Module 2 (LE 2, pt. 1)

• —> a lot of factors are contributing to the survival or resistance of microorganisms (before processing, during processing, until consumption) - we have to consider the intrinsic, extrinsic, and implicit parameters 
• —> The microbiota of different foods depend on the particular commodities and the means of preservation employed 

  —> Quality deteriorations may occur or be initiated at any stage between the acquisition of raw materials and consumption
• —> Food deterioration due to microorganisms 

  1. Commercially undesirable changes (but still safe from public health point of view) = changes in color, flavour, appearance 
  2. The presence or growth of infectious and toxigenic (pathogenic) microorganisms represent the worst forms of quality deterioration
• —> The first (commercially undesirable) talks more about the food quality and the perception of the consumer about the food, however it doesn’t pose as a public health concern (the second is more problematic because this is a concern of food safety) - because of the growth or presence of infectious or pathogenic microorganisms 
• —> food spoilage can take place anytime from farm to fork, spoilage can be due to physical, chemical, and microbial factors and also enzymic factors (most preservation methods are designed to inhibit the growth of microorganisms) — there are also preservation methods where we combine (hurdle concept) different technologies so that one of the aims of the preservation technique or preservation method is to preserve the food while keeping its quality

  —> preservation methods are based on microbial growth reduction due to unfavourable environmental conditions 
• —> Some preservation methods:

  • Reduction and inhibition of growth of microorganisms - has a lesser degree of effect (ex: low temperature, low aw (water activity), restriction of nutrients, low oxygen, raised CO2, acidification, alcoholic fermentation, use of preservatives) -= control intrinsic or extrinsic parameters 
  • Inactivation of microorganisms - try to kill microorganisms (heating, irradiating, pressurising) 
• —> there are three distinct temperature ranges for low-temperature stored foods

  1. Chilling temperatures (5-15 C)
  2. Refrigerator temperatures (0-7 C)
  3. Freezer temperatures (at or below -18C) (growth of all microorganisms are prevented at freezer temperatures) - some can still grow at a freezer range but at an extremely slow rate 
• —> The use of low temperatures to preserve food is based on the fact that the activities of microorganisms can be slowed at temperatures above freezing and generally stopped at sub-freezing temperatures 
• —> All the metabolic reactions of microorganisms are enzyme catalysed (the rate of enzyme catalysed reactions is dependent on temperature, that’s why lower temperatures slow down the enzyme catalysed reactions) 

  —> In a rising temperature, there is an increase in reaction rate 
• —> Low-temperature food preservation has become important in prolonging the shelf life of minimal processed foods (commodities which have been subjected to milder than traditional food preservation techniques) - psychotropic and mesophyllic microorganisms are of concern to these commodities
• Fats freezing (Quick freezing) vs. Slow freezing

  —> Fast freezing may be achieved by direct immersion or indirect contact of food with the refrigerant and it uses air blasts or frigid air blown across the food being frozen (the temperature is lowered to -20C within 30 mins causing small ice crystals)

  —> Slow freezing is often utilised in the home — it refers to the process whereby the desired temperature is achieved within three to seventy-two hours  
• —> Effect of freezing on microorganisms 

  1. Freezing results in a loss of cytoplasmic gases (loss of oxygen in aerobic cells surpassing the respiratory actions, the more diffused state of O2 may make for greater oxidative actions within the cells which may be detrimental to microorganisms)
  2. Freezing affect a concentration of cellular electrolytes (this effect is also a consequence of the concentration of water in the form of ice crystals)
• —> Effect of freezing on microorganisms +

  3. Freezing causes general alteration of the colloidal nature of the cytoplasm because many of the constituents of cellular protoplasm such as the proteins exist in a dynamic colloidal state in living cells (a proper amount of water is necessary to the well-being of the state)

  4. Freezing induces temperature shock and causes sublethal injury to microorganisms (for thermophiles and mesophyllic bacteria rather than psychrophiles)

