Module 2 (LE 2, pt. 2 & 3)

• Primary Effects of CO2 on Microorganisms

  • The inhibitory activity increases with decreasing storage temperature (due to the greater solubility for CO2 in water at low temp and the additive effect of a less than optimal growth temperature)
  • Inhibition is greater at the acidic pH range (CO2 is more effective in metas than in seafoods)
  • Gram negative are more sensitive than gram positive both in lag and log phases of growth 
  • CO2 Is more inhibitory (more anti-microbial) under higher pressure (the destructive action occurs when pressure is suddenly released) 
• —> Shows the species most sensitive to CO2 relative to Vacuum packaged and MAP (most sensitive on top - Pseudomonas spp., Aeromonas spp.) (clostridium app. Are more resistant to VP and MAP)
• —> As a general rule, foods to be subjected to MAP should possess one or more of the following antibotulinal hurdles:

  • Aw < 0.93 (water activity less than 0.93)
  • pH < or = 4.6 (low acid product)
  • Cured with NaCl or NO2
  • Contain high levels of commensals 
  • Maintained in frozen state or < or = 4.0 C
  • Have a definitive shelf life
• Preservation of Foods by Drying and Dehydration 

  —> drying to the point where the activities of food spoilage and pathogenic microorganisms are inhibited (since these microbes and enzymes need water in order to be active)

  Dried, desiccated, low-moisture  LM) foods are those with < or + 25% moisture and aw of 0.00-0.60

  • Food products that contain 15%-50% moisture and aw of 0.60-0.85 are intermediate-moisture (IMF) foods 
• Storage Stability of Dried Foods

  • At aw of 0.80-0.85, spoilage occurs readily by a variety of fungi in 1-2 weeks
  • At aw of 0.75, spoilage is delayed, with fewer types of organisms in the spoiled foods
  • At aw of 0.70, spoilage is greatly delayed and may not occur during prolonged holding
  • At aw of 0.65, very few organisms grow, and spoilage is most unlikely to occur even up to 2 years
• —> As a guide to the storage stability of dried foods, the alarm water content has been suggested so the alarm water content is the moisture level that should not be exceeded if mould growth is to be avoided 

  • The alarm water should be cautiously followed because only a rise of 1% may be disastrous
  • In freeze-dried foods, the rule of thumb has been to reduce the moisture level to 2%
• Intermediate-Moisture Foods (IMF)

  —> Aw of 15-50%

  • Have lowered aw values which are achieved by withdrawal of moisture by: absorption, adsorption, addition of salts and sugars, addition of humectants (Glycerol, glycols, etc.)
• Traditional IMFs —> dried fruits, cakes and pastries, frozen foods, sugars and syrups, some candies, fruit cakes, honey, fruit juice concentrates, sweetened condensed milk, some fermented sausages (aw = 0.60-0.85)
• —> IMF can be prepared by formulating the product so that the moisture level is 15-50%

  • Aw can be adjusted to below 0.86 by use of humectants (since s. Aureus is the concern here)
  • Growth of yeasts and moulds that can thrive at aw > 0.70 can be arrested by adding antifungal agents
  • Additional storage stability may be achieved by reducing the pH
• Raoult’s Law of Mole Fractions

  Aw = moles of H2O / moles of H2O + moles of solute
• Microbial Aspects of IMF:

  • The general aw range of IMF products makes it unlikely that Gram negative and gram positive bacteria will proliferate, except for some cocci, spore-formers and lactobacilli
  • In addition to the effects of lowered aw microbial growth inhibition results from the interaction of pH, Eh, preservatives, microbial interference, and processing and storage temperature
• Food preservation with chemicals

  • The safety of food additives is only applicable up to the time when scientific facts prove otherwise 
  • Food additives make food products affordable (long shelf-life = cheaper)
  • Consuming foods with chemicals is a gamble 
• • The use of chemicals to preserve foods are based in part on the abilities of such compounds to cure diseases of humans, animals, and plants
  • Although a large number of chemicals have been described to show potential as preservatives, only a small number are generally recognised as safe (GRAS)

  GRAS = Generally Recognised As Safe
• • Growth of an endospore into vegetative cells (showing the stages of minimum inhibitory restorations of some food preservatives)
  • Nisin, subtilin, diethyl pyrocarbonate: before swelling
  • Benzoate: before outgrowth
  • Sorbate, metabisulphite, sodium chloride: right after outgrowth
  • Tyrosine, polyphosphate: right before cell division
• GRAS Chemical Food Preservatives:

  Propionic acid/propionates, sorbic acids/sorbates, benzoic acids, benzoate’s, paragons, SO2/sulfites, ethylene/propylene oxides, sodium dictate, nisin, dihydroacetic acid, sodium nitrite, ethyl formate 
• —> Benzoic acids and benzoate’s were the first chemical preservatives permitted tin foods by the FDA 

  • Bemnzoates have better antimicrobial activities at lower pH where the molecules are undissociated (only used in high-acid products: apple cider, soda, tomato ketchup, salad dressing)
  • Parabens (P-hydroxybenozoic acid) are less sensitive to pH changes and are more effective against moulds than yeasts
  • Benzoates have been found to inhibit the uptake of substrate molecules
  • Benzoates can also alter proton concentration within the cell 
• —> NaNO3 and NaNO2 are used in curling formulas for meats because they stabilise meat color, inhibit spoilers and pathogens and contribute to flavour development

  • The nitrite ion is by the far more important than nitrate in preserved meats
  • In an acid environment, nitrite ionises to form nitrous acid (HNO2) that further decomposes to form nitric oxide (NO) 
• Color reaction in cured meats: (ex: bacon has a pink color)

