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Food Microbiology 1 Unit 2 Indicators in Food Microbiology II Yeasts and Molds o Grow slowly in the presence of bacteria o Proliferate when bacterial growth is restricted For instance in: Refrigerated temperatures High sugar/salt Dried foods High Acid Yeasts • Five times larger than bacteria • True yeast reproduce via sexual ascospores although they can also reproduce asexually • Sexual reproduction: two yeast cells fuse to form a zygote, ascospores are formed within a zygote Yeasts Those found in food reproduce asexually by Budding Budding: new cell forms as a small outgrowth of the old cell. The bud gradually enlarges and eventually breaks away. Yeasts • Grow aerobically (in presence of oxygen) and produce predominantly CO2 (bread makingleavening bread) • Cannot grow anaerobically but metabolize carbohydrates to predominantly ethanol (production of alcohol, beer, wine, industrial; alcohol) Saccharomyces cervisiae Wild Yeasts • Produce a range of undesirable products (especially in alcoholic fermentations) Hydrogen Sulfide Organic Acids Phenolics • Gas blowing in vacuum packed foods Molds • Filamentous fungi • Have no roots, stems, leaves or chlorophyll • Fundamental unit is the hypha, a mass of hyphae is mycelium •They reproduce sexually, asexually or by both Perfect fungi Imperfect fungi Molds • Strict aerobes (require O2) • Grow at low temperatures, low pH and low water activity Beneficial Molds • Provide an indication that the food is not fit for consumption • Fermentations Aspergillus: Oriental fermentations, produces citric acid & gluconate Penicillium roquerfortii: Cheese ripeming Food Safety Indicators Safety Indicators • Pathogens usually occur in low numbers and hard to pin down • May not necessarily be pathogenic but commonly associated with the origins of pathogens • Used as a measure of sanitation and history of the product Safety Indicators • Mesophilic Aerobic Plate Counts • Bacterial Spore Counts • Enterobacteriaceae •Total coliforms • Fecal coliforms • E. coli • Staphylococcus spp • Mold counts Mesophiles Grow best between 30oC40 oC Mesophilic Aerobic Plate Counts • Levels indicate bacteria that can proliferate at body temperature • High levels in cooked products or foods from plant origin could indicate presence of pathogens Bacterial Spore Count Spores (dormant form of the organism) are produced by Clostridium spp and Bacillus spp when conditions become unsuitable for growth (heating, freezing, drying, etc..) Bacillus Spores Clostridia Spores • Spores can remain dormant for years and are often found in soil and dust Sporulation In bacteria, is the process of forming an endospore. Transition between vegetative growth and sporulation is a response to external triggers • Exhaustion of nutrients • Accumulation of metabolic products From initiation until spore release can take > 10h • Not all cells will undergo sporulation •Typically occurs under sub-optimal growth conditions Step 1: A vegetative bacterium about to enter the endospore cycle Step 2: The nucleoid replicates Step 3: A spore septum forms Step 4: Each nucleoid becomes surrounded by its own cytoplasmic membrane. Step 5: The cytoplasmic membrane surrounds the isolated nucleoid, cytoplasm, and membrane from the previous step forming a forespore Step 6: The forespore is completed and the other molecule of DNA is eventually degraded Step 7:A thick protective layer of peptidoglycan called the cortex is synthesized between the inner and outer forespore membranes. Step 8: Another protective layer called the spore coat and composed of protein is synthesized Step 9: Sometimes a final layer called the exosporium is added. As the vegetative portion of the bacterium is degraded, the completed endospore is released Step 10: With the proper environmental stimuli, the endospore germinates. As the protective layers of the endospore are enzymatically broken down, a vegetative bacterium begins to form and emerge Step 11: The vegetative bacterium now begins to divide by binary fission Step 12 Spores are Very Resistant Treatment to inactivate 90% Spore Vegetative Cell Wet Heat 14 mins @ 95oC < 15 sec @ 65oC Dry Heat 33 mins @ 120oC 5 mins @ 90oC 10% Hydrogen Peroxide 40 mins < 2 mins Resistance of Endospores • Spores are packages of DNA • Structures of spores primarily function to protect DNA and provide resources for initial germination events • DNA of spores is contained within the core • Spores are the most resistant structures encountered Spore Structure Spore Germination Spore germination (to become vegetative cell) is stimulated by activation: • Mild heat (heat shock) @ 70o C for 10 mins • High pressure • Oxidizing or reducing agents Activation enhances permeability of spores to germinating agents. If none are present the spore returns to its dormant state Germinating Agents • L-alanine, fructose, glucose • Once initiated, spore germination cannot be reversed • Very rapid: initiation to growing cells < 40 mins • Not all spores germinate (10% from a super dormant population) Significance of Spores To inactivate spores, focus on damaging DNA Inactivation of spore germination systems result in spore death Spores are commonly used as indicators to verify sterilization process (thermal treatment, irradiation) Significant spores (in terms of food safety) encountered in foods: • Clostridium botulinum • Clostridium perfringens • Bacillus cereus