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7/28/2019 Factors That Influence Microbial Growth in Food
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FACTORS THAT INFLUENCE
MICROBIAL GROWTH IN FOOD
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FACTORS THAT INFLUENCE MICROBIAL
GROWTH IN FOOD
Characteristics of the food itself is calledintrinsic factors (substrate limitation)
Factors that are external to the food is calledextrinsic factors (environmental factors)
Implicit factors
Processing factors
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INTRINSIC FACTORS
Nutrient content
pH and buffering capacity
Redox potential Water activity
Antimicrobial constituents
Antimicrobial structures
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Nutrient Content
Foods contains nutrients: as a source of water,source of energy, source of nitrogen, source ofvitamins and related growth factors and source ofminerals.
Nutrients are chemical in nature (organic andinorganic)
Microorganisms need the nutrients in food for theirbiomass, essential nutrients for growth that theorganisms cannot synthesize, and substrate forenergy source.
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Nutrient content
An organisms that is unable to utilize a majorcomponent of a food material will limit itsgrowth in that food than those that can.
Simple substances can be utilized by themicroorganisms.
Microbes have enzymes to breakdown
complex chemicals (nutrients) in foods.
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Nutrient Content
Carbohydrates:
Monosaccharides: (Hexoses and Pentoses)
Disaccharides: Lactose, Sucrose, Maltose
Oligosaccharide:Raffinose: glucose + fructose + galactose)
Stachyose: glucose + fructose + galactose +
galactose
Polysaccharides: starch, glycogen, cellulose,hemicellulose, dextrins, pectins, gums and
mucilage, inulin
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Nutrient Content
Carbohydrates as source of energy through several
metabolic pathways
Metabolic end products to produce amino acids,
CO2, organic acids. Synthesize complex carbohydrates e.g. dextrins
(desirable), slime (undesirable)
Sugars are used in biochemical identifications of
microorganisms isolated from foods.
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Nutrient Content
Proteins:Simple proteins: polymers of amino acids (albumins,
globulins, lutelins, prolamins, and albuminoids.
Conjugated proteins: protein + ------
Metal (hemoglobin and myoglobin)
Carbohydrates (glycoprotein such as mucin)
Phosphate (phosphoproteins such as casein)
Lipids (lipoproteins)
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Nutrient Content
Peptides: amino acids (few)
Nonprotein nitrogenous (NPN) compounds:
(amino acids, urea, ammonia, creatinine,
trimethylamine)
Degree of solubility determines the ability of
microorganisms to utilize the protein. E.g. albumin
soluble in water; collagens insoluble in water.
Specific microbes.
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Nutrient Content
Lipids in foods:
Free fatty acids, glycerides, phospholipids,
waxes and sterols.
Plant sources vs animal sources.Lipids are in general less preferred as source of
energy and cellular materials.
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Nutrient Content
Many microbes produce extracellular lipases that
hydrolyzed fats.
Some produce extracellular lipid oxidase to produce
different aldehydes and ketones from unsaturatedFAs.
Some produce endocellular lipases and oxidases.
Food spoilage (rancidity), flavour development,
metabolize cholesterol (probiotic).
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Nutrient Content
Minerals and vitamins:
Minerals in small amount and present in foods.
Most microorganisms can synthesize B vitamins.
Some fastidious Lactobacillus species require some
essential amino acids, B vitamins that may be added
to foods.
It is not possible or practical to control microbialgrowth in food by restricting nutrients.
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Nutrient content
Amylolytic enzymes to breakdown starches.
Proteolytic enzymes to breakdown proteins.
Lipolytic enzymes to breakdown lipids andfats.
Pectinase?, Gelatinase? , Cellulases?
Specific microorganisms.
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How nutrients affect growth?
Addition of certain nutrients to certain will favorcertain microbes.E.g. adding fruits containing sucrose in yoghurtincrease the range of carbohydrates available and
allows the development of more diverse microflorae.g. yeasts.
The amount or concentration of nutrient available,
to some extent the determine the rate of microbialgrowth; more easily utilizable nutrients, the fasterthe growth rate.
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How nutrients affect growth?
