Intrinsic & Extrinsic Parameters Affecting Microbial Growth

Terrence Madhujith, PhD

Department of Food Science and Technology

University of Peradeniya

Intrinsic and Extrinsic parameters of foods that affect microbial growth

Foods are of plant or animal origin

Especially the plant foods are evolved with natural mechanisms to evade pests and Mos.

Since some plant foods contain living plant, we can make use of such mechanisms for protecting foods from Mos.

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Intrinsic Parameters

Inherent properties of plant and animal tissues

◦ pH

◦ Moisture

◦ Oxidation-reduction potential (Eh)

◦ Nutrient content

◦ Antimicrobial constituents

◦ Biological structures

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pH

The best is the neutral pH for most MOS. – 6.5-7.5

Few grow below 4

Bacteria are more sensitive than molds and yeasts

Pathogenic bacteria are even more sensitive

The boundaries depend on many factors

pH range depends on

◦ The microbial species 4

◦ Nutritional level

◦ Presence of other factors e.g. NaCl, toxicants

E.g. In the presence of NaCl, Alcaligenes fecalis grow over a wide range of pH

◦ Type of acid used

5

pH

Gro

wth

Rate

No additive

NaCl added

Na citrate added

5 7 9

pH tolerant pattern of Alcaligenes fecalis

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

molds

Lactic acid Bacteria

Satphylococcus aureus

Acetobactor spp

Salmonella spp

E coli

C botulinum

B cereus

V parahaemolyticus

Vibrio sp

pH growth ranges of some foodborne microorganisms

Food pH

Beans 4.6-6.5

Beets 4.2-4.4

Broccoli 6.5

Cabbage 5.4-6.0

Carrots 4.9-5.2

Cauliflower 5.6

Corn 7.3

Onions 5.3-5.8

Potatoes 5.3-5.6

Pumpkin 4.8- 5.2

Tomatoes 4.2-4.3

Fruit pH

Apples 2.9-3.3

Bananas 4.5-4.7

Lime 1.8-2.0

Melon 6.3- 6.7

Orange 3.6-4.3

Grapes 3.4 -4.5

Butter 6.2

Milk 6.5

Cheese 5.0 -6.0

Beef 5.00-6.00

Chicken 6.2

Fish 6.8

Crab 7

Tuna/Salmon 5.2-6.00

Shrimp 7.00

Fruits, fruit drinks, vinegar and wines fall below the pH that bacteria normally grow

Better keeping quality of these products are mainly due to the pH effect

Fruits generally undergo yeast and mold attacks

this is due to their ability to grow below pH 3.5

<3.5 pH values cannot be tolerated by most bacteria

Meat and seafood have a pH of approximately 5.6 and this is one of the major reasons why they are readily spoiled

They are usually attacked by both molds and bacteria as their pH optima well coincide with the pH of these foods

Most vegetables have a higher pH value than fruits and therefore they are more susceptible for bacterial attacks

Meat from fatigued animals spoils faster than the meat from well rested animals

This is due to the difference in ultimate pH in meat

Well rested animals have min. of 1% glycogen which produces enough lactic acid to bring pH down

After rigor mortis pH goes down from 7.4 to 5.6 in well rested animals

pH of meat varies between 5.1 and 6.2 depending of the rest given to the animal

Acids may be ◦ Inherent

◦ Produced by microbes ( bioacidity)

Cellular medium of microbes is generally neutral

Exceptions – Methanococcus and Sulfolobus

The cells need neutrality for functions and reproduction

When Mos are placed outside of neutrality their growth is dependent on their ability to bring the environment to neutrality

When the cells are placed in a high acidic environment the cells either have to prevent migration of H+ into the cell or pump out excess into the outside environment

Organic acids in the non-ionic form are more effective in destroying microbes.

