Biochemical Engineering II

EG-M11

Dr Jesús Javier Ojeda

j.j.ojedaledo@swansea.ac.uk
Sterilisation
(a reminder!)
 Sterilisation
Sterilisation is the elimination, by removal or killing, of all microorganisms
and the inactivation of viruses present in a product. According to this
definition, sterility is an absolute concept.

Except in cases where obvious physical destruction of microorganisms is

apparent, as for example, in flaming, the mechanism by which sterilising
agents induce death is by no means certain.

It is not possible, in practice, to kill or inactivate all viable organisms.

Commercial sterilisation therefore aims to reduce the risk of contamination

to an acceptable level.

Sterility: the absence of detectable levels of viable

organisms in a culture medium or in a gas.
 Sterilisation
Reasons for Sterilisation:
• Many fermentations must be absolutely devoid of foreign
organisms (otherwise production organism must compete
with the foreign contaminant organisms for nutrients).
• Foreign organisms can produce harmful (or unwanted)
products which may inhibit the growth of the production
organisms.
• Economic penalty is high for loss of sterility.
• Vaccines must have only killed viruses.

• Recombinant DNA fermentations: exit streams must be

sterilised.
 Sterilisation methods
• Chemical:
→ ethylene oxide (gas) for equipment
→ 70% ethanol-water (pH=2) for equipment/surfaces
→ 3% sodium hypochlorite for equipment

• Irradiation:
→ ultraviolet for surfaces
→ X-rays for liquids (costly/safety)

• Filtration
→ membrane filters having uniform micropores
→ depth filters of glass wool

• Thermal:
preferred for economical large-scale sterilisations
of liquids and equipment
 Thermal sterilisation
• Dry air or steam can be used as the heat agent.

• Moist (wet) steam can also be used as the heat agent

(eg: done at 121oC at 2 bar).

• Death rate of moist cells are higher than that of the dry
cells since moisture conducts heat better than a dry
system.

• Therefore moist steam is more effective than dry

air/steam.

• Thermal sterilisation does not contaminate the medium

of equipment that was sterilised (as in the case of use
of chemical agent for sterilisation).
 Thermal sterilisation using
dry heat
- Direct flaming

- Incineration

- Hot air oven

-170 °C for 1 hour

-140 °C for 3 hours

 Thermal sterilisation using
steam
- Pasteurisation (below 100oC)
Destroys pathogens without altering the flavour of the food.
High Temperature/Short Time (HTST) : 72oC; 15-20 sec
Ultra High Temperature (UHT) : 140oC; 4 sec

- Boiling (at 100oC)

killing most vegetative forms microorganisms
Requires 10 min or longer time
Hepatitis virus can survive for 30 min & endospores for 20 h

- Autoclaving (above 100oC)

killing both vegetative organisms and endospores
121-134oC; 15 min or longer
Thermal sterilisation using
steam

Tunnel Pasteurisation
Thermal sterilisation using
steam

Autoclaves
Thermal sterilisation using
steam

Autoclaves
Kinetics of Cell Death
dn
− = kd n
dt
dn
= − k d dt
n
nt dn t
no n = −k d o dt
nt
ln = −kd t
no
nt
= e −kd t
no
 Kinetics of Cell Death
nt
= e −kd t “survival factor”
no

kd also varies with temperature according to the Arrhenius

equation: Ed
−
kd = Ad e RT

- Activation energies for the death of the organisms (Ed) range from about 50
to 150 kcal/mol:

Bacillus stearothermophilus, Ed≈ 70 kcal/mol E. coli, Ed≈ 127 kcal/mol

 Kinetics of Cell Death

(a) Typical death-rate data for spores of Bacillus stearothermophilus in

distilled water. (b) Typical death rate for E. coli.
 Sterility assurance level
(SAL)
The desired sterility assurance level (SAL) varies according to
the intended use of the product.

For sterilisation of medical devices, implantable devices or

any products intended to come into contact with breached skin
or compromised tissue, the most rigorous SAL should be
selected. Such products should have a SAL of 10-6; that is,
no more than one viable microorganism in one million
sterilised items of the final product.

