antonio del casale microbion srl verona university...

Microbion srl Verona University Spin-off - Italy

Antonio Del Casale

Food Matters Live – 19 November 2014

Microbion – Verona University Spin-off

Biodiversity services & products

Finds the best performing microbes using innovative DNA-fingerprinting

Microbion – Verona University Spin-off

Established company in 2011 >200 peer-review research publications

>50 Successful industrial case-studies >20 Customers >6 Industrial sectors

Technology Transfer

Microbion – Verona University Spin-off

Service provider: •Analytical services •R&D services •Bio-bank service

Products developer: •Molecular diagnostics •Pro-tech microbial strains

What we do?

Microbion – Verona University Spin-off

Innovation: •Product development •Process optimization

Quality control: •Safety assessments •Regulatory compliance

What customers need?

Microbes Biodiversity

Biodiversity Who is the good? Can the good’s win?

Microbes: Smallest & most abundant living organisms

Pathogenic Harmful

Spoiling Unwanted Anti-pro

Pro-technologic Make products Health improver

Microbes Biodiversity

Pro-technologic Make products Health improver

Natural occurring microbes

Green Chemistry • Bio-fuels • Bio-plastics • Bio-preservatives

Food • Fermented

Health • Probiotics

Agriculture • Bio-fertilisers • Bio-pesticides

Industrial microbes - Manufactured - Controlled

Microbes Biodiversity

Food Microbes

Industrial microbes • Bacteria (Lactic Acid Bacteria) • Yeast (Saccharomyces spp.)

Benefits: • Shelf-life • Taste/Flavour • Digestibility • Nutrition

+Raw ingredients

Health Microbes

Benefit: • Competition to pathogens • Wellness & Nutrition • Immuno-regulator • Psychotropics

Industrial microbes • Bacteria (Lactic Acid Bacteria) • Yeast (Saccharomyces spp.)

Human Microbiome Top 10 emerging technologies The World Economic Forum (1 September 2014)

Probiotics

Health Microbes

DNA patters Molecular markers Signature sequences Genomes sequence Meta-genomic

Advantages of the molecular approach

High discrimination High accuracy High reliability ...and OVER the species level

Intra-specie biodiversity: Same specie Different abilities

Advantages of the molecular approach

DNA patters Molecular markers Signature sequences Genomes sequence Meta-genomic

High discrimination High accuracy High reliability ...and OVER the species level

Specie

Ge

nre

STRAIN

Saccharomyces cerevisiae (strain?)

Advantages of the molecular approach

DNA patters Molecular markers Signature sequences Genomes sequence Meta-genomic

•Improved quality wines •Cost efficiency

Local population study (1000+ yeasts)

Experimental wine production (10+ yeasts)

Scale-up winemaking (2 yeasts)

MASI Yeast Bio-Bank (100+ yeasts)

Distinctive flavour

Cost saving (and C02)

Seasonal consistency

Unique on the market!

Case study: Innovation in traditional winemaking

Case study: Microbes manufacturing

• Improved quality control • Regulatory compliance • Patent deposit

Molecular Fingerprinting Services

Intra-specie biodiversity study

Manufacturing: Batch validation

Regulatory: Genome stability reference

Legal: IP right defensibility

Species

Gen

ere

Strain

?

Consumer confusion by intra-species diversity:

•S. cerevisiae pathogenic, when adapted to human niche (temperature)

•Enterococcus faecium health promoter or marker for water pollution?

•Feed additive Bacillus cereus Vs. hemotoxic B. cereus

•Probiotic Escherichia coli (Nissle 1917) Vs. virulent E. coli

genus

species

strain

Advantages of the molecular approach

Random Amplified Polymorphic DNA (RAPD-PCR)

Pulsed Field Gel Electrophoresis (PFGE)

Random genome sampling Whole genome profiling

• Rapid • Not unambiguous

• Unambiguous • Time consuming

Genome analysis

Recent advancement of PFGE methodology: Optical Mapping

Linearized genome

Genome restriction

Fragments detection and measuring

Strain specific “DNA-barcode” • Strain tracking • Detection of genomic

rearrangement Genome sequencing scaffolding

Genome analysis

L. brevis ATCC 367 (NC 008497.1) (in-silico WGM)

L. brevis KB290 (NC 020819.1) (in-silico WGM)

L. brevis ATCC 367 (NC 008497.1) (in-silico WGM)

L. brevis LMG 11437 (4 independent replicates)

Genome analysis

•Strain specific “DNA-barcode” •Genome sequencing scaffolding

Genomic DNA restriction fragment (PFGE, ribotyping)

Profiling PCR-amplified DNA (RAPD-, ERIC-, REP-PCR)