E.g. malic, tartaric, citric, oxalic acids

H+ interact with enzymes in cytopolasmic membrane

Thus, when MOs grow on either side of the optimum range, an increase in lag phase will result

Lag phase represents the time required by the MOs to convert the environment suitable for the microbes

time

Pop

ula

tion

Adverse pH makes cells more vulnerable for other parameters

Cl. acetobutyliticum neutralizes acidity by converting butyric acid into butanol

Enterobactor aerogenes produces acetone from pyruvic acid

Effects of pH

Morphology of the cell is affected

Dysfunction of biomoleucles - ATP, DNA, proteins and enzymes

Absorption of cations is affected – e.g. K+ - glucose metabolism

High energy expenditure

Increased lag phase

Moisture content itself does not correlate well with microbial activity

Instead, water activity (aw) is a very good parameter that decides the microbial activity

Water Activity

aw = p/po

p = partial pressure of water of the food

po= partial pressure of pure water at the same temperature

Water activity is related to RH

ERH = 100 X aw

Pure water has aw of 1

22% NaCl solution = 0.86; saturated NaCl solution = 0.75

Microorganism aw

Cl. botulinum type E 0.97

Cl. botulinum type A and B 0.94

Pseudomonas 0.97

E coli 0.96

Acinetobactor 0.96

Bacillus subtilis 0.95

Vibrio parahaemolyticus 0.94

Staphilococus aureus 0.86

Penicilium patulum 0.81

Aspergillus 0.70

Rhizopus stolonifer 0.93

Mucor 0.93

Microorganism aw

Halophilic bacteria 0.75

Xerophilic molds 0.61

Osmophilic yeasts 0.61

Candida 0.92

Xeromyces 0.61

Most spoilage bacteria 0.90

Most spoilage molds 0.80

Most spoilage yeasts 0.88

Water activity of most fresh fruits and vegetables is above 0.99

In general, bacteria require higher aw than molds and yeasts

Most spoilage bacteria don’t grow below 0.91

G(-)ves need higher water activity than G(+)ve bacteria

However, spoilage molds can go up to 0.80

Staphylococcus aureus can grow at 0.86

Cl. botulinum does not grow below 0.94

Lowering aw results in increasing the length of the lag phase

All chemical and biochemical reactions depend on water and therefore microbial functions cease at low aw

With reduction of aw in the envt. The no. of groups of MO capable of growing reduces

Certain MOs can cope with the reduction of aw by generating osmo-regulatory compounds

E.g. many bacteria, molds and yeasts produce polyols

Some Mos need low aw envts. For optimum growth ◦ Halophilic

◦ Osmophilic

◦ Xerophilic

E.g. Halophilic bacteria (Halobacteria) need high salt concentration for their growth

E.g. Halobacteria and Halococci produce KCl as the osmo-regulator – they live in salt water, slat pans, salt lakes etc.

Some produce proline

The limiting value of aw for any MO is 0.60

Below this level, cells can not live

Cell wall of bacteria and fungi provides the necessary support to avoid cell rupture up to a certain extent

At the point where the cell can no longer cope with the water influx the cell enlarges and bursts

Interaction of aw with other

parameters aw interacts with

◦ Temperature

◦ pH

◦ Nutrient content etc.

Effect of temperature on aw is prominent

The ability of MOs to grow is reduced as aw is lowered at any temperature

The range of aw over which MOs can grow

is high at optimum temperature

Nutrients increases the range of aw

10 0 20 30 40 50

0.80

0.85

0.90

0.95

1.0

Temp

aw

Penicillium expansum

Aspergillus flavus

Effect of Low Water Activity

Prolongs lag phase

Reduce growth rate, size and final population

Effects depend on other factors such as temp, pH, Eh etc.

E.g. when temp and pH are made unfavorable the min aw for growth goes up.

Water activity of some foods

Food aw

Fresh F&V, meats, fish milk, beverages

>0.98

Evaporated milk, tomato paste, cheese, bread

0.93-0.98

Sweetened condensed milk 0.85 - 0.93

Dried fruits, flour, cereals, jam jellies, nuts, cakes, IMFs

0.6 – 0.85

Chocolates, candies, honey, biscuits, crackers, milk powder, dried vegetables

<0.60

Oxidation Reduction Potential

Called Redox potential (Eh)

Oxidation and reduction reactions happen as a result of electron transfer

The tendency of a medium to accept or donate electron is known as Redox potential

