lactic starter cultures and bacteriophages mainly used in dairy industry (yogurt, fermented milks,...
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LACTIC STARTER CULTURES AND
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)
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)
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)
Prevent product failure
Industrial scale production
Initially back slopping and natural fermentation
Still used in domestic manufacture of fermented milk.
Stock culture mother culture bulk culture (1-2 % of volume
Difficult to produce of consistent quality
Single strains of known
Starter failure due to bacteriophage
Combating against phage
Rotation of strains
Defined media to produce bulk starter to reduce phage attack
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.
Different types of starters
Cheese, yogurt, fermented sausage, some ethnic fermented
Freeze-dried concentrated cultures
Eliminate use of dry ice
Prevent accidental thawing
But some cells do not survive well in freeze-dried state.
Custom designed starter cultures
Understanding genetic background of some important desirable
traits, phage inhibition
TYPES OF STARTER CULTURES
LIQUID STARTER Scale-up: Liquid stock Mother Intermediate Bulk
(increase volume by successive subcultures)
Needs skillful personnel
Easily contaminated by bacteriophages
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)
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).
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
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.
Several additional steps:
Minimize acid damage
Low lactose level
Limit acid production
Reduce acid injury
Food grade ingredients
STARTER CULTURE PROBLEMS
In mixed culture (two or more strains)
Dominance strain due to different growth environment or
production of inhibitory metabolites (e.g., bacteriocins, acids,
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
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
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
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
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
Precipitate after pasteurization clarification step should be
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
Growth factors (if needed)
Tween 80 (polysorbate 80, polyoxyethylene monooleate):
nonionic surfactant and emulsifier
Oleic acids (C19, unsaturated FA, membrane fluidity)
Optimum growth temperature
Neutralizer: ammonium hydroxide
Harvesting time: end of log phase
Oxygen toxicity (due to constant agitation to maint