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INTRODUCTION TO FOOD SAFETY AND QUALITY MANAGEMENT CURRICULUM (M. Phil) Program at FCC Dr. Quratulain Syed Chief Scientific Officer Food & Biotechnology Research Centre, PCSIR, Labs, Complex ,Lahore Course : Food Microbiology & Toxicology Credit Hours: 3(2-1) Objectives: • To understand different microbial threats related to food safety and epidemiology of different food borne illness. • To understand the international microbial limits for safe foods, toxicological aspect of foods and their impact • Food microbiology including microbial analysis, growth, physiology and survival; • Interpret the Codex & ICMSF microbiological criteria regulations for foodstuff; • Role and ability of antimicrobial agents and their role in cleaning science; • Comprehend results relating to the detection, enumeration, identification and prediction of microorganisms; explain the etiology of common food borne illnesses/diseases; • Role of microbes in disease, food spoilage, food production, • Food preservation methods and biotechnology; • Importance of water quality, water chlorination (chemistry, methods, testing and interpretation of results) in food production. • Food toxicology: Overview - intrinsic and extraneous toxins; Principles, types, branches; Toxicity: curve, factors influencing potency, margin of safety, factors influencing toxicity; Dose-response relationship, manifestation of organ toxicity. • Measurement of toxicants and toxicity; Toxicokinetics: carcinogenesis, mutagenesis, teratogensis; Practicals • Safety in microbiological laboratory. • Basic functions and handling of laboratory equipments. Use of microscope. • Sterilization and disinfection of glassware. • Preparation of culture media. Staining of microorganisms and their structures. • Bacterial cultivation, growth measurement. Characteristics of bacterial colonies. Bacterial and fungal morphology. Micrometry. 略 Microbial physiology 6 The Domain of Life Comparative ribosomal RNA sequencing has defined the three domains of life: • Bacteria •Archaea •Eukarya Prokaryotic Diversity • Several lineages are present in the domains Bacteria and Archaea • Enormous diversity of cell morphologies and physiologies Cell Structure • Two structural types of cells are recognized: the prokaryote and the eukaryote. • Prokaryotic cells have a simpler internal structure than eukaryotic cells, membrane-enclosed organelles. lacking Bacterial Morphology Some typical bacterial morphologies include • Coccus, • Rod / Bacilli • Coccobacilli • Spirillum, • Spirochete, • Appendaged, • Filamentous. Cytoplasmic Membrane The cytoplasmic membrane • Highly selective permeability barrier • lipids and proteins • Forms a bilayer with hydrophilic exteriors and a hydrophobic interior. Cell Wall • Gram-negative Bacteria have only a few layers of peptidoglycan , but gram-positive Bacteria have several layers. • In addition to peptidoglycan, gram-negative Bacteria contain an outer membrane consisting of lipo-polysaccharide (LPS), protein, and lipo-protein. Cell wall • Surrounds the cyptoplasmic membrane • Directly reflects adaptive strategies involved with -Uptake of nutrients - Excretion of waste products -Movements -Protection -Adhesion • In some organisms >25 of the genome is devoted to its synthesis, regulation and maintenance Gram positive cell wall: • Rigid structure • Based on a crossed-linked polymer -Peptidoglycan • Also contains teichoic acids (2 types) -Wall teichoic acids Polymer consisting of ribitol and phosphate Confer antigenic specificity for the bacteria Gram negative membrane: • Consist of outer and inner (cytoplasmic) membrane separated by the periplasm • Outer membrane - Flexible outer phospholipid bilayer with an inner peptidoglycan layer Strong negative charge of phospholipid bilayer helps evade phagocytosis Also protects against some antibiotics - Outer membrane also contain hydrophobic lipopolysaccharides and lipoproteins • Periplasm: - Solution between the inner and ouetr membrane - Contains specific periplasmic proteins Usually involved in hydrolysis and transport of materials • Cytoplasmic (inner) membrane: - Feature of both Gram-positive and Gram-negative cells - Phospholipid bilayer - Allow the pasage of membrane components through - Has peripheral or integral proteins associated with it Chemically, bacteria consist of: • Water (75-85%) – bound water and free water • Dry matter (1525%) – organic part and mineral substances (inorganic part) Dry matter • Organic part • proteins – 50-80% • nucleic acid – 10-30% • carbohydrates – 12-18% • polysaccharides – 3-5% • lipids – 5-10%. • Inorganic part • nitrogen (N), carbon (C), oxygen (O), hydrogen (H), phosphorus (P), sulfur (S), sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), iron (Fe) and other Growth of Microbes • Increase in number of cells, not cell size • One cell becomes colony of millions of cells Growth of Microbes • Control of growth is important for – Infection control – Growth of industrial products and biotech organisms Cell Growth • Microbial growth involves an increase in the number of cells. Growth of most microorganisms occurs by the process of binary fission. • Alternative means Budding Conidiospores (filamentous bacteria) Fragmentation Generation Time • Time required for cell to divide/for population to double • Average for bacteria is 1-3 hours • E. coli generation time = 20 min – 20 generations (7 hours), 1 cell becomes 1 million cells! Fig. 7.14a Growth and survival 38 Standard Growth Curve Phases of Growth • Lag phase: adaptation to the environment making new enzymes in response to new medium • Exponential logarithmic growth: Desired for production of products . Most sensitive to drugs and radiation during this period • Stationary phase: nutrition exhausted, toxin increased – death rate = division rate • Decline: cell die (steady biomass) or lysis (decrease biomass) death exceeds division • Dormant as spore, non-viable state Measuring Growth • Direct methods – count individual cells • Indirect Methods – measure effects of bacterial growth Measuring Microbial Growth There are 4 surrogate ways: • Cell counting (direct), • Colony formation (direct) , • Biomass determination (indirect), • Turbidity (indirect) • Microbes are useful tools in research because of their rapid life cycle, their simple growth requirements, and their small size. Growth measurement • Cell count: microscopic observation; flow cytometer (direct) • Colony formation: Measure the living cell (direct) • Biomass determination: dry weight; essential cell component (indirect) • Turbidity (indirect) Counting the viable cells: Dilution Agar : Solidifying medium Indirect Counting • Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth. However, to relate a direct cell count to a turbidity value, a standard curve must first be established. Turbidity Metabolic Activity Dry Weight Factors Regulating Growth • Nutrients • Environmental conditions: • Generation time Nutrients (Chemical Requirements) • Water • Elements – C (50% of cell’s dry weight) – Trace elements • Organic – Source of energy (glucose) – Vitamins (coenzymes) – Some amino acids, purines and pyrimidines Water • Microbes require water to dissolve enzymes and nutrients required in metabolism • Water is important reactant in many metabolic reactions • Most cells die in absence of water – Some have cell walls that retain water – Endospores and cysts cease most metabolic activity in a dry environment for years • Two physical effects of water – Osmotic pressure – Hydrostatic pressure Osmotic Pressure • Is the pressure exerted on a semi-permeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution • Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst Osmotic Pressure • Hypertonic solutions have greater solute concentrations; cells placed in these solutions will undergo cremation (shriveling of cytoplasm) – This effect helps preserve some foods • Restricts organisms to certain environments – Obligate halophiles – grow in up to 30% salt – Facultative halophiles – can tolerate high salt concentrations Hydrostatic Pressure • Water exerts pressure in proportion to its depth – For every addition of depth, water pressure increases 1 atm • Organisms that live under extreme pressure are barophiles – Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape Environmental Effects on Microbial Growth Factors affecting Growth • The orderly increase in the sum of all the components of an organism Affected by: Nutrients pH: neutrophils, acidophils, alkalophils Temperature: psychrophils; mesophils; themophils, thermodurics Aeration Pressure Ionic strength and osmotic pressure: halophils, osmophils 1. Temperature • Temperature is a major environmental factor controlling microbial growth. • The cardinal temperatures - minimum, optimum, and maximum. • Microorganisms can be grouped by the temperature ranges they require. 2. Low or High pH • The acidity or alkalinity of an environment can greatly affect microbial growth. • Organisms that grow best at low pH are called acidophiles; those that grow best at high pH are called alkaliphiles. • Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8. • The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. • Acid (below pH 4) good preservative for pickles, cheese pH • Many bacteria and viruses survive low pH of stomach to infect intestines • Helicobacter pylori lives in stomach under mucus layer 略 3. Air requirement:O2 Aerobe: A microorganism whose growth requires the presence of air or free oxygen Anaerobe: A microorganism that grows only or best in the absence of free oxygen. Organisms utilize bound oxygen Microaerophile: A microorganism that grows best in the presence of low concentrations of oxygen Facultative anaerobe/aerobe: A microbe that adjusts its metabolism to depending on the oxygen concentration in which it is growing. can live with or without oxygen. Aerotolerant anaerobe: an organism that always grows in an anaerobic mode -- can tolerate oxygen and grow in its presence even though they cannot use it. Capneic microbe: An organism that requires 3 to 10% CO2 for growth Growth pattern 1. 