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Classification of Microbes in the Food Industry

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Food Science
FOOD 3230
Keith Warriner

Food Microbiology F-13 Unit 1 – Classification of Microorganisms Only __% of bacteria are pathogenic Only __5% can be cultured Religion and Food Safety • In the absence of science, religion was the main contact route for populations • Most early religion banned pork (key source of trichinosis parasite) • Separation of milk and meat, in addition to slaughter techniques were advanced food safety initiatives Food Insecurity • Availability of foods • Spoilage o Potato famine o ~40% of food wasted • Farming & preservation o Chemical and natural preservatives Food Preservation • Fundamental techniques in food preservation were known over 10,000yrs ago o Drying o Heating o Freezing o Fermentation o Chemical preservatives Microbiology as a Science • 1680: bacteria first observed by Leeuwenhoek using a compound microscope (x300 mag) • 200yrs later until same observations were repeated • Indirect evidence provided the existence of microbes Theories of Life • Spontaneous generation of life o Survived until early 1800s o Pasteur using swan neck flasks proved that bacteria were carried in air  He went on to develop milk pasteurization • Microbes are carried/transferred and grow on foods • Other advances o 1873- Lister develops first antiseptic o Koch- selective agar and isolation  Koch postulates Pathogens • 1800s- typhoid, plague, cholera, dysentery, TB • High population density • Poor sanitation • Limited treatments • Foodborne illness just one of many causes • Start of water chlorination in cities leads to decrease in TB 20 Century • Sanitation • Antibiotics • Freezing/refrigeration • Aseptic technologies • Irradiation • Epidemiology • Food safety regulation Even with the introduction of HACCP, cases of foodborne illness have been higher than in the 1990s. Why? Possibly due to the “organic craze” and farmers market where there are no regulations. Also because of new strains of certain microbes and the antibiotic resistant strains. st 21 Century • Continue to reduce incidence of foodborne illness • Alternative preservation technologies • Risk analysis • Food safety culture • Modernization of food and drugs regulations • GI tract microflora and health, probiotics Prokaryotes (Bacteria) vs Eukaryotes (plant/animal cell) • 1940s- electron microscope- bacteria have no nucleus Broad Classification • Good (fermentations) o Food production  Lactic acid bacteria- yogurt, cheese  Yeasts- bread, beer  Mold- cheese, single cell protein (SCP) o Antibiotics o bioremediation • Bad (spoilage) o Food spoilage o Cans/packaging blowing up o Off odours and flavour o Pigmentation o Slime formation • Very bad (foodborne illness) o Disease causing bacteria o Bad mold o Protozoa o Virus Taxonomy- the study of the classification, organization and naming of living things (similarity) Phylogeny- relationships between organisms (evolutionary links) Biochemistry (catabolic and anabolic pathways) • Specific enzymes – catalase, oxidase • Metabolic products – eg fermentation • Sugar utilization (API identification kits) • Growth characteristics (temperature, an/aerobic) Serology (specific antibodies) • Flagella • Receptors • Toxins • Phage typing (specific binding and lysis of antibodies) Amino acid sequencing (SDS page) – fatty acids Flow cytometry – differences in electrical conductivity Development of more sophisticated techniques has led to reevaluation of bacterial taxonomy -Bergey’s Manual of Systematic Bacteriology still regarded as the foundation for classifying bacteria Genotyping • How similar microorganisms are at the DNA level • Evolutionary links o Mutations in DNA increase evolutionary distance • DNA hybridization o Complementation of DNA strands • DNA sequencing –eg 16S rRNA o The 16S rRNA gene: most useful molecule for determining evolutionary relationships o General sequence highly conserved but point mutations slowly occur over time o Sequence provides a measure of evolutionary distances Naming of Bacteria • Phenotypic characteristic • Place name • Source of original isolation • Person who discovered Classification Ex • Kingdom- prokaryote • Phylum- gracilicutes • Class- scotobacteria • Family- enterobacteriaceae • Genus- Escherichia • Species- coil Nomenclature of Bacteria Escherichia coli ATCC 13704 GENUS SPECIES ID # • Names usually derived from latin or greek • Coli – belonging to colon • Escherichia – named after Escherich who discovered the bacterium in 1895 • Abbreviated to E. coli • Underline or italicized, species name not capitalized • Salmonella is an exception since there is only one species (enterica) o Salmonella composed of closely related serovars o Serotypic names are used in place of species Sources of Microorganisms • Primary sources are any environment that permits growth and survival o Soil, plants water o Animals, hide, skin o Intestinal tract • The ability of microorganisms to utilize nutrient sources, compete with other microflora and survive environmental stresses is key. • Routes of transfer- air, water, contact What do food