  Note: more cells die when there’s an immediate drop in temperature above freezing compared to a slow drop
• —> It is well known that freezing is one means of preserving microbial cultures (low freezing temperatures of about -20 C are less harmful to microorganisms than the median range of temperatures such as -10 C) (for ex: more microorganisms are destroyed at -4C than at -15C or below)
• —> Intracellular free water freezes resulting to dehydration (the difference of slow and fast freezing is that, in slow freezing, ice crystal are extracellular, in fast freezing, ice crystals are intracellular so the freezing of cells depletes them of usable liquid water because water is essential for the enzyme catalysed reactions and when water freezes, intracellular water freezes and it is then depleted in cells and this preservation technique dehydrates the microbial cells
• —> freezing results to increase in viscosity of cellular matter (this is a direct consequence of water being concentrated in the form of ice)

  —> Intracellular pH changes have also been reported (from freezing)
• —> Microorganisms undergo cold adaptation through:

  1. Membrane composition modifications to maintain membrane fluidity for nutrient uptake 
  2. Structural integrity of proteins and ribosomes
  3. Production of cold shock proteins
  4. Uptake of compatible solutes 
• —> To maintain membrane fluidity, microorganisms increase the proportion of shorter and/or unsaturated fatty acids (among the multiple roles of fatty acids, they have structural functions as constituents of phospholipids which are the building blocks of the cell membrane) - shorter and unsaturated fatty acids present lower melting points when compared to their respectively longer and saturated counterparts 
• —> To maintain membrane fluidity, microorganisms increase the proportion of shorter and/or unsaturated fatty acids

  • For example: In E. Coli the proportion of cis-vaccenic acid (C18:1) increases at the expense of palmitic acid (C16:0) 
  • For example: Listeria monocytigenes have increased C15:0 and decreased C17:0 at low temperature 
• —> Uptake of compatible solutes like betaine, proline, and carnitine may be needed for osmoprotection and cold adaptation 

  —> solutes prevent water leakage and cell freezing
• —> cold shock proteins (CSP’s) (typically 7 kilo Daltons in size (kDa)) are synthesised and are involved in the synthesis of other protein molecules and mRNA folding (these cold shock proteins are found in most bacteria, the functions of the cup’s have not been fully elucidated, however a number of trends have been proposed including their possible role in protecting microorganisms during freezing and in this regard, anti-freeze proteins could lower the freeze temperature and inhibit recrystallisation and growth of ice crystals during frozen storage) — these ice crystals harm microorganisms
• —> Pasteurisation implies either the destruction of all pathogens (as in milk) or reduction in the number of spoilage organisms (as in vinegar)

  —> The pasteurisation of milk is achieved by heating at one of the following time-temperature combinations: (63 C for 30 mins (LTLT=low temperature, long time), 72C for 15 s (HTST=high temperature, short time), 89C for 1 s, 90C for 0.1 s, 94 for 0.1 s, 100C for 0.01 s)

  —> The temperature in pasteurisation does not exceed 100C
• —> Milk pasteurisation is also able to destroy, in addition, all yeasts, moulds, gram (-), and many gram (+) bacteria, however there are still bacteria that can survive the pasteurisation process such as thermoduric microorganisms
• —> Thermoduric microorganisms(can survive exposure to high temperatures, but they don’t grow at these temperatures = not optimal temp) (Streptococcus and lactobacillus) and thermophilic (micro-organisms survive and require high temps for their growth and metabolic activities) (found in milk) pasteurisation (Bacillus and clostridium) are able to survive the pasteurisation process 
• —> Sterilisation involves the destruction of all viable organisms as maybe measured by an appropriate plating or enumerating technique 

  —> canned foods are sometimes called commercially sterile (number of survivors is so low to be of significance under the conditions of storage and distribution + does not pose public health concern) to indicate that the number of survivors is so low to be of significance under the condition of storage and distribution
• —> sous-vide products are vacuum packed, undergo a mild heat treatment (90C, 10 mins) and have very carefully controlled chilled storage — raw food is placed in high barrier bags and cooked under vacuum (sous=under, vide=vacuum)
• —> Most vegetative cells are destroyed but bacterial spores survive in this process (Clostridium botulinum = produces spores + is anaerobic, which is the concern in sous-vide products)
• Heat assisted high hydrostatic pressure processing

  • The current interest is due to consumer demands for minimally processed foods (underwent mild heat treatments) — to lower the costs and greater availability of processing equipment
  • High hydrostatic pressure of 300-500 MPa can inactivate vegetative bacterial cells while >1000 MPa is required to inactivate spores 
  • This process can synergistically combined (teamed up) with heating such that lower pressures of 100-200 MPa are enough to safely process a food commodity 
• —> the targets for heat inactivation of microbes are the intrinsic stability of macromolecules 

