LACTIC STARTER CULTURES AND

BACTERIOPHAGES

INTRODUCTION

Selected strain of food-grade microorganisms of known and

stable metabolic activities and other characteristics that is used

to produce fermented foods of desirable appearance, body,

texture, and flavor.

Some are also used to produce

food additives (organic acids, bacteriocins)

probiotics

drug delivery.

Give better product than those produced through natural

fermentation of the raw materials.

During successive transfer of mother starter to fresh medium

often face bacteriophage infection that brings product failure if

careful procedures are not taken.

Mainly used in dairy industry (yogurt, fermented milks, cheese)

LAB

STARTER FUNCTION

Fermentation of sugars organic acids pH decrease

clotting, reduction and prevention of adventitious microflora

Protein hydrolysis texture change, taste enhance

Synthesis of flavor

Synthesis of texturing agents

Production of inhibitory components (i.e., bacteriocin, etc)

Consistency

Prevent product failure

Industrial scale production

HISTORY

Initially back slopping and natural fermentation

Still used in domestic manufacture of fermented milk.

Back slopping

Before 1950’s

Stock culture mother culture bulk culture (1-2 % of volume

of milk)

Difficult to produce of consistent quality

Small business

Single strains of known

Starter failure due to bacteriophage

After 1950’s

Combating against phage

Rotation of strains

Multiple strains

Defined media to produce bulk starter to reduce phage attack

Consistent quality

After 1960’s

Introduction of frozen concentrate culture that have high cell

number (1011-12 cells/ml).

Could be directly inoculated into milk (direct vat set, DVS)

unnecessary to produce bulk starter.

Air transported in dry ice.

Phage inhibitory media (PIM): high concentration of phosphate

(PO4-) to chelate calcium (Ca2+) in milk unable phage to

adsorb on bacterial cell.

After 1970’s

Demand increased

Different types of starters

Cheese, yogurt, fermented sausage, some ethnic fermented

foods

Freeze-dried concentrated cultures

Eliminate use of dry ice

Prevent accidental thawing

But some cells do not survive well in freeze-dried state.

Recently

Custom designed starter cultures

Understanding genetic background of some important desirable

traits, phage inhibition

TYPES OF STARTER CULTURES

TRADITIONAL STARTER

LIQUID STARTER Scale-up: Liquid stock Mother Intermediate Bulk

(increase volume by successive subcultures)

Expensive

Laborious

Needs skillful personnel

Easily contaminated by bacteriophages

Still practiced

FREEZE-DRIED CULTURE

The same as liquid culture for preparation

Used when small amount of starter is needed

Excellent preservation (@ - 25°C for several months)

Mixed stain should be separately freeze-dried to maintain

balance between strains

CONCENTRATED CULTURES (DIRECT VAT SET,

DIRECT VAT INOCULATION)

CONVENTIONALLY

In controlled fermentation, starter is added @ ca. 106-7 cells/ml.

In conventional bulk starter with ca. 108-9 cells/ml needs to be

added at 1% (v/v) to the milk

For 100,000 gal of milk, > 1000 gal (1% of 100,000 gal) of bulk

starter is needed daily.

Too much volume to handle!!

This large volume is produced from mother culture through

several intermediate subcultures (more handling at processing

facilities) vulnerable to phage infection since phages are more

abundant in processing environment (Fig. 13-1).

SOLUTION

Instead, more handling is done by culture producers under

controlled environmental conditions, minimize phage problems.

Let the pros take care of the job!!

FROZEN CONCENTRATE AND FREEZE-DRIED CONCENTRATE

Skip mother, intermediate, and bulk

Possible to direct inoculation to production process

Easy to use

Good starter activity

Less labor

Bacteriophage problem is limited

Significant savings in labor, material costs

Drawbacks: Require low temperature during shipping and

storage (dry ice in Styrofoam box)

Concentrate the cell by centrifugation up to 1012 cells/ml add

cryoprotectant (DMSO, glycerol) and freeze store in dry ice (-

78°C) or liquid nitrogen (-196°C) distribute in Styrofoam box @

-20°C or below thaw in warm (45°C) de-chlorinated boiled

water before use.

Only 360 ml of frozen concentrate culture in DVS system can be

added to 5,000 gal milk to get desired 106-7 cells/ml.

Or the concentrated cell can be freeze dried plastic bag under

vacuum distribute to factory store < 5°C or in refrigerator

(use it within expiration date, usually for 3 - 12 mo) use.

BULK CULTURE

Dairy industry

Several additional steps:

Phosphate buffer

Minimize acid damage

Protect bacteriophage

Low lactose level

Limit acid production

Reduce acid injury

Food grade ingredients

STARTER CULTURE PROBLEMS

STRAIN ANTAGONISM

In mixed culture (two or more strains)

Dominance strain due to different growth environment or

production of inhibitory metabolites (e.g., bacteriocins, acids,

peroxide).

Use compatible strains.

LOSS OF DESIRED TRAITS

Plasmid-linked traits (lac+, cit+, muc+, bac+, R/M, suc+) are usually

lost during storage, subculturing, and under some nonselective

growth conditions.

