Intrinsic and extrinsic factors affecting microbial growth in foods

Intrinsic and extrinsic characteristics of foods affecting microbial growth
ALONZO A. GABRIEL, PHD

Department of Food Science and Nutrition College of Home Economics University of the Philippines at Diliman
From Farm to Fork
Ackerman (2002)
Factors influencing the re-emergence of infectious diseases
Adaptation: From Farm to Fork
Cohen (2000)
Gabriel (2012)
RNA Polymerases: Prokaryote
RNA Polymerases: Prokaryote
Electrophoresis Gel Separation by Urea

Weaver, 2001 Weaver, 2001
Transcription: the ! factor

In prokaryotes, the sigma (!) factor is the subunit of the RNA polymerase that chooses which SPECIFIC gene to transcribe.
Weaver, 2001

!70(RpoD) (!A), "housekeeping" sigma. Relative to the DNA base corresponding to the start of the RNA transcript, the consensus promoter sequences are characteristically centered at 10 and 35 nucleotides before the start of transcription (10 and 35).
Weaver, 2001

!19 (FecI), ferric citrate sigma factor, regulates the fec gene for iron transport !24 (RpoE), extracytoplasmic/extreme heat stress sigma factor !28 (RpoF), flagellar sigma factor
!32 (RpoH), heat shock sigma factor. Some of the enzymes that are expressed upon activation of !32 are chaperone, proteases and DNArepair enzymes.
Weaver, 2001
Weaver, 2001
Protein folding energy funnel
!38 (RpoS), starvation/stationary phase sigma factor !54 (RpoN), nitrogen-limitation sigma factor
Weaver, 2001
Horwich, 2002
Protein folding energy funnel
Protein folding: Chaperones
kinetic trap! native state

http://splette.deviantart.com/art/Protein-Folding-Funnel-150094122
!! !!
Prevent improper interactions. Untangle aggregated proteins.
Alberts et al, 2010
Protein folding: Chaperones
Protein digestion: Proteasomes
!!
Aided by the Hsp 40.

Alberts et al, 2010
Alberts et al, 2010
Factors influencing the re-emergence of infectious diseases
Microorganisms and the Food Supply

!! Plants
and animal food sources have all developed mechanisms of defense against the invasion and proliferation of microorganisms.
Cohen (2000)
Intrinsic parameters
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pH
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The characteristics inherent to a food system are referred to as intrinsic parameters.
! pH ! Moisture content ! RedOx potential ! Nutrient content ! Antimicrobial constituents ! Biological structures
Most microorganisms grow best at pH values around 7.0 (6.6-7.5), whereas few grow below 4.0. Generally, bacteria are more fastidious in their relationships to pH than yeasts and molds
!!
Frazier and Westhoff (1988); Jay (2000)
Approximate pH growth range of some foodborne microorganisms

1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
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Molds Yeasts Salmonella spp. Acetobacter spp. Vibrio parahaemolyticus Vibrio cholera
Microbial growth however, is not solely dependent on pH alone. In the presence of 0.2 M NaCl, Alcaligenes faecalis has been shown to grow over a wider pH than in the absence of NaCl.
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pH
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pH and keeping quality of foods

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The pH minima of certain lactobacilli have been shown to be dependent on the type of acid used in adjusting growth medium pH. Citric, hydrochloric, phosphoric and tartaric acids were found to favor growth at lower pH than acetic or lactic acid.
Fruits generally undergo mold and yeast spoilage because these organisms grow at pH <3.5. Meats and seafoods have final pH of about 5.6 and above, making them susceptible to both bacterial and fungal spoilage.
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pH of some foods
High Acid 1.0
2.0 3.0
Organic acids commonly used in meat and poultry products

14.0
8.5 9.0
4.5
5.0 6.4
7.0
ACIDIC
beef veal pork chicken milk
BASIC
egg white soda crackers
limes pickles vinegar mayonnaise
Clostridium botulinum
Meat pH and keeping quality

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Biological acidity
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Meat from fatigued animals spoils faster than from those of rested ones. Such difference could be attributed to the presence of glycogen in the meat which could be degraded into lactic acid upon the completion of rigor mortis. Meats are also able to resist changes in pH than other products like fruits and vegetables.
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Some food products are preserved through the production of acidic compounds due to the actions of certain microorganisms. Fermented milks, sauerkraut, pickles, etc.
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Action of pH against foodborne microorganisms

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Cell Membrane Structure
Adverse pH changes could affect at least 2 aspects of a respiring cell:
!The functioning of cellular enzymes !Transport of nutrients into the cell !Protein Denaturation!!!
Frazier and Westhoff (1988); Jay (2000) Krulwich et al. (2011)
Microbial cell internal pH

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Reactions of microorganisms to changes in environmental pH

