1

What is a starter (culture)

2

Where is a starter used

3

Why is a starter culture

used

4

Sacco Srl wwwsaccosrlit

5

First of all

Who is Sacco

since 1872

In Cadorago

Dairy ndash Food Industry

Mainly Lactic Acid Bacteria

Research Laboratories

Production Plants

A family company

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

2

Where is a starter used

3

Why is a starter culture

used

4

Sacco Srl wwwsaccosrlit

5

First of all

Who is Sacco

since 1872

In Cadorago

Dairy ndash Food Industry

Mainly Lactic Acid Bacteria

Research Laboratories

Production Plants

A family company

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

3

Why is a starter culture

used

4

Sacco Srl wwwsaccosrlit

5

First of all

Who is Sacco

since 1872

In Cadorago

Dairy ndash Food Industry

Mainly Lactic Acid Bacteria

Research Laboratories

Production Plants

A family company

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

4

Sacco Srl wwwsaccosrlit

5

First of all

Who is Sacco

since 1872

In Cadorago

Dairy ndash Food Industry

Mainly Lactic Acid Bacteria

Research Laboratories

Production Plants

A family company

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

5

First of all

Who is Sacco

since 1872

In Cadorago

Dairy ndash Food Industry

Mainly Lactic Acid Bacteria

Research Laboratories

Production Plants

A family company

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

1872 1908

1948

1962

1995

1994

1984

2006 1998 2013

A continue evolution

Clerici-Sacco history

6

1972

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Locations

7

ITALY

OTHER

COUNTRIES

Kemikalia AB Skurup

Production plant Sweden

CSL France Claix ndash Grenoble

Commercial Office France

Sacco amp Caglificio

Clerici Cadorago

3 production plants Italy

Sacco Caslino al Piano Production plant

Italy

CSL Zelo Buon Persico Production plant

Italy

CSL Pasturago

Production plant Italy

Sacco India Ashish home Liaison Office

India

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Some figures

7

Production volumes 37 tyear pharma-nutra 106 tyear frozen cultures 56 mill UCyear freeze-dried cultures

Exporting to more than 100 countries

60 mill euro turnover 2012

240 total employees

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

9

Global presence ndash Market division

56

44

Starter

cultures

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

10

Global presence ndash Export division

3

45

10

26

11

5

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Products range

Dairy starter

cultures

Nutraceuticals

Probiotic cultures

Rennet and cheese

enzymes

Moulds and yeasts

Protective cultures

Bread cultures

Fish cultures Meat cultures

Vegetables cultures

Starter media

Product range

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

12

Research amp Development

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Investments

13

INVESTING 6-7 OF THE BUSINESS UNIT TURNOVER

Strains isolation

New products development Process improvements

Phages robustness

Genotypic and phenotypic characterization

Europe has the objective to invest 3

of the GNP in research

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

14

Host Master

and PhD

students

giving them the

possibility to

experience life

from an

ldquoindustrialrdquo

point of view

Partecipate to

courses and

meetings as

well as

International

Conferences

and symposia

Financially

support Master

and PhD

programs both

at National and

International

level

Updates

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

15

Collaborations ndash External RampD

University of Piacenza

Prof Piersandro Cocconcelli

TUM ndash Weihenstephan

Munich

Prof Rudi Vogel

Prof Peter Schieberle

Prof Ulrich Kulozik

KU ndash LIFE

Copenhagen

Prof Finn Vogensen

University of Bari

Prof Marco Gobbetti

Prof Maria De Angelis

University of Parma

Prof Marco Ventura

FH Flensburg

Prof Detlef Goumllling NIZO food research Moorepark Food Research

Center

Prof Paul Ross

Dr Olivia McAuliffe

Dr Mary Rea

University College Cork

Prof Douwe Van Sinderen

DeFENS University of Milan

Prof Diego Mora

Dr Simone Guglielmetti

CRA-FLC

Dr Giorgio Giraffa

Dr Domenico Carminati

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

16

Heterogeneity

Different nationality

- Italy

- Denmark

- Venezuela

- Romania

Different background

- 30 laboratory technicians

- 70 with Master degree in

Food Science 50

Biotechnology 26

Biology 24

Whereof 19 with a PhD

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

17

Internal RampD

Non-stop research of

novel and

interesting

LAB

Study and

characterization

of LAB colture

collection

Scale-up and

production

processes

Blending

packaging

and

shipment

Application in

clients

production

plants

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Microbial species available for products

18

Bacteria

Bifidobacterium sp

Carnobacterium sp

Enterococcus sp

Lactobacillus sp

Lactococcus lactis subsp lactis

Lactococcus lactis subsp cremoris

Lactococcus lactis subsp lactis biovar diacetylactis

Leuconostoc sp

Pediococcus sp

Propionibacterium sp

Staphylococcus sp

Streptococcus thermophilus

Surface ripening strains

Fungi

Geotrichum

Penicillium

Saccharomyces

Kluyveromyces

Candida

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Isolation

Identification

Phage profile

Activity determination

Selected strains are inserted into the strain collection

Culture collection insertion

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Physiologic characterization ndash Part 1