  —> The exact primary cause for cell death due to heat exposure is not fully understood

  —> Thermotolerance of bacteria increases when exposed to sublethal heating, phage infections and chemical treatments including ethanol and streptomycin 
• —> Microbes can adapt to mild heat treatments through:

  1. Alteration of cell membrane composition to maintain optimal cell membrane fluidity and activity of intrinsic proteins (same mechanism of heat shock and cold shock proteins)
  2. Accumulation of osmolytes to enhance protein stability
  3. Production of spores and heat shock proteins 

  —> When bacterial cells are exposed to higher temperatures, a set of heat shock proteins or HSP’s is induced rapidly to cope with increased damage in proteins 
• • Thermal Death Time (TDT) = the time necessary to kill a given number of organisms at a a specified temperature 
  • Thermal Death Point (TDP) = temperature necessary to kill a given number of microorganisms in a fixed time (10 Mins)
  • For example: the TDP of E. Coli in cream is hotter (higher temp) because it contains more protective layers such as the lipids of the cream 
• —> factors affecting the heat resistance of microorganisms: moisture

  • Heat resistance of microbial cells decreases with increasing humidity, moisture and aw (colligative properties) 
  • Protein denaturation occurs at a faster rate when heated in water than in air (it is either a mechanism of death by heat or is closely associated with it)
• —> factors affecting the heat resistance of microorganisms: moisture

  • heating of wet proteins causes the formation of free —SH, sulfhydryl groups with a consequent increase in the water-binding capacity of proteins)
  • The presence of water allows for the thermal breaking of peptide bonds (a process that requires more energy in the absence of water and converts a greater refractivity to heat)
• Properties of mixtures: 

  Freezing point: temp at which liquids are solidified (affected by the amount of solute dissolved but eh solvent) (freezing point depression)

  Delta Tf = Kfmi (m=molality mol/kg, i = van’t hoff factor)

  Boiling Point: Pbar=Pvap (affected by altitude and the amount of solute dissolved by the solvent) (boiling point elevation)

  Delta Kb = Kbmi (m=molality mol/kg, i = van’t hoff factor) 
• —> factors affecting the heat resistance of microorganisms: Fats and salts 

  • General increase in heat resistance is observed with increasing fat content (called fat protection) (long-chain fatty acids)
  • Salts can exhibit variable effect on the heat resistance of microorganism depending on  the effects on aw 
• —> factors affecting the heat resistance of microorganisms: Carbohydrates

  • The presence of sugars in the suspending medium causes an increase in the heat resistance of the suspended microorganisms (the effects is in part due to the increasing water activity, adding solutes = free water bounds to solutes and it lowers the water activity - caused by the high concentrations of sugar)
  • Among sugars and alcohols, relative to their effect on heat resistance (from most resistant to least resistant)

  Sucrose > glucose > sorbitol > fructose > glycerol
• —> factors affecting the heat resistance of microorganisms: pH

  —> microorganisms are most resistant to heat at their optimum growth pH (7.0) 

  • High acid foods have considerably less severe heat treatment than low acid foods (for ex: fruit juices have lower pH and require less severe heat treatment)
• —> factors affecting the heat resistance of microorganisms: pH

  • Alkaline foods like eggs are neutralised prior to heat treatment (pH of the egg white is 9, and when this food is subjected to pasteurization conditions of 60 to 62 C for 3-4 mins, coagulation of proteins occur with a marked increase in viscosity) — which is why neutralisation is necessary prior to heat treatment 
• —> factors affecting the heat resistance of microorganisms: proteins

  • Proteins in the heating medium provide protection to microorganisms 
  • High-protein foods must be heat treated more severely than those with less (in order to achieve the same end results)
  • For identical microorganisms, the presence of colloidal particles in the heating medium increases microbial heat resistance (for ex: under identical conditions of pH, intake slinger to sterilise a puree rather than nutrient broth)
• —> factors affecting the heat resistance of microorganisms: population 

  • Initial population and heat resistance are directly related
  • The mechanisms of heat protection by large microbial population is due to the production of protective substances excreted by the cells and cell clumping (when there’s a lot of microbes on the cells, cell clumping occurs which adds to the protective layer)