Cheese starter cultures
A thousand years of tradition
wrapped in a century of
microbiological science
Ian Powell
Dairy Innovation Australia
In the beginning …..
How old is cheese-making?
• Various animals were domesticated by
10,000 years ago
• ‘Bone assemblages’ suggest slaughter of
calves and older females
- evidence of dairying?
• Fragments of ‘cheese strainers’ up to
7,000 years old have been found across
Europe, Africa and the Middle East
How old is cheese-making?
• Fragments of ‘cheese strainers’
Traces of organic molecules indicate presence of milk fats
Salque et al., Nature 493, 522–525 (2013)
Dunne et al., Nature 486, 390–394 (2012)
Evershed et al., Nature 455, 528-531 (2008)
How old is cheese-making?
• We don’t know much about early cheeses
• 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
How old is cheese-making?
• 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)
– Some probably interpreted it as a magical
transformation
How old is cheese-making?
• Whatever they did ….
– They didn‟t realise that they were some of the
earliest adopters of enzyme processing and
microbial biotechnology
– They didn‟t realise that they were making use of
temperature control, acidification,
partial dehydration and lowering of water activity
as components of a prototype multiple-hurdle food
preservation and safety plan
6,000 years later
• Many kinds of cheese were made and traded
• Regional cheeses were made across Europe, the Near East
and North Africa
• Pliny the Elder1 wrote of many kinds of cheese available
in Rome
- ‘…that which is made in Gaul has a strong taste, like that of medicine‟
• Coagulants had come a long way
• Columella2 described alternative coagulants and cheesemaking processes
– Lamb, kid, thistle flower, fig sap, pine needles, etc
1 Gaius
2 De
Plinius Cecilius Secundus, Naturalis Historia (ca 79 CE) see http://www.muellerscience.com/SPEZIALITAETEN/Schweiz/Pliny_caseus.htm
Re Rustica (Lucius Iunius Moderatus Columella, ca 4 – ca 70 CE) see http://www.archive.org/stream/onagriculturewit02coluuoft/onagriculturewit02coluuoft_djvu.txt
Columella
Process diagram based on the description given by Columella.
Fox et al, Cheese – Chemistry, physics and microbiology (Elsevier, 2004)
1000 years after that …
By the middle ages there
were many established
regional cheese styles (and
manufacturing techniques)
and extensive trade …..
1000 years after that …
But where are the
starter cultures?
http://www.wga.hu/art/zgothic/gothic/3a/2trade03.jpg
Starters were part of the magic
Poorly controlled, highly variable, risky
Description
Comments
Artisanal. Milk is incubated under Low cost. Undefined composition.
conditions that favor the growth of Highly variable composition and
„Natural‟ milk cultures naturally occurring thermophilic performance. Prone to undesirable
lactic acid bacteria, then used as contamination; microbiologically
starter.
hazardous.
Back-slopped starter
Low cost. Undefined composition.
Artisanal. Use some of a previous Highly variable composition and
product batch as starter.
performance. Microbiologically
hazardous.
Whey cultures
Low cost. Undefined composition.
Artisanal. Starter is produced by Variable composition and
incubating cheese whey under
performance. With careful handling
conditions that favor the growth of and quality control testing, whey
desirable (typically thermophilic) cultures are used on an industrial
lactic acid bacteria.
scale for making some traditional
Italian and Swiss cheese types.
Powell, Broome and Limsowtin, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier, 2011)
‘Natural microflora’ of milk
Hassan and Frank, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier ,2011)
‘Natural microflora’ of milk
• Is any of the flora ‘natural’ to milk? Not really
• It is natural to the animal and to the environment
• „natural contaminants‟ with possible local characteristics
• A huge mix of organisms
• Variable in acid production and flavour impacts
• Some pathogenic
• Mostly suppressed in an acidic, salty, low-water-activity
cheese environment (not suppressed in high-pH,
high-moisture, or low-salt cheeses)
• Traditional starter preparation and cheesemaking attempt to control the microflora
• No magical balance of organisms, but targeted process control
to influence the development of desirable cheese microflora
..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
Description
Comments
Undefined mixed
cultures (propagated in
cheese factory)
Cultures (typically descended from
artisanal cultures with desirable
properties) are sequentially
subcultured at the cheese factory.
