Cheese starter cultures

Ian Powell Dairy Innovation Australia 23rd October 2014

Cheese-making is not new Fragments of cheese strainers

Traces of organic molecules indicate presence of milk fats in strainers from 7,000 years ago

Salque et al., Nature 493, 522525 (2013) Dunne et al., Nature 486, 390394 (2012) Evershed et al., Nature 455, 528-531 (2008)

What were early cheeses like? Historical assumptions:

No deliberate use of coagulant Enzymes from use of stomachs as vessels to carry milk?

No deliberate use of starter cultures Natural milk flora Micro-organisms from GI tract

Of the animal the stomach came from or from human drinkers

Micro-organisms from skins used as vessels to carry milk Some carry-over from batch to batch

In storage vessels and cheese-making equipment

What knowledge did they have? Whatever they did .

They learnt that following certain procedures usually led to an edible, tasty product with better keeping qualities than liquid milk

They learnt that failing to follow the correct procedures often led to a foul-tasting, inedible product (or worse)

What knowledge did they have? Whatever they did .

They did not realise that they were some of the earliest adopters of enzyme processing and microbial biotechnology to generate a food with physical, nutritional and hedonic functionality

They did not realise that they were making use of temperature control, acidification, removal of fermentable sugars, partial dehydration and lowering of water activity as components of a prototype multiple-hurdle food preservation and safety plan

What knowledge did they have? Whatever they did .

They just did it.

Process diagram based on the description given by Columella. Fox et al, Cheese Chemistry, physics and microbiology (Elsevier, 2004)

Lucius Iunius Moderatus Columella ca 4 ca 70 CE

By the middle ages there were many established regional cheese styles (and manufacturing techniques) and extensive trade ..

But where are the starter cultures?

Starters became part of the magic Poorly controlled, highly variable, risky

Natural milk cultures Milk is incubated under conditions that favor the growth of naturally occurring (thermophilic) lactic acid bacteria, then used as starter.

Back-slopped starter Use some of a previous product batch as starter.

Whey cultures Starter is produced by incubating cheese whey under conditions that favor the growth of desirable (thermophilic) lactic acid bacteria.

Powell, Broome and Limsowtin, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier, 2011)

No magical balance of organisms, but targeted process control to influence the development of desirable cheese microflora (even if no-one knew)

..and then the 19th century came

Louis Pasteur. Archives Photographiques, Paris Joseph Lister, 1st Baron Lister. Wikimedia Commons Lactobacilli. Bottazzi and Bianchi

Building on work by Henle, Schwann, Latour and others, in 1856-57 Pasteur connects yeasts and bacteria with alcoholic fermentation and its spoilage

1873-77 Lister isolates Bacterium lactis and demonstrates its role in acidification and curdling of milk

1884-90 Storch researches starters for butter and cream, leading to commercial supply of starter cultures in 1891

20th Century starters

Powell, Broome and Limsowtin, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier, 2011)

Undefined mixed cultures (propagated in cheese factory)

Cultures (typically descended from artisanal cultures with desirable properties) are sequentially subcultured at the cheese factory.

Undefined mixed cultures (maintained in laboratory)

Cultures (typically descended from artisanal cultures with desirable properties) are preserved and propagated under controlled laboratory conditions.

Defined-strain starters

Laboratory-purified selected strains, free of microbiological contaminants, preserved and propagated under controlled laboratory conditions, then blended to give a mixed culture with desired properties.

The rituals are old, but the science is

relatively new.

http://www.wga.hu/art/zgothic/gothic/3a/2trade03.jpg

What is a starter culture? ..in 50 words or less

A cheese starter culture is a prepared culture that contains one or several strains of microorganisms at high counts, being added to bring about a desirable enzymatic reaction (e.g. fermentation of lactose resulting in acid production, degradation of lactic acid to propionic acid or other metabolic activities directly related to specific product properties). ISO 27205:2010 (IDF 149: 2010) Fermented milk products - bacterial starter cultures - standard of identity.

Photo modified from Bottazzi and Bianchi (1984)

What do cultures do?

Photo modified from Bottazzi and Bianchi (1984)

What do cultures do?

We now have enzyme studies and metabolic maps, genome sequences, transcriptome analysis, proteome studies ...... What does the cheese taste like? How has culture use changed?

