Module 1 - Topic 2 (LE 1, pt. 2)

• 1. 3M Petri film —> a rapid method that involves 3 simple steps: inoculate, incubate, interpret (this method is advantageous than other standard plating techniques because it contributes 85% less space the agar, it has 45% reduced labor cost (no media prep), 80% increased technical efficiency, and obtained data in half the time — the disadvantage: expensive (4K or more for 50 pieces)
• 1. Enzyme Linked Immunosorbent Assay (ELISA) —> it is a plant-based assay technique designed for detecting and quantifying soluble substances (such as peptides, proteins, antibodies, and hormones) - development of color = presence of the antibody being identified (antigen is used to identify the antibody)
• —> ELISA is both a quantitative and qualitative test: Color change of positive or negative = qualitative, the production of a standard curve = quantitative, with the comparison of antigens of known concentration, the concentration of antigen in your sample can then be calculate using the optical density 
• ATP Bioluminescence Techniques and Hygiene Monitoring —> Adenosine Triphosphate is found in all living cells, therefore the presence of ATP indicates that living cells are present (so it can be used for hygiene monitoring) — the limit of detection is around one picogram (pg) ATP - approx 1000 bacterial cells based on 10^-15 g ATP per cell — this process is faster than traditional colony counts for detection of bacteria, yeast, and fungi 

  • ATP is detected using the Luciferin reaction 
  • This method is primarily used for hygiene monitoring (the luciferase is from fireflies)
• Protein Detection by Biuret Reaction —> this is an alternative to ATP detection or ATP bioluminescence (more rapid than culture-based methods and is less expensive than ATP bioluminescence, however it is less sensitive than the ATP bioluminescence) for hygiene monitoring — basically, the surface is sampled by swabbing:

  green color = clean, hygienic surface

  grey = for caution

  purple = dirty surface 
• 4 Molecular Techniques

  PFGE, RAPD, LAMP, Metagenomics 
• 1. Pulsed-Field Gel Electropphoresis (PFGE) —> a technique used for the separation of large deoxyribonucleic acid molecules by applying to a gel matriculates or the agarose gel — an electric field that periodiclally changes direction (has a hexagonal platform and a changing current/electric field) 
• 1. Random Amplified Polymorphic DNA (RAPD) —> A PCR based technique in which arbitrary primers are used to randomly amplify segments of target DNA under low stringency PCR conditions — this process leads to the amplification of one or more DNA sequences and generates a set of finger printing patterns of different sizes specific to each strain — RAPD does not require any specific knowledge of the DNA sequence of the target organism 
• 1. Loop-mediated Isothermal Amplification (LAMP) —> can be used for the detection of salmonella in food
• 1. Metagenomics —> a molecular tool used to analyse DNA extracted from environmental samples in order to study the community  of microorganisms present without the necessity of obtaining pure cultures 
• —> Accreditation of Analytical Techniques: used to set standards  - the method must be validated against a standard test using collaborative studies 

  International validation bodies include: 

  AOAC (USA) - used in PH

  UKAS (UK)

  EMMAS (Europe)

  AFNOR (France) - used to lead and coordinate the standards development process and to promote the application of those standards, 

  DIN (Germany) 
• How do prokaryotes adapt?

  —> Prokaryotes use the same RNA polymerase to transcribe all of their genes (for ex: in E. coli, the polymerase is composed of 5 polypeptide subunits: 2 are identical, 4 of these units are denoted as alpha 1, alpha 2, beta, and beta prime — these four subunits comprise of the polymerase core enzyme 

  —> The polymerase active form = Holoenzyme (composed of the core enzyme + sigma factor)
• Sigma factor —> Subunits of all bacterial RNA polymerases that are responsible for determining the specificity of promoter DNA binding (meaning they are the ones responsible for what gene will be transcribed and are the ones that control how efficiently RNA synthesis is initiated 
• —> E. Coli has seven sigma factors the first one is sigma 70 or what we call “housekeeping sigma” — it is involved in the transcription of all major genes 
• —> There is also sigma 19 or the ferric citrate sigma factor that regulates the  fed gene for iron transport 

  —> Sigma 24 or the extracytoplasmic or extreme heat stress sigma factor (means that this dictates what gene is transcribed for the E. Coli to adapt to this extreme heat stress)

  —> Sigma 28 is the flagellar sigma factor 
• —> sigma 32 is the heat shock sigma factor (focused on in food micro because E. Coli prevention through thermal or heat processing is often studied) 

  Plants and animals have developed defense mechanisms against the invasion and proliferation of microorganisms 
• —> The characteristics inherent to a food system are referred to as intrinsic parameters (ex: pH, moisture content, RedOx potential, Nutrient content, Antimicrobial constituents, Biological structures)
• —> Healthy cells may be subjected to a number of stresses before the application of  a processing kill step in this phase, these stresses may include intrinsic food characteristics and extrinsic environmental conditions — these stresses may individually or interactively influence the induction of adaptive mechanisms or inactivation of the microorganisms (similarly, the efficacy of the eventual food processing against the pathogens may be dependent on individual and/or interactive influences of food and environmental parameters 
• Microbial responses - can be bacterial injury, lag time, growth rate (Kg), Max population, and Inactivation (D values)
• Outbreak —> When two or more people get the same illness from the same contaminated food or drink (food borne illness outbreak)
• • Most microorganisms grow best at pH values around seven (6.6-7.5) whereas few grow below four
  • Generally, bacteria are more fastidious (choosy/picky) in their relationships to pH than yeasts and moulds 
• Three categories based on pH: acidophiles (acid-loving), neutrophiles, (neutral) and alkalophiles (alkali-loving)
• —> With respect to pH minima and maxima of microorganisms, these range of values should not be taken to precise boundaries as the actual values are known to be dependent on a lot of factors, but in general, moulds have wider pH ranges for their growth followed by yeast and then bacteria