microbiologists do? • Study microbes that inhabit, produce or contaminate foods • Establish criteria to establish if a food or an environment is acceptable • Develop and review policy to ensure the safety of foods • Validate and verify intervention methods to ensure the stability and safety of foods • Provide info to the public on food safety Unit 2 – Microbes of Importance in Food Microbiology Indicators • Screening for specific spoilage or pathogenic bacteria is like looking for a needle in a haystack • In food micro, an index (or indicator) is needed to confirm that the food is safe or has an adequate shelf life • Provide a gauge of product shelf life • Highlight potential hazards • An assessment of the previous history of food product • Evaluation of the efficacy of control measures to prevent and/or inactive microbial activity Spoilage Indicators • Types of counts will depend on the nature of the product o Total aerobic count  An assessment of the general lvls of bacteria  High numbers typically indicate significant bacterial activity  Conditions plates are incubated under reflect the food environment • Ex low incubation temps for samples derived from chilled foods o Psychrotrophic count  Grow at low temps  Responsible for spoiling refrigerated foods  Numbers provide an estimate of shelf life  Includes • Yeasts and molds • Main concern is Pseudomonas spp o Lactic acid bacteria o Yeast and molds Pseudomonads • Aerobic, Gram negative rods • Non-fermentative • Simple nutrition requirements and can metabolize a wide range of substrates (even crude oil) • A number form extracellular polysaccharide (biofilms) at low incubation temps and/or in high sucrose environments • Enzymes produced by pseudomonas: o Proteinases  Enzymes secreted into foods break down proteins leading to generation of ammonia, sulfur, and/or organic acids (butyric, acetic) o Lipolytic  Enzyme secreted by cells hydrolyze triglycerides and accelerate lipid oxidation leading to rancidity o Pectolytic  Enzymes breakdown plant cell walls leading to loss of turgor pressure • Pigmentation o Fluorescent pseudomonas release siderophores to assimilate iron o Siderophores are pigmented (fluorescent) • Important features o Quorum sensing o Biofilms Quorum sensing- bacterial cell to cell communication to talk to each other and coordinate activities, cell density affect. It increases spoilage potential of populations, stimulates biofilm formation and increases virulence of pathogens. It depends on the activation of a sensor kinase or response regulator by a small diffusible signal molecule. Signal molecule for gram negative = N-acylhomoserine lactones, gram positive=cyclic peptides. Only by pooling the activity of a quorum of bacterial cells can a bacterium be successful. Quorum sensing blockers- regulation of multiple gene expression by LuxR/LuxI homologues. Algae produce quorum sensing blocking agents. Advantages • Prevent cell communication and detrimental functions • Reduce generation of antibiotic resistant strains • Prevent biofilm formation Recognition & Communication in the Unicellular Bacterial World • Sexual exchange – conjugation • Protecting your niche • Combating host defenses • Population migration Two basic strategies for controlling bacteria: 1. Kill the pathogen 2. Modify cellular function Biofilms • Slow growing Pseudomonas (due to temp or stress) produce extracellular polysaccharides • Method for the bacteria to utilize energy sources without growing • Primary colonizers form a slime layer on a surface • Secondary grow in the slime o Often use the polysaccharides and waste products made by the primary for carbon and energy sources • Nutrition and energy o Energy production in the bacterial cell becomes difficult o Bacterial stress mechanisms switched on: Sigma B etc o Inessential functions are switched off • Why do cells become integrated into biofilms? o Nutrients are pulled from the environment o Synergistic growth with other microbes o Genetic exchange to enhance fitness o Protected from environmental stress • Significance of biofilms o Cause slime layers o Enable cells to resist desiccation and sanitizers o Potentially can embed pathogenic bacteria that subsequently become protected from environmental stress and sanitizers Representatives • Opportunistic pathogens o P. aueruginosa • Spoilage o P. fluorescens o P. putrefacines o P. fragi Lactic Acid Bacteria • Gram positive non spore forming rods or cocci • Facultative anaerobes • Genera of significance: o Lactococcus o Lactobacillus o Leuconostoc o Pediococcus o Streptococcus o carnobacterium o bifidobacteria • widespread in the environment (plants and GI tract of animals) • fastidious (complex nutritional demand) • tolerate low pH and high ethanol • can be beneficial or spoiling depending on fermentation products and food type • homofermentative (ferment carbs to predominantly lactate) o lactobacillus planetarium o lactobacillus delbrueckii • Facultative homofermentors (prefer homolactic fermentation but can perform heterolactic) o Lactococcus lactis subsp lactis • Heterofermentative (ferm carbs to a mix of products ie lactate, acetate and ethanol) o Leuconostoc paramesenteroides o Lactobacillus brevis