The present and the future Understanding what cultures are

and what they do (strains alone and together in mixed cultures, different species in core or on surface, etc) is fundamental to future targeted culture and cheese innovation

...but, frankly, we just want good

cheese at a reasonable price. How do starters contribute to that?

Useful (?) definitions

Primary culture Primary starter, acid producer, starter Responsible for acid production in the

cheese make Contributes to flavour development Some have notable secondary characteristics

(e.g. heterofermentative cultures)

Examples of cheeses and related primary starters

Gobbetti et al. 2007

Useful (?) definitions

Secondary culture Secondary starter, adjunct, ripening culture No significant contribution to early acid production Influence / dominate flavour development, gas production, etc Characteristic cheese properties develop over time Added at same time as primary starter Incorporated into curd Added later Applied to surface, spiked etc

Examples of cheeses and

related secondary starters or

adventitious secondary microflora

Useful (?) definitions

How useful are these definitions? Traditional cultures include everything in one

culture .. except for the environmental contaminants

Modern cultures tend to split up the functions into multiple functions Why?

Practical (and profitable) solutions

Complex mixes are difficult to grow Variable, inconsistent Impossible to change growth conditions

without altering the balance of culture components

Single components can be grown more reliably (and cheaply) and then blended

but that makes complex blends expensive So ... Defined cultures are the trend

but the loss of complexity .......

How do we restore complexity?

Starter + secondary cultures Starter for acid and basic flavour

Trade activity for flavour? Secondary for

Specific flavour notes Surface ripening (bacteria, fungi)

This gives you complexity and control but is it as complex as it should be? industrial cheddar is a good example

What about raw milk flora?

An excellent source of non-starter organisms Are these significant contributors to flavour in

raw milk cheese? Can we capture them and tame them (and

turn them into starters/adjuncts)? ...without losing complexity

Regional/farm-specific milk flora

Repeatedly propagate to encourage acidifiers and other acid-tolerants

Check purity

Establish seed stocks

Trial cheese-making Laboratory / cheese manufacturer

Product assessment

Commercialisation

Regional/farm-specific milk flora

Microbial composition analysis Community DNA profiling

Purification of representative safe organisms Purify and identify

Assembly of defined culture blend(s) Assemble, check and establish seed stocks

Production of trial defined culture blend(s) Prototype cultures of commercial quality

Trial cheese-making Laboratory / cheese manufacturer

Product assessment Sensory and instrumental analysis; community profiling

Commercialisation

What about raw milk flora?

Collection, selection, application and management of bacterial starter cultures

Culture collectionA biodiverse archive from which cultures are drawn for characterisation, development and industry use

Single (pure, defined) strains Undefined mixed-strain cultures

Culture composition Conventional microbial analysis and DNA methods Species and strain analysis of dominant types

Strain identification DNA-based methods Species classification Strain relationships

Strain blends, rotation and replacement Design blends, rotations and potential replacements Confirm performance, flavour and texture impacts Trial cheese manufacture Advice to culture users

Strain characterisation Performance testing Flavour impact Texture/appearance impact Antibiotic resistance Biogenic amine production Assessment of growth, survival and activity under production/storage conditions

Phage sensitivity Insensitive (known phages) Select resistant variants Phage-host infectivity table

Culture characterisation Performance testing Flavour impact Texture/appearance impact Antibiotic resistance Biogenic amine production Assessment of growth, survival and activity, and batch composition variability under production/storage conditions Trial cheese manufacture

Phage sensitivity No inhibition (known phages) Select resistant variants Phage-host infectivity table

Culture rotation and replacement Design rotations and potential replacements Advice to culture users

Sample and data feedback from cheese-maker to culture supplier

Phage detection Whey testing

Phage collection Archive of phage diversity Used in phage sensitivity screening and selection of phage-resistant variants

Phage analysis Host range testing and virulence assessment Identification, grouping and evolution analysis

Phage-resistant strains or mixed cultures

Emerging genomic analysis techniques can be used to characterise defined and undefined cultures

Current trends in cultures Increasing separation of culture functions

Use of adjuncts to achieve flavour definition Novel blends

Streptococcus/Lactococcus Higher activity at lower cost (phage & flavour effects)

Blending to re-create traditional complexity Without the quality and safety variability

Unique blends Your cheese, your culture

Demand for greater definition Species and strain typing Biochemical (flavour) potential Safety concerns, especially for new species

