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.

Lucius Iunius Moderatus Columella

ca 4 ca 70 CE

Process diagram based on the description given by Columella.

Fox et al, Cheese Chemistry, physics and microbiology (Elsevier, 2004)

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.

No magical balance of organisms, but targeted

process control to influence the development of
desirable cheese microflora (even if no-one knew)
Powell, Broome and Limsowtin, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier, 2011)

..and then the 19th century came

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

Louis Pasteur. Archives Photographiques, Paris

Joseph Lister, 1st Baron Lister. Wikimedia Commons

Lactobacilli. Bottazzi and Bianchi

20th Century starters

Cultures (typically descended from artisanal

Undefined mixed cultures

cultures with desirable properties) are
(propagated in cheese factory) 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.

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

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.

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?

Photo modified from Bottazzi and Bianchi (1984)

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 collection
A biodiverse archive from which cultures are drawn for characterisation, development and industry use

Single (pure, defined) strains

Undefined mixed-strain cultures

Strain identification

Culture composition

DNA-based methods
Species classification
Strain relationships

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

Phage-resistant strains
or mixed cultures

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

Strain blends, rotation

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

Culture characterisation
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 detection
Whey testing

Sample and data feedback

from cheese-maker to
culture supplier

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

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 cheesemaking 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 stocks

Laboratory scale
Pure strains or undef ined cultures
Quality tested

Scale-up
Sequential scale-up of growth volume (mother/intermediate cultures)
Can be quality tested and f rozen/f reeze-dried f or later use

In-house inocula

Bulk starter inocula

For use by culture supplier

Supplied to cheese f actory

Growth of large-volume starter

Preparation by culture supplier

Concentration of culture

Growth of large volume starter

Bulk starter preparation in cheese f actory
Blending of strains at point of inoculation
or af ter separate growth
Chilled, quality tested bef ore use

Centrif ugation

Freezing/freeze-drying
Culture blending if required
Perf ormance and quality tested
Supplied to cheese f actory

Cheese manufacture

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?