SBO meeting 29 09 2011 by DTAvkE99

VIEWS: 79 PAGES: 127

									                         SBO PROJECT
New Butyric Acid Producing Bacterial
Strains for Gastro-intestinal Disorders

                       Het Pand – Ghent
                      September 29th 2011
                              Strictly confidential
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    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




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                                          Problem

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                       State-of-the-art

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    Butyrate as possible treatment
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    Delivery of butyrate into the gut

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                                  Goal




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         Structure of the project




YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
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    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




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                     Workpackage 1:

Isolation, identification and characterization
  of butyrate producing Clostridial cluster IV
   and XIVa strains from human and animal
                  fecal samples



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                               Objectives:
1.   Isolation of a collection of representative strictly anaerobic
     bacteria from human and animal stools that produce high
     levels of butyrate in batch cultures
2.   Identification and phylogenetic classification of the isolated
     butyrate producers using 16S rRNA gene sequence
     determination
3.   Determination of the biochemical and morphological
     characteristics of the isolated butyrate producers




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1. Isolation of butyrate producing strains from chicken
       caecal content and human faecal samples
               5 min




     HPLC




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2. Identification and phylogenetic classification of the
   isolated butyrate producers using 16S rRNA gene
                sequence determination
•   Determination of SCFA production by HPLC
•   Cut-off value: 5 mM butyric acid
•   Identification by 16s rRNA gene sequencing
•   Results:
    – First isolation: 11 butyrate producing isolates  1 new species
      1.66: Roseburia faecis M6/1 (Cluster XIVa) 95% homology
      1.20: Butyricicocccus pullicaecorum 99% homology
    – Second isolation: 18 butyrate producing isolates  2 new genera
      2.12: Blautia coccoides (Cluster XIVa) 92% homology
      2.33: Eubacterium oxidoreducens (Cluster XIVa) 92% homology
    – Third isolation: 20 butyrate producing isolates to be identified
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                                  Clostridium perfringens
                                   Clostridium viride
                                   Eubacterium desmolans
                                  1.20
                                  Butyricicoccus pullicaecorum
                                  Clostridiales bacterium 7-4c
                                  1.31-32
                                  Gemmiger formicilis strain ATCC 27749
                                  2.25
                                                                                           Cluster IV
                                  1.57
                                  Faecalibacterium prausnitzii strain ATCC 27768
                                  Faecalibacterium prausnitzii M21/2
                                  .
                                  2.53
                                  Butyrate producing bacterium ART55/1- E.ruminantium
                                  1.67
                                  Butyrate-producing bacterium SM6/1- E.halii
                                  Anaerostipes caccae strain L1-92
                                  Eubacterium hadrum DSM 3319-clone1
                                  2.1
                                  2.48
                                  Butyrate-producing bacterium SSC/2- C.indolis
                                  Anaerostipes sp. 5_1_63FAA
                                  2.27
                                  Eubacterium hadrum DSM 3319-clone2
                                  2.18
                                  2.24
                                  2.33
                                  Butyrivibrio fibrisolvens 16.4
                                  Roseburia hominis A2-183
                                  1.27
                                  Roseburia intestinalis
                                  2.30
                                  2.16
                                  Roseburia faecis gene
                                                                                           Cluster XIVa
                                  Eubacterium rectale
                                  Butyrate-producing bacterium M104/1- E.rectale
                                  2.17
                                  2.8
                                  2.52
                                  1.66
                                  Eubacterium oxidoreducens- DSM3217T
                                  Coprococcus comes ATCC 27758
                                  Butyrate-producing bacterium A2-231
                                  1.44
                                  2.3
                                  2.29
                                  2.21
                                  Clostridiales bacterium L2-14
                                  2.12
                                  Blautia coccoides
                                  2.43
                                  Ruminococcus obeum
                                  2.19
                                  Firmicutes bacterium DJF_VR50

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                              Planned experiments

• Biochemical and morphological characteristics
  –   API microtest
  –   H2 - CO 2 production
  –   Sporulation
  –   Scanning electron micrograph


• Genomic DNA extraction:
  –   1.20
  –   2.12
  –   2.33
  –   New isolates from 3rd isolation

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1 & 2 & 3. Isolation, identification and characterisation of
 butyrate producing strains from chicken caecal content



 16 butyrate producing chicken isolates:
    • 1 isolate: 99,1% homology with Clostridium lactatifermentans
    • 10 isolates: > 94% homology  new species
    • 5 isolates: < 94% homology  new genus




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                                Clostridium viride T2-7 DSM 6836T (X81125)
                          isolate 53-4c
                              isolate 24-4c
                               isolate 30-4c
                                 Pseudoflavonifractor capillosus ATCC 29799T (AY136666)
                                   Flavonifractor plautii DSM 6740T (Y18187)
                                  - Eubacterium desmolans ATCC 43058T (L34618)
                                   Clostridiales strain A2-207 (AJ270471)
                                                                                                    cluster IV
                                 isolate 7-4c
                                isolate 25-3b Butyricicoccus pullicaecorum CCUG 55265T (EU410376)     (6 isolates)
                                   isolate 40-4c
                                   Subdoligranulum variabile DSM 15176T (AJ518869)
                                  Faecalibacterium prausnitzii 1-84 (AY169429)
                                    Faecalibacterium prausnitzii L2-6 (AJ270470)
                                     Faecalibacterium prausnitzii M21/2 (AY305307)
                       Clostridiales strain SS2/1 (AY305319)
                        isolate 35-7e Anaerostipes butyraticus DSM 22094T (FJ947528)
                            Anaerostipes sp. IE4 (AY960568)
                        Anaerostipes caccae L1-92 DSM 14662T (AJ270487)
                               isolate 33-7e
                                 Eubacterium hallii L2-7 (AJ270490)
                                Eubacterium hallii DSM 3353T (L34621)
                                                                                                    cluster XIVa
                       isolate 77-5d
                        Clostridium populeti ATCC 35295T (X71853)                                      (3 isolates)
                         Roseburia hominis A2-183 DSM 16839T (AJ270482)
                          Roseburia intestinalis M50/1 (AY305308)
                             Butyrivibrio fibrisolvens 16/4 (AJ250365)
                         Clostridiales strain K10 (EU305624)
                        Clostridiales strain M62/1 (AY305309)
                          Clostridiales strain SS3/4 (AY305316)
                         Clostridium neopropionicum DSM 3847T (X76746)
                         Clostridium propionicum DSM 1682T (X77841)
                                                                                                    cluster XIVb
                         Clostridium lactatifermentans DSM 14212T (AY033434)
                         isolate 21-4c                                                                  (1 isolate)
                                          Erysipelotrichaceae strain 5_2_54FAA (NZ_GG749091)
                                             Eubacterium dolichum DSM 3991T (L34682)
                                             Eubacterium tortuosum ATCC 25548T (L34683)
                                             isolate 60-7e
                                         isolate 10-3b
                                               Eubacterium biforme DSM 3989T (M59230)
                                                Eubacterium cylindroides ATCC 27803T (L34617)
                                                                                                    cluster XVI
                                                Eubacterium cylindroides T2-87 (AY305306)
                                              isolate 65-2a                                           (6 isolates)
                                               isolate 20-2a
                                               Streptococcus pleomorphus ATCC 29734T (M23730)
                                                   isolate 41-2a
                                                isolate 37-2a
              Clostridium perfringens ATCC 13124T (M59103)

0.02
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                          WP1. Deliverables
• A collection of at least 15 bacterial isolates from both human
  and chicken fecal samples that produce high concentration of
  butyric acid in batch cultures (Month 4)

• A species-level identification and positioning of the
  butyrate-producing isolates in a phylogenetic tree (Month 8)

• A detailed list of biochemical and morphological properties of
  the butyrate-producing strains (Month 11)




     YOUR TOP EXPERTS FOR INNOVATION      info@food2know.be
           Feed - Food - Health           www.food2know.org
    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




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                     Workpackage 2


   Detailed characterization of the in vitro
behaviour of the butyrate-producing isolates




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                              Objectives:
1.   Evaluation of the anti-inflammatory properties of the butyrate-
     producing strains on mononuclear blood cells and intestinal
     epithelial cells
2.   Evaluation of the effects of the butyrate-producing strains on
     repair of mucous epithelial layers in vitro
3.   Short-term evaluation of the survival and metabolic activity of
     butyrate-producing strains under gastrointestinal conditions
4.   Colonization efficiency of butyrate-producing strains at the
     mucosal interphase and the luminal colon environment
5.   Strategies to stimulate endogenous butyrate-producing strains
     and optimize the colonization efficiency of supplemented
     butyrate-producing isolates


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 1. Evaluation of the anti-inflammatory properties of the
butyrate-producing strains on mononuclear blood cells and
                  intestinal epithelial cells
                                           CaCo2 cells
                                                                  Cytokine expression levels:
                                           PBMC
                                                                   pro- or anti-inflammatory
                                                                           response ?
                                                                         e.g. IL10 vs IL12
                                                                             IL1, TNF-α, IL8




Bacteria, supernate and
      precipitate

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 1. Evaluation of the anti-inflammatory properties of the
butyrate-producing strains on mononuclear blood cells and
                  intestinal epithelial cells
  The purpose of these experiments is to test whether supernatants (SN)
  of Butyricicoccus cultures can protect epithelial cells from the effect of
  inflammation.
  We look specifically here at permeability of Caco-2 cells, by measuring
  transepithelial resistance (TER) in inflamed conditions and –as a
  control- in normal conditions.