B) Acidificationgrowth profile

A) Colony morphology

39

4

41

42

43

44

45

46

47

48

49

5

51

52

53

54

55

56

57

58

59

6

61

62

63

64

65

66

67

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960

Time (min)

ST366 1

ST336 005

ST07 1

ST07 005

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

C) EPS production

D) Citrate fermentation

On plates

In milk

Physiologic characterization ndash Part 2

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

E) Antibiotic resistance

F) In vitro GIT transit

bull Oxgall

bull Bovinepig bile

bull HCl pH 30

bull Pepsin

bull Pancreatin

LA 2 LA 4

OXGALL 100 OXGALL 98

Bovine bile 100 Bovine bile 30

Pig bile 41 Pig bile 29

HCl 39 HCl 37

Pepsin 48 Pepsin 29

Pancreatin 43 Pancreatin 46

Technical guidance prepared by the Panel on

Additives and Products or Substances used in Animal

Feed (FEEDAP) on the update of the criteria used in

the assessment of bacterial resistance to antibiotics of

human or veterinary importance The EFSA Journal

(2008) 732 1-15

According to EFSArsquos recommendation

Physiologic characterization ndash Part 3

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

G) Antimicrobial activity

Screening against LAB and pathogenic microrganisms

Agar plate assay

Determination of the compounds responsible

for inhibitory activity

Physiologic characterization ndash Part 4

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

G) Phage profile

Phages for S thermophilus

Phages for Lc lactis

Phages for L bulgaricus

Phages for Leuconostoc

Phages against L helveticus

pH (pH multiscan)

Plaque assay

Physiologic characterization ndash Part 5

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

25

Continuous collection of information and samples from all over the

world

NEVER ENDING MONITORING Client service

Whey samples Communications Alerts

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Product development

According to the desired product selected strains are

combined and the final blend is evaluated regarding

- Activity

- Viscosity

- Overall texture

- Sensory

- Post-acidification

- Shelf lifestability PAPER FOR ORGANOLEPTIC TEST OF FERMENTED MILK PRODUCTS

VISCOUS MESOPHILIC CULTURES

Product name

Batch ndeg

Media

Temperature

Date

Assessor

STIRRED

TEXTURE ON SPOON

0=thin 4= very thick

0 1 2 3 4

0=no ropy 4= very ropy

0 1 2 3 4

0=no lumps 4= very lumpy

0 1 2 3 4

MOUTHFEEL

0=thin 4= very thick

0 1 2 3 4

0=smoothness 4= grainyness

0 1 2 3 4

0=not ropy 4= very ropy

0 1 2 3 4

COMMENTS

LUMPINESS

THICKNESS

ROPINESS

THICKNESS

ROPINESS (how easy to swallow)

GRAININESS

Fresh Milk

Soy milk Lab milk (SMP)

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Factory

Development pilot production medium production closed

transport systems with CIP fermentors separation freeze-

drying harvest QC

Starter culture production

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

THE ENEMY The most widely distributed and diverse entities in the

biosphere

BACTERIOPHAGES

The major cause of fermentation failure in

the dairy industry

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

29

What is the real mechanism

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

about bacteriophages

Bacterial viruses

Very simple structure

Where do they come from

- raw materials

- the AIR

Lytic cycle

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

31

45

55

65

0 1 2 3 4 5 6

pH

ore

the attack from the

application point of view

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Dairy Application

1 Use of starters containing phage-unrelated or phage-

insensitive strains (see example later)

2 Production of phage-free bulk starter

bull Aseptic propagation systems

bull Use of phage-inhibitory media

bull Segregation of starter room and cheese process equipment

CIP-systems

bull Removal of deposits on bulk starter vessels

bull Ensuring the head space is minimised in bulk starter vessels

and sterilised

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

3 Minimising the concentration of phage in processing

plants

bull Culture rotations

bull Air conditioning

bull Aerosol generationfumigation

bull Cleaningchlorination of vats between refills

bull Good factory design Location of whey storage tanks whey

handling systems

4 Other measures

bull Direct inoculum

bull Inspection of jacketsagitators for pin holing

bull Early renneting

Dairy Application

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Production of phage-free bulk starter

Aseptic techniques for the propagation of starters

Maintenance of the starter room under positive pressure

with filtered air

Aerosol control

Location of the bulk starter facility

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Bacteriophage inhibitory media

