Biodiversity in new starter cultures as a response to ...

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

2Copyright © 2018 DuPont or its affiliates.

All rights reserved.

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)



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



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



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

Copyright © 2018 DuPont or its affiliates.


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



Blocking of

DNA

injection

Abortive infection

(phage-induced

apoptosis)

Restriction-

modification

CRISPR-Cas

Prevention

of adsorption

1 2 3 4 5 6 7 8 9 10

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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



Culture development

• A new concept



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



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



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!



Agenda



Culture development

• A new concept



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



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



Geneticbiodiversity

Resistancetophages

Strain Safety

Strain features

FunctionalStrain

equilibrium

DefinedMultiple Culture

15

Defined multiple starter cultures



Geneticbiodiversity

Resistancetophages

StrainSafety

FunctionalStrain

equilibrium


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




Strain features

17


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


Strain features

Geneticbiodiversity

Resistancetophages

Strain Safety

Strain features

FunctionalStrain

equilibrium


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




Inoculum

Culture

Geneticbiodiversity

Resistance tophages

Strain Safety

Strain features

FunctionalStrain

equilibrium


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




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


216 décembre 2017

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