  As butyrate is suspected to be the effective compound, the effect of
  butyrate as such is also tested.



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                              Caco-2 Model
• Caco-2 cells are grown on transwells for
1-2 weeks before use. → high trans
epithelial resistance (TER).
• Supernatant (SN) will be tested in
    –“normal conditions” and
    – “infammed conditions” (i.e. after addition of
    100 ng/ml TNF and 100 ng/ml IFNg in
    basolateral compartement)
• SN are added in the apical                             Protocol:
compartment and are diluted in caco-2                    t=0
medium                                                   Inoculation of SN                   Harvest medium
                                                         Stimulation with TNF + IFN          and cells
(4 part SN + 6 part caco-2 medium)
• Experiment performed in quadruplicate
                                                      TER at start         TER at 24h    TER at 48h

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Results (non inflamed conditions):

    Non inflamed conditions:
    Without TNF and IFN,


                           SN of bacteria




                                                                                                                                                                                          10 exp 7 lyoph. Buty.
                                                    Apical:




                                                                                                    Fresh bact. Medium




                                                                                                                                              Buty. Supernatant A
                                                                                                                         Fresh bact. Medium
                                                                              Fresh bact. medium




                                                                                                                                                                    Buty. Supernatant B
                                                              Caco-2 medium




                                                                                                    + 20mM butyrate

                                                                                                                         + 40mM butyrate
Data are expressed as percentage of TER at start.

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    Results (inflamed conditions):
                                                                                                                                       *
                                                                                                                        *
                                                                                                    *
Inflamed conditions:                                                                          *
With TNF and IFN to mimic
inflammation

                       SN of bacteria




                                                                                                                                                                                             10 exp 7 lyoph. Buty.
                                                                                                                                                 Buty. Supernatant A
                                                                                                   Fresh bact. Medium


                                                                                                                            Fresh bact. Medium
                                                                         Fresh bact. medium




                                                                                                                                                                       Buty. Supernatant B
                                                         Caco-2 medium
                                            Apical:




                                                                                                   + 20mM butyrate


                                                                                                                            + 40mM butyrate
                     TNF + IFN

•P˂0.01, unpaired student t test.
See note about statistics!                Basolateral:
                                                                                                            TNF + IFN
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        Results – IL-8 secretion:

Inflamed conditions:
With TNF and IFN to mimic
inflammation

                   SN of bacteria




                                                                                                                                       Buty. Supernatant A
                                                                                             Fresh bact. Medium


                                                                                                                  Fresh bact. Medium
                                                                       Fresh bact. medium




                                                                                                                                                             Buty. Supernatant B
                                                       Caco-2 medium




                                                                                                                                                                                   10 exp 7 lyoph. Buty.
                                         Apical:




                                                                                             + 20mM butyrate


                                                                                                                  + 40mM butyrate
                 TNF + IFN


                                        Basolateral:                                                  TNF + IFN

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                            Results
Summary of non-inflamed conditions:
•Inoculation of bacterial medium containing butyrate or SN of
Butyricicoccus seems to slightly (although not significantly) increase
the TER in non-inflamed conditions.
REM: the cells are already differentiated with high values of TER,
which can hardly be increased  it is not surprising that the SN can
not increase TER more.
•10 Exp7 lyophilized Butyricicoccus were added to caco-2 medium to
see whether bacterial components (cell wall, …) could mediate the
effect. The bacteria were not living in caco-2 medium (no change in pH
observed, not anaerobe conditions). If the effect is mediated by
butyrate, it is normal that no effect is seen.
•Data at 24h are also available and are similar to the ones observed at
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                                        Results
Summary of inflamed conditions:
•Stimulation with TNF-α and IFN-γ decreases of TER with about 40 %
as expected. This decrease is already visible after 24h.
•When cells are stimulated with butyrate or supernatant of
Butyricicoccus, no decrease of TER is observed, meaning that cells are
protected from the effect of inflammation.
•Furthermore, secretion of IL-8 is also reduced.
•The lyophilized Butyricicoccus did not protect cells from the effect of
TNF + IFN suggesting that the active compound must be secreted by
living bacteria.



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                    Future experiments
A second set of experiments were performed, but unfortunately failed
due to problems with the cell incubator.
In a second set of experiments, we will repeat the experiment with
Butyricicoccus SN and add Butyricicoccus SN depleted of butyrate, to
check whether the effect is lost.

In addition to the measurement of permeability, other parameters
could be checked.
-Expression of genes in epithelial cells. (cells were lysed in buffer for
RNA extraction and thus RT-PCR can be performed.)



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2. Evaluation of the effects of butyrate-producing strains on
          repair of mucous epithelial layers in vitro
 • How can mucosal damage be repaired?
      – continuous regeneration from stem cells and precursor cells in the crypts
      – restitution, allowing for the rapid repair of superficial mucosal lesions via
        cell migration




 1) Make standardized
 scratch in cell culture
 2) Incubate with
 bacterial supernate



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 3. Short-term survival and metabolic activity of butyrate-
producing bacterial strains under gastrointestinal conditions
  Batch incubations – experimental design
                                             Butyrate
                                         producing isolates
                                                         Variation digestive parameters
                                                                    pH
                                                                    [enzyme]
                                                                    [bile salt]
                                                                    O2
                                                                    Eh
               CT                                                   residence time
                                                                    fed/fasted
      CA
      CA                   CD
                                                                   Microbial background
                                                                   IBD vs. healthy control

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 3. Short-term survival and metabolic activity of butyrate-
producing bacterial strains under gastrointestinal conditions
    Batch incubations – experimental design
    - Cultivation
       Anaerobic conditions 8% CO2 - 8% H2 - 84% N2
    - Quantification:
       Plating M2GSC 8.54 LOG CFU/mL
       Flow cytometry: anaerobic live/dead staining (+ cystein*HCl)
           Concentration cystein*HCl (g/L)   LOG living cells/mL
                             0                      8.76
                           0.25                     8.76
                           0.50                     9.00
                           1.00                     8.49

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                                             Flow Cytometry

                fluorescent stain (DNA,
                surface antibody, etc.)

                                                                                               detector 3:
                                                 flow cell
                                                                  cells        filter 2
                                                                                               fluorescence signal 520 nm


                                                                 filter 1
                                                                                               detector 2:
                                                                                               side scatter 488 nm

        detector 1: forward scatter 488 nm                                                           1
                                                                                                     argon laser
                                                                                                         488nm
                                                             glass capillary
•   total cell count in < 15 minutes
•   up to 1000 cells/second counted
•   detects all bacteria, also those that                    (cell sorting)
    can not be cultured
•   multi-parameter analysis (multiple
    signals from a single cell)
                                               waste


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 3. Short-term survival and metabolic activity of butyrate-
producing bacterial strains under gastrointestinal conditions

                   Batch incubations – Results stomach - Plating
                   Butyricicoccus pullicaecorum (LMG 24109T) 7Log CFU/ml

                             Survival stomach 1 h
                                                                                                       Survival stomach 2 h
              9
                                                                                  9
              8
                                                                                  8
              7
                                                                                  7
              6
 Log CFU/mL




                                                                                  6
                                                                     Log CFU/mL
              5
                                                                                  5                                               Fed conditions
              4                                                                   4                                               Fasted conditions
              3                                                                   3
              2                                                                   2
              1                                                                   1
              0                                                                   0
                  inoculum     pH2    pH3     pH4    pH5       pH6                    inoculum   pH2     pH3   pH4    pH5   pH6




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 3. Short-term survival and metabolic activity of butyrate-
producing bacterial strains under gastrointestinal conditions

 Batch incubations – Results stomach – Plating vs Flow cytometry
 Butyricicoccus pullicaecorum (LMG 24109T) 107/mL

                             Stomach fed 1 h                                                        Stomach fed 2 h
             10                                                               10
              9                                                                9
              8                                                                8
              7                                                                7
                                                                 Log CFU/mL
Log CFU/mL




              6                                                                6
              5                                                                5                                              Plating
              4                                                                4                                              Flow cytometry
              3                                                                3
              2                                                                2
              1                                                                1
              0                                                                0
                  inoculum    pH2    pH3     pH4    pH5    pH6                     inoculum   pH2    pH3    pH4   pH5   pH6



                                            Viable but nonculturable state?
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 4. Colonization efficiency of the colon environment of the
                 butyrate-producing strains
 Long term colonization assay – conventional SHIME
       -     Colonization efficiency
       -     Metabolic activity
       -     Interaction endogenous microbiota




Selected butyrate
producing isolates
    (> Task 3)