Most but not all lactococcal phages require divalent cations

in particular calcium for multiplication

Calcium-free substrate

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Culture rotations

Cheese whey which may contain very high titers of phage is

regarded as the primary source of phage infection during cheese

manufacture

If the whey contains very high levels of phage ie 109-1010 PFUml

then there may be sufficient phage in the air of the creamery to

contaminate the cheese milk and utensils If cheese were made with

the same starter next day then slow acid production or even failure of

the fermentation could occur

Anderson and Meanwell (1942) rotation of phage unrelated starters

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Single culture ripetitive use

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Lo

g C

FU

Lo

g P

FU

9

8

7

10

9

8

7

6

5

4

3

2

Culture rotations

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Culture rotations limits

Where the cross phage relationships have not been

determined the new starter could provide a link allowing a

faster developing phage to attack an existing starter

Need for defined cultures with similar behavior

Need for a excellent phage collection

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

Cleaningchlorination of vats between refills

In modern cheesemaking plants vats can be filled and

refilled many times a day With this practice there is potential

for significant phage build up This can be controlled by

rinsing followed by a chlorine rinse

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

41

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

ADSORPTION

(phage attachment to host)

UPTAKE (phage genome release into chytoplasm)

HOST TAKEOVER (host death for more vir phages)

MATURATION (phage progeny formation)

RELEASE (of phage progeny host death if less vir)

PHAGE ATTACK STEP

BACTERIAL RESISTANCE

ADSORPTION RESISTANCE

(eg capsules phage-receptor loss)

UPTAKE BLOCKS

(eg plasmid of prophage encoded)

RESTRICTION

(eg restriction enzymes CRISPR)

ABORTIVE INFECTION

(plus reduced infection vigor)

REDUCED INFECTION VIGOR

(egdarrburstuarrlatent period)

INTERFERENCE WITH DISSEMINATION

(eg extracellular polymers) From Adv Appl Microbiol 201070217-48 Epub 2010 Mar 6

Bacteriophage host range and bacterial resistance

Hyman P Abedon ST

43

Inibizione dellrsquoassorbimento

bull BIMs mutanti insensibili al batteriofago ndash Modificazione o mascheramento del sito di attacco del fago

bull Polisaccaridi della parete (galattosio e ramnosio) e LAM ndashloosely associated material- Lc lactis

bull Surface layer Lb helveticus

bull Sistemi plasmidici

ndash Produzione di EPS

ndash Produzione di antigeni di superficie

44

45

Plasmidi multiresistenza in Lc lactis

Lc lactis presenta plasmidi con piugrave sistemi di difesa Ads RM Abi

Plasmidi coniugativi coinvolti nella resistenza ai fagi pNP40 2 Abi e 1 inibizione dellrsquoiniezione

46

Uso dei sistemi naturali di scambio genetico per il trasferimento dei sistemi di resistenza ai batteriofagi

Anni rsquo80 Prima applicazione industriale

Plasmidi con multi-resistenze

Plasmidi con differenti meccanismi nella stessa cellula

Ads Inj RM Abi

Miglioramento genetico di L lactis

47 Moineau Ant Van Leeeuvenhoek 76 377-382 1999

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

44

45

Plasmidi multiresistenza in Lc lactis

Lc lactis presenta plasmidi con piugrave sistemi di difesa Ads RM Abi

Plasmidi coniugativi coinvolti nella resistenza ai fagi pNP40 2 Abi e 1 inibizione dellrsquoiniezione

46

Uso dei sistemi naturali di scambio genetico per il trasferimento dei sistemi di resistenza ai batteriofagi

Anni rsquo80 Prima applicazione industriale

Plasmidi con multi-resistenze

Plasmidi con differenti meccanismi nella stessa cellula

Ads Inj RM Abi

Miglioramento genetico di L lactis

47 Moineau Ant Van Leeeuvenhoek 76 377-382 1999

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

45

Plasmidi multiresistenza in Lc lactis

Lc lactis presenta plasmidi con piugrave sistemi di difesa Ads RM Abi

Plasmidi coniugativi coinvolti nella resistenza ai fagi pNP40 2 Abi e 1 inibizione dellrsquoiniezione

46

Uso dei sistemi naturali di scambio genetico per il trasferimento dei sistemi di resistenza ai batteriofagi

Anni rsquo80 Prima applicazione industriale

Plasmidi con multi-resistenze

Plasmidi con differenti meccanismi nella stessa cellula

Ads Inj RM Abi

Miglioramento genetico di L lactis

47 Moineau Ant Van Leeeuvenhoek 76 377-382 1999

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

46

Uso dei sistemi naturali di scambio genetico per il trasferimento dei sistemi di resistenza ai batteriofagi