Other sterilised products are generally thought to be safe for

use with a SAL of 10-3 per whole batch; or the probability of
one nonsterile unit in a total of a thousand.

note that SAL refers to the total number of viable cells (not concentration)
 Problem 1
10 litres of broth containing 105 of viable spores of Penicillium
chrysogenum per litre need to be sterilised prior disposal. The
medium is to be autoclaved at 121°C for 20 minutes. At this
temperature, the specific thermal death rate, kd, is 1.0 min-1 and
the activation energy for the death of the spores, Ed, is 90 kcal
mol-1.
However, the autoclave malfunctioned and the temperature
reached only 119.5 °C. What is the probability that the medium
was sterile, if the sterility assurance level (SAL) should be 10-3 for
the whole batch?

nt Ed
−kd t −
=e kd = Ad e RT
no
 Problem 1 (cont...)

T1 = 121 °C = 394 K, and T2 = 119.5 °C = 392.5 K

Arrhenius equations for kd at 394 K , T = 394 K and kd at 392.5 K , T = 392.5 K
can be expressed as follows:
− Ed − Ed
k d at 394 K = Ad e R  394 K
and k d at 392.5 K = Ad e R  392.5 K

Ed Ed
ln k d at 394 K = ln Ad − and ln k d at 392.5 K = ln Ad −
R  394 K R  392.5 K

Rearranging:
Ed Ed
ln Ad = ln k d at 394 K + and ln Ad = ln k d at 392.5 K +
R  394 K R  392.5 K
 Problem 1 (cont...)
Ed Ed
ln k d at 394 K + = ln k d at 392 .5 K +
R  394 K R  392 .5 K

Ed  1 1 
ln kd at 392.5 K = ln kd at 394 K +  − 
R  394 K 392 .5K 
Substituting:
kcal
90
( )
ln k dat 392.5 K = ln 1.0 min −1 + mol
kcal
(− 9.697  10 −6
)
K −1 = −0.4392
−3
1.987  10
mol  K
kd at 392.5 K = 0.644 min −1

nt − k d at 392 .5 K t − 0.644 min −1  20 min

Survival factor at 20 mins: =e =e = 2.514  10 − 6
no
Number of viable spores:
spores
𝑛𝑡 = 2.514 × 10−6 × 105 l × 10 l = 2.51 spores ∴ does not meet SAL
Biosafety
 Biohazard

Biohazard: Any organism or its toxin that is known to cause

disease in humans or animals or that is a potential hazard to
humans, animals or the environment.

Examples:
Microorganisms such as viruses, bacteria, fungi, and
parasites and their toxins. Blood, body fluids and tissues
from humans and animals.
 Biosafety

Biosafety: the combination of measures employed when

handling biohazardous materials to:

Protect personnel from exposure to infectious agents

Prevent environmental contamination
Comply with applicable federal, provincial and municipal
requirements
 How is Biosafety achieved?

Administrative controls:
Training, Inspections, Permits and Certificates

Engineering Controls
Biological Safety Cabinets, Ventilation

Personal Protective Equipment

Practices and Procedures

Medical Surveillance
Immunization when necessary
Biosafety
 Biosafety risks
Biosafety risks are defined as a function of the likelihood of
infection by the agent and the likelihood of exposure through an
infectious route, based on the procedures and work practices,
and the consequences of disease assuming infection.

“What you have,

Ms Bates, sounds
highly contagious.
You’d better see
another doctor”
 Biosafety risks

4 scenarios
• Risk to individuals in the workplace
• Risk to an individual outside the workplace
(the human community)
• Risk to animals outside the workplace (the
animal community)
• Risks to humans and animals resulting
from a secondary exposure
 The law

Primary legislation
The Health and Safety at Work Act 1974

The Anti-Terrorism Crime and Security Act 2000

Secondary legislation & approved Codes of Practice

The Control of Substances Hazardous to Health Regulations 2002 –
general and biological agents provisions