                                     Stomach   Small intestine   Ascending   Transverse     Descending colon

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 4. Colonization efficiency of the colon environment of the
                 butyrate-producing strains
 Long term colonization assay – mucosal SHIME
                                                                                                            pH controller
                                                      Headspace connections
                                                                                                                 Acid/base
                                                      Liquid connection
                                                                                                      pH electrode
                                                      Pump
                                                                                                                                              Conventional
                                                                                                                                             ascending colon
                                                                                                                                              = “L-SHIME”
                                                                                                                                               (only luminal
                                                                                                                                                microbes)
                                              N2
Selected butyrate                                                                                                            pH controller

producing isolates                                                                                                                Acid/base


    (> Task 3)             Pancreatic juice
                                                                                                     Addition of mucin-
                                                                                                  covered microcosms to
                                                         Stomach         Small intestine              ascending colon
                                                                                                       = “M-SHIME”
                                                                                                   (luminal and mucosal
                                                                                                         microbes)
                                                                  Microcosms
                                                              7 mm
                                  SHIME feed                                                         Coated with mucin
                                                                                           9 mm         type II-agar



                                                             Side view      Front view


                                         Van den Abbeele et al., 2011. ISME journal (submitted), LabMET

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4. Colonization efficiency of the colon environment of the
                butyrate-producing strains
Long term colonization assay – mucosal SHIME
                  Higher taxonomic Group                       Inoculum                    Lumen                         Mucus
              Bacteroidetes                                  47.36 ± 5.50a              24.93 ± 3.66b                 3.53 ± 1.08c

              Total Firmicutes                               50.09 ± 5.21a              64.11 ± 3.21a                94.20 ± 1.68b

                     Clostridium cluster I                   0.04 ± 0.00a                0.53 ± 0.33a                10.45 ± 3.75b
                                                             28.47 ± 2.60a              15.77 ± 1.53b                18.72 ± 2.52ab
                     Clostridium cluster IV
                     Clostridium cluster XIVa                13.09 ± 2.47a              40.17 ± 2.48b                58.67 ± 3.82c


    Higher taxonomic Group                                                            Percentual Abundance (%)                        Code*1
            Firmicutes
                                      Bacterial group               Inoculum                Lumen                     Mucus
      Clostridium cluster I               Clostridia               0.03 ± 0.00 a          0.31 ± 0.18 a             6.08 ± 2.50 b      2C
                                  Anaerotruncus colihominis        0.47 ±   0.09 a        1.94 ±   0.30 b           0.86 ±   0.25 a    1A
                                     Clostridium cellulosi         1.12 ±   0.06 a        0.54 ±   0.10 b           0.49 ±   0.10 b    3B
                                   Clostridium orbiscidens         1.49 ± 0.21 a          1.76 ± 0.39 a             5.17 ± 0.86 b      2C
                                    Eubacterium siraeum            0.14 ± 0.08 a          0.02 ± 0.01 b             0.02 ± 0.01 b      3B
     Clostridium cluster IV      Faecalibacterium prausnitzii      10.92 ±   1.20 a       0.56 ±   0.25 b           2.20 ±   0.50 c    2B
                                 Papillibacter cinnamivorans      1.00± 0.21 a            0.27 ±   0.07 b           4.31 ±   0.73 c    2B
                                    Ruminococcus bromii            0.48 ± 0.07 a          0.29 ± 0.07 ab            0.17 ± 0.03 b      3B
                                    Ruminococcus callidus          1.25 ± 0.19 a          0.29 ± 0.07 b             0.14 ± 0.03 b      3B
                                  Subdoligranulum variable         1.61 ±   0.63 ab       2.53 ±   0.43 a           0.94 ±   0.15 b    1C
                                             Van den Abbeele et al., 2011. ISME journal (submitted), LabMET

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4. Colonization efficiency of the colon environment of the
                butyrate-producing strains
Long term colonization assay – mucosal SHIME
      Higher taxonomic Group                                                                    Percentual Abundance (%)                          Code*1
          Firmicutes
                                           Bacterial group               Inoculum                 Lumen                        Mucus
                                          Anaerostipes caccae                 0.15 ±   0.05 a         1.66 ±    0.14 b       0.34 ± 0.06 c          1A
                                       Bryantella formatexigens               0.52 ±   0.04 a         3.52 ±    0.38 b       1.97 ±   0.08 c        1A
                                         Butyrivibrio crossotus               1.58 ±   0.53 a         0.35 ±    0.08 b       4.48 ±   1.00 c        2B
                                          Clostridium colinum                 0.42 ± 0.09 a           0.08 ± 0.01 b          1.38 ± 0.32 c          2B
                                           Clostridium nexile                 0.60 ±   0.07 a         2.06 ±    0.16 b       2.17 ±   0.22 b        3A
                                        Clostridium sphenoides                0.64 ±   0.07 a         2.92 ±    0.34 b       2.74 ±   0.20 b        3A
                                        Clostridium symbiosum                 1.83 ±   0.32 a         15.54 ±   0.98 b      10.95 ±   0.22 c        1A
                                         Coprococcus eutactus                 1.73 ± 0.21 a           1.65 ± 0.25 a          4.83 ± 0.72 b          2C
                                        Dorea formicigenerans                 0.80 ±   0.07 a         5.19 ±    0.67 b       3.71 ±   0.28 b        3A

      Clostridium cluster XIVa            Eubacterium hallii                  0.17 ±   0.02 a         0.24 ±   0.04 ab       0.57 ±   0.19 b        3A
                                          Eubacterium rectale                 2.33 ±   0.56 a         0.22 ±    0.06 b       8.55 ±   1.47 c        2B
                                        Eubacterium ventriosum                0.78 ± 0.21 a           0.11 ± 0.04 b          1.68 ± 0.27 c          2B
                                         Lachnobacillus bovis                 1.45 ±   0.35 a         0.14 ±    0.02 b       3.85 ±   0.51 c        2B
                                       Lachnospira pectinoschiza              1.40 ±   0.29 a         3.19 ±    0.41 b       3.90 ±   0.54 b        3A

                                 Outgrouping Clostridium cluster XIVa         0.39 ± 0.04 a           1.80 ± 0.42 b          1.52 ± 0.19 b          3A

                                         Roseburia intestinalis               1.62 ± 0.49 a           0.08 ± 0.01 b          6.74 ± 0.59 c          2B
                                         Ruminococcus gnavus                  0.36 ±   0.06 a         3.94 ±    0.71 b       2.16 ±   0.51 b        3A
                                         Ruminococcus lactaris                0.12 ± 0.02 a           0.27 ± 0.12 a          0.83 ± 0.18 b          2C
                                         Ruminococcus obeum                   1.17 ±   0.14 a         3.96 ±    0.57 b       4.03 ±   0.47 b        3A
 *11: more abundant in lumen, 2: more abundant in mucus, 3: no difference between lumen and mucus – A: more abundant in SHIME lumen than in inoculum, B: more abundant in inoculu
 than in SHIME lumen, C: equally abundant in SHIME lumen and inoculum


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5. Strategies to enhance the metabolic activity of added or
   supplemented clostridial cluster IV and/or XIVa strain

 Prebiotics
    Stimulation endogenous/supplemented butyrate producers
    Stimulation acetate/lactate producers (Cross feeding)
    Inulin, XOS, AXOS


 Probiotics
    Acetate/lactate producers stimulate butyrate production
    Bifidobacteria, Lactobacilli

       YOUR TOP EXPERTS FOR INNOVATION    info@food2know.be
             Feed - Food - Health         www.food2know.org
                          WP2. Deliverables

• First generation anti-inflammatory Clostridial cluster XIVa
  isolates that are capable of surviving the upper digestive tract
  (Month 12)

• Second generation Clostridial cluster XIVa strains that
  effectively colonize the large intestinal environment over a
  long timeframe (Month 16)

• Strategy to promote butyrate production using functional
  foods on the short term (Month 18) and long term (Month 48)


     YOUR TOP EXPERTS FOR INNOVATION       info@food2know.be
           Feed - Food - Health            www.food2know.org
    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




          YOUR TOP EXPERTS FOR INNOVATION                  info@food2know.be
                Feed - Food - Health                       www.food2know.org
                       Workpackage 3:

Efficacy of administration of butyrate-producing
 Clostridial cluster IV and XIVa isolates to control
              broiler necrotic enteritis




    YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
          Feed - Food - Health        www.food2know.org
                               Objectives:
1.   To evaluate the efficacy of butyrate-producing Clostridial cluster
     IV and XIVa isolates to decrease inflammation and mucosal
     damage in a broiler necrotic enteritis model
2.   To evaluate the effects of administration of butyrate-producing
     Clostridial cluster IV and XIVa isolates on broiler performance in a
     field-like trial




     YOUR TOP EXPERTS FOR INNOVATION           info@food2know.be
           Feed - Food - Health                www.food2know.org
1. Evaluation of the efficacy of B. pullicaecorum in a broiler
                   necrotic enteritis model

                 Onset of necrotic enteritis



                                         +
                                             not necessarily!!

