Factors Affecting Microbial Growth and Survival in Foods

Learning Outcomes

• Draw and label the growth curve of bacterial batch culture

• Explain the factors that affect microbial survival and growth in foods

• Describe the impacts of the survival and growth of microorganisms in foods

• Apply the principles of microbial growth in food processing, preservation, and fermentation

Survival and Growth of Bacteria

• Classical growth curve comprises four phases: lag, logarithmic growth, stationary, and logarithmic death.

• Exponential growth rule

Phases of Growth

• Lag phase: relatively flat, no apparent growth, viable cells produce necessary enzymes for use of new medium

• Exponential phase: doubling of cells at a steady rate, limited by absence of nutrients or increase in harmful metabolites, generation time (doubling time)

• Stationary phase: no net increase in microbial population, very little cell division, variation in cell morphology, spores may be produced, toxins are produced

• Death phase: rapid decline in population, marked difference between viable and total count

Factors Affecting Growth, Survival, and Death of Microbes in Food

1. Intrinsic factors (substrate limitations)

2. Extrinsic factors

3. Processing factors

4. Implicit parameters

Intrinsic Factors

• Moisture content (water activity)

• pH and acidity

• Nutrient content

• Biological structure

• Redox potential

• Anti-microbial barriers and constituents

pH

• Each microbial species has a definite pH growth range and growth optimum.

• Acidophiles (opt. 0-5.5), neutrophiles (opt. 5.5-8.0), alkalophiles (opt. 5-11.5)

• Effect of pH on microorganisms: energy required to maintain cell's internal pH, cell membrane integrity, affect/denature membrane transport proteins, affect enzyme activity, denaturation of proteins, DNA, and other molecules, ionization of nutrients, microorganisms have mechanisms for maintaining internal pH close to neutral

Oxygen Concentration

• Aerobes: grow in the presence of atmospheric O2

• Anaerobes: grow in absence of O2

• Facultative anaerobes: do not require O2 for growth but grow well in its presence

• Aerotolerant anaerobes: ignore O2 and grow well whether it is present or not

• Strict or obligate anaerobes: do not tolerate O2 at all and die in its presence

• Microaerophiles: require O2 levels below atmospheric levels

Water Activity (aw)

• Aw = Psoln/Pwater = 1/100 x Relative humidity

• Microorganisms vary in their aw requirement, most grow well at values around 0.98 or higher

• Aw is affected by solute concentrations

• Halophiles: capable of living in salty environments

• Osmophiles: organisms able to live in environments high in sugar

• Xerophiles: able to live in very dry environments

Minimum Levels of AW Permitting Growth at Near Optimum Temperatures

• Moulds: Aspergillus chevalieri (0.71), Aspergillus ochraceus (0.78), Aspergillus flavus (0.80), Penicillium verrucosum (0.79), Fusarium moniliforme (0.87)

• Yeasts: Saccharomyces rouxii (0.62), Saccharomyces cerevisiae (0.90)

• Bacteria: Bacillus cereus (0.92), Clostridium botulinum (proteolytic) (0.93), Clostridium botulinum (non-proteolytic) (0.97), Escherichia coli (0.93), Salmonella (0.95), Staphylococcus aureus (0.83)

Range of aW in Foods and Their Microbial Flora

• Aw > 0.98: Fresh meats, fresh fish, fresh fruits, fresh vegetables, canned vegetables in brine

• Aw 0.93 - 0.98: Fermented sausages, processed cheese, bread, evaporated milk, tomato paste

• Microbial flora: C. perfringens, Salmonella, Pseudomonas, lactobacilli, bacilli, and micrococci

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• Range of aW in foods and their microbial flora:

  • 0.85 - 0.93:

    • S. aureus

    • Mycotoxin producing moulds

    • Spoilage yeasts and moulds

    • Dry fermented sausages

    • Raw ham (17% salt, saturated sucrose)

  • 0.6 - 0.85:

    • Xerophilic fungi

    • Halophiles

    • Osmophilic yeasts

    • Dried fruit

    • Flour

    • Cereals

    • Salted fish

    • Nuts

  • < 0.6:

    • No growth but may remain viable

    • Confectionery

    • Honey

    • Noodles

    • Dried egg, milk

• Aw range:

  • Foods

  • Microbial flora

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• NaCl and glucose concentrations and corresponding Aw values at 25°C:

  • 1.00:

    • 0.00% w/w NaCl

    • 0.00% w/w Glucose

  • 0.99:

    • 1.74% w/w NaCl

    • 8.90% w/w Glucose

  • 0.98:

    • 3.43% w/w NaCl

    • 15.74% w/w Glucose

  • 0.96:

    • 6.57% w/w NaCl

    • 28.51% w/w Glucose

  • 0.94:

    • 9.38% w/w NaCl

    • 37.83% w/w Glucose

  • 0.92:

    • 11.90% w/w NaCl

    • 43.72% w/w Glucose

  • 0.90:

    • 14.18% w/w NaCl

    • 48.54% w/w Glucose

  • 0.88:

    • 16.28% w/w NaCl

    • 53.05% w/w Glucose

  • 0.86:

    • 18.18% w/w NaCl

    • 58.45% w/w Glucose

• AW

• % w/w NaCl

• Glucose

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• Effect of water activity on lag time of S. aureus in UHT milk at 12°C:

  • Lag time (h)

  • Water activity (aw)

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• Effect of salt concentration on time to botulinum toxin production:

  • Salt Concentration (%)

  • 0:

    • 10°C

    • 14°C

    • 18°C

    • 24°C

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• Redox reactions:

  • Oxidation is the removal of electrons from an atom or molecule. Usually generates energy

  • Reduction is the gaining of one or more electrons by an atom or molecules

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• Redox potential (Eh):

  • A measurement of the ease by which a substance gains or loses electrons

  • Measured in terms of millivolts

  • Major groups of microorganisms based on Eh for growth are aerobes, anaerobes, facultative aerobes, and microaerophiles

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• Redox potential (Eh):

  • Eh ranges for growth:

    • Aerobes: +500 to +300 mV

    • Facultative anaerobes: +300 to -100 mV

    • Anaerobes: +100 to <-250 mV

  • Relationship of Eh to growth can be significantly affected by the presence of salt and other food constituents

  • Eh of foods vary with several factors, e.g. pH and buffering capacity, microbial growth, packaging, the partial pressure of oxygen in the environment, chemical composition.

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• Redox potential (Eh):

  • The O/R potential of a food is determined by the following:

    • The characteristic O/R potential of the original food

    • The poising capacity; that is the resistance to change in potential of the food

    • The oxygen tension of the atmosphere about the food

    • The access that the atmosphere has to the food.

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• Micro-organisms require nutrients for growth

• Main elements required by the active cells of micro-organisms are carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus

• Elements needed in smaller quantities are Mg, Mn, Ca, Na, K, Cl, Cu, Fe, Zn, Co, Mo

• The inability of an organism to utilize a major constituent of a food material will limit its growth. It will thus be at a competitive disadvantage to those microbes that make use of the food constituents

• Intrinsic factors - Nutrients

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• Intrinsic factors - Nutrients:

  • Proteolytic microbes (e.g. Pseudomonas) grow well in protein-rich food (e.g. Meat, fish, milk)

  • Glycolytic microbes grow well in food with high sugar-/ carbohydrate-content (e.g. Fruits)

  • Nutrients required by one microbe can be provided by others if not present in food (e.g. peptides produced by Streptococcus thermophilus used by Lactobacillus bulgaricus)

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• Naturally occurring and added antimicrobials:

  • Plant-based antimicrobial constituents:

    • Essential oils, e.g. eugenol in cloves, allicin in garlic prevents general bacteria growth

    • Tannins

    • Glycosides

    • Resins

    • Phytoalexins

    • Lectins

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• Naturally occurring and added antimicrobials:

  • Animal-based antimicrobials:

    • Lactoferrin, conglutinin and lactoperoxidase system in cow’s milk

    • Lysozyme in eggs and milk prevent growth of Gram negative bacteria

  • Antimicrobials formed during processing:

    • Smoking

    • Browning

    • Fermentation

    • Chemical preservatives – Cranberry has benzoic acid which prevent growth of fungi

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• Extrinsic factors (Environmental limitations):

  • Relative humidity

  • Temperature

  • Time

  • Gaseous atmosphere

  • Types of packaging/atmospheres

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• Temperature:

  • Microorganisms are poikilothermic – temperature varies with that of the external environment.

  • Microbial growth has a fairly characteristic temperature dependence with 3 distinct cardinal temperatures

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• Temperature:

  • Minimum, optimum and maximum. The optimum is always closer to the maximum.

  • The cardinal temperatures vary greatly between microorganisms

  • Growth temperatures for particular organisms usually span over 30 ˚C

  • Stenothermals have smaller ranges

  • Eurythermals have wider ranges

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• Temperature:

  • How temperature affects growth rate of a bacterial population:

    • C (Minimum)

    • B (Optimum)

    • A (Maximum)

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• Temperature:

  • Five classes of microorganisms based on their temperature requirements:

    • Psychrophiles – Can grow at 0 ˚C; Optimum temp. 15 ˚C or lower; Maximum temp. around 20 ˚C.

    • Psychrotrophs or facultative psychrophiles – can grow at 0 ˚C, but their optima are between 20 and 30 ˚C

    • Mesophiles – Minimum temp. 15-20 ˚C; Optima temp. around 20-45 ˚C. Maxima is about 45 ˚C.

    • Thermophiles – Minimum temp. around 45 ˚C; Optima between 55 and 65 ˚C;

    • Extreme (Hyper) thermophiles –Have optima between 80 and about 110 ˚C.

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• Temperature affects bacteria:

  • Lag phase

  • Growth rate

  • Final cell numbers

  • Enzymatic and chemical composition of cells

  • Membrane structure & Nutritional requirements

  • Limits for other factors influencing growth through the change in

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• CARDINAL TEMPERATURES FOR BACTERIAL GROWTH:

  • Thermophiles

  • Hyperthermophiles

  • Mesophiles

  • Psychrotrophs

  • Psychrophiles

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• Temperature range for growth of pathogenic bacteria:

  • Min.