Physical (e.g., freezing and thawing) and chemical stress also

result in loss of desirable traits.

Genetic studies (i.e., integration of plasmid mediated genes on

chromosomal DNA) are being conducted to understand the

mechanism.

CELL DEATH AND INJURY

The effectiveness of freeze-dried concentrated or frozen

concentrate starter depends on two important factors:

Culture has to have large number of viable cells

Cells should have a short ‘lag’ phase so that they can multiply

quickly in food.

To minimize cell damage (cryoinjury) to cell:

Addition of cryoprotectant

Rapid freezing at very low temperature

To minimize cell viability loss, avoid:

Repeated freezing and thawing

Thawing long before use

Mixing starter with curing salts, spice for a long time (sausage

fermentation)

Long storage @ -20°C or higher temperature.

INHIBITORS IN RAW MATERIALS

Antibiotics and sanitizers in milk

Phosphate or nitrite in sausage fermenting factories

INDUSTRIAL SCALE PRODUCTION OF

STARTER CULTURE

A. FERMENTATION

GROWTH MEDIUM

Should be cheap but contain enough nutrients for the growth

Should contain some milk solids to ensure the synthesis of

necessary enzymes for starter to perform well in milk.

Cheese whey and whey permeate w/ supplements

Cheap medium (waste product)

Limitation for some nutrients for maximum growth

Need partial hydrolysis by proteolytic enzyme to improve

growing

Precipitate after pasteurization clarification step should be

followed

Not considered adequate for maximum growth

Skim milk supplemented w/ sodium citrate (solubilize milk

proteins helps harvesting of cells)

Same composition of cultured milk products

Good choice medium

Contains milk solids

Maintaining balance among strains in multiple strain starter

Yeast extract

Peptone

Growth factors (if needed)

Tween 80 (polysorbate 80, polyoxyethylene monooleate):

nonionic surfactant and emulsifier

Oleic acids (C19, unsaturated FA, membrane fluidity)

GROWTH CONDITIONS

Optimum growth temperature

Optimum pH

Neutralizer: ammonium hydroxide

Cooling

Harvesting time: end of log phase

Oxygen toxicity (due to constant agitation to maintain constant

pH):

Reducing agents

Catalase

CO2 sparging

PH CONTROL

Cells held at pH 5 or below for even modest period of time will

lose viability and behave sluggish when inoculated into

fermentation tank.

Preventing acid injury during the production and propagation of

starter culture is essential.

Also increase cell density.

Two approaches

External pH control: monitoring pH and addition of

neutralizing agents (NaOH or KOH) maintain pH 5.8 - 6.2

Internal pH control: addition of buffering salts (NaCO3,

Mg3PO4) slowly become soluble in medium maintain pH

> 5

HARVESTING CELLS (CONCENTRATION)

Centrifugal separation or membrane process

Ultrafiltration

Microfiltration w/ ceramic filter

HANDLING AND STORAGE

Cryoprotectant

Lactose

Sucrose

DMSO (inapplicable to food)

Glycerol

Low temperature (-196 C for LN2, deep freezing, etc)

The lower the temperature, the longer the shelf life

Monosodium glutamate (MSG) provide some protection to

cultures during freeze-drying

ENCLOSED STARTER PROCESSOR

This specially designed process vessel provides a high degree of

protection for the production of bulk starter and minimizing

chances for bacterial and phage contamination. It is a completely

sealed unit and includes the following:

An air-tight, insulated cover with stainless steel hinge and cam

latch, an O-ring gasket snaps on the cover.

A removable inoculating port with live steam ring and sealed

cover for protective inoculating technique.

Easily attached, high-efficiency filter for controlled entrance of air

and protection from airborne contamination.

Sealed agitator port with two-piece rotary agitator seal. Complete

seal can be removed quickly and easily without removing agitator

shaft.

FUTURE TRENDS

SEVERAL STRAINS (MIXED STRAINS)

Relationship

Symbiotic and no inhibitory phenomena between strains

(compatible)

GENETICS

Good flavor + acid + bacteriocin producer enhance safety

Uncooked fermented foods (fermented sausages, kimchi,

sauerkraut)

Identification of corresponding genetic determinant of certain

desirable traits

Stable gene transfer methods

Customized starter

Isogenic gene transfer (within the same species via conjugation)

no GMO. GRAS

Full genomic sequence of lactic starters

BACTERIOPHAGES OF LAB

Major commercial problems in dairy fields:

Mostly cheese fermentation (open system)

Phage survive pasteurization of milk

Once contaminated in dairy environment, easily spread by air,

whey residue

Product failures (symptoms):

Unacceptable low production of lactic acid and flavor

Reduced proteolysis

Slow vat

Dead vat

Severe economic losses (cheese industry is more serious,

open fermentation system)

20 temperate and virulent bacteriophages have been found and

their genomes have been completely sequenced.