                           PREDISPOSING FACTORS
       YOUR TOP EXPERTS FOR INNOVATION             info@food2know.be
             Feed - Food - Health                  www.food2know.org
1. Evaluation of the efficacy of B. pullicaecorum in a broiler
                   necrotic enteritis model
                  Experimental in vivo model for sub-clinical NE
                                      d16 d17    d18   d19   d20    d21     d22      d23   d24

 Gumboro vaccine                        x

 Feed + fishmeal (30%)                       x    x     x     x       x          x    x     x

 10-fold dose of Paracox-5                        x

 Oral inoculation with
                                             x    x     x     x
 C. perfringens

 Autopsy                                                                         x    x     x

                     40-70% of infected birds present necrotic lesions

           YOUR TOP EXPERTS FOR INNOVATION                   info@food2know.be
                 Feed - Food - Health                        www.food2know.org
1. Evaluation of the efficacy of B. pullicaecorum in a broiler
                   necrotic enteritis model
  Necrotic lesions




      YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
            Feed - Food - Health        www.food2know.org
1. Evaluation of the efficacy of B. pullicaecorum in a broiler
                   necrotic enteritis model
                   Percentage animals having lesions (%)




                     B. pullicaecorum             probiotic B                 probiotic D
            positive ctrl             probiotic A               probiotic C                 negative ctrl




        Lyophilized and added ± 10exp9 cfu/kg feed

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             Feed - Food - Health                                  www.food2know.org
2. Effect of administration of B. pullicaecorum on broiler
                       performance
  Future experiment

 • As feed additive
 • Either or not in ‘challenged’ conditions (suboptimal conditions)
 • Trials at ILVO (6 replicates for each treatment group)

 • Monitoring of BWG and FCR
 • Samples for IH and morphometric analyses
 • Samples for quantification of different bacterial populations



      YOUR TOP EXPERTS FOR INNOVATION      info@food2know.be
            Feed - Food - Health           www.food2know.org
                           WP3. Deliverables

• Data on the efficacy of butyrate-producing isolates to decrease
  inflammation and mucosal damage in a broiler necrotic
  enteritis model (Month 24)

• Data on the effect of butyrate-producing isolates on
  zootechnical parameters in broilers (Month 30)

• A butyrate-producing Clostridial cluster IV or XIVa strain that
  affects performance and prevents necrotic enteritis in broiler
  chickens (Month 30)



      YOUR TOP EXPERTS FOR INNOVATION      info@food2know.be
            Feed - Food - Health           www.food2know.org
    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




          YOUR TOP EXPERTS FOR INNOVATION                  info@food2know.be
                Feed - Food - Health                       www.food2know.org
                   Workpackage 4:

 Efficacy of administration of butyrate-
 producing Clostridial cluster IV and XIVa
       isolates in IBD rodent models




YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
      Feed - Food - Health        www.food2know.org
                                  Objectives:
To evaluate the efficacy of butyrate-producing isolates from
fecal samples to decrease the severity of lesions in:

   1.     TNBS induced colitis model in rats
   2.     DSS-induced colitis model in mice
   3.     IL10 -/- mice
   4.     TNFDARE mice




        YOUR TOP EXPERTS FOR INNOVATION        info@food2know.be
              Feed - Food - Health             www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model

 First experiment:
Human versus chicken isolate
     Faecalibacterium prausnitzii ↔ Butyricicoccus pullicaecorum
          Anaerostipes caccae ↔ Anaerostipes butyraticus

Clostridal cluster IV versus Clostridial cluster XIVa
        Faecalibacterium prausnitzii ↔ Anaerostipes caccae
      Butyricicoccus pullicaecorum ↔ Anaerostipes butyraticus



      YOUR TOP EXPERTS FOR INNOVATION        info@food2know.be
            Feed - Food - Health             www.food2know.org
 1. Evaluation of efficacy of B. pullicaecorum to decrease
            severity of lesions in a TNBS model

60 male Wistar rats (n = 10)
group 1: 109 cfu B. pullicaecorum orally+ 10mg TNBS intrarectally
group 2 : 109 cfu A. butyraticus orally+ 10mg TNBS intrarectally

group 3: 109 cfu F. prausnitzii orally+ 10mg TNBS intrarectally

group 4: 109 cfu A. caccae orally+ 10mg TNBS intrarectally

group 5: PBS orally + 10mg TNBS intrarectally (pos ctrl)

group 6: PBS orally + PBS intrarectally (neg ctrl)

        YOUR TOP EXPERTS FOR INNOVATION        info@food2know.be
              Feed - Food - Health             www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model

                     Colon index (mg/g)                                    Colon weight (mg/cm)
                         colon weight / body weight                             colon weight / colon length
 15
                                                                 310
 13
                                                                 260
 11
                                                                 210
 9
                                                                 160
 7
                                                                 110
 5

 3                                                               60
      B. pullicaecorum   F. prausnitzii    pos ctrl                    B. pullicaecorum   F. prausnitzii       pos ctrl
               A. butyraticus       A. caccae         neg ctrl                 A. butyraticus      A. caccae              neg ctrl




            YOUR TOP EXPERTS FOR INNOVATION                                      info@food2know.be
                  Feed - Food - Health                                           www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model
    B. pullicaecorum                       Positive control




     YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
           Feed - Food - Health        www.food2know.org
     1. Evaluation of efficacy of B. pullicaecorum to decrease
                severity of lesions in a TNBS model

               Wallace score                                                        Ulceration (%)
                                                                                  lesion length/ colon length
12
                                                            35
10                                                          30

8                                                           25

                                                            20
6
                                                            15
4
                                                            10
2                                                           5

0                                                           0
 B. pullicaecorum F. prausnitzii      pos ctrl                   B. pullicaecorum F. prausnitzii      pos ctrl
             A. butyraticus    A. caccae         neg ctrl                   A. butyraticus   A. caccae         neg ctrl




              YOUR TOP EXPERTS FOR INNOVATION                                info@food2know.be
                    Feed - Food - Health                                     www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model

 Second experiment:
Preventive versus curative
     before TNBS administration ↔ after TNBS administration




      YOUR TOP EXPERTS FOR INNOVATION     info@food2know.be
            Feed - Food - Health          www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model

20 male Wistar rats (n = 5)

 group 1: 109 cfu B. pullicaecorum orally day 1 till day 14
              + 10mg TNBS intrarectally day 7
 group 2 : 109 cfu B. pullicaecorum orally day 7 till day 14
               + 10mg TNBS intrarectally day 7
 group 3: PBS orally
              + 10mg TNBS intrarectally day 7 (pos ctrl)
 group 4: PBS orally
              + PBS intrarectally day 7 (neg ctrl)
      YOUR TOP EXPERTS FOR INNOVATION         info@food2know.be
            Feed - Food - Health              www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model
                    %


                  Colon index (mg/g)                            Ulceration (%)
                      colon weight / body weight              lesion length / colon length




                                                          pos ctrl                                 neg ctrl
 pos ctrl                                      neg ctrl
            B. pullicaecorum   B. pullicaecorum                  B. pullicaecorum   B. pullicaecorum
                d7-d14           d1 – d 14                           d7-d14           d1 – d 14




            YOUR TOP EXPERTS FOR INNOVATION                 info@food2know.be
                  Feed - Food - Health                      www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model

 Third experiment:
Probiotic versus therapeutic
       Butyricicoccus pullicaecorum ↔ anti-inflammatory drug
                                    ↔ corticosteroid
                                    ↔ immunosuppressant
                                    ↔ anti-TNFα




      YOUR TOP EXPERTS FOR INNOVATION      info@food2know.be
            Feed - Food - Health           www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model
65 male Wistar rats (n = 10)
group 1: 250mg/kg 5-ASA (Mesalazine) orally day 1 - 7
                + 10mg TNBS intrarectally day 1
group 2: 5mg/kg corticosteroids (SoluMedrol) IM day 1 - 7
       + 10mg TNBS intrarectally day 1
group 3: 33mg/kg immunosup (Mycophenolate mofetil) orally day 1 - 7
       + 10mg TNBS intrarectally day 1
group 4: 3mg/kg anti TNFalfa (Infliximab) IV once
       + 10mg TNBS intrarectally day 1
group 5 : 109 cfu B. pullicaecorum orally day 1 - 7
       + 10mg TNBS intrarectally day 1
group 6: PBS orally + 10mg TNBS intrarectally day 1 (pos ctrl)
group 7: PBS orally + PBS intrarectally day 1 (neg ctrl)
       YOUR TOP EXPERTS FOR INNOVATION         info@food2know.be
             Feed - Food - Health              www.food2know.org
1. Evaluation of efficacy of B. pullicaecorum to decrease
           severity of lesions in a TNBS model
                                  Colon index (mg/g)
                                     colon weight / body weight




        pos ctrl    5-ASA       corticosteroids   immunosup        anti TNF-α        probiotic      neg ctrl
                   mesalazine    solu medrol      mycophenolate    infliximab    B. pullicaecorum
                                                    mofetil

                       Macroscopic (Wallace score)




        pos ctrl     5-ASA      corticosteroids   immunosup         anti TNF-α      probiotic      neg ctrl
                   mesalazine     solu medrol      mycophenolate    infliximab    B. pullicaecorum
                                                   mofetil

     YOUR TOP EXPERTS FOR INNOVATION                                       info@food2know.be
           Feed - Food - Health                                            www.food2know.org
                                Planned experiments

