intrinsic & extrinsic parameters affecting microbial growth · effect of low water activity y...
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Intrinsic & Extrinsic Parameters Affecting Microbial Growth
Terrence Madhujith, PhD
Department of Food Science and Technology
University of Peradeniya
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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
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◦ 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
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pH
Gro
wth
Rate
No additive
NaCl added
Na citrate added
5 7 9
pH tolerant pattern of Alcaligenes fecalis
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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
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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
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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
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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
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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
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<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
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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
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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)
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Cellular medium of microbes is generally neutral
Exceptions – Methanococcus and Sulfolobus
The cells need neutrality for functions and reproduction
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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
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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
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Lag phase represents the time required by the MOs to convert the environment suitable for the microbes
time
Pop
ula
tion
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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
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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
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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
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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
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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
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Vibrio parahaemolyticus 0.94
Staphilococus aureus 0.86
Penicilium patulum 0.81
Aspergillus 0.70
Rhizopus stolonifer 0.93
Mucor 0.93
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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
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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
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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
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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
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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
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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
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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
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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
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10 0 20 30 40 50
0.80
0.85
0.90
0.95
1.0
Temp
aw
Penicillium expansum
Aspergillus flavus
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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.
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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
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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
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◦ 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
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More oxidized materials – positive potential +mv
More reduced materials – negative potential –mv
When neutral – 0mv
Aerobes require – positive Eh
Anaerobes require – negative Eh
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Eh of food is determined by
Redox couples
Poising capacity
Oxygen tension of atmosphere
Access to atmosphere
Microbial activities
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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
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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
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Eh = 2.303 RT [RH-2pH]
F
R = 8.315 J
F = 96,500 C
T = abs. temp.
N = no. of electrons transferred
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Thus, Eh is pH dependent and pH is to be mentioned
Eh is measured at pH 7.0 and at 25C
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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
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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
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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
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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
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Nutrient Content
Water
Energy – sugars, alcohols, AA, starch, cellulose, fats
Nitrogen source – AA, nucelotides, peptides
Vitamins and minerals – e.g. Vit B
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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
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Biological Barriers &
Antimicrobials First line of defense is biological
structures
They contain ◦ low water
◦ less available nutrients
◦ Antimicrobials
Secondary line of defense is antimicrobials
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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)
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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
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Turmeric, pepper, rosemary, sage, onion, oregano, ginger, lemon, mace, mustard
Eugenol
Allicin
Thymol
Cinnamic aldehyde
humulones
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Eggs – lysozyme - act against G(+)
peptidoglycans
avidin – sequester Biotin
milk lactoferrin
lactoperoxidase
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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
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Oleuropein in green olives prevents fermentation of green olives