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Developments in Cheese Starter Technology Michael Pinches

DuPont Nutrition & Health

Society of Dairy Technology Annual Dinner 2017

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Using Starter cultures

The practicality of employing cultures in cheese making has been tested.

Upon the belief that lactic acid bacteria are the main agents in the curing of cheese this species has been suggested as the “Starter” under the following conditions:

The organism should be a pure lactic germ

Free from undesirable aroma

Adapted for vigorous development in milk

The advantages accruing from the use of such a “Starter”

Saves time in the process of manufacture

Aids development of a proper amount of acid for a typical cheese

The flavour and quality of cheese is preferable to that which has not been thus produced

Bacteriology of Milk 1903 by Harold Swithinbank

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Lactococcus lactis Magnification 20,000X. Scanning electron micrograph

1903 2017


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Physical forms of starter cultures

• Daily Propagation

• Frozen Pints

• Immediate Inoculation to the bulk starter tank

• Your starter for 1000

• Direct Vat Inoculation

• Frozen cans

• Frozen pellets

• Freeze dried

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Lactic Acid culture collections

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Ripening Cultures

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Product Composition Application

CB2 Penicillium roqueforti Strong Blue Tast - Blue Green

KL71 Kluyveromyces lactis Flavour in soft cheese and hole formation

Linens W Brevibacterium linens, Orange colour Aroma, fast growth

MGE Arthobacter nicotianae Strong aminopeptidase activity, fast growth

MVA Staphylococcus xylosus Stimulation of lactic acid bacteria

Mycodore Trichothecium domesticum Saint Nectaire Aspect

OFR 20Brevibacterium casei, Brevibacterium linens,

Debaromyces hansenii, Candid utilsBlend for colouration of smear cheese

OFR 9Brevibacterium casei, Brevibacterium linens,

Candida utils, Geothricium candidum

Appearance and flavour of all the surface

ripening

PA Penicillium roqueforti Mild Blue Taste - Dark Green

PC 02 Penicillium Candidum Ultra Filtered and stabilised cheese

PC 33 Penicillium Candidum Anti Mucor activity, use every day

PC 53 Penicillium Candidum Stabilised and Traditional

PC 12 Penicillium Candidum Stabilised Cheese, Longer Shelf life

PC 42 Penicillium Candidum Traditional cheese, Normal shelf life

PJ Penicillium roqueforti Typical Blue Taste - Middle Green

PLABrevibacterium linens, Geotrichum candidum,

Debaromyces hensenii, Arthrobacter nicotianae

Complex blend for the appearance and

flavour of the main French cheese

PV Penicillium roqueforti Strong Blue Taste - Blue Green

R2R Rhodosporidium infirmominiatum Flavour and colour in mixed smear cheese

Debaromyces hansenii Neutralisation for mix and sear cheese

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CASEIN

Chymosin /Plasmin

Intermediates

peptides

Free

Amino Acids

Enzymatic & Chemical

modifications

Proteinases

(Lactococcus)

Short Peptides

Volatile Flavour

Compounds

Bitterness

Savoury

Sulphur

Into the pathways of ripening in Flavour Creation

Peptidases of

NSLAB

0

50

100

% M

obile P

hase

100

150

mV

olts

20 30 40

Minutes

c:\gilson\unipoint\lab-an\peptides.007\peptides.gdt : waters uv/vis 214nm : NSLAB 18: Inj. Number: 10



Comparison of Levels of Casein Hydrolysis Cheddar with

Flavour Adjuncts - HPLC of intact casein at 6 months.

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50

100

% M

obile

Phase

50

100

150

mV

olts

10 20

Minutes

c: \gilson\unipoint\ lab-an\caseines.010\caseines.gdt : waters uv/vis : 01: Inj. Number: 7



b-caseinas1/1 casein

para k casein

Lactobacillus helveticus 1994C and two controls, no significant difference observed.Casein Hydrolysis Quantified by HPLC of

cheese protein and rates of

b-casein breakdown

in buffered solution.

Peptide Hydrolysis Quantified by HPLC of soluble

peptides in cheese and enzyme

assays, pepN and pepX on the

strains.