   • Effect of B. pullicaecorum in comparison with #VSL-3 on the
     intestinal microflora in a rat TNBS model for ulcerative colitis
   • 5 groups of 15 rats
                                      Day 7 10 mg TNBS in                          PBS
                                           400µL EtOH




              B.pullicaecorum     B.pullicaecorum                                  Negative
Day 1-9          (chicken)           (human)
                                                      #VSL-3   Positive control
                                                                                    control




                                            Faecal samples


          YOUR TOP EXPERTS FOR INNOVATION                      info@food2know.be
                Feed - Food - Health                           www.food2know.org
                                Planned experiments
• Effect of administration of butyrate-producing clostridium cluster
  IV and XIV isolates on the intestinal microflora in a rodent TNBS
  model for ulcerative colitis
• 5 or more groups of 15 rats
                                      Day 7 10 mg TNBS in                            PBS
                                          400µL EtOH




                                                                                     Negative
Day 1-9           Strain A           Strain B         Strain C   Positive control
                                                                                      control




                                            Faecal samples


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                Feed - Food - Health                             www.food2know.org
2. Evaluation of efficacy of B. pullicaecorum to decrease
            severity of lesions in a DSS model

       Results: curative DSS treatment

• Clinically most relevant
• Potential effect of butyrate on recovery
• Using Butyricicoccus pullicaecorum




     YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
           Feed - Food - Health        www.food2know.org
2. Evaluation of efficacy of B. pullicaecorum to decrease
            severity of lesions in a DSS model
  Intragastric admission of 100 ml B.p. culture (~108 cfu) or PBS

                                                                                                  †
    Day:
    0       1     2     3      4      5      6     7        8       9      10      11   12   13   14   15




   4% DSS                                         STOP DSS


    Sample prelevation (feces, colon, liver, spleen, serum):




    No                                            Peak                    Early                      Late
   DSS                                       inflammation               recovery                   recovery
  control


        YOUR TOP EXPERTS FOR INNOVATION                                      info@food2know.be
              Feed - Food - Health                                           www.food2know.org
2. Evaluation of efficacy of B. pullicaecorum to decrease
            severity of lesions in a DSS model

                   5
                                                   Results: weight loss
                   0
                        0      2         3     4     5    7   8   9   10     11     12    13   14


                   -5
  % weight loss




                  -10

                              PBS
                              Butyricicoccus
                  -15




                  -20




                  -25




                  -30




                        YOUR TOP EXPERTS FOR INNOVATION                    info@food2know.be
                              Feed - Food - Health                         www.food2know.org
2. Evaluation of efficacy of B. pullicaecorum to decrease
            severity of lesions in a DSS model
                    Results: colon length
                      8


                      7


                      6


                      5
  colon length (cm)




                      4                                                               Butyricicoccus
                                                                                      PBS

                      3



                      2


                      1


                      0
                              no DSS                 D7     D10            D14




                          YOUR TOP EXPERTS FOR INNOVATION         info@food2know.be
                                Feed - Food - Health              www.food2know.org
                                         To do

 Determine histological inflammation, inflammatory parameters and
recovery parameters at day 14

 Follow weight evolution until final recovery in a new curative DSS
experiment

 Perform a preventive treatment in DSS colitis

 Test B.pullicaecorum in chronic models (IL10-/- and TNFDARE)



       YOUR TOP EXPERTS FOR INNOVATION           info@food2know.be
             Feed - Food - Health                www.food2know.org
                          WP4. Deliverables

 A butyrate-producing strain that is capable to improve clinical
  parameters and inflammation markers in the TNBS-induced
  colitis model, the DSS-induced colitis model in mice and/or
  colitis in IL10-/- mice, ileitis in TNFDARE mice (Month 24)




     YOUR TOP EXPERTS FOR INNOVATION       info@food2know.be
           Feed - Food - Health            www.food2know.org
    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




          YOUR TOP EXPERTS FOR INNOVATION                  info@food2know.be
                Feed - Food - Health                       www.food2know.org
                     Workpackage 5:

Exploration of the host response to butyrate




  YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
        Feed - Food - Health        www.food2know.org
                               Objectives:

1.   To determine the expression of genes that are regulated by
     butyric acid in a collection of biopsies of IBD patients and healthy
     controls and in animal models
2.   To determine whether the expression of these genes is
     influenced by treatment with butyric acid producing bacteria in
     animal models




     YOUR TOP EXPERTS FOR INNOVATION           info@food2know.be
           Feed - Food - Health                www.food2know.org
          Effects of butyrate in the intestine




Canani et al. 2011

             YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
                   Feed - Food - Health        www.food2know.org
Is the lack of butyric acid a target for therapeutic intervention
                             in IBD?




    YOUR TOP EXPERTS FOR INNOVATION      info@food2know.be
          Feed - Food - Health           www.food2know.org
1. Determination of butyric acid regulated gene expression

                      Results: intestinal gene expression
                                      Control            CD                      UC
                                       N=24             N=50                    N=27
                                    Nbiopsies=36     Nbiopsies=95            Nbiopsies=50
       Gender (M/F)                       9/15          26/24                   17/10

       Age mean (range)                 50 (22-69)    37 (8-73)               39 (17-57)

       medication (no/5-ASA/IS)          24/0/0        36/10/4                 7/13/2

       colon/ileum                        21/15         52/43                   36/14

       healthy/inflamed                   36/0          54/41                   30/20



                                                                       ROC curve analysis
                                                                       IL8 - Macroscopic inflammation




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1. Determination of butyric acid regulated gene expression

                 Results: intestinal gene expression
             P-values                COLON                ILEUM       Effect of butyrate

                                  CD         UC                CD

             Ion and molecule transport
             MCT1               0.019      0.021      0.681                   +
             CFTR               <0.001     <0.001     0.090                   +
             PEPT1              0.465      0.637      0.087                   +
             GLUT1              0.092      0.648      0.037                   -
             Immune regulation
             ICAM1              <0.001     0.001      0.948                   -
             ALPI               0.092      0.558      0.033                   +
             Cell growth
             CCDN1              0.085      0.002      0.386                   -
             Barrier function
             TJP1               0.000***   0.000***   0.004**                 +
             CLDN1              0.000***   0.025*     0.039*                  -
             OCLN               0.001**    0.003*     Low abundance           +

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                             Conclusion


 Butyrate-regulated genes are impaired in (colonic) IBD

 Administering butyrate can provide benefits by stabilizing the
downstream genes and their processes




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What are the effects of the butyric acid producing isolates
            on gene expression in the gut?




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            Host response to the bacterial isolates



•   Colonic biopsies and HT29 colonic epithelial cells seeded

•   Incubated for 24 hours with 1:25 of

              • B. pullicaecorum supernatant
              • B. pullicaecorum supernatant minus butyric acid (Vivaspin)
              • medium supplemented with 20 mM butyric acid

•   Gene expression measured by qRT-PCR and ELISA




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                                 Results: intestinal gene expression
                                          Biopsies:                                                                       HT29:
                                                                                               1200




                                                                         normalized MCT1mRNA
normalized MCT1 mRNA




                                                                                               1000
                        12                                                                      800
                        10
                         8                                                                      600
                         6                                                                      400
                         4                                                                      200
                         2
                         0                                                                            0




                                                                                                             (negative)




                                                                                                                                                    SN - BZ
                                                                                                                             medium+BZ




                                                                                                                                            SN
                                                               SN-BZ
                                            medium+BZ
                             (negative)




                                                          SN




                                                                                                              medium




                                                                                                                              (positive)
                              medium




                                             (positive)




                                                                                               1600
normalized GLUT1 mRNA




                                                                                               1400




                                                                       normalized GLUT1 mRNA
                        20
                                                                                               1200
                        15
                                                                                               1000
                        10
                                                                                               800
                         5
                                                                                               600
                         0                                                                     400
                                                               SN-BZ
                                            medium+BZ
                             (negative)




                                                          SN
                              medium




                                             (positive)




                                                                                               200
                                                                                                 0
                                                                                                           medium         medium+BZ        SN    SN - BZ
                                                                                                          (negative)       (positive)



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                             Results: intestinal gene expression
                       5                                                                    1800
                                                                                            1600




                                                                      normalized IL8 mRNA
                       4                                                                    1400
normalized CFTR mRNA




                                                                                            1200
                       3                                                                    1000
                                                                                             800
                       2                                                                     600
                                                                                             400
                       1                                                                     200
                                                                                              0
                       0




                                                                                                                                      SN-BZ
                                                                                                                   medium+BZ
                                                                                                   (negative)




                                                                                                                                 SN
                                                                                                    medium




                                                                                                                    (positive)
                                           medium+BZ




                                                              SN-BZ
                              (negative)




                                                         SN
                               medium




                                            (positive)




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                        Results: IL8 secretion
                                                        hIL-8 genormaliseerd
HT29 cells stimulated with SN of:        140.000



Butyricicoccus pullicaecorum             120.000

E. coli MC1061
                                         100.000
E. coli DH5α
E.coli AIEC (pathogenic)                  80.000
Lactococcus lactis (GRAS)
                                          60.000


