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  • 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 maint

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