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Industrial Microbiology

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Level of sterilisation is determined by probability of ... Breakdown of heat-labile constituents (e.g. amino acids, vitamins) (b) STRATEGIES/ SOLUTIONS ... – PowerPoint PPT presentation

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Title: Industrial Microbiology


1
  • Industrial Microbiology
  • INDM 4005
  • Lecture 4
  • 13/02/04

2
Importance of Sterilisation
  • Loss of productivity
  • Contaminate final product
  • Troublesome during downstream processing
  • Contaminant may cause degradation of product
  • Cause lysis of culture e.g bacteriophages

3
Steps to avoid contamination
  • Use pure inoculum
  • Media sterilisation
  • Fermenter sterilisation
  • Sterilise all raw materials
  • Maintain aseptic conditions during process

4
Sterilisation
  • Level of sterilisation is determined by
    probability of contamination and the nature of
    its consequences
  • Termed protected used e.g brewing
  • Vast majority not protected
  • Sterilisation more important in penicillin
    production than in brewing

5
Sterilisation
  • Fermentaton media is universally sterilised by
    steam (any exceptions?)
  • Moist heat treatment carried out at 121oC for 15
    mins for the sterilisation of culture media,
    vessels and connecting pipework
  • Microorganisms are not killed instantly on
    exposure to a lethal agent
  • Population decreases exponentially by a constant
    fraction at constant intervals
  • Several factors influence the effectiveness of
    any antimicrobial treatment

6
Factors that influence the effectiveness of any
antimicrobial treatment
  • 1. Population size
  • 2. Population composition
  • 3. Concentration of the antimicrobial agent or
    intensity of the treatment
  • 4. Period of exposure to the lethal agent
  • 5. Temperature
  • 6. Environmental conditions

7
Terms relating to heat sterilisation used in
fermentation industries
  • Thermal Death Time (TDT) is the shortest time
    required to kill all microrganisms in a sample at
    a specific temperature and under defined
    conditions
  • Decimal reduction time (D-value) is the time
    required to kill 90 of the microorganisms in a
    sample at a specific temperature
  • Z-value is the rise in temperature required to
    reduce D to 1/10 of its previous value
  • F-value is the time in mintues at a specific
    temperature (usually 250oF or 121.1oC) necessary
    to kill a population of cells or spores

8
2.2. PREPARATION and STERILIZATION OF MEDIA
  • CASE STUDY
  • Prepare a flow-sheet for WORT PREPARATION.
  • Contrast with media preparation for penicillin
    production.

9
2.2.1. BASIC KINETICS OF DESTRUCTION (/GROWTH)
  • The destruction of microorganisms by steam may be
    described as a first order reaction
  • first-order reaction A chemical reaction
    involving only one chemical species, in which the
    rate of decrease of the concentration of the
    reactant is directly proportional to its
    concentration.
  • Represented by the following equation -dN / dt
    k .N
  • where N number of viable organisms present
  • t time of the sterilisation treatment
  • k reaction rate constant

10
BASIC KINETICS
  • Integration gives Nt / N0 e -kt
  • Where
  • N0 initial number of viable microorganisms,
  • Nt no. of viable m-organisms present after
    treatment period t
  • t time,
  • k destruction coefficient (B.
    stearothermophilus)
  • On taking natural logs, equation is reduced to
  • ln (Nt / N0) -kt

11
Calculation
  • Can now calculate treatment required to bring
    about a
  • required level of destruction
  • (what value of "t" will give the required value
    to ln Nt /N0).
  • Example - require 10 -3
  • probability of survival with an initial value
    (N0) of 10 11
  • ln (10-3 / 1011)
  • ln10 -14 ( -32.2 ) kt

12
Graphical representation of previous equations
Nt No
Slope -k
Nt No
ln
Time Time
Plots of the proportion of survivors and the
natural logarithm of the proportion of survivors
in a population of microorganisms subjected to a
lethal temperature over a period of time
13
Kinetics
  • This kinetic description makes two predictions
    which contradict each other
  • i) An infinite time is required to achieve
    sterile conditions
  • ii) After a certain time there will be less than
    one viable cell remaining
  • Thus a value of Nt of less than one organism
    remaining is considered in terms of the
    probability of an organism surviving a treatment
  • e.g If a treatment reduced the population to 0.1
    of a viable organism, implies a probability of
    one in ten batchs becoming contaminated

