1. Death of micro-organisms and microbial populations Agents that kill micro-organisms Cessation of growth and the death of a microbial population can be associated with : Nutrient depletion pH changes in the cell environment The build-up of toxic bi-products of metabolism Lack of oxygen if the organism is an obligate aerobe Various combinations of the above

2. Agents causing death of micro-organisms and microbial populations : wet heat dry heat chemical agents- these include sanitizers (disinfectants), chemical preservatives and antibiotics extremes of pH radiation – ultraviolet light, ionizing radiation, microwaves low water activity freezing chilling – ‘cold shock’ ultrasonic sound hydrostatic pressure

3. Microbial cell damage can be associated with : cell wall structure cell membranes cell protein, including enzymes RNA DNA mechanisms associated with cell wall formation and cell growth

4. HOW DO WE KNOW WHEN AN ORGANISM IS DEAD? The criterion for death of a micro-organism is failure to grow and reproduce because of irreversible cell damage. Ability to grow and reproduce is shown by : formation of colonies on agar plates turbidity in broth cultures chemical changes produced by metabolic activity that are easily observed

5. Death due to heating (thermal death) 열에 의한 사멸 To determine the heat resistance of m/o’s experimentally with any degree of accuracy is quite tricky. Heating is not instantaneous so that there are problems involving time lags in heating and cooling.

6. Z –VALUES D-value 를 1/10 로 만드는데 필요한 온도 변화치 ( 상승치 ) Moulds, yeasts and the vegetative cells of bacteria normally have z-values ranging from 5 to 8 ℃ (normally 5 ℃ ). Bacterial spores have z-values ranging from 6 to 16 ℃ (normally about 10 ℃ ).

7. The major groups of chemical antimicrobial agents( 물질, - 제 ) phenols and phenolic compounds( 화합물 ) heavy metals( 중금속 ) and their salts( 염 ) alcohols aldehydes anionic detergents( 음이온 계면활성제 ) quaternary ammonium compounds alkylating gases peroxides organic acids alkalis inorganic acids ozone antibiotics( 항생물질 ) synthetic antimicrobial drugs( 화학합성 항균제 )

8. TERMINOLOGY USED TO DESCRIBE CHEMICAL AGENTS THAT KILL MICROORGANISMS (1) Disinfectants( 소독제, 살균제 ) and sanitizers( 위생제 ) - applied to inanimate subjects : floors, walls, working surfaces, processing equipments - unsafe for human tissue / not safe for ingestion - to reduce m/o’s in food industry environment to acceptable level

9. (2) Antiseptics ( 방부제 ) - to kill or inhibit m/o’s on skin or mucous membranes - not for internal use (3) Chemotherapeutic agents ( 화학치료제, 화학요법제 ) - disease control in humans, animals and plants - antibiotics / sulpha drugs ( 설파제 ) (4) Preservatives ( 보존제 ) - added to foods / produced by m/o’s - extend shelf-life of foods

10. Undissociated hypochlorous acid ( 비해리 차아염소산 ) :  oxidize double bonds in fatty acids : membrane damage  oxidize sulphhydryl groups in protein : enzyme inactivation Chlorine levels used in the water and food industries : Production of potable (drinking-quality water) : 0.4 ppm residual chlorine Cooling water used in the canning process : 5 ppm residual chlorine Dairy industry - vats, tanks, pipework : 100-300 ppm

11. FACTORS THAT INFLUENCE THE ACTIVITY OF SANITIZERS (1) Concentration - the higher the concentration, the more effective the sanitizer (2) Contact time - the longer, the greater - manufacturers of sanitizers frequently give minimum contact times for their products (3) pH - sanitizer solution / the environment - undissociated hypochlorous acid increases with an increase in acidity - D value for Bacillus cereus at 25 ppm chlorine : 2.5 min. at pH 6.0 20 min. at pH 9.0

12. (4) Hardness ( 경도 ) of water - calcium and magnesium ions in the water used to dilute the sanitizers  block damaging process (5) Numbers of organisms - the larger, the more difficult to kill (6) Types of organisms - QUATs for G(+) bacteria - chlorine is active against all m/o’s depending on the concentration used

13. (7) Presence of organic matter - protective effect - chlorine required to oxidize any organic matter present - break point chlorination  residual chlorine ( 잔류염소 ) (8) The temperature at which the sanitizer is used - the chemical reaction increases with temperature - Q 10 value

14. NITRATE ( 질산 HNO 3 ) AND NITRITE ( 아질산 HNO 2 ) - cured meats ( 염지육 ) : red color, flavor, inhibit m/o’s - NaNO 3 ( 질산나트륨, 칠레초석 ) - nitrate  nitrite by Micrococcus spp nitrite + myglobin  nitrosomyoglobin  (heating)  nitrosohemochrome ( 심홍색 ) - inhibit wide range of bacteria : Clostridium spp / Bacillus spp / Staphylococcus aureus - not very effective against lactobacilli / Enterobacteriaceae (salmonellae) - Cl. Botulinum : proteolytic strain  inhibited by 100ppm/pH6.2/NaCl 5% non-proteolytic  inhibited by 100ppm/pH6.2/NaCl 3% - Antimicrobial activity of nitrite increases as the pH is lowered.