Presence of antimicrobial factors, orprocessing methods that may cause deficientin essential nutrients, may limit microbial
growth.
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Nutrient metabolism
Starch hydrolysis, sugar fermentation, production ofhydrogen sulphide and indole, or nitrate reductioncan be used to identify microorganisms.
Also measurements of microbial metabolism or end
production of end products can be used to estimatethe bacterial population.
E.g. use of dyes (methylene blue, resazurin,tetrazolium). The rate at which the indicator changecolour, is related to the metabolic rate of theorganisms in the sample. The greater the number ofbacteria, the faster is the colour change. Useful indairy industry.
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pH and Buffering Capacity
pH = -log (aH) = log 1 / (aH)~ log 1 / [H+]
Where aH is hydrogen ion activity
[H+] is hydrogen ion concentration
pH values below 7 is acidic, above 7 is alkaline
environment.
Difference in pH of 1, 2 and 3 units correspond to10- , 100- , and 1000- fold differences in hydrogen
ion concentration.
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Importance of pH of an
environment
Effect the activity and stability of enzymes. Plotting microbial growth rate against pH produces a
approximately symmetrical bell-shaped curve. In general:
bacteria grow fastest in the pH range 6.0
8.0 Yeasts 4.5 to 6.0 Filamentous fungi 3.5 to 4.0 Exceptions: lactobacilli and acetic acid bacteria, can
grow at low pH, optimum usually pH 5.0 to 6.0. Food with alkaline pH: egg white, gamat extract.
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pH and microbial ecology
plant products e.g. vegetables havemoderately acid pH, spoilage organisms aresoft-rot producing bacteria such as Erwinia
carotovoraand pseudomonads Fruits lower pH prevents bacterial growth and
spoilage dominated by yeast and moulds.
Fresh milk? Meat? pH growth range of microorganisms on food
(Jay pg 36)
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Keeping quality of meat
Meat from rested keeps longer than meat fatiguedanimals
Direct result of final pH of meat attained uponcompletion of rigor mortis.
Upon death of well-rested animals, 1% of glycogenin meat is converted to lactic acid, pH reduces fromabout 7.4 to 5.6 (depending on type of animals)
Fish: pH attained upon complete rigor mortis is
about 6.2 to 6.5. Meat have better keeping qualities than fish,
spoilage mainly by bacteria.
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Inherent vs added acidity
Inherent acidity by activity of microorganisms e.g.fermented foods, means of preservation, presenceof weak organic acids.
Partial dissociation of weak acids plays an importantrole in their ability to inhibit microbial growth.
E.g. of common food acids: acetic, propionic, lactic,sorbic, citric, benzoic, phosphoric, carbonic, nitrous
and sulfurous.
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Microbial inhibition by weakorganic acid
weak organic acids do not dissociate completelyinto protons and conjugate base in solution butestablish an equilibrium.
HA H+ + A-
equilibrium constant Ka= [H+] [A-] / [HA]
Rearrange: 1/ [H+] = 1/Ka. [A-] / [HA]
Log base 10: pH = p Ka + log [A-] / [HA]
Henderson-Hasselbach equations
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1. Organic acids enter the cell only in the
protonated form (undissociated) form. Onceinside the cell, they dissociate, releasing H+(proton) into the cell cytoplasm. The cytoplasmbecomes acidic, accumulation of anions that
cannot passed back across the cell membrane.
2. Change in pH results in a change in trans-membrane proton gradient (difference in pH
between inside and outside of cell), can serve assensor to stop or start energy-dependentreactions.
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1. The changes in acidity inside the cellmay result in protonation ordeprotonation of amino acids inproteins. May alter secondary andtertiary structures of proteins,changing functions and signalingchange of pH to the cell.
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pH
When the pH is equal to acids pKa, then half
of the acid present will be undissociated.
If pH is increased then dissociation of the
acid will be increased.
Weak acid is used to inhibit growth of
microorganisms.
High extracellular H+ and concentration of
undissociated acid.
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Three mechanisms bacteriamaintain the internal pH (pHi)
E.g. Salmnella entericaserovar Typhimurium.