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The cytoplasmic membrane is relatively impermeable to H+ and OH. The internal pH of most microorganisms appears to be near neutrality despite the changes in environmental pH. The intracellular pH of S. cerevisiae was found to be 5.8.
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When placed in an acid environment, the cells must keep the H+ from entering or expel H+ as rapidly as they enter. Cellular metabolic activities also adjust the pH of the environment
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Reactions of microorganisms to acidic environments

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Cl. acetobutylicum, when placed in acidic medium, reduces butyric acid to butanol. Enterobacter aerogenes produces acetoin from pyruvic acid In acidic medium (pH 4.0), amino acid decarboxylases are activated.
Krulwich et al. (2011)
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!!
Glutamic Acid: Predict the product after amino acid decarboxylase
Glutamic Acid Decarboxylation

$" #" !"
Glutamic Acid Decarboxylation

$" #" !"
Reactions of microorganisms to alkaline environments

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A group of amino acid deaminases are activated at pH 8.0 and cause spontaneous adjustment of pH as a result of organic acid accumulation.
Various Amino Acids
Substrate pH and microbial nutrient transport

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Bacterial cell membranes tend to have a residual negative charge. At neutral or alkaline pH, organic acids cannot enter the cells, whereas at acid pH, these compounds are non-ionized and can enter the negatively charged cells .
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Le Chateliers Principle and Antimicrobial Action of Organic Acids
pKa, pH and acid dissociation

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If a change is made to the conditions of a chemical equilibrium, then the position of equilibrium will readjust so as to minimize the change made.
The pKa denotes the pH value when the dissociated and undissociated species of the acid are in equal concentrations.
AH
!! !!
A-
H+
pH < pKa: Backward reaction favored pH > pKa: Forward reaction favored
Beales (2004)
pKa and pH: Sample problem

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pKa and pH: Sample problem

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A food technologist would like to use benzoic acid (pKa=4.2) as an additive to prolong the shelf life of coconut water beverage (pH=5.5, dominant acid, malic acid). Benzoic acid is generally regarded as safe (GRAS) food additive.
What step should the FT do before he can effectively use benzoic acid as additive? Illustrate the antimicrobial mechanism of benzoic acid against a spoilage yeast cell with an internal pH=6.8.
!!
Lm
David Goodsell, The Machinery of Life
Pear
+ 0.6 MA

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Other environmental factors interacting with pH

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The denaturation of membrane and transport enzymes on either side of neutrality also affects the nutrient transport of the microbial cells.
The pH of a substance decreases as the temperature is raised. Adverse pH values sensitize the cells to toxic agents. Younger cells are more susceptible to pH changes than older or resting cells.
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Moisture content and aw

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Water activity (aw)

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The preservation of foods by drying is a direct consequence of removal or binding of moisture, without which microorganisms do not grow. It is now generally accepted that water requirements of microorganisms for water should be described in terms of water activity (aw).
aw is defined by the ratio of water vapor pressure of the food substrate to the vapor pressure of pure water at the same temperature. p aw = po RH = aw x 100
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Relationship between aw and concentration of salt solutions

aw
0.995 0.99 0.98 0.96 0.94 0.92 0.90 0.88 0.86
Approximate minimum aw values for growth of microorganisms

Organisms Groups Most spoilage bacteria Most spoilage yeast Most spoilage molds Specific Organisms Cl. botulinum (E) Escherichia coli Cl. Botulinum (A and B) Vibrio parahaemolyticus Candida utilis Rhizopus stolonifer
aw
Organisms Groups
aw
NaCl Concentrations Molal 0.15 0.30 0.61 1.20 1.77 2.31 2.83 3.33 3.81 % w/v 0.9 1.7 3.5 7.0 10.0 13.0 16.0 19.0 22.0
0.90 0.88 0.80
Halophilic bacteria Xerophilic molds Osmophilic yeasts Specific Organisms
0.75 0.60 0.61
0.97 0.96 0.94 0.94 0.94 0.93
Candida scottii Staphylococcus aureus Penicillium patulum Aspergillus conicus Zygosaccharomyces Xeromyces bisporus
0.92 0.86 0.81 0.70 0.62 0.61
aw of some foods
candy flour
soft cheeses
Relationships among aw, temperature and nutrition

!!
0.1
0.67
0.85
1.0
0.95
At any temperature, microbial growth rates decrease when aw is reduced. The range of aw over which growth occurs is greatest at the optimum temperature for growth. The presence of nutrients increases the range of aw over which microorganisms can survive.
PHF
0.50 0.75 0.92 0.98
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crackers crisp bacon Staphylococcus aureus

raw bacon meats poultry
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Action of lowered aw against foodborne microorganisms

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Reactions of microorganisms to reduction in aw

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The general effect of lowering aw below optimum are:
! increase in the length of the lag phase ! decrease in the growth rate ! decrease in the size of the final population
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Microorganisms tend to accumulate (by transport or synthesis) solutes that serve as osmoprotectants to counterbalance the increase in osmotic pressure outside cell.
! K+, glutamate, glutamine, proline, !aminobutyrate, alanine, glycine betaine, sucrose, trehalose and glucosylglyserol
Essential biochemical reactions occur in an aqueous milieu.

