Control of Microbial Growth

1. 28 November 2005 M.S. Peppler Dept of MMI mark.peppler@ualberta.ca 1-69 Medical Sciences Building Control of Microbial Growth

2. Control of Microbial Growth Objectives. After today’s session you will understand: 1. The definition of microbial death. 2. The kinetics of death by heat with special consideration for food. 3. Other inhibitors of microorganisms in food.

3. Objective 1a. Defining Microbial Death. Recall "death phase" of growth curve: a natural phenomenon. But note that each phase refers to the state of the entire population, not to the state of individual cells. i.e., the apparent "death" MAY only be non-viability in the lab (e.g., if the growth curve is being determined by plate counts; concept of viable but non-culturable; VBNC). Injured cells MAY recover, depending on treatment (i.e., the recovery medium). SO, we need to remember the distinction between individual cell unculturability, individual cell death, and population “death”.

4. Objective 1b. Defining Microbial Death. How do we achieve cell DEATH?? 3 general ways: (1) heat (flame sterilization/incineration; moist heat). (2) irradiation (UV, gamma rays). (3) chemicals. (a) chemicals for inanimate surfaces e.g., phenol; heavy metals. (b) chemicals for food use and tissues e.g., antibiotics.

5. Lower temperature Log CFU/mL Higher temperature TIME Objective 2a. Kinetics of Death by Heat. DRY HEAT DEATH (pyrolysis/incineration) e.g., Flame inoculating loop. No real kinetics to study. MOIST HEAT DEATH most common type of growth control/killing. e.g.,canned foods; hospital sterilization; boiling water. Empirical data: heat cells at a temperature known to be lethal to the organism in question (e.g., boil E. coli). For cells suspended in dilute liquid (e.g., buffer, water), the relationship between death and time exposure to heat is linear on semi-log graph. i.e. EXPONENTIAL DEATH

6. a. b. 2 min 2 min 2 min 103 cfu ml-1 102 cfu ml-1 101 cfu ml-1 1 cfu ml-1 900 cells die 90 cells die 9 cells die Objective 2b. Kinetics of Death by Heat. What does this mean? • A single event leads to cell death (no complex curves). • A constant PROPORTION of cells dies per unit time • A higher absolute NUMBER of cells are killed in the first time interval than in subsequent intervals. a. b. why is this important to know? Because you need extended heat exposure times to ensure sterilization (to kill "the last few cells"). This is expensive, and may harm product quality.

7. N0 = original CFU k = death rate constant log CFU/mL Nt = CFU after treatment Objective 2c. Kinetics of Death by Heat. How do you determine product "sterilization"?? Can you simply extrapolate to 0 cells? NO There is no "0" on log scale; you actually have killed 0.9 cell. Can you measure 0.1 cell? NO So you calculate as 1cfu/10 ml or 1 cfu/10 cans (etc.). Therefore, you "overkill" i.e., continue heat treatment beyond 100 (1) CFU/mL.

8. Objective 2d. Kinetics of Death by Heat. BY HOW MUCH should you "overkill"??? It depends on ultimate use: e.g., pasteurized beer: if 1 viable yeast cell survives/100 bottles beer (i.e. survival of 10-2) Is it a problem??? NO e.g., SPAM (canned meat product) if 1 viable Lactobacillus survived/100 cans (i.e. survival of 10-2) Is it a problem??? NO e.g., canned green beans if 1 viable Clostridium botulinumsurvived/100 cans (i.e. survival of 10-2) Is it a problem??? YES!! lethal food poisoning!!! (recall production of potent exotoxin)

9. Objective 2e. Kinetics of Death by Heat. How many contaminating organisms is acceptable? 10-8? How do you sample 108 cans of SPAM for 1 CFU ?? Obviously you can’t, so you need to be able to calculate (predict) death using kinetics of heat treatment. This is possible if the RATE of killing of particular species is determined experimentally in that particular product at that particular temperature. These data can then be used to extrapolate to a “safe” time for heat killing. The equation used for moist heat death is very similar to that for population growth, except that the term for doubling (natural log; Log2) is not necessary.

10. N0 = original # CFU Nt = # CFU after heat treatment kd= death rate constant in logs/time unit (e.g., min-1) (do not confuse with growth rate constant) t = time of heat exposure/treatment (e.g., min) kd = log N0 - log Nt t Objective 2f. Kinetics of Death by Heat. The equation used for moist heat death

11. Practical example: Starting with 106 CFU/ml and a death rate constant of 0.5 min-1, how long will it take to achieve 10-2 survivors in clear chicken broth soup?? (idealized example). kd = log N0 - log Nt then… 0.5 min-1 = log106 - log10-2 t t t = 6 - (-2) = 8 = 16 min 0.5 min-1 0.5 min-1 (Reminder: what does 10-2 survivors mL-1 actually mean? 1 survivor per 102 mL, (100 mL) of soup. Objective 2g. Kinetics of Death by Heat.

