biodiversity in new starter cultures as a response to
TRANSCRIPT
Biodiversity in new starter cultures as a response to increasing bacteriophage pressures
Mette Winther Børsting,Cheese Application Specialist, DuPont
Danmarks Mejeritekniske Selskab 5. april 2018
Agenda
Bacteriophage – Know the enemy
Bacteriophage - Impact on cheese production
Culture development
• A new concept
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Bacteriophages
Bacteriophages are bacterial parasites (viruses)
They need bacteria cells to multiply!
Mainly made of proteins (head and tail)
Contains genetic material (DNA)
Their structure makes them very robust
and difficult to destroy
Head shell DNA
Long tail fiber
Tail spike
Short tail fiber
Baseplate
Portal
Neck & Collar
Whisker
Sheath (outside)
Tube (inside)
(Wedge outside,
plug inside)
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0.001 0.01 0.1 1.0 10 100 1000 µ m
Electron Microscope
Optical Microscope
Visible to the Eye
RELATIVE SIZE OF COMMON MATERIALS
Salt Solution
Metal Ion
Sugars
Whey Protein Milled Flour
Pollen
MistTobacco Smoke
Coal Dust
PhageBacteria
Yeast Cells
Paint PigmentHuman Hair
Beach Sand
Phage Size
Relative size of common materials
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Very small (10 times smaller than bacteria)
and therefore not visible to the eye or
in a normal microscope
but … they spread as fast and
easily, as smoke !!
Bacteriophage multiplication cycle death of bacterial cell fermentation problem
Bild der Wissenschaft; 4.2002; p21-22; Ute Eberle
The cycle of bacteriophages
1. Attachment to cell membrane
2. Injection of genetic material (DNA)
3-5. Reproduction and assembling of new
bacteriophages in the cell
6. Lysis of the cell and release of new
bacteriophages
Life cycle (1-6) takes between 25-80 minutes
One bacteriophage can produce up to 10-400 phages
(burst size) in one bacterial cell
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6
Bacteriophages multiply faster than bacteria
= phage particle
Bacteria Moderate Phage Virulent Phage
1 cell 2 cells
2 cells 4 cells
4 cells 8 cells
8 cells 16 cells
….
1 10
10 100
100 1000
1000 104
…..
1 100
100 104
104 106
106 108
…..
The cycle of bacteriophages
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1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
1,00E+09
1,00E+10
0 1 2 3 4 5 6
log
n (
cfu
, p
fu)
Hours
Phage Growth vs. Cell Counts
Cell count product
Phage Low Virulence burstsize 5
Phage High Virulence Burstsize 35
[pfu =plaque forming units]
Bacterial Defense Systems against Phages
To resist bacteriophage attack strains have developed
many different strategies
Among the classic strategies are:
• Prevention of absorption
• Blocking injection
• Abortive infection (death of the infected cell)
• Restriction/modification (DNA-degrading enzymes)
• ...etc
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Blocking of
DNA
injection
Abortive infection
(phage-induced
apoptosis)
Restriction-
modification
CRISPR-Cas
Prevention
of adsorption
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Strain 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 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 3
Strain 2 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 1
Strain 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 # # 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
Strain 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 # 2 2 # 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
Strain 5 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 1 1 1
Strain 6 4 4 4 4 4 4 4 4 4 4 4 # 4 4 4 4 4 4 4 4 4 4 4 4 # 4 # 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 # 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 #
Strain 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 # 2 # 2 2 2 2 2 2 2 2 2 2
Strain 8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 # 2 2 2 2 1 2 2 2 2 2 2 2 2
Strain 9 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 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
Strain 10 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 1 1 1
Strain 11 # 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 # 2 2 2 2
Strain 12 # 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 1 1 1
Bacteriophages are species specific
Agenda
Bacteriophage – Know the enemy
Bacteriophage - Impact on cheese production
Culture development
• A new concept
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All rights reserved.