  Myoglobin + NO = purplish red

  Nitrosomyoglobin = bright red

  + heating

  = Nitrosohemochrome = pink
• • Because nitric oxide is known to be capable of reacting porphyrin-containing compounds (catalyses, peroxides, cytochromes, etc.) it is conceivable that its antimicrobial action maybe through this mechanism
  • Heating a medium with nitrite produces the perigee factor - a substance/agent 10 times more inhibitory than nitrite alone
• Antibiotics and Bacteriocins

  • Antibiotics are secondary metabolites produced by microorganisms that inhibit or kill a wide-spectrum of other microorganisms (Natural preservatives)
  • Most of the useful antibiotics are produced by moulds and bacteria of the genus Streptomyces
  • Some antibiotic-like substance are produced by Bacillus spp. and at leas one, nisin is produced by lactococcus lactic
• —> the mode of action of Nisin is that it interacts with the Lipid II

  • Affects the integrity of the cell wall
  • Creates pores on cell membranes 
  • This compound is effective against gram-positive bacteria, primarily spore-formers and is not effective against fungi and gram-negative bacteria
• General consideration in Using Antibiotics

  • The antibiotic should kill not inhibit the flora and should ideally decompose into innocuous products (not harmful), or be destroyed by cooking
  • It should not be inactivated by food components or products of microbial metabolism
  • But should not readily stimulate the appearance of resistant strains
  • It should not be used in foods as therapeutic or an animal feed additive
• Microbial Challenge Testing

  • Useful in the validation of processes that are intended to deliver some degree of lethality against a target organism or group of target organisms
  • Validation of an associated performance standard that process must deliver (5D reduction in E. Coli O157:H7 for fermented meats)
  • Fruit juices: 5D, meats: 12D
• • Microbiological challenge testing is a useful tool for determining the ability of a food to support the growth of spoilage organisms or pathogens
  • microbiological challenge testing is useful in determination of potential shelf life of certain refrigerated or ambient-stored foods
• Factors to be considered in microbiological challenge testing:

  1. Selection of appropriate pathogens or surrogates
  2. Level of challenge inoculum (how much inoculum) 
  3. Inoculum preparation and method of inoculation
  4. Duration of the study
  5. Formulation and storage conditions
  6. Sample analyses 
• Selection of challenge organisms 

  Knowledge of the food formulation and history of the food (for example, association with known illness outbreaks and/or evidence of potential growth)

  • Clostridium botulinum is a concern in certain  MAP products
  • Staphylococcus aureus in products with low aw
• Selection of challenge organisms

  • The ideal organisms for challenge testing are those that have been previously isolated from similar formulations
  • Additionally, pathogens from known food borne outbreaks should be included to ensure the formulation is robust enough to inhibit those organisms as well 
  • Clostridium, E. coli, salmonella: affect fruit juices (high acid)
• What if multiple organisms cannot be challenged? A single challenged strain with a set of well-defined characteristics may be used to screen products similar in formulation (multiple strains, then a single challenged strain should be used in testing)
• —> Performance standard should be based on the inactivation of most resistant strain 
• Change in population = final population - initial population (log CFU/mL or g)

  • Higher change in population = higher death rate
• —> Multiple specifics strains of the target pathogens should be included in the challenge study 

  —> It is typical to challenge a food formulation with a cocktail or mixture our composite of multiple strains in order to account for potential strain variation 
• —> How are cocktails prepared?

  • Organisms are individually propagated, and harvested
  • Individual test species are combined just before inoculation 
• Exponent e = negligible difference in values
• --> Consideration must also be given to adapting the challenge suspension to the environment of the food prior to inoculation (acclimitization makes the microorganism adapt to its environment)

  —> Acid-adpatation of E. Coli O157:h& and salmonellae cells prior to inoculation can greatly influence their ability to survive when inoculated into acidic food 
• Two adaptations:

  1. Homologous
  2. Heterologous

  Ex: stress factor = acidity

  —> can either become dead cells or adapted cells 

  —> Adapted cells may either have homologous adaptation or heterologous adaptation

  1. Homologous adaptation - occurs when the survive population of cells developed resistance to the inactivating agent that it was first exposed to
  2. Heterologous adaptation -  occurs when cells develop resistance other than the first stressor 
• Stressed vs. Steady state of microorganisms (there are many stressors prior to food processing)

  —> in nature, microorganisms are exposed not only to one but several stressful conditions 
• —> For certain applications, surrogate microorganisms may be used in challenge studies in place of specific pathogens (surrogate = replacement)

  • It is usually not possible to introduce pathogens into a processing facility, therefore it is desirable to use surrogate microorganisms in those cases
• —> An ideal surrogate is a strain of the target pathogen that retains all other characteristics excepts its virulence

  1. Clostridium sporogenes (surrogate) for Clostridium botulinum
  2. Listeria innocua for Listeria monocytogenes
  3. Escherichia coli K12 for Escherichia coli O157:H7 
• Surrogate microorganisms

  1. Nonpathogenic
  2. Inactivation characteristics and kinetics that can be used to predict the target pathogen
  3. Behavior similar to the target pathogen when exposed to formulation and/or processing parameters (pH stability, temperature, oxygen) 
• Surrogate microorganisms

  1. Stable and consistent growth characteristics
  2. Easily prepared to yield high-density populations
  3. Once prepared, population remains stable until utilised
  4. Easily enumerated using rapid, sensitive, and inexpensive detection systems