PCR-amplification of restriction fragments (AFLP)

Restriction analysis of DNA fragments (PCR-RFLP)

DNA Multi-Locus Sequence Typing (MLST)

House-keeping Multi-Locus Sequence Analysis (MLSA)

Amplified Ribosomal DNA Restriction Analysis (ARDRA)

rRNA gene sequencing analysis (SSU, LSU)

Genomic DNA-DNA Hybridisation (DDH)

Genomic DNA mol% G+C

Whole genome sequencing analysis

Genome analysis

• Tremendous advancement of DNA sequencing = Next Generation Sequencing

Reduced time and lower cost Improved downstream analysis

• Regulatory authorities suggest or require “state-of-the-art-technology = genome sequencing:

FDA 2012 - Guidance for Industry: Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information

EFSA 2012 - Guidance on the safety assessment of Enterococcus faecium in animal nutrition

Strain-specific diagnostics and tracking

Safety assessment

Genome analysis

• All molecular markers become available (also currently unknown) development of specific diagnostic methods

• Gene copy number and regulators of health claims relevant genes Adaptation to the intestinal niche Genes involved in the interactions with the host Probiotic factors (e.g. serpin) able to suppress inflammatory responses Antimicrobial compounds for pathogen inhibition

• Assessment of health risks Biogenic amine production Virulence genes Antibiotic-resistance gene

Genome analysis

(EFSA, 2012)

Comparative Genomics the study of the relationship of genome structure and function across different biological species or strains

Genome analysis

Antibiotic-resistant strains

Team

Antonio Del Casale – CEO •MSc Biotechnology

•Tech Transfer

•Open Innovation

Sandra Torriani – Scientific Adv. •Full Professor Microbiology

•Food-tech Innovation

• 150+ Peer review publications

Fabio Fracchetti – COO •PhD Biotechnology

•Molecular Microbiology

•Patents

Valter Carturo – CFO •MEc Business Administration •Chattered Accountant •Start-up Operations

Giovanna Felis – Scientific Adv. •Associate Professor Microbiology

•Lactic Acid Bacteria

•Taxonomy & Genomics

The leading microbiology innovation provider (micro-organisms making mega-impact)

Antonio Del Casale a.delcasale@microbion.it www.microbion.it

Meet us at:

Milestones & Use of funds

Seed €10K Spin-off

University grants

First €10K customer

First tech partner

First conference invitation

Veneto Region grant

First commercial partner

“Impresa per Impresa” ConfindustriaProgramme

First EU-H2020 project

First patent

2011 2012 2013 2014

2015 2016 2017

Spin-out from University facilities

First patent licensed

Second patent

Marketing team

SME Instrument EU-H2020 project

First strain licensed

Third patent

Royalties > Sales Second patent licensed

Microbion Agro Microbion Health Microbion Food-tech Microbion Energy

Vitafoods Europe 2013

Golden standards for identification: Multi-locus sequence typing

Actinobacteridae putative proteomes

All vs All

BLASTp

Core genes of

Bifidobacteria

In-silico selection

One copy in every genome Dinucleotide frequencies, GC content and codon usage consistent with genome

18

Within a species, ribosomal subunit are not discriminant

9 Candidate

markers analysis (partial sequences) (Ventura et al. 2006)

7 Molecular markers

typing scheme

• High resolution between species

• Discriminate strains of same species

ROI Strategy

Potential acquisition paths: A – Management buy-back B – Partial asset sale C – Diagnostic brand co-development

•Lactic Acid Bacteria ID •Yeasts ID

Lumora – Cambridge Spin-off R&D of BART tech raised $2.1M by Tate&Lyle now licensed to 3M

Acquisition examples benchmark:

ROI Strategy

Potential acquisition paths:

Acquisition examples benchmark:

A – Management buy-back A – Management buy-back B – Partial asset sale

•Diagnostics patents •Microbial Strains

BioFire sold FilmArray asset

to bioMérieux’s for $450M

BioGaia receive €10M/year for L. reuteri from Nestlé infant nutrition

Key Advantages

2012-2013 Service and Products

20+ Customers success cases 6 Industries

Probiotics

Consumer products

Agri-food

Dairy

Wine

Universities

2013 Sales Unique advantage:

50+ collective years of experience

Market needs & solutions

Unique microbes bio-bank

Non-patented trade secrets

Open innovation network

Business Scouting…

Institutional

Key partners

Definition of Species: Group of strains sharing high level of genomic and phenotypic similarity

• Foundations for microbial characterization

• Exchange of information among scientific community • Laws regulations and guidelines FDA – Generally Regarded As Safe (GRAS) EFSA – Qualified presumption of safety (QPS)

Genome analysis