3 mechanisms:
1. homestatic response
2. acid tolerance response (ATR)
3. synthesis of acid shock proteins
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Homeostatic response
At external pH is >6.0, salmonella cell adjustthe internal pH; the homeostatic responsemaintains the pH by increasing the activity of
proton pumps to expel proton fromcytoplasm. The mechanism is always on andfunctions in the presence of protein synthesis
inhibitors.
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ATR
ATR is triggered by a pH outside of 5.5 to 6.0. Thismechanism is sensitive to protein synthesisinhibitors.
ATR appears to involve the membrane-boundATPase proton pump and maintains internal pH at>5.0, even external pH values as low as 4.0.
Loss of ATPase activity abolish by gene mutation or
or metabolic inhibitors abolishes the ATR but not pHhomeostatic mechanism.
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Synthesis of acid shock proteins
The synthesis of these proteins is triggeredby outside pH of 3.0 to 5.0.
A set regulatory proteins.
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Buffering capacity
those that tend to resist changes in pH aresaid to be buffered.
In general meats are more highly bufferedthan vegetables. Proteins in meat contributeto the buffering capacity.
Vegetables are gen. low in protein, lack
buffering capacity to resist changes in pHduring growth of microorganisms.
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Approximate pH values of some foodsFOOD pH
Milk
Cheddar cheese
Ground beef
Chicken
FishShrimps
Cabbage
Spinach
Tomatoes
Apples
Bananas
Honeydew
Limes
6.3 to 6.5
4.9; 5.9
5.1 to 6.2
6.2 to 6.4
6.6 to 6.86.8 to 7.0
5.4 to 6.0
5.5 to 6.0
4.2 to 4.3
2.9 to 3.3
4.5 to 4.7
6.3 to 6.7
1.8 to 2.0
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Minimum pH values for the growth of somefoodborne bacteria
Microorganisms pH
Clostridium botulinumGroup I
C. botulinumGroup II
C. Perfringens
Escherichia coliO157:H7Lactobacillus brevis
Listeria monocytogenes
Pseudomonas fragi
SalmonellasppStaphylococcus aureus
Vibrio parahaemolyticus
Zygosaccharomyces bailii
4.6
5.0
5.0
4.53.16
4.1
ca 5.0
4.054.0
4.8
1.8
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refer to Jay Table 3-1, 3-2 and 3-3 pp 38 and39.
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Redox Potential Eh Oxidation-reduction (redox) reaction occurs as a
result of transfer of electrons between atoms ormolecules.
[Oxidant] + H+
+ ne [Reductant]
Where nis number of electrons e transferred
e.g. electron transport chain, and generation ofrelease of energy by oxidative phosphorylation
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Redox
Is the tendency of a medium to accept or donate electronsi.e to oxidize or to reduce.
When electrons are transferred from one compound toanother, a potential difference is created between the two
compounds. This difference can be measured byinstrument and expressed as millivolts (mV).
Positive potential indicates oxidizing environment;negative potential indicates reduced environment.
Zero potential when concentration of oxidant andreductant is equal.
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Factors influencing themeasured Eh of foods
Redox couple present
Ration of oxidant to reductant
pH Poising capacity i.e. the resistance to change
in potential of the food
Availability of oxygen (physical state,packaging)
Microbial activity
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Redox potential of some food materials
Food E(mV) pH
Raw meat (post-rigor)
Raw minced meat
Cooked sausages and canned
meatsWheat (whole grain)
Spinach
PearGrape
Lemon
-200
+200
-20 to -150
-320 to -360
+74
+436+409
+383
5.7
5.9
ca 6.5
6.0
6.2
4.23.9
2.2
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Hydrogen ion concentration affect the Eh
For every unit decrease in pH the Ehincreases by 58 mV.
E.g. fruit juice high positive Eh reflection oftheir low pH.
Couples in foods:
Gluthione and cystein in meats, ascorbic acidand reducing sugars tend to establish
reducing conditions.