14
Arrhenius Constant
  • As with any first order reaction, the reaction
    rate increases with increase in temperature due
    to an increase in the reaction rate constant,
    which in sterilisation of media is k
  • Relationship between temperature and the reaction
    rate constant was demonstrated by Arrhenius and
    is represented by
  • In k In A - E /RT
  • E activation energy
  • R gas constant The constant factor in the
    equation of state for perfect gases
  • T absolute temperature Temperature measured
    from absolute zero. In the Fahrenheit scale it is
    gauge temperature plus 460 degrees and is called
    Rankine temperature in the Centigrade scale it
    is gauge temperature plus 273 degrees and is
    called Kelvin temperature.
  • A Arrhenius constant

15
Kinetics
  • From this equation a plot of ln k against a the
    reciprocal of the absolute temperature gives a
    straight line
  • Plot is called an Arrhenius plot
  • Enables a calculation of the activation energy
    and prediction of the reaction rate for any
    temperature
  • Thus a plot of the natural logarithm of the time
    required to achieve a certain Del factor, against
    the reciprocal of the absolute temperature will
    yield a straight line, the slope of which is
    dependant on the activation energy.
  • Same degree of sterilisation may be obtained over
    a wide range of time and temperature regimes

16
Del Factor
  • Del factor ( V ) In (No/Nt),
  • where No is the number of organisms at the start
    of sterilisation
  • and Nt is the number remaining after time t.
  • Therefore, the Del factor is a measure of the
    fractional reduction in viable organism count
    produced by a certain heat and time regime.
  • The larger the Del factor, the greater the
    sterilisation regime required.
  • Term commonly used in the fermentation industry

17
2.2.2. STERILIZATION CHART
  • (a) This equation can be expressed in chart form
    or as a MODEL.
  • This kinetic description of bacterial death
    enables the design of procedures, giving certain
    Del factors for the sterilisation of fermentation
    media
  • By choosing a value for Nt, procedures may be
    designed having a certain probability of
    achieving sterility
  • The probability (p) that all organisms are killed
    or the probability (1 - p ) of N organisms
    surviving is expressed
  • p (N0 - N ) / N0
  • 1 - N / N0
  • 1 - e k.t
  • or ln ( 1 - p ) - k.t
  • Note N0, k and t affect the probability of
    survival / kill

18
  • (b) Approach
  • ? DETERMINISTIC - can measure the effect e.g.
    count survivors as in Thermal Death Point
    experiment in second year practicals
  • ? PROBABILISTIC - probability of survival very
    small, would need numerous experiments to count
    one cell, Therefore must calculate probability of
    survival. Applies to sterilisation (what does
    0.001 cells surviving mean !!)

19
(c) INFLUENCE OF TEMPERATURE ON DESTRUCTION
  • This particular model does not allow for
    variation in temperature (this would affect "k" )
    LIMITATION. In fact Batch sterilization on
    an industrial scale results in a
  • TEMPERATURE - TIME profile
  • ? Heating
  • ? Holding
  • ? Cooling
  • Must evaluate effect of process TEMP as well as
    TIME on destruction.
  • ? Achieved using ARRHENIUS EQUATION
  • (modifies the value of "k")

20
  • The DEL FACTOR v achieved at the different
    temperatures during the treatment cycle must be
    calculated
  • Thus the DEL FACTOR of the whole process is equal
    to the sum of the del factors of each of its
    constituents
  • v.t v.h v.m v.c
  • v.t del factor whole process
  • v.h " " heating up period
  • v.m " " holding period (maintained)
    period
  • v.c " " cooling-down period
  • Design conditions may specify the fraction of
    destruction at each stage e.g. v.h/v.t 0.2,
    v.m/v.t 0.75, v.c/v.t 0.05

21
2.2.3. DESTRUCTION OF NUTRIENTS DURING HEATING
  • Results in loss of yield
  • (a) TYPES
  • (b) STRATEGIES/ SOLUTIONS
  • (c) TEMPERATURE - TIME PROFILE