2. 3. 4. 5. Obligate aerobe Obligate anaerobe Microaerophile Aerotolerant anaerobe Facultative anaerobe/aerobe Two types of biofilms when cells grow as biofilm Environmental Disease-associated Symbioses Termite, ruminant digestion Sewage treatment bioreactors Water pipes Dental units Contact lens cases Dental plaque Endocarditis Cystic Fibrosis Otitis media Urinary catheter Implants • Special techniques are needed to grow aerobic and anaerobic microorganisms. • Several toxic forms of oxygen can be formed in the cell as the result of respiration, but enzymes are present that can neutralize most of them. Hydrogen peroxide is one of those forms that can be neutralized by catalase. 4. Salinity • Some microorganisms (halophiles) have evolved to grow best at reduced water potential, and some (extreme halophiles) even require high levels of salts for growth. extreme halophiles can live in solutions of 25 % salt; seawater = 2% salt Extremophiles • Definition - Lover of extremities • Temperature extremes – Boiling or freezing, 1000C to -10C – Chemical extremes – Vinegar or ammonia (<5 pH or >9 pH) – Highly saline, up to x10 sea water • we sterilize & preserve foods today Extreme Temperatures • Thermophiles - High temperature – Thermal vents and hot springs – May go hand in hand with chemical extremes • Psychrophiles - Low temperature – Arctic and Antarctic • 1/2 of earth’s surface is oceans between 1-40C • Deep sea –10C to 40C • Most rely on photosynthesis Chemical Extremes • Acidophiles - Acidic – Again some thermal vents & hot springs • Alkaliphiles - Alkaline – Soda lakes in Africa and Western U.S. • Halophiles - Highly saline – Natural salt lakes and manmade pools Extremozymes • Enzyme from Extremophile – Industry & Medicine • What if you want an enzyme to work – In a hot factory? – Tank of cold solution? – Acidic pond? – Sewage (ammonia)? – Highly saline solution? – Pay a genetic engineer to design a “super” enzymes... – Extremophiles have the enzymes that work in extreme conditions “Compatible Solute” Strategy • Cells maintain low concentrations of salt in their cytoplasm by balancing osmotic potential with organic, compatible solutes. • They do this by the synthesis or uptake of compatible solutes- glycerol, sugars and their derivatives, amino acids and their derivatives & quaternary amines such as glycine betaine. • Energetically synthesizing solutes is an expensive process. – Autotrophs use between 30 to 90 molecules of ATP to synthesize one molecule of compatible solute. – Heterotrophs use between 23 to 79 ATP. Osmoregulation • Halophiles have adapted to life at high salinity in many different ways. – Structural modification of external cell walls- posses negatively charged proteins on the outside which bind to positively charged sodium ions in their external environments & stabilizes the cell wall break down. Halophiles • The “salt-in” strategy uses less energy but requires intracellular adaptations. Only a few prokaryotes use it. • All other halophiles use the “compatible solute” strategy that is energy expensive but does not require special adaptations. Mean Generation Time and Growth Rate • The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes. 1 • Growth rate (min-1) =mean generation time • Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation Classification of bacteria based on nutritional requirements • Autotrophs are free-living, most of which can use carbon dioxide as their carbon source. The energy can be obtained from: • sunlight – protoautotrophs (get energy from photochemical reactions) • inorganic compounds, by oxidation – chemoautotrophs (get energy from chemical reactions) • Heterotrophs are generally parasitic bacteria, requiring more complex organic compounds than carbon dioxide, e.g. sugars, as their source of carbon and energy. Methods of laboratory diagnosis 1. 2. 3. 4. 5. 6. Bacterioscopical (Microscopic examination) Bacteriological (Culture method) Detection sensitivity of bacteria to antibiotics Serological Biological DNA-technology test (PCR) In the clinical laboratory it is necessary: • isolate bacteria in pure culture; • obtain sufficient growth of bacteria for demonstration their properties such as study of morphological, cultural, biochemical, antigenic and pathogenic properties, bacteriophage and bacteriocin susceptibility; • determine a sensitivity to antibiotics. Methods of the cultivation • Streak culture (surface plating). The inoculum is spreaded thinly over the plate of a culture media in series of parallel lines in different segment of the plate. On inoculation well separated colonies are obtained over the final series of streaks. Methods of the cultivation • Lawn or carpet culture. Lawn cultures are prepared by flooding the surface or plate with