When Eh is measured against an external inert metal electrode (Pt) under standard conditions – E`0

◦ 25C

◦ 1M concentrations

Expressed in mV

When substrate lose electrons – substrate is said to be oxidized

When substrate gains electrons – substrate is said to be reduced

Substrates that readily donate – good reducing agents

Substrates that readily accept – good oxidizing agents

More oxidized materials – positive potential +mv

More reduced materials – negative potential –mv

When neutral – 0mv

Aerobes require – positive Eh

Anaerobes require – negative Eh

Eh of food is determined by

Redox couples

Poising capacity

Oxygen tension of atmosphere

Access to atmosphere

Microbial activities

Facultative anaerboes can grow in both aerobic or anaerobic envts.

Some aerobic bacteria grow better at slightly reduced conditions – microaerophiles

E.g. Lactobacilli and Streptococus

Most yeasts and molds are aerobes with few facultatively anaerobic (can grow under both) species

Clostridia require -36mv or lower Eh

When aerobes grow ◦ Oxygen depletes

◦ Reducing compounds increase

◦ E.g. –SH groups, H2S

Thus, Eh gradually goes down

Eh can go down up to -300mv

Eh = 2.303 RT [RH-2pH]

F

R = 8.315 J

F = 96,500 C

T = abs. temp.

N = no. of electrons transferred

Thus, Eh is pH dependent and pH is to be mentioned

Eh is measured at pH 7.0 and at 25C

Eh value of some common

foods Food Eh (mv) pH

Raw meat (post-rigor) -200 5.7

Raw minced meat +225 5.9

Canned meat -25 -150 6.5

Barley (ground) +225 7

Spinach +74 6.2

grapes +409 3.9

Lemon +383 2.2

Some dye reduction tests are based on Eh reduction

Strict aerobes that need high Eh predominate on food surfaces exposed to oxygen

◦ E.g. G(-) rods producing slime on meat surfaces

◦ Bacillus subtilis producing rope in bread

◦ Acetobactor on alcoholic beverages and vinegar

Oxygen has its own toxic effects on Mos

E.g. Clostridium acetobutylicum can grow at Eh of +370 mv maintained with ferricyanide can not grow at +110 mv in an aerated culture medium

These are called aero-intolerant anaerobes

They do not have capacity to scavenge oxygen radicals

They lack SOD or catalase enzyme systems

Deep meat tissues

Vacuum packaged products

Canned products

These situations favor strict anaerobes

Strict = obligate anaerobes die in the presence of oxygen

Nutrient Content

Water

Energy – sugars, alcohols, AA, starch, cellulose, fats

Nitrogen source – AA, nucelotides, peptides

Vitamins and minerals – e.g. Vit B

G(+) are more sensitive to nutrient content e.g. Vit B

G(-) and molds can synthesize their requirements

Thus, G(-) and molds can grow under low vit B conditions

Biological Barriers &

Antimicrobials First line of defense is biological

structures

They contain ◦ low water

◦ less available nutrients

◦ Antimicrobials

Secondary line of defense is antimicrobials

Essential oils stored in cells may come out in case of an injury

Separately stored enzyme and a substrate may come together

In mustard, horsehoeradish, cabbage, other brassicas – isothiocyante

In garlic, onion and leks – thiosulfinates (allicin)

Many plants produce phytoalexins in response to microbial invasions

Herbs and plant parts used in foods do contain many antimicrobials

E.g. spices, herbs

Basil, bay leaves, cardamom, celery, dill weed

Cinnamon, cloves, coriander, fennel, paprika, thyme

Turmeric, pepper, rosemary, sage, onion, oregano, ginger, lemon, mace, mustard

Eugenol

Allicin

Thymol

Cinnamic aldehyde

humulones

Eggs – lysozyme - act against G(+)

peptidoglycans

avidin – sequester Biotin

milk lactoferrin

lactoperoxidase

Not a guaranteed way to control Mos in food

Sometimes they introduce Mos.

Most often the concentration of active ingredients is not enough

Resistance

Sometimes these compounds interfere with processing

Oleuropein in green olives prevents fermentation of green olives