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Oxygen is a powerful oxidizing agent; if sufficient airis present in a food, a high positive potential willresult, the redox couples present are in oxidized
state. E.g. chopping, grinding, or mincing increase access
of air to food, increase Eh Modified vacuum packaging or canning will reduce
Eh . Microbial growth in food reduces Eh. (oxygen
depletion, production of hydrogen bymicroorganisms). E.g. testing quality of dairyproducts use methylene blue or resazurin (redoxdyes).
Yeast appear colourless (reuced, viable), or blue(non-viable) when stained with methylene blue.
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Effect of redox on microrganisms
Physiological groups:
Obligate or strict aerobes: oxidativephosphorylation, oxygen as final electron acceptor.
Pseudomonas fluorescensand other gram negativebacteria rods, slime and off-odours on meatsurfaces Eh of +100 to +500 mV.
Bacillus subtilisproduces rope in bread
Acetobactergrows on the surface of alcoholicbeverages, oxidize ethanol to acetic acid.
Eh
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Obligates anaerobes: oxygen must beabsent, grow at low or negative redox.
Clostridium botulinum causing botulinumgrow in anaerobic conditions such as deep inmeat tissues and stews, in vacuum packs and
canned foods.Anerobically vs aerobically grown
Saccharomyces cerevisiae showeddifferences in lipid and sterol contents.
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Aerotolerant anaerobes are incapable ofaerobic respiration but can grow in the
presence of air.
E.g. lactic acid bacteria, lack catalase andsuperoxide dismutase, but able to in the
presence of oxygen because they have themechanism to destroy superoxide.
Superoxide dismutase: convert free radicals
+ hydrogen to water and oxygen catalase break hydrogen peroxide to water
and oxygen
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Antimicrobial Barriers andConstituent
Physical barriers: skin, shell, husk, rinds ofproduct. (dry, antimicrobial compounds onsurfaces).
Physical damage to these barriers allowsmicrobial invasion of nutrient rich tissues.
Certain plant antimicrobials produced as a
result of physical damage eg isothiocyanate,allicin
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Natural antimicrobials
Mustard oils
Alicin
Euganol
ThymolCinnamic
aldehyde
Benzoic acid
Sorbic acid
Oleuropein
Mustard, cabbage, brassicas
Garlic, onions
Allspice, cloves, cinnamon
Thyme, oreganoCinnamon
Cranberries
Moutain ash berries
Green olives
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Antimicrobials in hens egg a
Albumin Milk
Nutrient Status:
High pH
Low levels of available nitrogen
Antimicrobials:
Ovotransferrin (conalbumin)
Lysozyme
Avidin
Ovoflavoprotein
Ovomucoid &ovoinhibitors
Moderate pH
High levels of protein,carbohydrate and fat
Lactoferrin
Lysozyme
--
-
Lactoperoxidase
Immunoglobulin
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Lysozymes most effective against Gram positivebacteria, also Gram negative bacteria if their outer
membrane is damaged. Ovotransferrin and lactoferrin scavange iron. Avidin and ovoflavoprotein sequester biotin and
riboflavin Milk generate antimicrobial in the presence of
hydrogen peroxide. Lactoperoxidase in milk catalysethe oxidation of thiocyanate by hydrogen peroxideto produce hypothiocyanate, kill Gram-negativebacteria and inhibit Gram-positives. Theantibacterial effect increase with acidity, target thecytoplasmic membrane.
Lactoperoxidase system can be used to preserveraw milk where refrigeration is uncommon.
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Water activity
Water requirements of microorganisms is bestdescribed in terms of water activity aw in theenvironment.
Defined as ratio of the partial pressure of water inthe atmosphere in equilibrium with the substrate, P,compared with partial pressure of the atmosphere inequilibrium with pure water at the sametemperature, P
0 This is numerically to the equilibrium relative
humidity (ERH) expressed as:
Aw = P/P0 =1/100 ERH
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Pure water has aw of 1.00,
22% NaCl (w/v) has aw of 0.86 Saturated NaCl has aw of 0.75
Depends on the number of molecules or ions
present in solution rather than their size. Salt better than sucrose at reducing water
activity on mole-to-mole basis.