Genetically Modified Organisms (Contained Use) Regulations 2000

The Management of Health and Safety Work Regulations 1999

The Reporting of Injuries, Diseases and Dangerous Occurrences

Regulations 1995
 Health and Safety at Work
Act 1974

• Key duties of the employer

- Ensure the Health and Safety of their
employees
- Ensure the Health and Safety of others

• Key duty of the employee

- Co-operate with the employer
 COSHH

“hazardous
substances”
• Chemicals (incl toxins)
• Carcinogens
• Biological agents
 COSHH - biological agents

Reg. 6: Assess health risks

Reg. 7: Prevent or control exposure Schedule 3
Reg. 8: Use control measures
Reg. 9: Maintain, examine and test control measures
Reg. 10: Monitor exposure
Reg. 11: Health surveillance
Reg. 12: Information, instruction and training for persons
who may be exposed
Reg. 13: Arrangements to deal with accidents, incidents
and emergencies
COSHH - biological agents
 COSHH

All biological agents must be classified in one of four

Hazard Groups

Hazard Hazard Hazard Hazard

Group 1 Group 2 Group 3 Group 4

Increasing hazard to human health

 COSHH

Definition of Hazard Groups

Group 1 - unlikely to cause human disease

Group 2 - can cause human disease and may be a hazard to
employees; but is unlikely to spread to community
and there is usually effective prophylaxis or
treatment available
Group 3 - can cause severe human disease and may be a
serious hazard to employees; it may spread to the
community, but there is usually effective prophylaxis
or treatment available
Group 4 - causes severe human disease and is a serious
hazard to employees; it is likely to spread to the
community and there is usually no effective
prophylaxis or treatment available
 Risk Group 1 Agents

• E.coli K-12
• Transgenic Plants
• Fungi
• Mould
• Yeast
 Risk Group 2 Agents

• Human or Primate
Cells
• Herpes Simplex Virus
• Replication
Incompetent
Attenuated Human
Immunodeficiency
Virus
• Patient specimens
 Risk Group 3 Agents

• Human
Immunodeficiency
Virus
• Mycobacterium
tuberculosis
• Coxiella burnetii
 Risk Group 4 Agents

• Lassa Fever Virus

• Ebola Hemorrhagic
Fever Virus
• Marburg Virus
• Herpes B Virus
 Risk Group 4 Agents

Biosafety Level-4
• Dangerous/exotic agents
• Life threatening disease
• Aerosol transmission
• Agents of unknown risk
of transmission or health affects
• No known treatment
Available at
http://www.hse.gov.uk/pubns/misc208.pdf
 Containment measures

The term containment is defined as the

confinement of a biohazardous agent that is being
cultured, stored, manipulated, transported, or
destroyed in order to prevent or limit its contact
with people and/or the environment. Methods
used for containment include physical and
biological barriers and inactivation using physical
or chemical means.

Containment is integrated into the distinguished

biosafety levels.
 COSHH

Classification of biological agent USUALLY determines

minimum containment level required

Hazard Hazard Hazard Hazard

Group 1 Group 2 Group 3 Group 4

Containment Containment Containment Containment

Level 1 Level 2 Level 3 Level 4
COSHH
Containment measures
Containment measures
(cont…)
Containment measures
(cont…)
Containment measures
(cont…)
Risk assessment strategy
 COSHH
1st fundamental principle

COSHH requires that exposure is

If prevention
prevented not possible
1
controlled
2
 Prevention of exposure by
segregation

If substitution is not possible then start by

considering whether the work is adequately isolated
from other employees, contractors or visitors

• Prevent unnecessary and unauthorised access

• Keep doors locked/ secure
• Use appropriate signage (Biohazard)
• Permit-to-work system
 Risk assessment
• risk assessment is CAREFUL judgment
• risk assessment is proactive rather than reactive
• assessments can be qualitative, semi-quantitative or
quantitative
• risk assessment methodology should be:
– identified
– implemented
– maintained
• risk assessment is first step to control (reduce or eliminate)
the hazards
• risk assessment will provide a guide for the selection of
appropriate
- biosafety levels needs
- microbiological practices
- safety equipment
- facility safeguards
 New risk assessment or
review of an existing one when:

• the introduction of new biological agents

• new work or changes to the programme of
work
• alterations to work flow or volume
• new construction / modifications to
installations, equipments or their operation
• introduction of altered and unplanned
staffing arrangements
 New risk assessment or
review of an existing one when:

• significant alterations to Standard

Operating Procedures (SOPs) or working
practices (e.g. disinfection/waste
management methodologies, PPE
provision / usage entry/exit protocols,
etc.);
• unexpected events that may have
relevance for the management of
biological risks
 New risk assessment or
review of an existing one when:

• actual or potential non-conformity with

internal / external rules and regulations is
identified
• as part of the existing management
system review process (e.g. annually or at
another appropriate and predetermined
frequency)
 HACCP and GMP

Hazard Analysis and Critical Control Points (HACCP) is a system

used by the food industry to ensure the safety of food. It is a
preventative, risk-based system to identify critical control points
(CCP) for:
• physical (e.g. glass),
• chemical (e.g. pesticides) and/or
• microbiological (e.g. food poisoning bacteria) hazards.

By law, all food businesses in Europe must implement and

maintain procedures based on HACCP principles.
Seven Principles of HACCP
 HACCP and GMP

Before implementing HACCP, good manufacturing practices (GMP) must be in

place. Examples include:
• personnel hygiene and training;
• cleaning and sanitation;
• maintenance and services;
• pest control;
• maintenance and services of plant, equipment, premises and structure;
• storage, distribution and transport;
• waste management.
 HACCP principle 1:
Conduct a hazard analysis

Those involved in designing food processing equipment and production

lines must proactively analyse designs to identify potential food safety
hazards. If the hazard analysis reveals contaminants are likely to find their
way into food products, then preventive measures are put in place in the
form of design revisions.

EXAMPLE 1: A food processing machine is designed, and hazard analysis

reveals that food can accumulate in areas where cleaning is difficult or
impossible. This accumulation will rot with time, and the bacteria-laden
glop can fall onto uncontaminated food passing through production lines.

EXAMPLE 2: A piece of metal tooling may have been designed with the
intent to form food products into a certain shape, but hazard analysis
reveals that the tooling is too fragile and cannot withstand the repeated
forces imposed on it by the mass production process. There is a strong
likelihood that small metal parts can break off and enter the food on the line.

http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-1
 HACCP principle 2:
Identify critical control points
A critical control point (CCP) is a step in the design process at which a control
can be most effectively introduced to prevent or eliminate hazards. In this
context a “control” would be a design revision to eliminate hazards identified
during the Principle 1 stage.

EXAMPLE 1: hazard analysis revealed that food can accumulate in a food

processing machine in areas where cleaning is difficult or impossible. Design
engineers would work to address this hazard by identifying a CCP within the
design process, that is, the best place where a preventative measure can be
added to the machine setup to facilitate removal of the accumulation (e.g.
include “easy to remove” access covers).

EXAMPLE 2: hazard analysis revealed that the metal tooling as designed for
our food production machine was too fragile and would not withstand the
repeated forces imposed on it by the mass production process. To correct this
situation, design engineers must identify the juncture within the design process
at which a CCP is identified and a preventative measure can be introduced(e.g.
more robust metal to be used in the tooling).
http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-2
 HACCP principle 3:
Establish critical limits for each CCP
A critical limit is a boundary of safety for each critical control point (CCP). Critical
limits can be obtained from regulatory standards, scientific literature,
experimental studies, as well as information provided by consultants. These
critical limits come into play with issues as varied as machine design, raw
material temperatures, and overall safe processing times.
EXAMPLE 1: Commercial machinery providing limited access for cleaning.
Design engineers introduce minimum and maximum parameters for the critical
limit dimensions of a removable covers: enough space is provided so that
personnel can fully access all aspects of machinery with tools for cleaning, but
removable cover dimensions aren’t too large and heavy to be manipulated by
hand.
EXAMPLE 2: Raw meats can contain hazards like Salmonella, E. coli, or other
pathogens dangerous to human health. One of the ways the commercial food
industry can use to ensure that these contaminants aren’t unleashed on the
public is to install programmable control systems into processing machinery that
essentially cooks the meat at an established minimum temperature for a
minimum amount of time.
http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-3
 HACCP principle 4: Establish critical
control point monitoring requirements