15. Drawbacks of storage-life testing : Results will not give any indication as to whether the product is safe at the end of its storage life. Pathogens and indicators may be introduced into a product on a haphazard basis, e.g. via a new batch of raw materials or a contamination event resulting from poor factory hygiene, so that even if microbiological testing for pathogens and indicators is carried out, negative results have no value. Batches of raw materials may vary in terms of their content of spoilage organisms so that samples taken from a particular product run may be unreliable. Products need to be produced in the factory environment before testing can be carried out. Formulations produced in the laboratory or test kitchen are unreliable as materials for testing as they may not be subject to the same sources of contamination as the factory product.

16. CHALLENGE TESTING( 유발 검사 ) - Artificial inoculation of the food with spoilage m/o’s or pathogens (indicator m/o’s) - Considerable variables for challenge test : What is the expected storage life of the product? At what stage in the development of a new product is the challenge test going to be used, e.g. test kitchen or full production run? Which organisms are going to be used for the challenge? How are the challenge organisms going to be cultured? How is the organism going to be inoculated into the food? Are any preservatives used evenly distributed in the product? What storage conditions are going to be used and do these take into account possible abuse during distribution, retailing or handling by the consumer?

17. PREDICTIVE MODELLING ( 예측모델링 ) - mathematical modeling / computational microbiology - computer program by USDA  Gompertz equation  parameters : temperature, pH, Aw, salt, etc.  for major foodborne disease causing bacteria - not in practical use

18. Food Spoilage The nature of food spoilage Spoiled food  smell, taste, appearance, texture, foreign substance Reasons for food spoilage : Organoleptic changes ( 관능적 변화 ) by microorganisms Chemical changes  chemical oxidation / browning Physical damage Freezer burn ( 냉동상해 ) Staling Ripening Presence of foreign bodies Contamination with chemical agents - hanging game / over-ripened bananas

19. COMPOSITION OF THE CONTAMINATING MICROFLORA - Bacteria from fresh carcass meat : Gram-nagative rods and coccobacilli Acinetobater, Aeromonas, Alcaligenes, Citrobacter, Enterobacter, Escherichia, Flavobacterium, Moraxella, Proteus, Pseudomonas, Salmonella, Shewanella, Yersinia Gram-positive rods Bacillus, Brochothrix, Clostridium, Corynebacterium, Latobacillus, Listeria Gram-positive cocci Enterococcus, Lactococcus, Micrococcus, Pediococcus, Staphyllococcus Moulds : Mucor, Rhizopus, Thamnidium, Cladosporium, Geotricum, Sporotrichum Yeast : Candida spp

20. What determines the composition of the spoilage microflora? - spoilage flora  dominated by a few m/o’s “Which component of the microflora becomes dominant is determined by a complex interaction between the components of the contaminating microflora (implicit factors), the storage environment (extrinsic factors) and the physicochemical properties of the food (intrinsic factors) ”

21. INTRINSIC FACTORS nutrient content of the food natural antimicrobial substances pH of the food and its buffering capacity oxidation reduction potential (Eh) water activity of the food mechanical barriers to microbial invasion

22. EXTRINSIC FACTORS temperature at which the food is stored gaseous atmosphere surrounding the food relative humidity of the atmosphere surrounding the food time ex) high Aw foods with pH above 5.0 vs. high Aw foods with pH below 4.2

23. INTERACTIONS BETWEEN COMPONENTS OF THE CONTAMINATING MICROFLORA - interactions between mixed population of m/o’s - growth of a dominant organism in a food is not a static situation  “succession” occurs ex) milk at ambient temperature : streptococci  lactobacilli  mould  putrefactive anaerobes

24. Koch’s postulates applied to food spoilage : The organism suspected of causing spoilage is isolated from the spoiled food, cultured in pure form and identified in the laboratory. A pure culture of the suspect organism is introduced into the food. To confirm the organism as the spoiler, symptoms produced must be the same as those originally described and the suspect organism isolated from the experimentally spoiled food.

25. “Under certain circumstances spoilage symptoms may be evident in a food but no organisms can be isolated.” e.g.) flat sour spoilage by Bacillus stearothermophilus - Detection of dead cells  microscopy, immunoassay, PCR (Polymerase Chain Reaction)

26. Changes in foods caused by spoilage micro-organisms Spoilage symptoms : general appearance color texture odor or flavor a mixture of the above GENERAL APPEARANCE - mouldy : typical fluffy appearance of fungal hyphae - slimy : bacterial growth

27. COLOR DUE TO ORANISMS - mould spores / mould hyphae - pigment by bacteria e.g.) Halobacterium salinarium : red pigment, salted fish Pseudomonas fluorescens : greening in poultry Rhizopus spp : black spot on frozen meat COLOR ASSOCIATED WITH CHEMICAL CHANGES - blackening of egg : hydrogen sulfide, Proteus spp - greening of processed meat : hydrogen peroxide, lactobacilli - brown rot of apple : polyphenol oxidase, Sclerotinia fructigena

28. TEXTURE - pectinase  Erwinia carotovora, Penicillium citrinum cf. parenchyma cells ( 식물 유연조직 ) - proteinase  Pseudomonas fluorescens