Dr Bill SimpsonThe Alfred Jørgensen Laboratory

Brewing yeast capabilities

Business overview Yeast activities and competences

Our yeast collection Yeast storage capabilities Yeast supply capabilities Yeast selection capabilities Trial fermentation capabilities Analysis capabilities Product and process development

Management, staff and quality systems

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Located in Copenhagen, Denmark Facilities include yeast storage

facility, dried yeast culture production plant, liquid yeast production laboratory, yeast testing laboratory

Facility founded by Alfred Jorgensen in 1891

Been supplying breweries with pure culture yeast for almost 130 years

Purchased by Cara Technology in 2009

The Alfred Jørgensen Laboratory

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We aim to establish the world’s most useful collection of validated, proven commercial strains of brewing yeast, housed within world-class facilities,

and supported by world-class analysis techniques and specialist

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Approximately 200 brewery customers in more than 70 countries

Services include Secure storage of yeast Assured yeast supply Yeast strain selection Yeast strain improvement Product and process prototyping

Both UK and Danish operations are accredited to BS EN ISO 9001:2008

Yeast activities and competencies

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Our yeast collection Largest collection of production brewing

yeasts in the world Almost 850 yeast strains (mostly lager yeast,

though some ale and wheat beer yeasts) All stored in liquid nitrogen at -196oC Continuously supplied to breweries for almost

130 years

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Our yeast collection Detailed fermentation information available

on about 200 of the strains Strain key describes important properties

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Unusual flavour production, eg very high isoamyl acetate production

Unusual sedimentary characteristics, eg ‘dust’ yeasts

Unusual attenuation properties, both low and high degrees of attenuation

High rate of attenuation Exceptionally good beer filtration performance Strains suited to production of low- and non-

alcoholic beers Strains associated with low levels of beer loss or

extract lossyeas

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Store approximately 100 yeast strains on behalf of customers

Held either for archiving purposes or for routine supply

Liquid nitrogen on a minimum of two sites

Electrical freezer storage as additional back-up in Copenhagen

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We supply yeast cultures in three forms: Slopes (slants) Liquid cultures (various

volumes) Ultra-pure dried yeast (50 g)

All cultures are quality assured prior to dispatch

Assured yeast supply

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Yeast supplied six times per year - four slopes per brewery per shipment - 24 slopes per brewery per year

All cultures are quality assured prior to dispatch Cell- and colony-morphology Carbohydrate utilization Growth temperature PAD gene activity Flocculation Multiplex PCR DNA fingerprinting Checks for contaminant microorganisms

Only cultures which have passed all tests are released to brewery customers

Phytosanitary Certificates and Certificates of Origin provided on request

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Two approaches

1. Select candidate yeasts with the desired traits from within our own collection

2. Select clones and variants from within the customer’s existing yeast culture, eg Consistent colony morphology on indicator

media Increased or reduced flocculation Higher ethanol tolerance Improved flavour (esters, sulphurs etc) Improved flavour stability (SO2)

Yeast strain selection

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We occasionally use additional techniques to increase our chances of success

1. Single cell isolation by micromanipulation

2. Mutation with physical or chemical agents

3. Rare mating

Yeast strain improvement

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Culture purity is key Phenotypic purity

Colony type Fermentation behaviour Flocculation behaviour Flavour formation

Genotypic purity Fingerprinting of chromosomal DNA

Yeast strain selection and improvement outputs

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We have the capability to perform trial fermentations at several scales 10 ml McCartney tubes 500 ml Schott bottles 1 litre AJL tall tubes 1.5 litre flasks 5 litre buckets 10 litre stirred Carlsberg flasks 1 hl lab-scale CCVs

Through partner organizations we can scale up to ‘laboratory’ pilot scale or ‘commercial’ pilot scale

Trial fermentationsye

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Various microbiological (phenotypic) tests

Multiplex PCR DNA fingerprinting

Yeast analysis capability

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DNA fingerprint pattern

% yeast strains with this fingerprint type

We have yeast analysis labs in Leatherhead and Copenhagen

Our analysis lab is based in our Leatherhead facility SPME-GC-MS Headspace GC SCABA and other wet chemistry equipment

We have a network of partner laboratories to supplement our in-house capabilities

Analytes include: basic physical parameters, higher alcohols and esters, volatile sulphur compounds, anions and cations etc

Chemical analysis capability

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We have a well-trained expert taste panel which is capable of Quality assessment Identification of non-

conformances Detailed descriptive

profiling Visual profiling

Sensory analysis capability

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Browser-based sensory software about to be implemented in our new taste room

Drivers of differentiation

Malt Adjunct Hops Water Salts Oxygen Yeast

Brewhouse practices Fermentation practices Maturation practices End-processing practices Packaging practices Hygiene control Oxygen control Heat transfer control Temperature / time control

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Primary influences of yeast on beer composition

Alcohol

Apparent extract

Real extract

pH

Colour

Bitterness

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Beer flavours whose levels are increased or decreased by yeastAcetaldehyde Acetic

Astringent

Bitter

Burnt rubber

Butyric

Caprylic

Caramel

Citrus

Diacetyl

DMS

Ethyl acetateEthyl butyrate

Ethyl hexanoate

Floral

Grainy

Grapefruit

H2S

Honey Indole

Isoamyl acetate

IsovalericLeathery

MaltyMercaptan

Metallic

Methional

‘Yeast bite’

Worty

Woody

Sweet

Solvent alcoholic

Smoky

Rotten vegetablePhenolic (4-VG)

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The brand flavour specification

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Bitter

Carbonation

Astringent

Sweet

DMS

Sour

After-bitter

Spicy kettle hop

Grainy

Isoamyl acetate

Malty

Solvent alcoholic

Ethyl hexanoate

Critical

Tolerable

Important

Target brand profile for a new pale lager beerParameter Target

OE 12.2oP

PE 2.9oP

RE 4.6oP

Alcohol 5.0% vol/vol

Apparent attenuation 76.6%

Bitterness 16 BU

pH 4.40

Colour 6.6oEBC

Isoamyl acetate 3.5 mg/l

Ethyl acetate 18 mg/l

DMS <0.04 mg/l

Diacetyl <0.01 mg/lprod

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Process constraints for a new pale lager beer

Parameter Target

Fermentation temperature 9 - 15oC

Primary fermentation time Max 9 days

Fermenters 6,000 hl CCVs – 18 metres high

Wort dissolved oxygen Max air saturation

Wort OE Max 15oP

Wort FAN Max 190 mg / l @ 15oP

Total fermenter residence time Max 12 days

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Develop a detailed understanding of

current brands and processes

Develop a detailed specification for the

new product

Identify changes needed to current production process and

bill of materials to make the new product

Identify suitable yeast strains for

producing the new product

Select the best yeast strain in

laboratory trials

Propagate yeast for

trials

Carry out brewery trials – review - revise

Production brews

Product and process development

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Seven of our 22 staff directly service our yeast business

Good mix of experience – all well trained and qualified

Leatherhead operation accredited to Investor in People (IiP) standard since 2001

All operations accredited to ISO 9001:2008

HACCP implemented throughout Disaster recovery systems in place and

regularly tested

Management, staff and quality systems

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Facilities and processes in place and well established for supporting product and process development through exploitation of brewing yeast

Specialist teams in UK and Denmark Good track record of developing

profitable new products and processes, and modifying existing ones on time and within budget

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Contact detailsCara Technology Limited

Leatherhead Enterprise Centre

Randalls Road

Leatherhead

Surrey

KT22 7RY

UK

Tel +44 1372 822218 Fax +44 1372 821599

www.cara-online.com bill.simpson@cara-online.com