Current trends in cultures Genomics, proteomics, metabolomics

Genes, gene expression, predictive biochemistry Aid to strain selection and blending

Still relatively new and expensive Examples are in the public domain Not economic to do for every strain/blend/culture Practical verification of predictions needed

Metagenomics etc Prediction of properties of mixed cultures on the basis

of the genomic potential of constituent strains Better understanding of the complexity and

dynamics of traditional cultures

Current trends in cultures

Safety of cheese flora Culture composition largely based on traditional

practices; non-starter adventitious flora undefined Most organisms have never been subjected to

formal safety assessment Most culture types in current use have

demonstrated their safety through years of use (GRAS/QPS) rather than through pre-release laboratory or clinical analysis

Current trends in cultures

Safety of cheese flora Rational basis for use

Generally Regarded as Safe (US FDA) either through scientific procedures or, for a substance used in food before 1958, through experience based on common use in food.

Qualified Presumption of Safety is being adopted by European Food Safety Authority (EFSA): rational risk assessment for the continued use of many traditional types of microorganisms in foods in the absence of any formalised safety testing program.

Current trends in cultures

Genetically modified strains Laboratory GM work only for over 30 years Useful for defining important culture properties

Provides the analytical tools for new methods of culture characterisation

A guide to potentially useful natural variants Requires long-term commitment to get from

experiment to application of knowledge Approved release of GM strains for cheese-

making not likely in the foreseeable future

Culture delivery to the cheese vat

Bulk starter or direct-to-vat concentrate Starters must grow and survive the process Composition, convenience, capability, cost

Culture batch reproducibility important Acid-producing activity Flavour development etc

Culture delivery to the cheese vat Recent innovations

Frozen pelletised concentrates Cheaper delivery of blended cultures

Membrane systems to remove growth inhibitors Old idea, new technologies (e.g. electrodialysis) for

better culture growth Aerobic growth systems for LAB

Applicable to some species; supplemented conditions High-density growth without inhibitors

Preparation and long-term storage of frozen/freeze-dried seed stocksLaboratory scale

Pure strains or undef ined culturesQuality tested

Growth of large-volume starterPreparation by culture supplier

Concentration of cultureCentrifugation

Freezing/freeze-drying Culture blending if required Performance and quality tested Supplied to cheese factory

Cheese manufacture

Scale-upSequential scale-up of growth volume (mother/intermediate cultures)

Can be quality tested and f rozen/f reeze-dried for later use

Growth of large volume starter Bulk starter preparation in cheese factory Blending of strains at point of inoculation or af ter separate growth

Chilled, quality tested before use

In-house inoculaFor use by culture supplier

Bulk starter inoculaSupplied to cheese factory

Sample and data feedback from cheese-maker to culture supplier

Bulk starter or Direct-to-Vat? The real question is about outsourcing

Do you DIY? Which parts of your production process do you

outsource? Why? Cost? Safety? Reliability? How much control do you want, and what

limitations of design are you willing to accept? Does the scale of your business justify the

capital expenditure needed to grow bulk starter with high levels of control?

How long-term is your investment strategy?

Culture application

A partnership of culture supplier and cheese-maker Communication essential

Understand each other (needs and capabilities) Both require expertise Risk: Fundamental knowledge and available

culture products are growing but practical expertise to apply them is not keeping up?

Slide Number 1Cheese-making is not newWhat were early cheeses like?What knowledge did they have?What knowledge did they have?What knowledge did they have?Slide Number 7Slide Number 8Starters became part of the magicPoorly controlled, highly variable, risky..and then the 19th century came20th Century startersThe rituals are old,but the science is relatively new.What is a starter culture?..in 50 words or lessWhat do cultures do?What do cultures do?The present and the futureUseful (?) definitionsExamples of cheeses and related primary startersUseful (?) definitionsExamples of cheeses and related secondary starters or adventitious secondary microfloraUseful (?) definitionsPractical (and profitable) solutionsHow do we restore complexity?What about raw milk flora?Slide Number 25Slide Number 26What about raw milk flora?Collection, selection, application and management of bacterial starter culturesSlide Number 29Current trends in culturesCurrent trends in culturesCurrent trends in culturesCurrent trends in culturesCurrent trends in culturesCulture delivery to the cheese vatCulture delivery to the cheese vatSlide Number 37Bulk starter or Direct-to-Vat?Culture application