Heat Sterilization Module- 4 Lec- 4 Dr. Shishir Sinha Dept. of Chemical Engineering IIT Roorkee
Sterilization is the total elimination of all microorganisms including spores Typically the last things to die are the highly heat- and chemical-resistant bacterial endospores Instruments used for invasive procedures must be sterilized prior to use Moist heat or steam, radiation, chemicals (e.g., glutaraldehyde), and ethylene oxide (a gas) are employed for sterilization Sterilization by autoclaving, which uses moist heat, is used in most hospital and microbiology laboratory settings Heat Sterilization Applying heat to foods to decrease the concentration of the viable microorganisms to such a level that would only allow growth of microorganisms and spores in the food under defined storage conditions to an acceptable level (commercial sterility). In heat processing, to achieve microbial stability and eating quality both: 1. The temperature of heating and 2. The duration of the thermal process are important. An optimum balance needs to be found to avoid over- and underprocessing. To design a heat process it is necessary to determine: 1. The heat resistance of the spoilage microorganisms (target microorganism) 2. The temperature history of the food at the slowest heating point. (thermal center) Thermal destruction of bacteria Bacteria have a logarithmic order of death when subjected to high temperatures. Log of viable bacteria concentration vs. time of exposure is a straight line relationship called a survivor or a thermal destruction curve.
Survivor or thermal destruction curve For the targett microorganism, if the initial viable cell concentration is N, viable cell i concentrationn at time t can be estimated by: log (N/N ) = Slope (t-) i The slope of the survivor curve is defined as -1/D, log (N/N ) = -t/d i D is called the decimal reduction time which is constant at a given temperature. D = D(T) D is the time period needed to decrease viable cell concentration 1-fold at a given temperature. The decimal reduction time, D is determined for each type of target microorganism in certain types of food (growth medium, a, ph, composition etc.) for different w temperatures. It is strongly dependent on temperature. From survivor curve equation: N = N x 1 i -(t/d) N only if t
An infinite time will be required for the destruction of all viable microorganisms. Basis for defining commercial sterility. Product will be accepted as commercially sterile when the concentrationn of the viable cells of the target microorganism is reduced below a certain level N just low enough that the spoilage hazard it presents is commercially acceptable within the period of suggested shelf life. A reduction exponent is defined as: m = log(n /N ) i Effect of varying temperatures During a thermal process temperature varies with time at the thermal center of the food. Since D = D(T), an integrationn w.r.t. time is necessary: log N N i t dt D T = T(t), D = D(t)
N, N : initial and final viable cell concentrations, i f t : duration of the thermal process needed to achieve commercial sterility. f log N f N i tf dt D Condition for commercial sterility: N N f log (N /N ) log (N /N ), f i i since m = - log (N /N ) i tf dt m D condition for commercial sterility becomes: The processing time t can be estimated by graphical integrationn of 1/D versus t f Steps: Generatee T vs. t dataa find D versus T dataa from literature plot 1/D versus t.
Modeling temperature dependency of D The variation in the logarithm of the decimal reduction time D could be well correlated as a linear function of temperature. If at temp. the decimal reduction time is D, then at T, D will be:
z-value is the temperature differencee required to change the decimal reduction time tenfold. From the equation above: Plugging into condition for commercial sterility: tf dt D m letting: L 1 ( )/ 1 T z For commercial sterility: L is defined as the lethal rate tf 1 1 D ( T )/ z dt m log D ( T ) D z 1 D 1 1 ( T )/ z D tf Ld dt md For each kind of microorganism z-values can be found in literature.
is called the reference temperature. For sterilization operations it is taken as 25 (121.1 C), the max. temperature experienced by the food in retorts. The value of the integral t Ldt and it is denoted by F. The equivalent time values are estimated for certain target microorganisms with known z-values at a fixed reference temperature. Therefore, equivalent time needed for commercial sterility is denoted as is called the equivalent time of the heat process z F F Since most target microorganisms have z-values close to 1 and since temperature is usually taken as 121.1 C, for this specific case: the reference F = F (F ) 121.1 is used. For commercial sterility: F md
Example Problem: Heat penetration data on a vacuum packed corn are given in the Table. The target organism for this food is C. Sporogenes (D =. 8). What is the minimum processing time the necessary to achieve commercial sterility for this food assuming instantt cooling after process? Time(min) T (Deg.F) L 2 4 6 8 11 14 17 2 82 217 23 233 233 228 232 237 24.146.775.114.114.6.1.215.278
24 242.36 29 245.526 32 246.599 35 247.68 Formula method for thermal process evaluation This method aims to perform the integral analytically time. Let T be the constant temperature of the medium r dimensionless temperature V is defined as: to estimate the equivalent where the food is heated. A V = ( T r T) / ( T r T ) T = initial temperature at the thermal center, T = temperature at the thermal center at time t at t = ; V = 1., as t, T Tr, V. A plot of logv vs. time can be approximated with a straight line.