Free Amino Acid

concentration Measured by TNBS assay in

cheese samples.

Absence of Amine Production

in optimised media.


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Impact of NSLAB cultures

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Changing Flavours

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Blue Cheese

Cheddar Types

pâte molle

Starter cultures

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Crucial roles to play during all phases of the cheese making and maturation process.

As the culture grows in the milk, it converts lactose to lactic acid. This ensures the correct pH for coagulation and influences the final composition of the cheese.

The rate of acid production is critical in the manufacture of certain products, e.g. Cheddar cheese.

In mechanized operations, starters are often required to produce acid at a consistently fast rate through the manufacturing period each and every day.

Biggest threat to satisfactory performance is:

Bacteriophage


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Phage Description

Phages are bacterial parasites (viruses), they requires bacteria to multiply.

Phages are very small living entities (10 times smaller than bacteria).

Phages are mainly made of proteins (head and tail). The phage head contains genetic material.

Their structure makes them very robust and difficult to eliminate

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Bacteriophage are very small and omnipresent

Whitehead and Cox first demonstrated the role of phage in the failure of dairy

fermentations in 1935


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The Phage Biology : Phage Propagation

An “explosive” phage proliferation that kills bacteria

• Phages adsorb to bacteria

• Phages inject their genetic material

• Bacterial biosynthetic machinery and energy are used to multiply

the phage from this genetic material.

• A single bacteria generates many phage particles (from 10 to 500)

• Phage release occurs upon bacterial death (lysis)

• Newly synthesized phages infect growing bacteria


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The Phage Biology : Rate of Propagation

Phages proliferates faster than bacteria (possibly 50–70 times faster)

Time

Population

10E10

10E9

10E6

1

Amount of bacterial cells multiplies by 2 at each generation time

Time

30min

1h

1h 30min

2h

2h 30min

3h

3h 30min

4h

4h 30min

5h

5h 30min

Phages

1

5. 101

2.5 103

1.2 105

6.2 106

3.1 108

1.6 1010

Bacteria

1 106

2 106

4 106

8 106

1.6 107

3.2 107

6.4 107

1.3 108

2.6 108

5.2 108

1.4 109


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2. Sources of Phages

Phages present in the environment where they encounter bacteria.

In milk (low level since there is little bacterial growth)

In tankers (very low level if only milk is transported, higher risk if tankers are

used for whey)

In tanks and pipes (especially those containing fermented products)

In atmosphere (spray)

In plants (floor, wall (concrete), soil ...)

In not well maintained CIP (make sure to maintain appropriate level of virucide

products)

In fermentation by-products (whey powders, whey cream...)


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The ‘Phage Biology

Development of expertise on bacteriophage to study the lysotype of strains

Phages are collected from dairy plants world wide over years (about 7000 up to date)

analyzed to determine their biodiversity (host spectrum and genetic

analyses) and classified

analyzed to determine their potential technological impact (virulence, heat

résistance)

used to determine the lysotype of bacteria

used to select new bacterial strains for the formulation of new starters

Development of single colony isolates

Strains of known biological relationships

Construction of culture rotations to minimize risk of attack


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3. Fighting phages : Starter Rotation

The starters need to have different lysotype when used in rotation.

Using its collection of phages, Danisco determines the lysotype of each of its

starters to make sure that they are appropriate rotation

Bacteriophage evolves quickly. There is a

constant need for vigilance

Example :

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Culture A 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 # 1 3 3 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Culture B 1 1 1 1 # 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 # 1 1 1 1 1 1 Culture C 3 3 3 3 3 3 3 3 # 3 3 3 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 # 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 # 3 3 3 3 3 3 # 3

Competitor1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

Competitor 2 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?


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Fighting phage : Starter Rotation

The use in rotation of 2 or more starters helps to stabilise the level of phages in a

dairy plant.

