Destruction of microorganisms

Destruction of microorganisms When the environment has a lethal effect on the microorganisms (high temperature, irradiation, disinfectant) cells are injured and killed as key cellular components are destroyed and cannot be replaced. The change of the viable cell count in time is directly proportional to the concentration of living cells: dN/dt = -k·N N: viable cell count, dN/dt: rate of the change of viable cell count k : death rate coefficient

Destruction of microorganisms The death of the microbes caused by a lethal effect of constant intensity is an exponential process. The concentration of the living cells exponentially decreases in time: N = N0·e(-k·t) Turn on logarithmic form, we can get the equation of the survival curve:

Destruction of microorganisms If the intensity of the lethal effect (temperature, disinfectant concentration, activity of the irradiation) is constant, the survival curve is linear. The death rate coefficient (k) can be calculated from the slope of this curve. In microbial practice instead of the death rate coefficient the decimal reduction time (D) is used. Decimal reduction time (D) is the time for the surviving population to be reduced by 1 log cycle. The surviving fraction is 1/10, the killed fraction is 9/10 of the initial living cell concentration.

Destruction of microorganisms Survival curve (heat treatment)

Destruction of microorganisms Connection between the decimal reduction time (D) and death rate coefficient (k) D = 2.303/k In case of irradiation the survival curve represents the logarithm of living cell number as a function of absorbed dose (Kgy), and the D value (Decimal reduction dose) means the dose necessary to reduce the survival cell number with one log unit.

Destruction of microorganisms Survival curve (irradiation)

Destruction of microorganisms Microbe 6D dose E.coli 1.5-3.0 Lactobacillus spp 2-7.5 Salmonella enteritidis 3-5 Micrococcus spp Salmonella typhimurium Deinococcus radiodurans >30 Vibrio parahaemolyticus < 0.5-1 Aspergillus flavus 2-3 Pseudomonas fluorescens 0.5-1 Penicillium notatum 1.5-2 Bacillus cereus 20-30 Saccharomyces cerevisiae 7.5-10 Cl. botulinum Type A Viruses

Destruction of microorganisms The measure of the resistance of microorganism to the lethal effect is the decimal reduction time, which is highly affected by the environmental factors (temperature, water activity, pH, redox potential). The higher the intensity of the lethal effect, the shorter the decimal reduction time. In food industry, there are three methods for the destruction of microbes; heat treatment, irradiation, disinfection.

Destruction of microorganisms Effect of temperature on the survival curves

Destruction of microorganisms Effect of temperature on D values

Destruction of microorganisms 12D concept Food safety requirement for every cases when the growth and toxin production of Clostridium botulinum is possible (pH>4.5, no preservative additives present) that the heat treatment has to result in minimum 12 log unit reduction in the Cl. botulinum spores. According to the 12D concept requirement, in the sterilization processes the heat treatment time means minimum 12 D of Clostridium botulinum.

Destruction of microorganisms Time equivalent of the heat treatment (F0) The heat treatments in the food industry, apart from a few exceptions, are not isotherm processes; they consist of heating, temperature keeping and cooling sections. Destruction of microbes takes place in every section and the result is the sum of the partial destruction. In order to compare the several heat treatment processes, their microbial effectiveness is given by an isotherm heat treatment for F0 minutes at 121.1 °C, referring to the thermal destruction of Clostridium botulinum.

Destruction of microorganisms Heat treatment Heat treatment is the most important technique in food preservation. The aim of a heat treatment technology is to reach a microbiological safety without degradation of vitamins, sugars, proteins etc. in foods. The planning of a proper heat treatment in the industry is based on the Bigelow model, which describes the thermal death time (TDT) as a function of temperature.

Destruction of microorganisms Thermal death time (TDT) is the time necessary for n decimal reductions of the population. The survival fraction is 10-n. As decimal reduction time means 1 decimal reduction in survivors, TDT = n·D Bigelow model. The connection between the thermal death time and temperature:

Destruction of microorganisms Heat destruction curve (TDT = 12D)

Destruction of microorganisms F value: the thermal death time (TDT) at 121.1 °C (250 F) z value: The temperature change, which results in a tenfold (1 log) change in TDT. Mathematically the z value is the negative reciprocal of the slope of the heat destruction curve. Vegetative cells: z = 3-7 °C, average of 5 °C Bacterial endospores: z = 10 °C Physical-chemical changes in foods: z = 30-32 °C

Destruction of microorganisms The z value is the measure of the temperature dependence of heat destruction rate, similarly to the Q10 value, which gives the effect of a 10 °C temperature increasing on the reaction rate. Vegetative cells: Q10 = 100 Endospores: Q10 = 10 Chemical reactions: Q10 = 2

Destruction of microorganisms Comparing the Q10 values of heat destruction to that of the chemical reactions, it is obvious that the temperature dependence of the thermal death is far more higher than that of chemical reaction. Increasing the temperature with 10 °C, the rate of chemical reaction will be twofold, while the heat destruction rate grows up to 10-100-fold. This recognition has led to the introduction of the UHT (ultra high temperature, 135 °C, 1 second) process in the heat treatment.

