactate
`
`23.72
`—28.42
`23.66
`9,52
`
`Butyrate
`31.73
`22.77
`7.97
`12.94
`0.08
`4.84
`1.57
`18.02
`19.31
`18.00
`3.56
`18.38
`1.84
`18.23
`1.75
`18.68
`0.52
`7.60
`37.00
`44.78
`
`
`
`
`
`*clone library sequence, uncultured (Hold et al., 2002)
`+clone library sequence, uncultured (Suau et al., 1999)
`#NDnot determined
`
`EXAMPLE3
`
`A. caccae and other Human Colonic Bacterial
`Isolates Consumes Lactic Acid and Acetic Acid and
`
`Produces Butyric Acid when Grown in Rumen
`Fluid Free Medium
`
`[0052] Table 2a shows the utilisation and production of
`formate, acetate, butyrate, succinate and lactate, on this
`occasion performed using the rumen fluid-free medium
`YCFA (Duncan et al. 2002) containing no added energy
`source, or with 32 mM lactate (YCFAL)orlactate plus 23
`mM glucose (YCFALG) as added energy sources. Sepa-
`rately Table 2b reveals the levels of the two isomers of
`lactate (D and L) remainingat the end of the incubations and
`the concentration