tf dt m D
Thermal destruction of microorganisms occurs to the most part when the linear asymptote forms a good approximation to the heating curve. The linear asymptote is specified by defining two parameters; the lag factor j ( j=1.6-1.4 for conduction-, j 1. for convection heating) and the slope 1/f. The equation for the asymptote is: -1/f = (logv-logj) / (t-) t/f = log(j/v) log j ( T r T ) / ( T r T ) = (1/f) t dt = f M dt/(tr-t), M = loge =.4343 Inserting dt = f M dt/(t r -T) into the integral for equivalent time (T- )/z F= 1 dt o (T- )/z F = 1 fm dt/(t -T) this integral is o r analytically solved in many steps to obtain: F = M f exp (T r - )/Mz -E i (-g/mz) + E i -(T r -T )/Mz g = T r -T at the end of the heating period (t=t h ) E i (-x) is an exponential function, values of which are read from mathematical tables. Since (T r -T )/Mz has a high value, E i -(T r -T )/Mz is very small, this term is usually neglected. F = M f exp (T r - )/Mz -E i (-g/mz) This equation relates the equivalent time to the processing temperature (Tr) and processing time (contained in g), for a given target microorganism of given z-value, for a certain food (heat transfer characteristics, contained in g and f) g = T -T r log j ( T r T ) / ( T r T ) = (1/f) t
Summary of heat process calculations Microbiological input D, z-values for the target microorg. F, the equivalent time necessary Heat penetration input T vs. time data f, j-values processing conditions: initial temperature heating medium temp. cooling medium temp. established process (processing time to meet microbiological, heat penetration and processing requirements)
Sterilization methods Mainly two methods: 1. Sterilization in containers, 2. Sterilization before placing into the container Selection of sterilization method largely depends on the packaging material used: - tin (metallic) cans - glass jars - film pouches Sterilization in containers Mostly carried out by heating the packaged foods in saturated steam
Sterilization of low acid foods is carried out at temperatures above 1 C, therefore pressurized vessels (retorts) are used. In retortt operations it is important to: a) have adequate venting of air from the retort and container surfaces to avoid air pockets, b) minimize thermal shock to the food, c) limit thermal and pressure strain on the containers by: 1. control of heat-up, cool-down rates. 2. use of pressurized air during cooling to balance increased internal pressure in the container. 3. processing jars immersed in water. Internal pressure increase ofcontainers: 1. Thermal expansion of food 2. Thermal expansion of headspace gas 3. Increased vapor pressure of water
A vertical batch retort Hydrostatic sterilizer
Sterilization of food outside container High temperature processing (T 15 C) by means of high speed heat exchangers reduces processing time substantially (to few seconds) and improves product quality. Such processes are called high-short processes (HTST -applied to sterilization of milk). Improved product quality is due to the fact that destruction of nutrients and flavor components in foods (vitamins, colors, antioxidants, enzymes, amino acids) are similar to destruction of bacteria. But the z-values of nutient compounds are considerably larger than that of the microorganisms. Example: For a certain food F 1 12 = 1 min is needed for commercial sterility. Two alternative procedures: 1. Heat food instantaneously to 12 C, hold at this temperature for 1 min and cool instantaneously. F=1 (T- )/z t = 1 (12-12)/1 x 1 = 1min. f 2. Heat food inst. To 14 C, hold at this T for.1min and cool inst. F= 1 (14-12)/1 x.1 = 1min. Suppose this food contains a valuable enzyme with a z-value of 5C which requires 4 min at 12 C for inactivation. At 14 C time required for inactivation will be: t = 4 x 1 (12-14)/5 = 1.6 min. Processing time needed Time needed for enzyme inactiv. Procedure 1: 1 min 4 min Procedure 2:.1 min 1.6 min Aseptic processing Sterilized food packed in sterile containers under aseptic conditions.