  • Opt.

  • Max.

  • Pathogenic bacteria

    • Salmonella

    • Campylobacter

    • E. coli

    • S. aureus

    • C. botulinum (proteolytic)

    • C. botulinum (non-proteolytic)

    • B. cereus

    • 1 = Mesophilic

    • 2 = Psychrotrophic

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• Temperature range for growth of toxigenic mould species:

  • Min.

  • Opt.

  • Max.

  • Penicill

Page 43: Growth of S. typhimurium at different temperatures

• Log Numbers of S. typhimurium at different temperatures over time

Page 44: Effect of temperature on time to botulinum toxin production

• Vacuum-packed hot smoked trout

• Salt concentration = 0.5%

• Relationship between temperature and time to botulinum toxin production

Page 45: Relative humidity

• Interrelation between relative humidity and water activity

• Condensation in high relative humidity environments

• Germination and growth of propagules in localized areas

• Influence of growth on water activity and other organisms

• Occurrence in grain silos and storage tanks

Page 46: Gaseous atmosphere

• Inhibitory effect of CO2 on microorganisms

• Variation in sensitivity among different microorganisms

Page 47: Gaseous atmosphere (MAP, carbonated beverages)

• Increase in inhibition effect with decrease in temperature

• Bacteriostatic effect of CO2 on pH

• Similar action to weak organic acid

Page 48: Reduced oxygen packaging 1

• Definition of reduced oxygen packaging

• Examples of packaging procedures

Page 49: Reduced oxygen packaging 2

• Cook-chill process

• Sous-vide process

• Comparison of cook-chill and sous-vide processes

Page 50: Vacuum packaging 1

• Process of vacuum packaging

• Absorption of residual oxygen in the bag

Page 51: Vacuum packaging 2

• Application of vacuum packaging to cooked meats, fish, and salads

• Prevention of aerobic growth due to high CO2 and low O2 tension

• Dominance of lactic acid bacteria over gram-negative aerobes

• Risk of Clostridium botulinum

Page 52: Modified atmosphere packaging (MAP)

• Flushing package with gas mixture containing CO2, O2, and N2

• Changes in gas composition during storage

• Consideration of gas composition for product stability

Page 53: MAP

• Role of oxygen in maintaining red appearance of oxymyoglobin

• Delay of oxidative rancidity by excluding oxygen

• Use of nitrogen to prevent pack collapse

• Inhibitory effect of CO2 on aerobes

Page 54: Mechanism of bacteriostatic effect of carbon dioxide

• Steps in the mechanism of CO2's effect on microbial cells

Page 55: Examples of gas mixtures used in MAP of food

• Gas mixtures used for different food products

Page 56: Controlled atmosphere storage

• Use of impermeable packaging for bulk storage and transport of food

• Examples of controlled atmosphere storage for fruits, vegetables, and lamb carcasses

Page 57: Effect of CO2 on microbial cells

• Impact on cell membrane, solute transport, enzymes, and proteins

Page 58: Implicit factors

• Influence of organism properties, growth rate, substrate affinity, physiological state, history, stress response, competitive microflora, and microbial interactions

Page 59: Specific growth rate

• Importance of microorganism in food microflora determined by specific growth rate

• Dominance of bacteria over moulds at low pH

Page 60: Affinity for growth limiting substrate

• Impact of substrate affinity on growth competition

Page 61: Physiological state of the organism

• Sensitivity of exponential phase cells to heat, low pH, and antimicrobials

• Relationship between growth rate and sensitivity to treatments

Page 62: History of the organism/stress response

• Pre-adaptation to adverse conditions

• Increase in heat resistance through culturing at higher temperatures

• Increase in acid resistance through pre-exposure to moderately low pH

• Reduction of minimum growth temperature through growth at progressively lower temperatures

Page 63: Microbial interactions

• Role of cell-to-cell communication in stress response

• Induction of stress response in nearby cells through molecules secreted by stressed cells

Page 64: Microbial interactions

• Mutualism and its effects on growth stimulation

• Safety implications of mutualism in certain food products

Page 65: Microbial interactions

• Nutrient availability and removal of inhibitory components through microbial interactions

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• Microbial interactions

  • Micro-organisms may be antagonistic towards one another.

  • Some produce inhibitory compounds or sequester essential nutrients such as iron to prevent other microbial populations from growing/surviving

  • Example: lactic cultures in fermented foods

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• Processing factors

  • Communition

  • Slicing

  • Packing

  • Irradiation

  • Pasteurization

  • Mixing

  • Washing

  • Storage

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• Ecosystem of Food

  • PROCESS FACTORS

    • Direct influence on number/type of micro-organisms

    • Indirect influence by change of intrinsic factors

  • Chemical and Physical conditions of food

    • INTRINSIC FACTORS

      • Microflora

      • Implicit parameters

        • Micro-organisms, growth characteristics, interactions

    • EXTRINSIC FACTORS

      • Temperature

      • Relative humidity (r