!!

!!
The ability of microorganisms to concentrate molecules within the cells prevents loss of water and allows the cells to extract moisture from the external environments.
Growth of some cells in high numbers can still take place at reduced aw to the expense of not producing some extracellular products or essential molecule loss.
! Cessation of enterotoxin B production by S.

aureus.
! Nonlethal alteration of the permeability of

Neurospora crassa cell membrane.
Frazier and Westhoff (1988); Jay (2000) Frazier and Westhoff (1988); Jay (2000)
Nutrient content
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Macromolecules
Macromolecules Polysaccharides Lipids Subunits Sugars Fatty Acids and Glycerol Amino Acids Examples Storage: starch, glycogen Structural: cellulose, chitin Storage: neutral fats Structural: phospholipids Functional: estrogen, testosterone Storage: storage proteins Structural: Keratin, Collagen Functional: enzymes, insulin Information storage: DNA Information processing: mRNA, tRNA, rRNA
In order to grow and function normally, microorganisms need the following:
! Water ! Source of energy ! Source of nitrogen ! Vitamins and related growth factors ! Minerals
Proteins
Nucleic Acids
Nucleic Acids
http://plantphys.info/principles/macromolecules.html
Microorganisms need water

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Microorganisms need energy sources

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Essential biochemical reactions occur in an aqueous milieu. Need for water: bacteria > yeasts > molds
Microorganisms may use sugars, alcohols, amino acids. Some microorganisms can degrade complex carbohydrates. A relatively small number can utilize fat as energy source.
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Microorganisms need nitrogen sources

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Microorganisms need nitrogen sources

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The primary N source utilized by heterotrophic microorganisms are amino acids. Other sources include peptides and complex proteins, nucleotides. In general microorganisms will assimilate the more simple substrates first than the complex ones.
Generally, food systems high in proteins are easily spoiled by microorganisms, particularly by bacteria.
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Microorganisms need vitamins and minerals

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Antimicrobial constituents of foods

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Particularly, microorganisms require Bvitamins, although microorganisms are able to synthesize their own needs. Gram negative bacteria and molds can synthesize needed vitamin and thus can thrive in foods low in such nutrient like fruits.
Natural antimicrobials can be found in food systems. Specific examples include eugenol in cloves, allicin in garlic, milk casein, lysozyme, conalbumin in eggs, and phenolic substances.
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The lactoperoxidase system

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Biological structures
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An inhibitory system that naturally occurs in bovine milk. The system is composed of the enzyme lactoperoxidase, thiocyanate (0.25 mM) and H2O2 (100 U/ml). The lactoperoxidase system can be used in preserving raw milk in the absence of refrigeration units.
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The natural covering of some foods provides excellent protection against the entry and subsequent damage by spoilage organisms.
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Extrinsic parameters
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Temperature of storage
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The extrinsic characteristics are those properties of the storage environment that affect both the foods and microorganisms.
The lowest temperature which a microorganism has been reported to grow is -34C. The highest temperature which a microorganism has been reported to grow is somewhere in excess of 100C.
!Storage temperature !Relative humidity of the environment !Presence and concentration of gases !Presence and activities of other
microorganisms
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Classes of organisms based on optimal growth temperature

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Classes of organisms based on optimal growth temperature
Psychrotrophs ! Optimal growth at 20-30C, can grow well at or below 7C. Mesophiles ! Optimal growth between 30-40C but can grow well between 20-45C Thermophiles ! Optimum growth between 55-65C and grow well at and above 45C
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Temperature Danger Zone
Psychrotrophs
Bacterial genera:
!Alcaligenes !Shewanella !Brocothrix !Corynebacterium !Flavobacterium

NRA (2004) Frazier and Westhoff (1988); Jay (2000)
!Lactobacillus !Micrococcus !Pseudomonas !Psychrobactrer !Enterococcus
Mesophiles and Thermophiles

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Foodborne fungi and storage temperature

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Mesophilic species and strains are known among all genera of foodborne bacteria. Most thermophilic bacteria of importance in foods belong to the genera Bacillus and Clostridium, although only a few species are thermophilic.
Just as molds are able to grow over wider ranges of pH, osmotic pressure, and nutrient content, they also grow over wider range of temperature as bacteria. Molds that grow on refrigerated temperature include Aspergillus, Cladosporidium and Thamnidium. Yeasts grow only in psychrotrophic and mesophilic temperature ranges.
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Storage Temperature and Food Quality