Homofermentative Food Fermentations Heterolactic Metabolism Probiotics • “live microorganisms that when administered in sufficient amounts confer a health benefit in the host” • Safe, viable, provide benefits, bile resistant, acid resistant Yeasts and Molds • Grow slowly in the presence of bacteria • Proliferated when bacterial growth is restricted o Refrigerated temps o High sugar/salt o Dried food o High acid • Yeasts o Aerobic growth produces predominantly CO2 (bread making) o Cannot grow anaerobically (exceptions) but metabolize carbs to predominantly ethanol o Saccharomyces cervisiae • Wild yeasts o Produce a range of undesireable products (esp in alcoholic fermentation)  Hydrogen sulfide  Organic acids  Phenolics o Gas blowing in vacuum packed foods • Molds o Penicilliumi spp • Beneficial molds o Provide an indication that the food is not fit for consumption o Aspergillus – oriental fermentations, produces citric acid and gluconate o Penicillin roquefortii- cheese ripening Food Safety Indicators • Pathogens usually occur in low numbers and hard to pin down • May not necessarily be pathogenic but commonly associated with origins of pathogens • Used as a measure of sanitation and history of the product • Mesophilic aerobic plate counts o Standard plate count 35’C o Levels indicate bacteria that can proliferate at body temp o High levels in cooked products or foods from plant origin could indicate presence of pathogens • Bacterial spore counts o Spores produced by;  Clostridium spp (anaerobic)  Bacillus app (facultative an)  Geobacillus  Alicyclobacillus • Enterobacteriaceae • Total coliforms • Fecal coliforms • E. coli • Staphylococcus aureus • Mold counts Sporulation • Transition btwn vegetative growth and sporulation is a response to external triggers o Exhaustion of nutrients o Accumulation of metabolic products • From initiation to spore release can take >10hrs • Not all cells will undergo sporulation • Typically occurs under sub optimal growth conditions • High population densities • Resistance of endospores: o Spores are packages of DNA o Structure of spores primarily function to protect DNA and provide resources for initial germination events o DNA of spores is contained within the core  Spore core is composed of dipicolinic acid complexed with calcium (DPA:Ca)  DPA:Ca dehydrates spore setting the DNA in a solid matrix (spores appear bright when viewed under a microscope)  Small acid soluble proteins (SASP) stretch the DNA to provide further rigidity and protection  Core also contains enzymes, RNA, NADH, and 3- phosphoglyceric acid required during germination  The structure is inert and no metabolic or catabolic activity occurs during dormancy o Spore cortex surrounds the core  Its composed of peptidoglycan but diff composition than found in vegetative cell  Function is not yet known but thought to help maintain core dehydration o Outer sporefore membrane is functional and highly impermeable o Spore coat composed of proteins forms an impermeable layer  Both function to stop diffusion of the antimicrobial molecules into the inner spore structures • Spore germination; o Stimulated by activation  Mild heat treatment (70’C for 10 min)  High pressure  Oxidizing or reducing agents o Activation enhances permeability of spores to germinating agents. If none are present, the spore returns to its dormant state o Germinating agents;  L-alanine (D-alanine competitive inhibitor)  Inosine, asparagine, glucose, fructose and KCI mix (IAGFR)  Once initiated spore germination cannot be reversed  Super dormant spores exhibit delayed germination o Sequence of Events;  Spore activation and germination activated  DPA:Ca released from core  Spores become phase dark with hydration of core  Enzymes within the cortex begin degradation of the structure enabling greater rehydration  Enzymes present within core degrade SASP to provide substrates for energy and redox (NADH) compounds  Stored 3-phosphoglyceric acid is metabolized to provide energy  After 10 minutes, external metabolites are used to produce energy  Resistance to heat, UV and sanitizers is lost  Spore elongates into a cell with the germ cell wall functioning as the outer membrane  Cell initiates DNA expression and replication  Cell outgrowth (division) occurs o Germination is very rapid: initiation to growing cells <40 min o Requires no exogenous energy sources or DNA expression during initial stages o Not all spores germinate (10% form super dormant population) • Significance of spores o Main modes of spore inactivation focused on damaging DNA o Inactivation of spore germination systems can also result in spore death o Spores are the most resistant structures encountered o They’re commonly used as indicators to verify sterilization process o Significant spores (in terms of food safety) encountered in food;  Clostridium botulinum  Clostridium perfringens  Bacillus cereus  Bacillus anthracis Enterbacteriaceae Family  Gram negative rods, aerobe or facultative anaerobe  Simple nutritional requirements  Ferment carbs to form gas and acid (not all ferment lactose, for ex Salmonella)  Enterics are all oxidase negative  Enterics reduce nitrates to nitrites  Perticious flagella if motile  