Advantages: 1. Product with higher organoleptic and nutritional quality, 2. Possibility to use large containers to pack the food, 3. Extended possibilities for using packaging materials of many package sizes, shapes and materials, 4. Handling of containers during subsequent sterilization is avoided, recontamination risk during cooling is minimized. Limitations: 1. Large capital investment. 2. Pumping at high pressures, product must be relatively homogeneous. 3. Need for specific design of systems for a specific product. 4. Complex operation requiring careful control and sophisticated instrumentation, need for highly trained personnel 5. Relatively limited filling rate (2 packages per min. versus 6 tin cans per min) There are two methods of commercial sterilization: Heating the food after placing it in a container. Heating and cooling the food then aseptically packaging and sealing. Heat Moist heat effective against all types of microorganisms degrades nucleic acids, denatures proteins, and disrupts membranes Boiling, autoclaving Dry heat sterilization less effective, requiring higher temperatures and longer exposure times oxidizes cell constituents and denatures proteins Flame, oven Endospores greatest resistance Vegetative cells differ in sensitivity to heat Higher temperatures allow shorter exposure times
Measuring heat-killing efficiency Thermal death time (TDT) shortest time needed to kill all microorganisms in a suspensionn at a specific temperature and under definedd conditions Decimal reduction time (D or D value) time required to kill 9% of microorganisms or spores in a sample at a specific temperature Figure 7.1 Other measures Z value increase in temperature required to reducee D by 1/1 F value time in minutes at a specific temperature needed to kill a population of cells or sporess
Moist heat Coagulation and denaturation of proteins halts cellular metabolism 4 methods: Boiling water Steam under pressure autoclaving Pasteurisation Non-pressurised steam Boiling water: o 1 C o Boiling water kills vegetative cells and spores of eucaryotes within 1 minutes o Bacterial endospores - resistant to boiling water and will not be sterilized o Method less effective with change in atmospheric pressure Autoclave use saturated steam under pressure to reach temperatures above boiling kill endospores Heat-resistantt materials glassware, cloth, metallic instruments, liquids,
o 121 C for 15 min - kills all endospores and vegetative organisms Wet steam generated under a pressure of 1 kpa/ 15 pounds per sq. inch to reach 121 o C destroys nucleic acids, enzymes and proteins in the cell Endospores of Bacillus stearothermophilus or Clostridium used to determine effectivity of heat sterilisation Pasteurization of milk flash pasteurization: high temperature short-term HTST: 72 C for 15 seconds then rapid cooling batch pasteurization: Low temperature high term LTHT: 63 C for 3 min ultrahigh-temperature (UHT) sterilization: 14 to 15 C for 1 to 3 seconds Tyndallisation Some products cannot withstand autoclaving temperatures Repeated heating at 9 1 C for 3 min on 3 successive days and in between incubation at 37 C Allows endospores to germinate into less resistant vegetative cells Temperature not sufficient to kill spores so multiple exposures required Eggs, carbohydrates, some canned foods Dry heat sterilization Incineration flame, incinerators Microbes reduced to ashes and gas Items are heated in an oven at 15 18 C for 2 to 4 h Destruction of spores Glassware, powders, oil
Low temperatures freezing stops microbial reproduction due to lack of liquid water - bacteriostatic some microorganisms killed by ice crystal disruption of cell membranes refrigeration slows microbial growth and reproduction Psychrophiles -7 C to -135 C preservation of cultures Dessication and osmotic pressure Dessication - dehydration of organisms not microbicidal, but microbistatic Used in food preservation Freeze-drying fast freezing, sublimation of water under a vacuum Increasing the osmotic pressure of the external environment High salt- or sugar concentrations Hypertonic environment cell lysis Filtration reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms also used to reduce microbial populations in air Filtering liquids Heat-sensitive liquids depth filters thick fibrous or granular filters that remove microorganisms by physical screening, entrapment, and/or adsorption
membrane filters porous membranes with defined pore primarily by physical screening sizes that remove microorganisms Figure 7.4a
Figure 7.5b polycarbonate membrane with.4 μmm pores Filtering air surgical masks cotton plugs on culture vessels high-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets Hospital theatres
Radiation Figure 7.6a Ultraviolet light (UV) - 26 nm Thymine dimers misreading of genetic code leads to cell death or altered growth Penetrates glass, water etc poorly Low penetrating ability used microorganisms Damaging to the skin and eyes to decontaminate surfaces or for airborne
Ionising irradiation: gamma rays and x-rays Destroys endospores and vegetative cells Effect on sugar-phosphate backbone of nucleic acids Sterilisation of plastic, antibiotics, medical products and some foods Outstanding form of sterilization and penetrates the specimen. Gamma irradiation with cobalt 6 is used for cold sterilization of antibiotics Sterilized food packed in sterile containers under aseptic conditions. Advantages: 1. Product with higher organoleptic and nutritional quality, 2. Possibility to use large containers to pack the food, 3. Extended possibilities for using packaging materials of many package sizes, shapes and materials,
4. Handling of containers during subsequent sterilization is avoided, recontamination risk during cooling is minimized. Limitations: 1. Large capital investment. 2. Pumping at high pressures, product must be relatively homogeneous. 3. Need for specific design of systems for a specific product. 4. Complex operation requiring careful control and sophisticated instrumentation, need for highly trained personnel 5. Relatively limited filling rate (2 packages per min. versus 6 tin cans per min)
References http://apps.who.int/phint/en/p/docf/ http://www.gibraltarlabsinc.com/gibraltarblog/difference-between-moist-heatsterilization-dry-heat-sterilization/ http://en.wikipedia.org/wiki/sterilization_%28microbiology%29 http://en.wikipedia.org/wiki/dry_heat_sterilization