Undefined composition. Variable
composition and performance, but less
variable than whey cultures. With careful
handling and some quality-control testing
these are still in limited use, but have
largely been replaced by laboratorymaintained cultures.
Undefined mixed
cultures (maintained in
laboratory)
Cultures (typically descended from
artisanal cultures with desirable
properties) are preserved and
propagated under controlled
laboratory conditions. Supplied to the
cheese factory in frozen or freezedried form.
Undefined composition. Variability greatly
reduced through maintenance of stable
stocks (usually frozen) and production of
each batch with minimal sequential
subculturing. Used as bulk starter or
direct vat inoculum.
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. Supplied to the
cheese factory in frozen or freezedried form.
Defined composition, usually of only a
small number of strains. This gives a high
degree of control over starter
performance parameters and product
properties, as long as strains are carefully
selected and managed. Novel blends with
specific properties can be made. Used as
bulk starter or direct vat inoculum.
Powell, Broome and Limsowtin, Encyclopedia of Dairy Sciences, 2nd edition (Elsevier, 2011)
What is a ‘starter culture’?
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)
Production of lactic acid
• pH influences
• Proteolysis and other
enzymatic reactions
• Whey expulsion
– Curd moisture
– Water activity
• Calcium retention
• Lactose
Lactate
• Inhibits growth of
undesirable flora
Broome et al. 2002
Production of flavour, aroma
• Citrate metabolism
• diacetyl, CO2
• Lactate metabolism
• propionate, acetate, formate,
ethanol, CO2
Broome et al. 2002
Production of flavour, aroma
• Proteolysis, peptidolysis
• bitter peptides, degradation
• Amino acid metabolism
• methanethiol, sulphides, esters,
alcohols, carboxylic acids, etc
• Lipolysis
• free fatty acids, 2º alcohols,
lactones, CO2
• Redox
• low redox potential influences
chemistry of maturation
Broome, 2007
Structure, texture, appearance
• CO2 production
• holes, splits (good/bad)
• Proteolysis
• softening, NH3
• pH changes
• calcium dynamics
• Fungal growth
• surface growth, veins
• Surface bacteria, yeasts
• aroma/flavour, colour
Easyart.com
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
• Probiotic culture
Probiotic starter
– Incorporation of probiotic organisms into cheese
• Experimental and commercial production
• Good delivery of cells to gut
– Added at same time as starter = incorporated into curd
– Effects on cheese flavour
• can be good, bad or have no impact
– Examples
• Bifidobacterium bifidum, B. infantis, B. lactis, B. longum
• Enterococcus faecium
• Lactobacillus acidophilus, Lb. casei/paracasei, Lb. fermentum, Lb. rhamnosus
When and where do cultures act?
• Cheese-making and maturation are
largely the result of microbial
biochemical events
• Events are due to a variety of organisms
• Different events due to various organisms
– Species and strains differ
– Microbial consortium; microbial succession
• Different events do not all happen at the same time
or the same place
– Over time in different parts of the cheese
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
Bacteriophages
• Viruses that infect bacteria
• Controlled through
(1) starter selection
• Not sensitive to known phages
• Rotation of dissimilar cultures
• Selection of variants and natural
transfer of phage-resistance
genes possible
(2) factory design and sanitation
50 nm
Phage consequences
Neve, 1996
example
a Bar =
50 nm
Phage
diversity
Example:
lactococcal
phage species
• Genetically distinct
species
• Many variants within
each species
(e.g. infecting different
host strains)
• „936‟ species is by far
the most common
Deveau et al. (2006)
Where do phages come from?
Can starters ever be totally resistant
to all phage infection?