suspension of bacteria. It provides uniform surface growth of bacteria. It is useful for bacteriophage typing and antibiotic sensitivity test. Methods of the cultivation • Stroke culture. It is made in tubes containing agar slopes. It is used for providing a pure growth of bacterium (for slide agglutination). Methods of the cultivation • Stab culture. It is prepared by puncturing with charged long straight wire (loop). Stab culture is employed mainly for cultivation of anaerobes. Pure plate culture • Methods of the cultivation Liquid culture in a tube, bottle or flask may be inoculated by touching with a charged loop • Identification of bacteria Microscopic examination: It helps to detect a shape, a size and an arrangement of microorganisms • Staining reaction: On gram staining we can have two groups of microorganisms: Gram positive and Gram negative. E. coli, Gram negative (A), Staphylococcus epidermidis, Gram positive (B) and Bacillus cereus, Gram positive • Identification of bacteria Motility: Some bacteria can move (Salmonella, E. coli, Proteus, Pseudomonas, Vibrions, Clostridia). Dark ground microscopy and Phase contrast microscopy, special culture media use for studying motility of bacteria Special stain for flagella Identification of bacteria • Culture character: Growth requirement, colonial characteristics in culture Colony morphology descriptions Colony morphology • Identification of bacteria Metabolism: Capacity to form pigment and power of haemolysis is help for classification of bacteria Staphylococcus aureus Micrococcus roseus Studying of haemolysis Colonies and pigments of bacteria • Identification of bacteria Biochemical reactions: The more important widely used tests are as under: • a) Sugar fermentation and • Identification of bacteria b) Indole production • c) Hydrogen sulfide production Identification of bacteria • d) Other tests: Citrate utilization; Nitrate reduction; Methyl red test; Urease test; Catalase test; Oxidase reactions. Positive Catalase Test on Staphylococcus aureus Negative Catalase Test on Streptococcus lactis API-20 "Bio Merieux" (France) strip test • Twenty tests are performed on this strip by a simple procedure, saving time and money. Escherichia coli Enterobacter agglomerans Edwardsiella hoshinae Identification of bacteria • Antigenic analysis: by using specific sera we can identify microorganism by agglutination reaction (Serologic Typing of Shigella). The clumping of the bacteria is seen in this circle No clumping of the bacteria is seen in this circle • Identification of bacteria Pathogenicity: For pathogenicity test commonly used laboratory animal models are guinea pig, rabbit, rat and mouse. • Resistance to antibiotics and other agents • Metabolism is the process of building up chemical compounds in the cell and their breaking down during activity to receive the required energy and the building elements. • Metabolism comprises of anabolism (assimilation) and catabolism (dissimilation) • • • • Classification of Media A. On the basic of consistency: Solid media Liquid media Semisolid media Classification of Media • Nutrient media can be subdivided: • 1. Simple media - meat-peptone broth (MPB), meat-peptone agar (MPA) • 2. Synthetic media • 3. Complex media • 4. Special media: a) Enriched media; b) Enrichment media; c) Selective media; d) Indicator and differential media; e) Sugar media; f) Transport media. • 5. Aerobic and anaerobic media according to type of respiration bacteria subdivided into 4 groups: • Obligate aerobes (Brucella) • Microaerophils (H.pylori) • Obligate Anaerobes (C.tetani) • Facultative Anaerobes (E.coli) Sterilization TREATMENT Incineration TEMPERATURE >500 C EFFECTIVENESS Boiling 100 C Thirty minutes of boiling kills vegetative forms of bacteria but may not kill bacterial endospores. There are also toxins that are not inactivated at 100C. Intermittent boiling 100 C Three 30-minute intervals of boiling, followed by periods of cooling kills bacterial endospores. Vaporizes organic material on nonflammable surfaces but may destroy many substances in the process. Autoclave 121 C for 15 Kills all forms of life including bacterial (steam under minutes at 15 endospores. The substance being sterilized pressure) p.s.i. must be maintained at the effective temperature for the entire time. Sterilization Dry heat (hot air oven) 160 C for 2 hours Used for materials that must remain dry. Good for glassware, metal, but not most plastic or rubber items. Dry heat (hot air oven) 170 C for 1 hour Same as above. Note that increasing the temperature by 10 C shortens the sterilizing time by 50 %. Pasteurizatio n (batch method) 63-66 C for 30 minutes Kills most vegetative bacterial cells, including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis. Pasteurizatio n (flash method) 72 C for 15 seconds Effect on bacterial cells is similar to batch method. For milk, this method has fewer undesirable effects on quality or taste. AUTOCLAVES (1) AND HOT AIR OVEN (2) 1 2