Monitoring activities are necessary to ensure that the critical limits

established at each critical control point (CCP) established under Principle 3
are working as intended.
EXAMPLE: The engineering manager is reviewing the logic in a
programmable controller for a cooker on a production line. She discovers a
problem with the lower critical limits established by her engineer at a CCP in
the design of a cooker temperature control loop. The time and temperature
in the logic is sufficient to thoroughly cook smaller cuts of meat in most of the
products that will be made on the line, however the larger cuts will be
undercooked. The time and temperature settings within the logic are
insufficient to account for the difference.

http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-4
 HACCP principle 5:
Establish corrective actions

When an established critical limit at a designated critical control point (CCP)

has been found not to be functioning as intended, design engineers must
enact corrective measures to resolve the issue as soon as possible.
EXAMPLE: An engineering manager has discovered a problem with the
lower critical limits established by her design engineer’s software logic as it
concerns a CCP established with regard to cooker temperature. The time
and temperature in the logic create a hazardous situation by not taking into
account that larger cuts of meat require more cooking time, resulting in them
being undercooked.

The engineering manager’s ongoing day-to-day monitoring has alerted her to

the error. She immediately provides feedback about it to the design engineer,
who makes corrections to the software logic.

http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-5
 HACCP principle 6: Establish procedures for
ensuring the system is working as intended

It follows up on the guidelines established in Principles 1 through 5,

organizing activities into written procedures. For each step, responsibilities of
key individuals involved must be clearly defined and sequentially ordered.
Training may be necessary, and management must decide what form that
educational process takes to be most effective.

EXAMPLE: Design engineers must routinely analyse important identified

stages within a design project, then write procedures, that is, a step-by-step
instruction guide, which encompasses them. In this way personnel involved
in the design process make best use of the safeguards put in place by
HACCP Design Principles 1 through 5. These steps include preparing design
proposals, analysing risks and hazards, creating preliminary designs,
conducting design reviews, building prototype equipment and tooling, running
tests, collecting test data, and analysing test results.

http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-6
 HACCP principle 7:
Establish record keeping procedures
This HACCP principle requires that all food manufacturing plants maintain records to
show they implemented a HACCP plan, are following all principles, and the plan is
working effectively.
EXAMPLE: In keeping with the directive of HACCP Design Principle 7, the
engineering department of a food manufacturing plant must keep records for each
design project. The design record for a new cookie forming machine would contain
engineering calculations to determine strength requirements of machine parts and
supports, power requirements for the electric motor that drives the machine. This
design record would also contain documentation concerning materials selected to
construct the machine, as well as dimensioned mechanical drawings of the machine
and its parts. These dimensioned drawings will show all physical dimensions of the
machine and its constituent parts.
The record would also contain test results and analysis of the results. Lastly, the
design record must include a risk analysis of potential hazards that could result.
Other activities include identification of CCPs, establishment of critical limits, and
other factors in accordance with HACCP Design Principles 1 through 5. In other
words, the record must be complete, bearing witness to an effective adherence to
HACCP Design Principles 1 through 5.
http://www.engineeringexpert.net/Engineering-Expert-Witness-Blog/food-manufacturing-challenges-–-haccp-design-principle-no-7
Additional problems about sterilisation
and biosafety (to be solved by you)

(Ans: t = 9.35 min)

(Ans: 223 kJ mol-1)

Additional problems about sterilisation
and biosafety (to be solved by you)

(−21.68 − 0)
Ans: Plot Ln(nt/no) vs time, then a) kd = -slope = − = 0.723 min-1 ;
(30 min − 0 min)
survival factor at 40 mins: 2.76x10-13 (no units) (∴ nt at 40 mins = 0.023 spores)
b) t = 44.4 mins