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Developments in Cheese Starter Technology Michael Pinches
DuPont Nutrition & Health
Society of Dairy Technology Annual Dinner 2017
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Using Starter cultures
The practicality of employing cultures in cheese making has been tested.
Upon the belief that lactic acid bacteria are the main agents in the curing of cheese this species has been suggested as the “Starter” under the following conditions:
The organism should be a pure lactic germ
Free from undesirable aroma
Adapted for vigorous development in milk
The advantages accruing from the use of such a “Starter”
Saves time in the process of manufacture
Aids development of a proper amount of acid for a typical cheese
The flavour and quality of cheese is preferable to that which has not been thus produced
Bacteriology of Milk 1903 by Harold Swithinbank
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Lactococcus lactis Magnification 20,000X. Scanning electron micrograph
1903 2017
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Physical forms of starter cultures
• Daily Propagation
• Frozen Pints
• Immediate Inoculation to the bulk starter tank
• Your starter for 1000
• Direct Vat Inoculation
• Frozen cans
• Frozen pellets
• Freeze dried
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Lactic Acid culture collections
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Ripening Cultures
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Product Composition Application
CB2 Penicillium roqueforti Strong Blue Tast - Blue Green
KL71 Kluyveromyces lactis Flavour in soft cheese and hole formation
Linens W Brevibacterium linens, Orange colour Aroma, fast growth
MGE Arthobacter nicotianae Strong aminopeptidase activity, fast growth
MVA Staphylococcus xylosus Stimulation of lactic acid bacteria
Mycodore Trichothecium domesticum Saint Nectaire Aspect
OFR 20Brevibacterium casei, Brevibacterium linens,
Debaromyces hansenii, Candid utilsBlend for colouration of smear cheese
OFR 9Brevibacterium casei, Brevibacterium linens,
Candida utils, Geothricium candidum
Appearance and flavour of all the surface
ripening
PA Penicillium roqueforti Mild Blue Taste - Dark Green
PC 02 Penicillium Candidum Ultra Filtered and stabilised cheese
PC 33 Penicillium Candidum Anti Mucor activity, use every day
PC 53 Penicillium Candidum Stabilised and Traditional
PC 12 Penicillium Candidum Stabilised Cheese, Longer Shelf life
PC 42 Penicillium Candidum Traditional cheese, Normal shelf life
PJ Penicillium roqueforti Typical Blue Taste - Middle Green
PLABrevibacterium linens, Geotrichum candidum,
Debaromyces hensenii, Arthrobacter nicotianae
Complex blend for the appearance and
flavour of the main French cheese
PV Penicillium roqueforti Strong Blue Taste - Blue Green
R2R Rhodosporidium infirmominiatum Flavour and colour in mixed smear cheese
Debaromyces hansenii Neutralisation for mix and sear cheese
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CASEIN
Chymosin /Plasmin
Intermediates
peptides
Free
Amino Acids
Enzymatic & Chemical
modifications
Proteinases
(Lactococcus)
Short Peptides
Volatile Flavour
Compounds
Bitterness
Savoury
Sulphur
Into the pathways of ripening in Flavour Creation
Peptidases of
NSLAB
0
50
100
% M
obile P
hase
100
150
mV
olts
20 30 40
Minutes
c:\gilson\unipoint\lab-an\peptides.007\peptides.gdt : waters uv/vis 214nm : NSLAB 18: Inj. Number: 10
c:\gilson\unipoint\lab-an\peptides.007\peptides.gdt : waters uv/vis 214nm : NSLAB 17: Inj. Number: 20
c:\gilson\unipoint\lab-an\peptides.007\peptides.gdt : waters uv/vis 214nm : NSLAB 26: Inj. Number: 18
Comparison of Levels of Casein Hydrolysis Cheddar with
Flavour Adjuncts - HPLC of intact casein at 6 months.
