factors that influence microbial growth in food

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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factors that influence microbial growth in food

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