The challenge: Starters in use must be
sufficiently resistant to phages currently
in the environment
and must make good cheese
Collection, selection,
application and management
of cheese starter cultures
Culture collection
A biodiverse archive f rom which cultures are drawn f or characterisation, development and industry use
Single (pure, defined) strains
Undefined mixed-strain cultures
• From traditional or industrial undef ined cultures, cheese or other
f ermented f oods
• Properties of each strain and relationships between strains can be known
• Descendents of earlier traditional starters of unknown composition
• Undef ined complex mixtures of strains/species, caref ully propagated to
reduce variability
Strain identification
Culture composition
• A variety of DNA-based methods available
• Species classif ication:
- targeted PCR, DGGE, gene sequencing
• Strain grouping:
- MLST, PFGE, AFLP, T-RFLP, RAPD
• Strain identif ication
- PFGE, genome (diagnostic) sequencing
• Conventional microbial analysis aided by
molecular methods
- isolate dominant organisms that can then be
classif ied, grouped and identif ied
- check levels and identity of characteristic
organisms (f lavour/aroma producers)
- assess levels of undesirable organisms
(pathogens, indicators, spoilage organisms)
• Direct DNA-based compositional analysis:
- quantitative PCR of targeted species;
f luorescence microscopy with specif ic probes
- semi-quantitative DGGE, T-RFLP
Strain characterisation
• Perf ormance testing (growth, acid production
activity, responses to temperature and salt)
• Flavour impact
- autolysis assessment
- genetic assessment of relevant properties
- biochemical prof iling of key enzyme activities
- assessment of f lavour production:
tasting panel assessment and/or
instrumental analysis of f lavour/aroma in
laboratory cheese/cheese analogue systems
• Texture/appearance impact
- proteolysis assessment
- substrate-specif ic gas production assays
- EPS assessment
• Antibiotic resistance
• Biogenic amine production
• Assessment of growth, survival and activity
under culture production/storage conditions
Phage-resistant strains and cultures
• Select resistant variants & transconjugants
• Preliminary assessment bef ore adding to
culture collection f or characterisation
Phage collection
• Historical archive of phage diversity
• Uses:
- f uture phage sensitivity screening
- selection of phage-resistant variants
Phage analysis
Phage sensitivity testing
• Find strains insensitive to known phages
• Find sensitive candidates f or selection of
resistant variants
• Compile phage-host inf ectivity table
- strains sharing sensitivity to related phages
Strain blends, rotation/replacement
• Design blends, rotations and potential
replacements based on properties of single
strains: perf ormance, f lavour potential,
phage sensitivity
• Conf irm perf ormance, f lavour and texture
impacts as f or single strains
• Trial cheese manuf acture
• Advice to culture users
Emerging genomic analysis techniques can be used to characterise
defined and undefined cultures
• Phage characterisation
- host range testing
( = bacterial phage sensitivity testing)
- virulence assessment
• Phage identif ication, grouping and evolution
- species classif ication: targeted PCR, gene
sequencing, MLST, genome sequencing
Phage detection
• Whey testing (phage detection)
- activity tests, plaque assays
Sample and data feedback from cheesemaker to culture supplier
• Purposes:
- testing f or phages, selection of resistant cultures
- ref inement of blends, rotations, inoculum rates, etc
Culture characterization
• Perf ormance testing (growth, acid production
activity, responses to temperature and salt)
• Flavour impact
- biochemical prof iling of key enzyme activities
- assessment of f lavour production:
tasting panel assessment and/or
instrumental analysis of f lavour/aroma in
laboratory cheese/cheese analogue systems
and/or cheese manuf acture
• Texture/appearance impact
- proteolysis assessment
- substrate-specif ic gas production assays
- EPS assessment
• Antibiotic resistance
• Biogenic amine production
• Assessment of growth, survival and activity,
batch composition variability under culture
production/storage conditions
• Trial cheese manuf acture
Phage sensitivity testing
• Find cultures showing no inhibition with
known phages
• Find sensitive candidates f or selection of
resistant variants
• Compile phage-host inf ectivity table
- cultures sharing sensitivity to related phages
Culture rotation/replacement
• Design rotations and potential replacements
based on properties of cultures: perf ormance,
f lavour potential, phage sensitivity
• Advice to culture users
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
– These organisms have never been subjected to
formal safety assessment
– Most culture types in current use have
demonstrated their safety through years of use
rather than through pre-release laboratory
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
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
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
Culture application
• A partnership of culture supplier and
cheese-maker
– Communication essential
• Understand each other (needs and capabilities)
– Both require expertise
– Risk: Has there been a historical increase in
fundamental knowledge and available culture
products but a decrease in available expertise
to apply them?
Cheese starter cultures
A thousand years of tradition
wrapped in a century of
microbiological science
Ian Powell
Dairy Innovation Australia