Level of

phages

CRITICAL LEVEL

fermentations

Cleaning

Starter A Starter B

Starter rotation keep phage for a longer time below the critical level


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Bacteriophage Management Strategy

Limit starter culture propagation

Appropriate use – dosage & rotation

Sanitation of equipment

Viricidal disinfection

Continuous selection of new strains resistant to phage

Renewal of starter cultures

Rotation of starter cultures

Disadvantages of resistance Selected strains are not 100% identical

Therefore starter cultures differ after their renewal

Rotations are not absolutely isofunctional

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The Swiss Knife approach

SIMPLE

COMPLEX

SELECTED MULTIPLE TOOLS

Geneticbiodiversity

Resistance to phages

Strain Safety

Devoid of antagonism

FunctionalStrain

equilibrium

DefinedMultiple Culture

Strain selection process

• 64 strains

Starter culture development

• More than 10 stains per starter

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Defined multiple starter cultures

The science behind the formulation

Devoid of antagonism

Ability to produce antagonistic

molecules

One to one strain testing

Antagonistic strains discarded

Genetic biodiversity

Genome sequencing for each strain

To make sure strains are distinct

To develop methods for the specific

detection of each strains


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DEFINED

STARTERS CULTURES

• Exact definition of the target starter properties

• High cheese process consistency

• Phage rotation program defined with the customer

COMPLEX

STARTERS CULTURES

• Interesting biodiversity but difficult to control

• Intensive industrial efforts to achieve a

cheese process consistency

• The phage alternative concept doesn’t exist

A new formulation approach : DEFINED MULTIPLE STARTER CULTURES

• Starting with selection of the target cultures properties

• Rotation concept is possible and has been developed

• No trade off on starter cultures properties

• Facilitate starters cultures performance and consistency

The Swiss Knife approach

CHOOZIT® 1000 series

Strain equilibrium

Upon production process

Risk to have some strains dominating

Analysis of strain ratio upon production

Thanks to genomics data and the

development of a q-PCR method

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Defined multiple starter cultures

The science behind the formulation

Resistance to phages

Thanks to biodiversity, little

impact of reasonable phage

attack

No dead-vat upon massive

phage attack on the contrary to

simple starter cultures

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5,5

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6,5

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0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200

pH

Time (min)

CHOOZIT® MC 1001Tests with Phages

5g /100L - 32°C

Fresh 1/2 skimmed milk

MC 1001 MC 1001 + 2 phages MC 1001 + 5 phages MC 1001 + 16 phages


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Bacteriophage resistance mechanisms

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CRISPR technology

Danisco uncovers mechanism behind bacterial immunity

Important findings from joint research project published in Nature

Joint research conducted between Danisco and Universitè Laval has shed light on the mechanisms behind the natural bacterial immune system CRISPR. The findings highlight the valuable potential as a simple natural way to generate more robust organisms with built in resistance to virus attacks. CRISPR research has generated results that can be used to generate more robust Starter cultures by improving the natural Defence systems in

Streptococcus thermophilus

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CRISPR Resistance to Bacteriophages

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Bacteriophage management - CRISPR

Immunization of cultures: Securing unchanged performance

The CRISPR system is more than a classic bacteriophage resistance mechanism

The CRISPR system acts as a restriction system by destroying bacteriophage DNA upon infection

It is an evolutive resistance mechanism; an immunization system

Upon encountering bacteriophage the CRISPR system acquires additional resistance capabilities

Each bacteriophage encounter allows the acquisition of additional resistances

Evolution of CRISPR system occurs spontaneously and naturally upon bacteriophage infection

Acquired bacteriophage resistances accumulate in a strain and are inheritable

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CRISPR Application to pizza cheese

Step 1: Definition of a series of representative phage

• 17 phage for strain C and 37 phage for strain D

Step 2: Selection of 3 CRISPR variants resistant to phage for each strain

• 3 independent variants for each strain each presenting different new spacers in their CRISPR loci

• All variants are totally resistant to the panel a representative phage

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Copyright © 2011 DuPont or its affiliates. All rights reserved. The DuPont Oval Logo, DuPont™, The miracles of science™ and all products denoted with ™ or ® are registered trademarks or trademarks of E. I. du Pont de Nemours and Company or its affiliates.

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THANK YOU

I have called this principle, by

which each slight variation, if

useful, is preserved, by the

term of Natural Selection.

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