0
50
100
% M
obile
Phase
50
100
150
mV
olts
10 20
Minutes
c: \gilson\unipoint\ lab-an\caseines.010\caseines.gdt : waters uv/vis : 01: Inj. Number: 7
c: \gilson\unipoint\ lab-an\caseines.010\caseines.gdt : waters uv/vis : 17: Inj. Number: 13
c: \gilson\unipoint\ lab-an\caseines.010\caseines.gdt : waters uv/vis : 26: Inj. Number: 15
b-caseinas1/1 casein
para k casein
Lactobacillus helveticus 1994C and two controls, no significant difference observed.Casein Hydrolysis Quantified by HPLC of
cheese protein and rates of
b-casein breakdown
in buffered solution.
Peptide Hydrolysis Quantified by HPLC of soluble
peptides in cheese and enzyme
assays, pepN and pepX on the
strains.
Free Amino Acid
concentration Measured by TNBS assay in
cheese samples.
Absence of Amine Production
in optimised media.
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Impact of NSLAB cultures
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Changing Flavours
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Blue Cheese
Cheddar Types
pâte molle
Starter cultures
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Crucial roles to play during all phases of the cheese making and maturation process.
As the culture grows in the milk, it converts lactose to lactic acid. This ensures the correct pH for coagulation and influences the final composition of the cheese.
The rate of acid production is critical in the manufacture of certain products, e.g. Cheddar cheese.
In mechanized operations, starters are often required to produce acid at a consistently fast rate through the manufacturing period each and every day.
Biggest threat to satisfactory performance is:
Bacteriophage
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Phage Description
Phages are bacterial parasites (viruses), they requires bacteria to multiply.
Phages are very small living entities (10 times smaller than bacteria).
Phages are mainly made of proteins (head and tail). The phage head contains genetic material.
Their structure makes them very robust and difficult to eliminate
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Bacteriophage are very small and omnipresent
Whitehead and Cox first demonstrated the role of phage in the failure of dairy
fermentations in 1935
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The Phage Biology : Phage Propagation
An “explosive” phage proliferation that kills bacteria
• Phages adsorb to bacteria
• Phages inject their genetic material
• Bacterial biosynthetic machinery and energy are used to multiply
the phage from this genetic material.
• A single bacteria generates many phage particles (from 10 to 500)
• Phage release occurs upon bacterial death (lysis)
• Newly synthesized phages infect growing bacteria
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The Phage Biology : Rate of Propagation
Phages proliferates faster than bacteria (possibly 50–70 times faster)
Time
Population
10E10
10E9
10E6
1
Amount of bacterial cells multiplies by 2 at each generation time
Time
30min
1h
1h 30min
2h
2h 30min
3h
3h 30min
4h
4h 30min
5h
5h 30min
Phages
1
5. 101
2.5 103
1.2 105
6.2 106
3.1 108
1.6 1010
Bacteria
1 106
2 106
4 106
8 106
1.6 107
3.2 107
6.4 107
1.3 108
2.6 108
5.2 108
1.4 109
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2. Sources of Phages
Phages present in the environment where they encounter bacteria.
In milk (low level since there is little bacterial growth)
In tankers (very low level if only milk is transported, higher risk if tankers are
used for whey)
In tanks and pipes (especially those containing fermented products)
In atmosphere (spray)
In plants (floor, wall (concrete), soil ...)
In not well maintained CIP (make sure to maintain appropriate level of virucide
products)
In fermentation by-products (whey powders, whey cream...)
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The ‘Phage Biology
Development of expertise on bacteriophage to study the lysotype of strains
Phages are collected from dairy plants world wide over years (about 7000 up to date)
analyzed to determine their biodiversity (host spectrum and genetic
analyses) and classified
analyzed to determine their potential technological impact (virulence, heat
résistance)
used to determine the lysotype of bacteria
used to select new bacterial strains for the formulation of new starters
Development of single colony isolates
Strains of known biological relationships
Construction of culture rotations to minimize risk of attack
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3. Fighting phages : Starter Rotation
The starters need to have different lysotype when used in rotation.
Using its collection of phages, Danisco determines the lysotype of each of its
starters to make sure that they are appropriate rotation
Bacteriophage evolves quickly. There is a
constant need for vigilance
Example :
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Culture A 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 # 1 3 3 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Culture B 1 1 1 1 # 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 Culture C 3 3 3 3 3 3 3 3 # 3 3 3 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 # 3
Competitor1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
Competitor 2 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
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Fighting phage : Starter Rotation
The use in rotation of 2 or more starters helps to stabilise the level of phages in a
dairy plant.