Destruction of microorganisms Do not confuse F and F0! F is a microbiological feature and it means the thermal death time at 121.1 °C. F0 is a technological feature and it means the effectiveness of the heat treatment. (The effect of the heat treatment on Clostridium botulinum spores is equal with an isotherm heat treatment for F0 minutes at 121.1 °C). As the 12 D121.1 of Clostridium botulinum spores is 2.52 minutes, the minimum requirement for a sterilization process is that F0 = 2.52 minutes.

Heat resistance of microorganisms Vegetative organisms The vegetative organisms including the vegetative form of the endospore forming bacteria have a relatively low heat resistance. Most of them could be destructed by pasteurization, which means a heat treatment below 80 °C. Some heat resistant ascospore forming moulds (Byssochlamys fulva, Neosartoria fisheri) have high heat resistance and sometimes cause problems in tomato juice and soft drinks. Their elimination claims a heat treatment near 100 °C for 20 minutes.

Heat resistance of microorganisms Heat resistance of vegetative microbes Vegetative organisms (z ~5 °C) D (min) Salmonella sp. D65 0.02 – 0.25 Salmonella senftenberg D65 0.8 – 1.0 Staphylococcus aureus D65 0.2 – 2.0 Escherichia coli D65 0.2 – 1.0 Yeasts and Moulds D65 0.5 – 3.0 Listeria monocytogenes D60 5.0 – 8.3 Campylobacter jejuni D55 1.1

Heat resistance of microorganisms The endospores of bacilli and clostridia Mainly due to their very low water activity (aw = 0.37) , are far more heat resistant than vegetative cells; thermophiles produce the most heat resistant spores while those of psychrotrophs and psychrophiles are most heat sensitive. The bacterium endospores can be killed with heat treatment over 100 °C.

Heat resistance of microorganisms Heat resistance of bacterium endospores Bacterial endospores (z~10°C) D121 (min) B. stearothermophilus 4 – 5 * Cl. thermosaccharolyticum 3 – 4 * Desulfotomaculum nigrificans 2 – 3* B. Coagulans 0.1 Clostridium botulinum types A & B 0.1 – 0.2 Cl. sporogenes D80 0.1 – 0.3 * Heat resistance is higher than that Clostridium botulinum

Heat destruction of microorganisms Heat resistance of bacterium endospores Some bacterial endospore have higher heat resistance than Cl. botulinum. The spores of these microbes could survive the F0 = 2.52 min value and spoil the food. In case of natural products (without preservative additives), the time equivalent of the sterilization process extends 2.52 min.

Heat destruction of microorganisms Some typical F0 values for canned foods Food F0 (min) Asparagus 2 – 4 Beans in tomato sauce 4 – 6 Carrots 3 – 4 Peas Milk pudding 4 – 10 Meats in gravy 8 – 10 Potatoes Mackerel in brine Meat loaf 6 Chocolate pudding

Heat destruction of microorganisms Effect of pH on heat resistance The heat resistance is the highest at pH optimum of the growth. Apart from this optimum, in acidic and alkaline range, the heat destruction rate (k) increases Effect of water activity on heat resistance Heat resistance increases with the decreasing water activity for every microorganisms. The high heat resistance of the bacterial endospores is due mainly to their low water activity (about 0.37)

Disinfection kinetics The concentration dependence of the death rate coefficient is expressed by the following equation: k2 and k1 are the death rate coefficients corresponding to c2 and c1 concentrations, n is the concentration exponent. It represents the sensitivity of the effectiveness on the dilution. The higher the concentration exponent, the higher the sensitivity to dilution.

Disinfection kinetics Determination of concentration exponent

Disinfection kinetics Concentration exponent of some disinfectants Compound n Hydrogen-peroxid 0.5 Hypochloric acid 1 AgNO3 0.9-1 Iodine 0.9 Formaldehyde Quaterner ammonium-base 0.8-2.5 Aliphatic alcohols 6-12.7 Chlor-dioxyde 1-2

Disinfection kinetics If the concentration exponent is known, from one concentration-time data-pair it is possible to calculate every other concentration-time combination: In practice, according to the standard European tests for antimicrobial activity, the concentration of a disinfectant is acceptable if it results in 5 log degradation in 5 minutes. That means that the decimal reduction time (D) of the population treated with the antimicrobial agent does not exceed 1 minute.