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Relative humidity of the environment (RH)

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Although it would seem desirable to store all foods at refrigerator temperatures or below, this is not always not best for maintenance of desirable quality of foods.
Generally, the RH of the food system and the RH of the storage environment should be compatible. A food system with aw= 0.60 will absorb moisture and encourage spoilage when stored at environments with RH higher then 60%.
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Presence and concentration of gases in the environment

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Presence and activities of other microorganisms

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CO2 is the single, most important atmospheric gas used to control microorganisms in foods. O3 is also becoming popular due to its antimicrobial properties. This substance is however, strongly oxidizing. 0.15-5.00 ppm in air is inhibitory to growth of bacteria and yeasts.
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Some foodborne organisms produce substances that are inhibitory or lethal to others: !Antibiotics !Bacteriocins (Nisin) !Hydrogen peroxide !Organic acids
General microbial interference

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Lactic antagonism
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The general non-specific inhibition or destruction of one microorganism by other members of the same niche. Interference could be due to (1) competition for nutrients, (2) competition for attachment/ adhesion sites, (3) unfavorable alteration of environment, (4) combination of these.
A specific example of microbial interference effected by the production of antimicrobial compounds, alteration of environmental conditions and nutrient depletion. Propionibacterium freudenreichii subsp. shermanii produces an ill-defined inhibitory system when cultured in skim milk. Lactobacillus reuetri produces reuterin.
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Protective cultures
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The Hurdle Concept

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Microorganisms that can be added to a product to effect preservation. Protective cultures should: !Present no health risks !Provide beneficial effects to the products !Have no negative impact on the sensory properties !Serve as indicators under abuse conditions
Also known as barrier technology and combination preservation. Involves multiple techniques to effect the control of microorganisms in foods. Control of proteolytic strains of Cl. botulinum: pH <4.6, aw <0.94, 10% NaCl, ~120 ppm NaNO2, storage at <10C, and a large aerobic biota.
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The Hurdle Concept

Intrinsic and extrinsic factors affecting microbial growth in foods

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Intrinsic and extrinsic factors affecting microbial growth in foods

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Авторское право:

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Intrinsic and extrinsic characteristics of foods affecting microbial growth

ALONZO A. GABRIEL, PHD

From Farm to Fork

Factors influencing the re-emergence of infectious diseases

Adaptation: From Farm to Fork

RNA Polymerases: Prokaryote

RNA Polymerases: Prokaryote

Electrophoresis Gel Separation by Urea

Transcription: the ! factor

Transcription: the ! factor

Transcription: the ! factor

Transcription: the ! factor

Transcription: the ! factor

Protein folding energy funnel

Protein folding energy funnel

Protein folding: Chaperones

kinetic trap! native state

Prevent improper interactions. Untangle aggregated proteins.

Alberts et al, 2010

Protein folding: Chaperones

Protein digestion: Proteasomes

Aided by the Hsp 40.

Alberts et al, 2010

Factors influencing the re-emergence of infectious diseases

Microorganisms and the Food Supply

The characteristics inherent to a food system are referred to as intrinsic parameters.

Frazier and Westhoff (1988); Jay (2000)

Frazier and Westhoff (1988); Jay (2000)

Approximate pH growth range of some foodborne microorganisms

Frazier and Westhoff (1988); Jay (2000)

Frazier and Westhoff (1988); Jay (2000)

pH and keeping quality of foods

Frazier and Westhoff (1988); Jay (2000)

Frazier and Westhoff (1988); Jay (2000)

Organic acids commonly used in meat and poultry products

egg white soda crackers

limes pickles vinegar mayonnaise

Frazier and Westhoff (1988); Jay (2000)

Meat pH and keeping quality

Frazier and Westhoff (1988); Jay (2000)

Frazier and Westhoff (1988); Jay (2000)

Action of pH against foodborne microorganisms

Cell Membrane Structure

Adverse pH changes could affect at least 2 aspects of a respiring cell:

Microbial cell internal pH

Reactions of microorganisms to changes in environmental pH

Frazier and Westhoff (1988); Jay (2000)

Frazier and Westhoff (1988); Jay (2000)

Reactions of microorganisms to acidic environments

Frazier and Westhoff (1988); Jay (2000)

Glutamic Acid: Predict the product after amino acid decarboxylase

Glutamic Acid Decarboxylation

Krulwich et al. (2011)

Krulwich et al. (2011)

Glutamic Acid Decarboxylation

Reactions of microorganisms to alkaline environments

Krulwich et al. (2011)

Frazier and Westhoff (1988); Jay (2000)

Various Amino Acids

Substrate pH and microbial nutrient transport

Krulwich et al. (2011)

Frazier and Westhoff (1988); Jay (2000)

Le Chateliers Principle and Antimicrobial Action of Organic Acids