Level of
phages
CRITICAL LEVEL
fermentations
Cleaning
Starter A Starter B
Starter rotation keep phage for a longer time below the critical level
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Bacteriophage Management Strategy
Limit starter culture propagation
Appropriate use – dosage & rotation
Sanitation of equipment
Viricidal disinfection
Continuous selection of new strains resistant to phage
Renewal of starter cultures
Rotation of starter cultures
Disadvantages of resistance Selected strains are not 100% identical
Therefore starter cultures differ after their renewal
Rotations are not absolutely isofunctional
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The Swiss Knife approach
SIMPLE
COMPLEX
SELECTED MULTIPLE TOOLS
Geneticbiodiversity
Resistance to phages
Strain Safety
Devoid of antagonism
FunctionalStrain
equilibrium
DefinedMultiple Culture
Strain selection process
• 64 strains
Starter culture development
• More than 10 stains per starter
7/25/2017 21
Defined multiple starter cultures
The science behind the formulation
Devoid of antagonism
Ability to produce antagonistic
molecules
One to one strain testing
Antagonistic strains discarded
Genetic biodiversity
Genome sequencing for each strain
To make sure strains are distinct
To develop methods for the specific
detection of each strains
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DEFINED
STARTERS CULTURES
• Exact definition of the target starter properties
• High cheese process consistency
• Phage rotation program defined with the customer
COMPLEX
STARTERS CULTURES
• Interesting biodiversity but difficult to control
• Intensive industrial efforts to achieve a
cheese process consistency
• The phage alternative concept doesn’t exist
A new formulation approach : DEFINED MULTIPLE STARTER CULTURES
• Starting with selection of the target cultures properties
• Rotation concept is possible and has been developed
• No trade off on starter cultures properties
• Facilitate starters cultures performance and consistency
The Swiss Knife approach
CHOOZIT® 1000 series
Strain equilibrium
Upon production process
Risk to have some strains dominating
Analysis of strain ratio upon production
Thanks to genomics data and the
development of a q-PCR method
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Defined multiple starter cultures
The science behind the formulation
Resistance to phages
Thanks to biodiversity, little
impact of reasonable phage
attack
No dead-vat upon massive
phage attack on the contrary to
simple starter cultures
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4,5
5
5,5
6
6,5
7
0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200
pH
Time (min)
CHOOZIT® MC 1001Tests with Phages
5g /100L - 32°C
Fresh 1/2 skimmed milk
MC 1001 MC 1001 + 2 phages MC 1001 + 5 phages MC 1001 + 16 phages
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Bacteriophage resistance mechanisms
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CRISPR technology
Danisco uncovers mechanism behind bacterial immunity
Important findings from joint research project published in Nature
Joint research conducted between Danisco and Universitè Laval has shed light on the mechanisms behind the natural bacterial immune system CRISPR. The findings highlight the valuable potential as a simple natural way to generate more robust organisms with built in resistance to virus attacks. CRISPR research has generated results that can be used to generate more robust Starter cultures by improving the natural Defence systems in
Streptococcus thermophilus
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CRISPR Resistance to Bacteriophages
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Bacteriophage management - CRISPR
Immunization of cultures: Securing unchanged performance
The CRISPR system is more than a classic bacteriophage resistance mechanism
The CRISPR system acts as a restriction system by destroying bacteriophage DNA upon infection
It is an evolutive resistance mechanism; an immunization system
Upon encountering bacteriophage the CRISPR system acquires additional resistance capabilities
Each bacteriophage encounter allows the acquisition of additional resistances
Evolution of CRISPR system occurs spontaneously and naturally upon bacteriophage infection
Acquired bacteriophage resistances accumulate in a strain and are inheritable
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CRISPR Application to pizza cheese
Step 1: Definition of a series of representative phage
• 17 phage for strain C and 37 phage for strain D
Step 2: Selection of 3 CRISPR variants resistant to phage for each strain
• 3 independent variants for each strain each presenting different new spacers in their CRISPR loci
• All variants are totally resistant to the panel a representative phage
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THANK YOU
I have called this principle, by
which each slight variation, if
useful, is preserved, by the
term of Natural Selection.
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