22
  • (a) TYPES
  • Interaction between nutrients e.g. reaction of
    carbonyl groups (possibly from sugars) with amino
    groups (amino acids / proteins).....browning
    reactions (Maillard type)
  • Breakdown of heat-labile constituents (e.g.
    amino acids, vitamins)
  • (b) STRATEGIES/ SOLUTIONS
  • First type - sterilize sugars, and/or some salts
    separately
  • Second type - Suitable temperature - time
    profile

23
  • (c) TEMPERATURE - TIME PROFILE
  • The plot of
  • "k" (destruction coef.) of microbial cells and of
    chemicals against temperature can vary.
  • Usual plot is lnk versus 1/T.
  • Consequently the ratio of microbial cell
    destruction to nutrient destruction varies with
    temperature
  • higher temp. less nutrient destruction, more
    cell destruction
  • lower temp. less cell destruction, more nutrient
    destruction
  • BASIS OF UHT - see starter culture

24
2.2.4. TECHNOLOGY OF MEDIA STERILISATION
  • (a) METHODS
  • ? MECHANICAL / PHYSICAL METHODS
  • Filtration
  • Irradiation
  • ? HEAT PROCESSING
  • EXPENSIVE PROCESS
  • Two criteria
  • ? Achieve required probability of sterility
  • ? Minimize loss of nutrients
  • MAIN HEATING METHODS
  • 1. BATCH
  • 2. CONTINUOUS - less destruction
  • Difference lies in the TEMPERATURE - TIME PROFILE

25
(b) COMPARISON OF CONTINUOUS BATCH
  • ADVANTAGES OF CONTINUOUS
  • Reduction of sterilization cycle time
  • Ease of scale-up
  • Superior maintenance of medium quality (less
    destruction)
  • Reduced surge capacity for steam (more efficient
    plant use)
  • ADVANTAGES OF BATCH
  • Lower capital cost (fermentor used as autoclave)
  • Lower contamination risk (less transfers of
    liquids)
  • Presence of solids (particles) less of a problem

26
2.2.5. TECHNOLOGY OF BATCH STERILIZATION
  • Can be carried out in
  • FERMENTATION VESSEL (in situ medium
    sterilization)
  • SEPARATE MASH COOKER
  • Advantages of separate medium sterilization
  • medium sterilized while fermenters are cleaned -
    less downtime
  • design conditions for sterilization more severe
    than fermentation
  • Disadvantages
  • Cost
  • More transfers - more pipework, more
    contamination risk
  • All fermenters would depend on cooker - fault
    render plant redundant

27
  • METHOD OF HEATING
  • ? Steam sparging direct through heating coils
  • ? Electric element
  • ? Heating jacket

28
2.2.6. TECHNOLOGY OF CONTINUOUS STERILIZATION
  • (a) STEAM INJECTION TYPE
  • ADVANTAGES
  • ?higher steam utilization efficiency
  • ?low capital cost
  • ?easy cleaning and maintenance
  • ?shorter heating and cooling cycle
  • DISADVANTAGES
  • ?foaming of media
  • ?media and steam have direct contact - chemical
    contamination
  • (b) CONTINUOUS PLATE EXCHANGER
  • Has longer heating and cooling period

29
  • CASE STUDY
  • Construct diagrams of both types and their
    respective temperature-time profiles. Give an
    example of an industrial process using each type.

30
Flow diagram of a typical continuous plate heat
exchange steriliser
Sterile media out
Steam Steam out in
Holding section
Cooling heat exchanger
Pre- heat heat exchanger
Heating heat exchanger
Cooling Cooling Unsterile water in
water out medium in
31
Flow diagram of a typical continuous injector
flash cooler steriliser
Cooling Cooling Unsterile water in
water out medium in
Steam in
Venturi valve
Holding coil
Cooling heat exchanger
Pre- heat heat exchanger
Expansion valve
Expansion chamber
Sterile medium out
32
Conclusion
  • Importance of Sterilisation
  • Kinetics of Asepsis
  • Del Factor
  • Technology of media sterilisation
  • Batch v Continuous sterilisation
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