                                          40.000


                                          20.000


                                           0.000
                                                   Bp    SN-BZ RCM+BZ MC 1061 AIEC   DH5a   Ll

                                         -20.000




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                              Conclusion


 Supernatant of B. pullicaecorum is able to “restore” gene
expression in human biopsies

 This is largely mediated by the butyric acid produced by the bacteria

 IL8 secretion is not induced upon treatment with B. pullicaecorum




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                                        To do


 Determine NF-kB status upon B. pullicaecorum treatment (pro- and
anti-inflammatory cytokines, phospho-p65 in nucleus, IkBa in cytosol)

 Functional effects of B. pullicaecorum in intestinal epithelial cells in
vitro (TER in Caco2 cells, MTT proliferation assay, ion transport,…)




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                          WP5. Deliverables

• Data generation on expression of specific genes, known to be
  regulated by butyric acid, in biopsies from IBD patients and
  healthy controls (Month 24)

• Data generation on the efficacy of butyrate-producing strains to
  neutralize aberrant gene expression in animal models of colitis
  (Month 32)




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    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert)
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




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                       Workpackage 6

Placebo-controlled randomized human trials to
   evaluate safety, and explore gut microbiota
      shifts and clinical activity after strain
                  administration




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                                Objectives
1.   Analyze safety of the butyrate-producing strains in OECD approved
     in vitro and in vivo toxicity tests
2.   Determine baseline prevalence of the selected butyrate-producing
     strains in the gut microbiota of healthy human volunteers and IBD
     patients
3.   Up scaling and production of the selected butyrate-producing
     strains in an easy to administer form
4.   Analysis of safety and tolerability after administration of the
     selected butyrate-producing strains in healthy human volunteers
5.   Analysis of safety and tolerability, including gut microbiota shifts
     and determination of easy clinical parameters after administration
     of the selected butyrate-producing strains to IBD patients


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                        Safety assessment
    according to the guidelines of the Organization for Economic Co-operation
                             and Development (OECD)



•    Mammalian erythrocyte micronucleus test
•    In vitro mammalian chromosome aberration test
•    Acute oral toxicity in rodents
•    Repeated dose 28-day oral toxicity study in rodents




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1. Evaluate safety of the butyrate producing strains in OECD
         approved in vitro and in vivo toxicity tests
   • Acute Toxicity (OECD 420)
      –   1 female rat: 109 B. pullicaecorum
      –   2 weeks observation
      –   4 additional females: 1010 B. pullicaecorum
      –   2 weeks observation


   • Repeated Oral Dose Toxicity (OECD 407)
      – 10 female rats (5 dosing group-5 control group)
      – 10 male rats (5 dosing group-5 control group)
      – Daily dose of 109-1010 B. pullicaecorum for 28 days

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1. Evaluate safety of the butyrate producing strains in OECD
         approved in vitro and in vivo toxicity tests

Observations and sample collection during the toxicity test
   •   Clinical observations               Blood sample:
   •   Body temperature                       Biochemistry

   •   Weight                                 Hematology

   •   Food conversion                     Gross necropsy
   •   Neurological toxicity               Histopathology
                                           Faecal samples
   4th exposure week:                      Liver , spleen:
   • Sensory reactivity                       Bacterial translocation

    Motor activity
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Clinical observations during the acute and repeated oral
                    dose toxicity test
Clinical observation        Parameters
General appearance          Fur appearance, skin appearance, mucous membranes, secretions
                            / excretions, lacrimation, salivation, diarrhea
Posture                     Lateralized posture, flattened posture, hunched back, rearing
Mobility                    Hypomobility, ataxic gait, circling
Handling measures           Ease of removal, ease of handling
Behaviour                   Passivity, hyperreactivity, irritability, arousal, vocalizations,
                            urination, defecation, palpebral closure
Bizarre behaviour           Walking backwards, selfmutilation, other
Stereotypies                Excessive grooming, repetitive circling, other
Neurological activity       Tremors, twitches, respiratory distress, piloerection, ptosis,
                            hypoalgesia, hyperalgesia, convulsions, paralysis, coma


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 Sensory and motor activity parameters during the 4th
                  exposure week

Clinical observation       Parameters
Reflexes                   Approach response, click response, touch response, tail pinch
                           response, pupillary reflex, righting reflex, forelimb and hindlimb
                           grip
Motor activity             Decreased body tone, forelimb flexion, decreased muscle strength,
                           body rotation, motor incoordination




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              Results from the acute toxicity test

                                        Clinical observations
                                        Body temperature
                                        Weight                         Normal
                                        Food conversion
                                        Sensory & motor activity
Acute toxicity
                                        Mortality
                                        Morbidity
                                        Neurological toxicity         Absent
                                        Bacterial translocation

                                        Blood samples: Between reference ranges
                                        Gross necropsy: No macroscopic lesions
                                        Histopathology (partial):



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     Results from the repeated oral dose toxicity test
                                                  Clinical observations
                                                  Body temperature
                                                  Weight                              Normal
                                                  Food conversion
                                                  Sensory & motor activity
Repeated oral                                     Mortality
 dose toxicity                                    Morbidity
                                                  Neurological toxicity               Absent
                                                  Bacterial translocation

                                                  Blood samples:
                                                  •   Between reference ranges
 Gross necropsy:
                                                  •   No difference between the dosing group and
 •    No macroscopic lesions                          the control group for all parameters involved
 •    One rat (D8) displayed mildly swollen MLN
 Histopathology (partial):

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1. Evaluate safety of the butyrate producing strains in OECD
         approved in vitro and in vivo toxicity tests


                              Planned experiments

      • Safety assessment in vivo

         – Further processing of samples from toxicity test




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2. Determination of baseline prevalence of the selected
           butyrate-producing strains in humans

• Quantify selected strain in fecal samples
   – 20 healthy subjects
   – 20 patients with Crohn’s Disease
   – 20 patients with Ulcerative Colitis
• Real-time PCR primers and probes
• Results will govern selection of the strain
   – prevalent in 90% of healthy subjects
   – depleted in IBD patients
   – dose to be administered: 100 x median log10 amount per g wet weight



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3. Production of sufficient quantities of the strains in an
              easy to administer formula

• SOP for fermentation and lyophilisation/spray drying (10 L
  fermentation)
• Upscaling (5000 L fermentation)




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                      WP6. Deliverables
• Report on the safety of selected butyrate-producing strains in OECD
  approved in vitro and in vivo models (Month 30)
• Baseline prevalence numbers of selected butyrate-producing strains in
  fecal material of healthy humans and IBD patients (Month 30)
• A protocol for production of selected butyrate-producing strains in large
  quantities for human administration (Month 30)
• Data on the safety and tolerability of the selected butyrate-producing
  strains in healthy human volunteers (Month 36)
• Data on the safety and tolerability of the selected butyrate-producing
  strains in IBD patients (Month 44)
• Data on the gut microbiota shifts and altered clinical parameters caused
  by uptake of the selected butyrate-producing strains in IBD patients
  (Month 48)

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    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert)
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




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                      Workpackage 7:

  Exploration of the microbiota response to
administration of butyrate-producing bacteria
      in human proof-of-concept study




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                               Objectives:
1.   Determination of the compositional changes of the microbiota
     and its functional consequences after administration of
     butyrate-producing Clostridial cluster IV and XIVa isolates to
     healthy humans
2.   Analysis of the effects of administration of butyrate-producing
     Clostridial cluster IV and XIVa isolates to IBD patients, and thus
     determination of the potential reversion of gut dysbiosis




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                              1. Quantification of different bacterial populations


                                          B. pullicaecorum                                                               F. prausnitzii
                              2,82%     3,73%     5,47%    0,63%        0,80%                                  2,93% 2,86%     3,51%       1,21%   0,39%
                                                                                                          12
                         12
Number (log10/g stool)




                                                                                 Number (log10/g stool)
                                                                                                          11
                         10
                                                                                                          10
                         8                                                                                9

                         6                                                                                8

                                                                                                          7
                         4
                                                                                                          6
                         2
                                                                                                          5
                                                                n=1        n=3
                         0                                                                                4
                                UC       UC    healthy   CD       CD                                             UC      UC     healthy       CD      CD
                              remission active         remission active                                        remission active            remission active




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1. Quantification of different bacterial populations:
               Butyricicoccus pullicaecorum

      Tm 78°      Healthy person
      Tm 79°      UC patient
      Tm 80°      UC patient
      Tm 80,5 Standard
      Tm 81°      Healthy person
      Tm 81,5° CD patient