Anni rsquo80 Prima applicazione industriale

Plasmidi con multi-resistenze

Plasmidi con differenti meccanismi nella stessa cellula

Ads Inj RM Abi

Miglioramento genetico di L lactis

47 Moineau Ant Van Leeeuvenhoek 76 377-382 1999

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

47 Moineau Ant Van Leeeuvenhoek 76 377-382 1999

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Use of starters containing phage-unrelated

or phage-insensitive strains

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

49

2007 Non-GMM approach

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

50

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Why is it a problem

1 MILK

TREATEMENT

2 INOCULATION =

STARTER COLTURES +

RENNET

3 CURD CUTTING

4 MOULDING

5 SALTING amp RIPENING Cheese p

rodu

ction p

rocess

What Sacco could do to solve

the problem

- Using multiple strains starters

- Rotations

- Resistant Mutants

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Size of the problem an example

Cheese factory Tom

5 different starters in rotation

Each starter made with two different strain

The weakest starter

- Using starter multiple strains

- Starters rotations

ITrsquoS NOT ENOUGH Production stop frequency in Tom

cheese factory

(years 2007-2009)

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Understanding the problem

1 ANALYSIS OF WHEY

SAMPLES

2 ISOLATION OF THE

PHAGE

3TESTING THE

SENSITIVITY OF

SINGLE STRAINS

SACCOrsquoS LABORATORY

ROUTINE

In ldquostarter 1rdquo Ab123

is the most sensitive

strain

0

05

1

15

2

25

3

35

4

45

2007 2008 2009

Ab123

Ab125

Production stop frequency

in Tom cheese factory

(years 2007-2009)

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

BIMs = Bacteriophages Insensitive

Mutants Moorepark Food Research

Center Fermoy

Prof Paul Ross

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Ab123 ΦAb123

Selective

pressure X20

Variables

- MOI (Multiplicity Of Infection) = ratio of infectious agents to infection

targets

-Strains number

-Phages number

-Passages number

STANDARD

PROTOCOL

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Main problems in BIMs creation

process

- Selective pressure induces big

changes in the strain

- The strain loses important

technological features (ie EPS

production strong acidification)

2) PRODUZIONE BIMs ST114

0150AA0CO618D XY OC01G50100AV6TC06EE0

+ - - - lt=- - - - =- - N+ - - 4

3amp4 - - - 6- - - NB010B78B7578E65TZ50EE0OC[ 65618O7AA05CHC17A6]+E6OC8C3amp4 - - lt=6- - - =

4

45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Necessary laboratory pre-tests

1 RESISTANCE TEST

2 STABILITY TEST

3 QUALITY CONTROL

4 IDENTITY CONTROL

Ab123 Ab123

BIM 1

BIM 3

BIM 5

BIM 4

BIM 2

BIM 6 6 out of 6 OK

X10 6 out of 6 OK

4 out of 6 OK

FIRST ROUGH SCREENING

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6 45

47

49

51

53

55

57

59

61

63

65

0 2 35 5

time (h)

pH level

Ab123

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

6 out of 6 OK

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Microbiology characterization

tests in Sacco

- Microscopy analysis

- Colony morphology

- First milk clotting tests

MICROBIOLOGY LAB

BACTERIOPHAGES LAB

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

STRAINS phage Ab123

whey 1 whey 2 bactometer pH Multiscan

Ab123 +++ +++ +++ +++

BIM 1 +- + ++ +

BIM 4 +- +- - +-

BIM 5 +- - - -

BIM 6 +- - + +-

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Pilot scale test production + RampD Lab

BIM 1

BIM 2

BIM 3

BIM 4

BIM 5

BIM 6

4500

5000

5500

6000

6500

0h

30

1

h1

h3

0

2h

2h

30

3

h3

h3

0

4h

4h

30

5

h5

h3

0

6h

6h

30

7

h7

h3

0

8h

8h

30

9

h9

h3

0

10

h1

0h

30

1

1h

11

h3

0

12

h1

2h

30

1

3h

13

h3

0

14

h1

4h

30

1

5h

15

h3

0

16

h

RampD activity control

Ab123

BIM 4

BIM 5

Strain Lag time Log time NH3 used (ml) Broth

CFUml

Concentrate

CFUg)

Lyo count

(CFUg)

Lyo (g) L

broth

Ab123 44 2h e 21 500 840E+09 140E+11 380E+11 147

BIM 4 45 2h e 40 520 720E+09 120E+11 390E+11 146

BIM 5 50 2h e 46 500 670E+09 140E+11 520E+11 147

Good (comparable) yield

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Test production + test application directly at Tom Cheese Factory

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

Non-stop research of

new interesting LAB

Study and

characterization of

LAB colture collection

Scale-up and

production processes

Blending

packaging and

shipment

Application in clients

production plants

Production stop frequency in Tom

cheese factory

(years 2007-2009)

0

2

4

6

8

10

12

14

starter 1 starter 2 starter 3 starter 4 starter 5

Production stop frequency in Tom

cheese factory in 2010

wwwsaccosrlit

Questions

wwwsaccosrlit

Questions