12. Objective 2h. Kinetics of Death by Heat. This leads to the concept of decimal reductions: The previous example calculated the time for a CFU reduction of 8 log cycles (i.e., 108-fold reduction in CFU). Knowing that the same proportion of cells are killedper unit time, we could have simply determined how long it takes to kill 1 log (i.e., 90% of the cells, leaving 10% [0.1] viable), then multiplied by 8. i.e., kd = 0.5 log min-1. Therefore, 1 log takes 2 min and 8 logs takes 16 min. The time to reduce the viable count by 1 logarithm is the DECIMAL REDUCTION TIME (D).

13. Objective 2i. Kinetics of Death by Heat. BUT is death ALWAYS exponential?? NO!!!! It is affected by the medium being heated: products with a high organic load and/or solids do NOT exhibit straight line kinetics but rather SIGMOIDAL: e.g. thick soup, canned meat or vegetables, etc. because it takes time for heat to penetrate through solids (or thick liquids), and the organic matter provides protection from heat killing for the last few cells…..

14. Objective 2j. Kinetics of Death by Heat. Death rate estimations are very difficult to perform at both ends of the sigmoid curve. Therefore, to be practical, industry adopts a standard of "12D" for safety i.e., 12 log cycle reductionin viable numbers (where D is the decimal reduction time) for the organism being considered under specified conditions of temperature, pressure, etc. e.g., if N0 = 106 CFU ml-1, 12D exposure time would result in 10-6 CFU ml-1 (i.e., 1 can of SPAM in a million) and you wouldn't have to sample 1 million cans of Spam; Do the calculation instead, knowing kd, N0 and calculating Nt for 12 log cycles of death. Note that “D” is different for each species, the temperature used and the medium being heated.

15. Objective 2k. Kinetics of Death by Heat. To be safe, D is calculated for the killing of endospores. “Endospores” are heat-resistant resting states of certain Gram positive bacilli like Bacillus spp. or Clostridium spp.) are much harder to kill than their corresponding “vegetative cells” (i.e., cells that are ready to grow and divide and are not in the endospores state). Killing of endospores also depends on temperature and medium, so it has to be determined empirically for each food and processing temperature. Endospore killing determined in buffer: 10 logs/360 minutes (kd = 0.03 log min-1). Endospore killing in corn juice (liquid at the bottom of the can) 5 logs/1140 minutes (kd = 0.004 log min-1). i.e., k has decreased almost 10-fold (i.e. takes almost 10 times longer to kill endospores in corn juice).

16. Objective 3a. Other Inhibitors of Microbes. Long heating times may compromise quality of product... what are the alternatives?? • 1.Add certain chemicals inhibitory to spoilage organisms, see Table 29.3 11E • Low pH (e.g., pickling with vinegar, lemon juice) pH<5 inhibits many spoilage organisms (but may not kill them); inhibits outgrowth of spores. • NaCl: addition of salt decreases available water and increases osmotic pressure. • c. Sugar: again, increase osmotic pressure to reduce spoilage organisms (except for some fungi).

17. Objective 3b. Other Inhibitors of Microbes. • 2.Combine certain chemicals with heatingso that lower temperatures are required to process the food/product, e.g., • a. Acidity: decreases temperature needed for heat killing. E.g., acidic tomatoes vs. neutral green beans in home canning deaths. • b. Sodium Nitrite (NaNO2)used in bacon, ham, wieners, deli sausages. nitrite + hemoglobin adduct is pink --> pink color. • Some sausages are fermented; made from a raw meat mixtures containing fat, blood, spices, salt, and usually also contains some bacteria from the grinding process. Before fermenting with desirable organisms (e.g., Lactobacillus) need to kill endospores of Clostridium botulinum (“botulus” means “sausage”) • Heat alone would require a long exposure time. So…

18. Objective 3c. Other Inhibitors of Microbes. …instead, add nitrite to medium plus low heat (60-70°C). The combination rapidly kills endospores. Lactic acid bacteria may survive; but if not enough do, inoculate more --> fermentation ---> deli sausage. PROBLEM: nitrite + protein + acid pH in stomach --> nitrosamines --> carcinogens in stomach--> cancer. Option: remove nitrites…but then a chance of botulism?

19. Objective 3d. Other Inhibitors of Microbes. • Other options for food processing: • 3) Irradiation on the electromagnetic continuum • (microwave ovens: heating causes death, not the microwaves). • • Ultraviolet (UV) is mutagenic; damages DNA. • • Ionizing radiation: • gamma rays (e.g., 60Co,137Cs) for foodstuffs. • Cause production of free radicals (recall early lectures) (e.g., hydroxyl radical [OH•], hydroxyl ion [OH–] which cause DNA damage.

20. Next time: Antimicrobial chemicals and antibiotics.