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     Cloning and sequencing of 125nt QPCR amplicon
Melt t°    coloni      Amplicon        Accesion nr   Isolate (16S rRNA gene sequence similarity)
             es        homology
Tm 78°      1-5           83%          AM693872      Uncultured bacterium clone AP16R.314 (100%)
            1&5           79%          HQ259866      Uncultured bacterium clone Ib16_27f (98%)
             2            77%          GU615844      Uncultured bacterium clone DF7667 (94%)
             3            85%          GQ897177      Uncultured bacterium clone C4-38 (98%)
Tm 79°
             4            79%          GU621816      Uncultured bacterium clone PF3921 (96%)
             6            78%          GU105632      Uncultured bacterium clone HFV05_327 (98%)
             1            77%          JF797918      Uncultured bacterium clone VT009-C10 (97%)
             2            80%          GU629408      Uncultured bacterium clone HF3899 (98%)
Tm 80°       3            79%          HQ259866      Uncultured bacterium clone Ib16_27f (96%)
             4            77%          JF781785      Uncultured organism clone B11d28bio2cold (98%)
Tm 81°       1            82%          FJ371196      Uncultured bacterium clone TS6_a02a09 (98%)
             2            81%          HQ910620      Uncultured bacterium clone NW_79 (97%)
Tm 81,5°    1-2           79%          HQ259866      Uncultured bacterium clone Ib16_27f (98%)
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                                       Clostridium perfringens ATCC 13124T (M59103)
  Anaerotruncus colihominis (DQ002932)
     Uncultured bacterium clone (GU105632)
               Uncultured bacterium clone (FJ371196)
             Ethanoligenens harbinense (AY833421)
         Acetanaerobacterium elongatum (AY518589)
         Acetanaerobacterium elongatum T (AY487928)
             Eubacterium siraeum (EU266550)
                Uncultured bacterium clone (HQ910620)
                   uncultured bacterium CHIMP1_aaj39b10; (EU462281)
                   uncultured bacterium p-1877-s962-3; (AF371805)
                    uncultured bacterium RL179_aao53a08; (DQ796674)
                         uncultured Clostridiales bacterium; YNP_ObP_B94; (DQ243771)
                        Clostridiales bacterium 7-4c (HQ452854)
                       Butyricicoccus pullicaecorum CCUG 55265T (EU410376)
                             Uncultured bacterium clone YO00041H06 (EU197231)
                          uncultured Firmicutes bacterium M0018_056; (EF071188)
                         Clostridiales bacterium DJF_B152; (EU728715)
                                uncultured bacterium nby636g06c1; (HM838952)                genus:
                                     uncultured bacterium C12_A06; (AY990070)
                                   uncultured bacterium WD3_aak03b03;(EU510220)
                       Eubacterium desmolansT (L34618)
                             uncultured bacterium L11-2; (AJ400270)                         Butyricicoccus
                        uncultured bacterium RL199_aaj43h12; (DQ793455)
                    butyrate-producing bacterium A2-207 (AJ270471)
                      uncultured bacterium SJTU_E_03_17; (EF399766)
                       uncultured bacterium B863; (AY984887)
                                                                                                                         family:
                           uncultured bacterium TS150_a04f06; (FJ364244)
                     uncultured bacterium BB93; (AY985169)
                       uncultured bacterium nbw1231c08c1; (GQ079310)                                                     Ruminococcaceae
                        uncultured bacterium BF0001C084; (AM697243)
                        uncultured bacterium PM8_a04e11; (GQ491576)
                      uncultured bacterium 16slp96-1b09.p1k; (GQ158589)
                        uncultured bacterium G-C-H03; (AB506192)
                           Clostridiales bacterium DJF_CP67; (EU728728)                                                            ↕
                              uncultured bacterium ncd1862h11c1; (JF161375)
                             Clostridium viride T2-7 DSM 6836T (X81125)
                             isolate 24-4c
                            isolate 30-4c
                                                                                                                       Clostridial cluster IV
                               Pseudoflavonifractor capillosus ATCC 29799T (AY136666)
                               Flavonifractor plautii DSM 6740T (Y18187)
                               Eubacterium plautii ATCC 29863T (AY724678)
                             Papillibacter cinnamivorans DSM12816T (AF167711)
                              Uncultured bacterium clone (GQ897177)
                                                   Uncultured bacterium clone (GU621816)
                      Ruminococcus bromii ATCC 27255T (L76600)
                              Uncultured bacterium clone (HQ259866)
                                       Faecalibacterium prausnitzii 1-84 (AY169429)
                                      isolate 40-4c
                                        Subdoligranulum variabile DSM 15176T (AJ518869)
                                      Faecalibacterium prausnitzii M21/2 (AY305307)
                                      Faecalibacterium prausnitzii L2-6 (AJ270470)
     Clostridium methylpentosum (Y18181)
                            Uncultured bacterium clone (AM693872)
                           Ruminococcus flavefaciens (X85097)
                           Uncultured bacterium clone (GU615844)
                                                                                         Uncultured bacterium clone (GU629408)
                                                                           Uncultured bacterium clone (JF781785)
                                                              Uncultured bacterium clone (JF797918)
0.1

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                          Designing new primers
Probe Match
Forward primer: 5’-ACCTGAAAATAAGCTCC-3’
         2538 hits / 1801521 all bacteria
                    1925 hits / 45890 Ruminococcaceae
                                601 hits / 758 Butyricicoccus
                                 + 846 hits / 15347 unclassified Ruminococcaceae
                                 + 382 hits / 2528 Acetivibrio
Reverse primer: 5’-TACGTAGGGAGCAAGCGTTATC-3’
         615 hits / 1801521 all bacteria
                    455 hits / 45890 Ruminococcaceae
                                431 hits / 758 Butyricicoccus
                                + 19 hits / 15347 unclassified Ruminococcaceae

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           Feed - Food - Health                            www.food2know.org
                                       Clostridium perfringens ATCC 13124T (M59103)
  Anaerotruncus colihominis (DQ002932)
     Uncultured bacterium clone (GU105632)
               Uncultured bacterium clone (FJ371196)
             Ethanoligenens harbinense (AY833421)
         Acetanaerobacterium elongatum (AY518589)
         Acetanaerobacterium elongatum T (AY487928)
             Eubacterium siraeum (EU266550)
                Uncultured bacterium clone (HQ910620)
                   uncultured bacterium CHIMP1_aaj39b10; (EU462281)
                   uncultured bacterium p-1877-s962-3; (AF371805)
                    uncultured bacterium RL179_aao53a08; (DQ796674)
                         uncultured Clostridiales bacterium; YNP_ObP_B94; (DQ243771)
                        Clostridiales bacterium 7-4c (HQ452854)
                       Butyricicoccus pullicaecorum CCUG 55265T (EU410376)
                             Uncultured bacterium clone YO00041H06 (EU197231)
                          uncultured Firmicutes bacterium M0018_056; (EF071188)
                         Clostridiales bacterium DJF_B152; (EU728715)
                                uncultured bacterium nby636g06c1; (HM838952)                genus:
                                     uncultured bacterium C12_A06; (AY990070)
                                   uncultured bacterium WD3_aak03b03;(EU510220)
                       Eubacterium desmolansT (L34618)
                             uncultured bacterium L11-2; (AJ400270)                         Butyricicoccus
                        uncultured bacterium RL199_aaj43h12; (DQ793455)
                    butyrate-producing bacterium A2-207 (AJ270471)
                      uncultured bacterium SJTU_E_03_17; (EF399766)
                       uncultured bacterium B863; (AY984887)
                                                                                                                         family:
                           uncultured bacterium TS150_a04f06; (FJ364244)
                     uncultured bacterium BB93; (AY985169)
                       uncultured bacterium nbw1231c08c1; (GQ079310)                                                     Ruminococcaceae
                        uncultured bacterium BF0001C084; (AM697243)
                        uncultured bacterium PM8_a04e11; (GQ491576)
                      uncultured bacterium 16slp96-1b09.p1k; (GQ158589)
                        uncultured bacterium G-C-H03; (AB506192)
                           Clostridiales bacterium DJF_CP67; (EU728728)                                                            ↕
                              uncultured bacterium ncd1862h11c1; (JF161375)
                             Clostridium viride T2-7 DSM 6836T (X81125)
                             isolate 24-4c
                            isolate 30-4c
                                                                                                                       Clostridial cluster IV
                               Pseudoflavonifractor capillosus ATCC 29799T (AY136666)
                               Flavonifractor plautii DSM 6740T (Y18187)
                               Eubacterium plautii ATCC 29863T (AY724678)
                             Papillibacter cinnamivorans DSM12816T (AF167711)
                              Uncultured bacterium clone (GQ897177)
                                                   Uncultured bacterium clone (GU621816)
                      Ruminococcus bromii ATCC 27255T (L76600)
                              Uncultured bacterium clone (HQ259866)
                                       Faecalibacterium prausnitzii 1-84 (AY169429)
                                      isolate 40-4c
                                        Subdoligranulum variabile DSM 15176T (AJ518869)
                                      Faecalibacterium prausnitzii M21/2 (AY305307)
                                      Faecalibacterium prausnitzii L2-6 (AJ270470)
     Clostridium methylpentosum (Y18181)
                            Uncultured bacterium clone (AM693872)
                           Ruminococcus flavefaciens (X85097)
                           Uncultured bacterium clone (GU615844)
                                                                                         Uncultured bacterium clone (GU629408)
                                                                           Uncultured bacterium clone (JF781785)
                                                              Uncultured bacterium clone (JF797918)
0.1

 YOUR TOP EXPERTS FOR INNOVATION                                                         info@food2know.be
       Feed - Food - Health                                                              www.food2know.org
                     WP7. Deliverables
• Expected variability in healthy and IBD flora determined from
  external data (Month 12)
• Simulation tools and metabolic models for functional interpretation
  of the gut microbiota sequence data developed (Month 24)
• Ecological measurements of gut ecosystem health developed
  (Month 36)
• Next-generation sequencing data on the microbial gut community
  composition collected (Month 44)
• Linking microbial structure and dynamics with functionality and
  health state completed (Month 48)