Effect of bacteriophages on the
Acidification
Different levels of phage virulence
- Phage A at 104 /ml stop the
acidification
- Phage B at 104 /ml has no or little
impact on the acidification curve
9
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4.5
5.0
5.5
6.0
6.5
Time
pHNo phages
Severe phage attack
Minor phage attack
Consequences of a bacteriophage attack
10
Bacteriophages are one cause of production failures and could lead to
• Acidification delay or failure (loss of production)
• Product defects
• Low or excessive gas and flavor development
• Texture problems
• Yield problems in cheese production
Control / Management of bacteriophages is key to ensure
• Productivity
• Production robustness
• Consistent product quality
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Slow Acidification =
Bacteriophages?Reason for fermentation problems = Bacteriophage in most cases
Inhibitors
• Antibiotics
• Disinfectants
• Preservatives
Other factors affecting culture activity
• Mixing / excessive aeration
• Old culture / wrong storage
• Wrong fermentation temperature
But don‘t forget!
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Agenda
Bacteriophage – Know the enemy
Bacteriophage - Impact on cheese production
Culture development
• A new concept
12Copyright © 2018 DuPont or its affiliates.
All rights reserved.
DEFINED STARTER CULTURES
• 1 to 4 strains
• Use of robust phage strains
• High cheese process consistency
• Suitable for cheese technology with a short
process time
• Phage rotation program defined with the
customer
The traditional culture offer
13
COMPLEX STARTER CULTURES
• Interesting biodiversity but difficult to control
• Robustness of phage strains is not known
• Difficulties in obtaining cheese process consistency
• Suitable for cheese technology with a long process
time
• No real phage alternative
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DEFINED MULTIPLE STARTER CULTURES
• Phage robustness -> multiplicity of strains and the quality
of the strains
• Possibility of rotations -> control of the complexity
• Functionalities of culture -> controlled by the use of
appropriate strains
• Reproducibility of productions
DEFINED STARTER CULTURES
The new concept: multiple defined cultures
14
COMPLEX STARTER CULTURES
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Geneticbiodiversity
Resistancetophages
Strain Safety
Strain features
FunctionalStrain
equilibrium
DefinedMultiple Culture
15
Defined multiple starter cultures
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Geneticbiodiversity
Resistancetophages
StrainSafety
FunctionalStrain
equilibrium
DefinedMultiple Culture
Genetic biodiversity
Genomic data of strains have been compared
Strains with identical or similar genomes are eliminated
Develop methods for the specific detection of each strains
16
Defined multiple starter cultures
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Strain features
17
Defined multiple starter cultures
Impact of strains on equilibrium
• Sensitivity to physico-chemical factors
• Optimal growth temperature
• Lactate sensitivity
• Competition for nutrients
• Nutritional needs
• Nutrient availability
• Ability to metabolize nutrients
• Quorum Sensing
• Communication between strains
• Bacteriocin
Geneticbiodiversity
Resistanceto phages
StrainSafety
FunctionalStrain
equilibrium
DefinedMultiple Culture
Strain features
Geneticbiodiversity
Resistancetophages
Strain Safety
Strain features
FunctionalStrain
equilibrium
DefinedMultiple Culture
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
18
• Examples of maintained versus lost biodiversity
Defined multiple starter cultures
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Inoculum
Culture
Geneticbiodiversity
Resistance tophages
Strain Safety
Strain features
FunctionalStrain
equilibrium
DefinedMultiple Culture
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
19
Defined multiple starter cultures
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4
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 phagesCulture Culture + 2 phages Culture + 5 phages Culture + 16 phages
A high rate of phage infection was
used (103 pfu/ml)
[pfu =plaque forming units]
20
Ready to use
• Very high phage resistance
• Reliable fast acidification time
• Formulated for high productivity
Easy to combine with flavor adjuncts for more intense/specific flavor
Can be combined with other cheese cultures to accelerate acidification
Good feedback from customers
Conclusion Geneticbiodiversity
Resistancetophages
Strain Safety
Strain features
FunctionalStrain
equilibrium
DefinedMultiple Culture
216 décembre 2017