  —> Gram-negative bacteria are more fastidious (choosy) 
• —> the pH minima of certain lactobacilli have been shown to be dependent on the type of acid used in adjusting growth medium pH such as citric acid, hydrochloric acid, and phosphoric, and tartaric acids were found to favour at lower pH than acetic and lactic acid
• —> commonly used organic acids in meat and poultry products: acetic acid, citric acid, lactic acid, propionic acid, magic acid, succinct acid, and tartaric acid 
• —> Microbial growth is not solely dependent on pH alone (there are a lot of factors)

  —> for example, in the presence of this soluble solid such as 0.2 M NaCl, Alcaligenes faecalis has been shown to grow over a wider pH than in the absence of NaCl (Alcaligenes faecalis favours higher soluble solids)
• —> Food that have a pH below 4.5 are termed a high acid foods (ex: fermented foods, wine and vinegar, mayonnaise)

  —> Food that have a pH greater than 4.5 are usually meat. Poultry, and crackers
• —> fruits generally undergo mould and yeast spoilage due to the capacity of these organisms to grow at less than 3.5 pH which is considerably below the minima for most food spoilage and all food poisoning bacteria 

  —> meats and seafoods have a final pH of about 5.6 and above making them susceptible to both bacterial and fungal spoilage as compared to fruits and vegetables
• —> Meat from fatigued animals spoils faster than from those of rested ones (upon the death of a well-rested animal, the usual one percent glycogen is converted to lactic acid, which directly causes a depression in pH values from 7.4 to 5.6 

  — if animals are not well-rested, there will be no glycogen converted into lactic acid meaning meat often have a higher pH than 5.6 and typically spoils faster
• —> Some foods are able to better resist pH changes than others (those that tend to resist changes in pH are said to be buffered, like the meats) - metas are more highly buffered than vegetables 
• —> Biological acidity: Some food products are preserved through the production of acidic compounds due to the actions of certain microorganisms (ex: fermented foods: milk, sauerkraut, pickles)
• —> Adverse pH against affects at least two aspects of our respiring microbial cell: first, the functioning of its enzymes and the transport of nutrients into the cell. Additionally, adverse pH changes also affect proteins and result in protein denaturation 
• • The cytoplasmic membrane of microorganisms is relatively impermeable to H+ and OH- ions — their concentration in the cytoplasm therefore remains reasonably constant despite wide variations that may occur in the pH of the surrounding medium 
  • The internal pH of most microorganisms appear to be near neutrality (ex: intracellular pH of S. Cerevisae (yeast) was found to be 5.8)
• —> when microorganisms are placed in environments below or above neutrality, (acidic or basic) their ability to proliferate or tow grow depends on their ability to bring the environmental pH to a more optimum value or range (so it’s either they must keep the H+ from entering or or expel H+ as rapidly as they enter) —> Cellular metabolic activities also adjust the pH of the environment 
• —> When most microorganisms grow in acid media, their metabolic activity results in the medium or substrate becoming less acidic (for ex: Clostridium acetobutylicum when placed in an acidic medium, reduces butyric acid to butanol)

  — whereas those that grow in high pH environments tend to affect a lowering of pH (ex: Enterobacter aerogenes produces acetone from pyruvic acid)
• — In acidic medium (ex pH 4.0), amino acid decarboxylates cause a spontaneous adjustment of pH toward neutrality when cells are grown in acid medium 

  — When amino acids are decarboxylated, the increase in pH occurs from the resulting amines
• For pH homeostasis in an E. Coli cell 

  —> During anaerobic acid challenge, E. Coli upregulates hydrogenase that catalyses H2 production from cytoplasmic protons contributing to survival at pH 2 to 2.5

  —> Glutamate and arginine decarboxylates are enzymes that are a cornerstone of acid resistance response of E. Coli and other enteric bacteria as they pass through our stomachs (our pH in the stomach is adverse, and this is the way of bacteria to proliferate inside our bodies and infect us)
• —> Glutamate decarboxylase (GadB), this enzyme is activated by gastric chloride ions and then it consumes a proton during decarboxylation reaction to form or yield GABA (Gamma-Aminobutyric) - the GadB partners with the anti porter that catalyses the eflux of resulting GABA (exit of GABA) in exchange for the glutamate for continued decarboxylation reaction — the consumption of the cytoplasmic proton supports the acid pH homeostasis of the E. Coli 

  —> Cyclopropane acids contributes for the pH homeostasis of the E. Coli and the anti porters, hydrogenase, and amino acid decarboxylase 
• —> If in acidic medium, decarboxylates are upregulated, in basic media, amino acid deaminase are activated — they cause spontaneous adjustment of pH as a result of organic acid accumulation (so, deamination is the removal of an amino group from a molecule)
• —> Conversely, challenges by alkaline conditions lead up to up regulation of amino acid deaminase or catabolic pathways that produce organic acids as shown for this up regulation of tryptophan deaminase in E. coli that yields indole and pyruvate