     YOUR TOP EXPERTS FOR INNOVATION         info@food2know.be
           Feed - Food - Health              www.food2know.org
    Agenda
•   11.30 – 12.00: Arrival of the Guests & Welcome in
    “Het Pand : De Oude Infermerie” (Filip Van Immerseel – Steffi Vervaet)
•   12.00 – 13.30: Lunch (Restaurant Het Pand)
•   13.30: Aims of the project (Filip Van Immerseel)
•   13.40: Workpackage 1 (Marjan Steppe)
•   13.55: Workpackage 2 (Clémentine Perrier & Annelies Geirnaert )
•   14.10: Workpackage 3 (Venessa Eeckhaut)
•   14.25: Workpackage 4 (Venessa Eeckhaut & Sarah Devriese)
•   14.40: Workpackage 5 (Sarah Devriese)
•   14.55: Workpackage 6 (Marjan Steppe & Kristin Verbeke)
•   15.10: Workpackage 7 (Venessa Eeckhaut)
•   15.25: Valorization aims and strategy (Benedikt Sas)
•   15.40: Discussions (All)
•   16.00: Final Remarks & Closing (Filip Van Immerseel)




          YOUR TOP EXPERTS FOR INNOVATION                  info@food2know.be
                Feed - Food - Health                       www.food2know.org
            Valorization aims and strategy




YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
      Feed - Food - Health        www.food2know.org
                         Patent Status
METHOD FOR ALLIVIATING INTESTINAL                 First Patent was filed on 23/2/2009
PROBLEMS AND NOVEL     BACTERIAL
STRAINS THEREFORE (UK090316.4)                    In July it moved to National Phase.

                                                  Covered countries are:
                                                  -Europe
                                                  -US
      Is mainly covering 2 strains:
                                                  -Canada
      -Butyricicoccus pulicaecorum
                                                  -India
      -Anaerostipes butyricus
                                                  -China



 In the near future we want to file a new patent that
 covers the technology in a more extensive way.
 For this patent we will invest more money in the
 national phase

        YOUR TOP EXPERTS FOR INNOVATION                 info@food2know.be
              Feed - Food - Health                      www.food2know.org
                                           Related Business Sectors
                                                 Follow Up Project
                                                 Started
High




                                 Feed
         PROBABLITY OF LAUNCH




                                Additive
                                            Pet Food
                                           Ingredient
Medium




                                                Functional Food
                                                 Nutriceutical


                                                        Vet Product
                                                            NE
Low




                                                                  Pharmaceutical
                                                                    IBD & IBS


                                             REVENUES
                                Low               Medium               High


                                YOUR TOP EXPERTS FOR INNOVATION                    info@food2know.be
                                      Feed - Food - Health                         www.food2know.org
     Follow Up Project: Feed
• Funding via Industrial Research Fund (IOF) as
  StepStone Project.
• Total funding of €392124.
• Period: 3 years
• Aim: to get Butyricicoccus pullicaecorum ready for
  the feed market:
      -pilot scale production (SOP)
      -tox package
      -mode of action
      -efficacy trials in broilers

     YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
           Feed - Food - Health        www.food2know.org
      Follow Up Project: Feed (continued)
A phased research program:
                                        SCALE-UP IN STABLE
                                              FORM
                                             Go/no-go        (>109 cfu/g of spores)


                            SAFETY TESTS         ZOOTECHNICAL STUDY
(acute toxicity >
1010 cfu/kg B.W.)   Go/no-go

                      EFFICACY STUDIES



                                                 Go/no-go
                                                             Performance better or equal to
                                                             Golden Standard
                                     MODE-OF-ACTION


           YOUR TOP EXPERTS FOR INNOVATION                    info@food2know.be
                 Feed - Food - Health                         www.food2know.org
   Follow Up Project: Feed (continued)
 Product Positioning:
• As a DFM (Direct Fed Microbial) for GI health.
• To be registered as a Feed Additive conform.
• Disruptive, second generation innovation.
  - better efficacy
  - more stable due to spore forming
  - ADME studies possible (cfr. EMEA, Vet)
  - LAB is fueling BAB
  - problems with LAB (differentiators), LA (cfr H2S
  production), BA as such (fast uptake, smell…)

      YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
            Feed - Food - Health        www.food2know.org
  Follow Up Project: Feed (continued)
Product Positioning:
Superior to current commercial available products,
  including the Golden Standard.
          Pos                             Biacton PoultryStar Probi-
                  Butyricicoccus Clostat Probi-
                                  Probi-           Probi-     Proflora
          ctrl        in feed             otic B
                                 (Kemin) (Chemvet) otic C
                                  otic A                      otic D
                                                   (Biomin) (Alpharma)
          50%          7,69%       15%      35%       35%        46%
                     P=0,003     P=0,011 P=0,372    P=0,263    P=0,781


   Butyricicoccus pulicaecorum is safe and more efficient in reducing the
   incidences of NE (p.o.10E9 CFU/day) in a chicken model




     YOUR TOP EXPERTS FOR INNOVATION              info@food2know.be
           Feed - Food - Health                   www.food2know.org
     Follow Up Project: Feed (continued)
Product Positioning:
                                                    SWOT ANALYSES

Strenghts                                  Weaknesses
-Very efficient                            -Scale up possible?
-Natural, non-GMO, non antibiotic          -Shelf life acceptable?
-Novel strain, new name

Opportunities                              Threats
-New generation DFM                        -Currently allowed antibiotics (vet)
-Replacement of butyrates/BA               -Butyric acid based products
-Replacement of antibiotics                -Other DFM/Probiotics

                                                    DIFFERENTIATORS

-Novel strain and MOA
-Excellent safety profile
-As efficient as AGPs - antibiotics

         YOUR TOP EXPERTS FOR INNOVATION            info@food2know.be
               Feed - Food - Health                 www.food2know.org
   Follow Up Project: Feed (continued)
Financial Valuation:
• Estimated non risk adjusted NPV (period = patent life
  time): €10 mio to €20 mio.
• This project will significantly reduce the risk and
  increase the commercial probability of success to
  >90%.
• Hence, pNPV will be: €9 mio to €18 mio.
• Top annual sales (estimate) : €15 mio to €40 mio



      YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
            Feed - Food - Health        www.food2know.org
       Follow Up Project: Feed (continued)
 Financial Valuation:
  •    The volume of compounded feed in the 7 major markets (US, EU, China, India, Brazil, Russia and Japan)
       reached 667 million MT in 2010.
  •    The application cost of a DFM can be estimated to be around €2 per MT of feed.
  •    We estimate to treat 0.01% to 0.03% of the total amount of compounded feed.
  •    The COGS for DFM is estimated @ 50% of the sales.
  •    The cost for marketing, sales and overhead is set @ 20% of the annual sales.
  •    Discount rate is set @ 12% and period to be considered is the patent life time.
  •    Product launch is estimated to occur after the project with the first year of sales to be 5% of the estimated
       top annual sales, the second year 20%, the third year 50%, the fourth year 75% and the following years
       100%.
                            E.G. NON RISK ADJUSTED NPV FOR THE LOWER END SALES
  (x€million) 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028
Project cost             0,12 0,12 0,12 0,12
Sales                                        0,65 2,6 6,5 9,75 13 13 13 13 13 13 13 13 13 13
COGS             0     0    0    0    0    0 0,29 1,17 2,93 4,39 5,85 5,85 5,85 5,85 5,85 5,85 5,85 5,85 5,85 5,85
Mkt, Sales , OV  0     0    0    0    0    0 0,13 0,52 1,3 1,95 2,6 2,6 2,6 2,6 2,6 2,6 2,6 2,6 2,6 2,6
P&L              0     0 -0,1 -0,1 -0,1 -0,1 0,23 0,91 2,28 3,41 4,55 4,55 4,55 4,55 4,55 4,55 4,55 4,55 4,55 4,55
Discounted       0     0 -0,1 -0,1 -0,1 -0,1 0,14 0,52 1,15 1,54 1,84 1,64 1,46 1,31 1,17 1,04 0,93 0,83 0,74 0,66
Accumulated      0     0 -0,1 -0,2 -0,3 -0,4 -0,3 0,25 1,41 2,95 4,79 6,43 7,89 9,2 10,4 11,4 12,3 13,2 13,9 14,6
NPV               € 14,6 million

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                  Feed - Food - Health                                       www.food2know.org
      Valorization - Feed Business
• Via outlicensing on a world-wide exclusive base.
• Different scenario’s can be discussed e.g.
  Production/Marketing agreement.
• First discussions will start in January 2012.
• Interested Board of Advisors Member: contact
  Benedikt Sas.
• Identified Licensee can provide input &
  guidance for the StepStone Project.

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          Feed - Food - Health        www.food2know.org
YOUR TOP EXPERTS FOR INNOVATION   info@food2know.be
      Feed - Food - Health        www.food2know.org

								
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