Caudovirales: tailed phages

Most of them carry 18 - 55 kb genome

Cells carrying prophages are resistant to attack by temperate

phage and thus will be dominant in the population

G + C content of the phage genome is similar to the G + C

content of the bacterial host chromosome reflecting intimate

phage/host relationship

PHAGE LIFE CYCLE

COMMON STEPS

PHAGE ADSORPTION (REVERSIBLE)

Phage specific receptors on host:

Carbohydrate components in cell wall (Rhamnose is

essential)

S-layer protein

Need Ca2+ or Mg2+

PHAGE PENETRATION (IRREVERSIBLE)

Mediated by phage infection protein (PIP) located in cell

membrane

Transmembrane protein required for phage DNA injection

CIRCULARIZATION

HOSTS DEFENSE MECHANISM AGAINST PHAGE

4 NATURALLY OCCURRING MECHANISMS

ADSORPTION INHIBITION

Masking/Shielding

Production of exopolysaccharide (EPS) or capsular

polysaccharide (CPS)

BLOCKING OF PHAGE DNA PENETRATION

Some mutant lack of phage infection protein (PIP) in cell

membrane

Still adsorb phage, but defective in penetration through

membrane

RESTRICTION/MODIFICATION (R/M) SYSTEM

Restriction endonuclease

Methylation

Plasmid linked

However, DNA progeny that has escaped restriction will reinfect

at higher efficiency

Efficiency of R/M depends on number of recognition sites in

phage DNA.

ABORTIVE INFECTION (ABI) SYSTEMS

Last crucial step for host to combat phage attack

Premature host cell death prevent ‘early’ or ‘late’ in lytic cycle

of phage development Absence of plaque or severe reduction

in plaque size (titer) and burst size

Plasmid encoded

Unusually low G+C content of their genes

Supplemented by R/M system incidence of cell death due to

Abi systems will be minimized

Some mutant phages overcome this system

ARTIFICIAL PHAGE RESISTANCE MECHANISMS (MOLECULAR)

ANTISENSE RNA STRATEGIES:

A second sequence of RNA complementary to the first strand

(mirror image of mRNA). The formation of double stranded RNA

can inhibit gene expression in many different organisms including

plants, flies, worms and fungi.

5 ́ C A U G 3 ́ mRNA

3 ́ G U A C 5 ́ Antisense RNA

Cloning of antisense RNA behind promoter will bind target

sense mRNA Prevent translation of phage proteins by either

destabilizing and making more susceptible for degradation by

RNase or by inhibiting loading of ribosome

Figure 1 Formation of antisense RNA blocks translation.

PER

Phage Encode Resistance

Utilization of phage origin of replication (ori, recognition site for

phage DNA replication))

Cloning of phage ori in multi-copy plasmid vector (multiple decoy

copies) transform host cloned ‘per’ competes in trans with

normal phage replication the greater the expression of the

plasmid, the more phage resistance Increase in insensitivity of

host to phage is enhanced

Highly effective but specific for distinct type of phages

CLONING OF PHAGE REPRESSOR GENE FROM TEMPERATE PHAGE

Protection against superinfection of the same temperate phage

But not against virulent phages

CONJUGAL TRANSFER OF PLASMID-LINKED PHAGE RESISTANCE SYSTEMS

Not regarded as GMO

CLONING OF “ABI” AND R/M USING PHYSIOLOGICAL SELECTION MARKER

Suicide trap

We need good selection marker

Bacteriocin (Nisin) resistance marker (nisr) is linked with phage

insensitivity determinant

Constructing plasmid containing Abi and R/M

Promoter of lactococcal phage:lactococcal R/M gene

Phage infection induction of R/M lyse host DNA single

cell death (altruistic death), programmed cell death (only infected,

not the whole population) but phage propagation is blocked

sparing remaining cell from more severe phage infection

PHAGE CONTROL (NON-MOLECULAR)

DEFINED STRAIN

Check the presence of prophage before use by mitomycin C

induction

Professional starter producer

PROPER SANITATION

CIP

chlorine, hypochlorite

Sterilization of whey (major bacteriophage reservoir)

Isolation of culture handling area from rest of processing facility

Maintaining positive pressure for culture handling area/ air

filtration

phages are frequently transmitted via air or airborne droplets

Do not let whey contaminate starter culture

Phage insensitive media (PIM):

Chelating Ca2+ by adding phosphate or citrate

Rotation of starters

Mixed strains

Slow-acid producing strain (prt-): generally less susceptible to

phage attack than fast-acid-producing strains (prt+)

pH-controlled system

Phage resistant starter strains: Grow the starter with lytic phage

select cells (spontaneous phage-resistant mutants) that were

not killed by phage apply to processing (Heap-Lawrence test)

Constant scrutinized phage monitoring

Detection of phage DNA in whey by dot blot technique

PCR

ELISA

Mitomycin C induction of temperate phage before use

Plaque

Validation of phage resistant strains at pilot and commercial scale. In

the trial, the phage resistant strain performed well under

manufacturing conditions and resulting 6-month-old cheeses

received flavor scores equal to, or better than, cheeses made with

the parent strain. This strain was subsequently validated in a

commercial cheese plant where it yielded a product of exceptional

quality. In addition, subsequently, the phage resistant derivative of

the strain DPC4932 (DPC5020) was validated in two commercial

cheese plants.

Phage therapy (숙제)