Mi i t ti iti t
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Minimum water activities atwhich active growth can occur
Groups of micro-organisms Minimum aw
Most Gram-negative bacteria
Most Gram positive bacteriaMost yeasts
Most filamentous fungi
Halophilic bacteriaXerophilic fungi
Osmophilic yeast
0.97
0.900.88
0.80
0.750.61
0.61
Approximate minimum a for growth of specific
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Approximate minimum aw for growth of specificorganisms
Names aw
Clostridium botulinumtype E
Escherichia coli
Bacillus subtilis
Clostridium botulinumtypes A and B
Vibrio parahaemolyticus
Staphylococcus aureus
Penicillium patulum
Aspergillus flavus
Aspergillus ochraceus
Aspergillus glaucus
Zygosaccharomyces roixii
Xeromyces bisporus
0.97
0.96
0.95
0.94
0.94
0.86
0.81
0.81
0.78
0.70
0.62
0.61
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Water activity
The limiting value of water activity for thegrowth of any microorganism is about 0.6and below this value spoilage of food is not
microbiological but may be due to insectdamage or chemical reactions such asoxidation.
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Effect of low water activity
General effect:
Lowering aw below the optimum is to increase thelength of lag phase of growth. Influenced of loweredwater on all metabolic activities (also influenced byother environmental factors: pH, temperature ofgrowth, and Eh.
As water activity is lowered, osmotic pressure in the
environment is increased, cytoplasm increaseosmotic pressure by increasing concentration ofcompatatible solutes that do not interfere withcytoplasmic function.
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E.g. polyols glycerol, arabitol, and mannitol infungi; amino acids and amino derivatives
accumulate in bacteria. Salt tolerant Staphyloccus aureusaccumulate
proline as a response to low water activity.
Listeria monocytogenes accumulatescarnitine, glycine betaine (osmotically andchilled stress)
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Germination ofBacillusand Clostridiumspores
were strongly inhibited when water activity wascontrolled by NaCL or CaCL but less inhibitionwhen glucose or sorbitol was used, very littleinhibition when glycerol, ethylene glycol,acetamide or urea was used.
Osmophilic yeasts accumulate glycerol understress. Reduce water activity results in cessation of
enterotoxin B production by S. aureuseventhough high numbers of cells are produced at
the same time.
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Lowered water activity affect nutrientavailability.
Affect the function of cell membrane whichmust be kept at in a fluid state. Drying ofinternal part of cell.
Ability to concentrate compatible solutes.
Methods to reduce water
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Methods to reduce water
activity
Adding solute
Adding ions
Adding hydrophilic colloids
Freezing and drying
Relationship between water
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Relationship between water
content and water activity
The relationship is affected by temperature,
and
May seem to depend whether the water is
added or remove from the substrate.
Adding water to the dry substrate will result in
higher water activity (adsorption).
Removing water from the substrate will resultin lower water activity (desorption)
Refer to
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Water activity of some foodsCereals, crackers, sugar, salt, dry milk
Noodles, honey, chocolate, dry eggs
Jam, jelly, dried fruits, parmesan cheese, nuts
Fermented sausage, dry cured meat, sweetened
condensed milk, maple syrup
Evaporated milk, tomato paste, bread, fruit juices,
salted fish, sausage, processed cheese
Fresh meat, fish, fruits, vegetables, milk, eggs
0.1 - 0.20
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Understanding water activity
Staphylococcus aureus min water activity is0.85, halophilic bacteria is 0.75.
Higher water activity is needed for bacteria to
sporulate and germinate and produce toxinsthan for the minimum water activity forgrowth.
Under ideal condition the minimum water
activity for growth is lower than in nonidealcondition e.g. ideal condition for bacteria pH6.8 min aw is 0.91; at pH 5.5 it can be 0.95 ormore.
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If aw is reduced below the minimum level for
growth of a microorganism, the cells remain
viable for a while;
If aw is reduced drastically, microbial cells
lose viability generally rapidly at first, then
more slowly.
Very important in food processing, controlling
spoilage and pathogen as well as isolating
microbes e.g. adding salt in cured meat, saltin media to isolate S. auereus
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