Representatives; o E. coli o Citrobacter o Enterobacter o Klebsiella o Proteus o Erwinia o Serratia o Salmonella o Shigella o Yersinia Coliforms  Special group of bacteria within the enterobacteriaceae family  All share common characteristics o Gram negative, non spore forming rods o Facultative anaerobes o Ferment lactose o Found in soil, water, GI tract of animals  Fecal coliform bacteria; o Subgroup of coliforms and includes those typically associated with fecal contamination o Selected against non-fecal coliforms by high incubation temperature used during cultivation (42’C for 24hr)(ie non-fecal coliforms adapted to lower temperatures) o Ex  E. coli  Some Klebsiella spp  Some Enterbacter spp o Indicative of fecal contamination if recovered from food products or contact surfaces Stapylococcus  Gram positive cocci  Mesophiles  Facultative anaerobes  Grow in the presence of 10% NaCl  Relatively resistant to desiccation  Catalase positive  Commonly found on the skin/nose of humans/animals  Significance; o Presence on food indicates extensive handling/mishandling o Inadequate sterilization of equipment and contact surfaces o Possible presence of toxin from S. aureus Moulds  Cause spoilage but certain strains also produce toxins (mycotoxins)  Significant in grains and pulses  Can accumulate in meat, esp poultry  Toxins very stable and resistant to heat  Accumulate in the body  Mycotoxins; o Broad class of highly toxic secondary metabolites from mold growth on specific substrates o Ex. Aflatoxin (Aspergillus flavus) is a potent carcinogen. Where it is most toxic and prevalent, can cause losses in animal production o ~200 recognized mycotoxins o Aspergillus flavus;  Found in stored grains, such as peanuts. It has also been long used in the orient in fermented foods such as sake, tofu and soysauce (A. oryzae)  Seldom produces alfatoxin when other fungi present  Greatest risk in peanuts  Aflatoxins; • Group of closely related compounds (B1,B2,G1,G2) produced by A. flavus and A. parasiticus • Found in cereals, nuts, spices, dried figs • Acutely and chronically toxic in animal studies. Recognised animal/human carcinogens, with aflatoxin B1 being the most potent. They should be reduced to the lowest levels technologically possible in animal and human food chains • Acute: have a rapid effect but does not persist • Chronic: accumulation of effects over time • Major problem in Ontario • Regulatory lvls 1million in Canada • Underestimation as only 1:10 cases are reported Estimating Cost of Foodborne Illness • Lost productivity: $495/day • Doctor visit: $75/visit • Hospital: $770/day • Chronic illness: $1.68mil • Mortality: $9.7mil Top Foodborne Pathogens in North America • Campylobacter • Salmonella • Clostridium spp • Staphylococcus aureus • E. coli O157:H7 • Bacillus cereus • Listeria monocytogenes Body Defense Systems • Body has evolved defense systems to counter pathogens • Symptoms of foodborne illness are caused by triggering the bodies defenses • Respiratory tract o Mucus o Ciliated epithelium o Antibody o Phagocytosis • Eyes o Washing by tears o Lysozomes • Digestive tract o Stomach acidity o Normal flora o Intestine-alkaline pH o Mechanical flushing o Enzymes o Bacteriocins • Skin o Anatomic barrier; sweat, sebum o Antimicrobial secretions; lactic acid, free fatty acids o Low PH (GS) o Commensal microbes • Genitourinary tract o Washing by urine o Acidity of urine o Lysozyme o Vaginal lactic acid Constitutive and Induced Defenses Defence Mechanisms • Epithelial barriers o Skin o Oropharyngeal mucosa o Respiratory tract • Bulk flow o Flow in GI tract o Flow in urinary tract • Hummoral Immunity o Antibodies o Complement (antibody mediated or non specific fixation) o Enhanced phagocytes of antibody or complement coated organisms • Phagocytes- kill by oxidation and defenses o Polymorphonuclear leukocytes o Macrophages • Neutrophils- white blood cells that release “bleach” to kill nearby cells and themselves • Natural killer cells- attack body cells that have been infected, do not attack microbes directly Inflammation • When an area of tissue is fighting off an infection • Blood vessels dilate and become more permeable • Characterized by redness, heat, and pain • Fever increases metabolism so that the defensive and repair functions occur more rapidly Foodborne Disease • Intoxication o Ingestion of toxins present in foods o Exotoxin- released into foods during cell growth and metabolism o Viable bacteria don’t need to be ingested o Symptoms can be displayed within hours or days o Mild symptoms – death o Key bacteria;  Staphylococcus aureus  Clostridium botulinum  Mold mycotoxins • Infection o Pathogenic bacteria are ingested and invade intestinal mucosa o Multiply within mucosa or spread to other organs o Incubation periods typically >2days since pathogens have to become established o Endotoxins- lipopolysaccharides associated with the outer cell wall o Symptoms include diarrhea, sickness, fever (response to fighting infection) o Key bacteria;  Salmonella  Campylobacter  Listeria monocytogenes  Yersinia • Toxico-infection o Pathogenic bacteria enter the body o Toxins produced dur
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