Doc.

5 A method for the assessment of in-line

steam sterilisation, July 2004

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 A METHOD FOR THE ASSESSMENT OF IN-LINE STEAM
STERILISABILITY OF FOOD PROCESSING EQUIPMENT *
(Second Edition)

T. Bénézech** (1), F. Bourion (2), B. Carpentier (3), G.J. Curiel (4), C. Faille (1), P.
Gustavsson (5), C. Hermon (6), J. Hofmann (7), J. Kastelein (8), J. Kold (9),
A.W. Timperley (10), G. Wirtanen (11)

(1) Laboratoire de Génie des Procédés et Technologie Alimentaires, INRA, 369 rue
Jules Guesde, B.P. 39, F-59651 Villeneuve D'Ascq Cedex, France.
(2) ASEPT, rue des Docteurs Calmette et Guérin, BP49/53020 Lavel Cedex, France.
(3) Laboratoire d'Etudes et de Recherches sur la qualité des aliments et sur les
procédés agro-alimentaires, AFSSA, 22 Rue Pierre Curie, BP332, F-94709
Maisons-Alfort Cedex, France.
(4) Unilever R&D Vlaardingen, PO Box 114, 3130 AC Vlaardingen, the Netherlands.
(5) SIK, Swedish Institute for Food and Biotechnology, P.O. Box 5401, S-402 29
Göteborg, Sweden.
(6) Centre Technique des Industries Mécaniques, CETIM, 74 Route de la Jonelière
BP 82617-44326 Nantes Cedex, France.
(7) Technische Univesität München, Lehrstuhl für Maschinen und Apparatekunde,
Am Forum 2, 85350 Freising, Germany.
(8) TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, the Netherlands.
(9) Biotechnological Institute, Holbergsvej 10, P.O. Box 818, DK-6000 Kolding,
Denmark.
(10) Campden & Chorleywood Food Research Association Group, Chipping
Campden, Gloucestershire GL55 6LD, United Kingdom.
(11) VTT Biotechnology, Tietotie 2, Espoo, P.O. Box 1500, FIN-02044, Finland.

* Update prepared by the Test Methods Subgroup of the European Hygienic

Engineering & Design Group (EHEDG), July 2004.
** Chairman

The production of EHEDG Guidelines is supported by the European Commission under

the Quality of Life Programme, Project HYFOMA (QLK1-CT-2000-01359).

© EHEDG Doc. 5 (Second edition) 1

 Table of Contents

1 Introduction............................................................................................................... 3

2 Definitions.................................................................................................................. 3

3 Materials .................................................................................................................... 3
3.1 Indicator Micro-organisms.......................................................................................... 3
3.2 Tripticase Soy Broth ................................................................................................... 4
3.3 Steam........................................................................................................................... 4
3.4 Test Equipment ........................................................................................................... 4

4 Test Procedure .......................................................................................................... 5

4.1 Equipment Soiling ...................................................................................................... 5
4.2 Sterilisation Procedure ................................................................................................ 5
4.3 Detection of Surviving Spores .................................................................................... 6
4.4 Interpretation of Results.............................................................................................. 6
4.5 Positive Controls......................................................................................................... 6

5 Discussion................................................................................................................... 6

6 References.................................................................................................................. 7

© EHEDG Doc. 5 (Second edition) 2

1 Introduction

The Test Methods subgroup of the European Hygienic Engineering & Design Group
(EHEDG) is responsible for producing standardised test methods for assessing the
hygienic and aseptic capability of food processing equipment. Methods for assessing in-
place cleanability (ref. 1), in-line pasteurisability (ref. 2) and bacteria tightness (ref. 3)
have been published.

This document details the test procedure for assessing the suitability for in-line steam
sterilisation of food processing equipment. It is advisable to conduct in-place cleanability
trials prior to this test in order to verify the equipment’s hygienic design. The method is
based on a Unilever R&D Vlaardingen procedure (ref. 4) and is designed to indicate
whether an item of equipment can be freed internally from viable micro-organisms by in-
line steam sterilisation. Additionally, it should be noted that if a piece of equipment
proves to be suitable for in-line steam sterilisability using this method, the test for
bacteria tightness can follow on directly using the same batch of broth and the same test
circuit.

2 Definitions

The definitions in the EHEDG Glossary (see www.ehedg.org/glossary.pdf) apply to this

guideline. The most relevant definitions specific to this test method are:

Sterilisability
The suitability of clean equipment to be freed from viable micro-organisms including
relevant bacterial spores (i.e. sterilised) by, for instance, a treatment with pressurised
water or saturated steam at 121°C for 30 minutes, or another sterilising fluid. (Alternative
conditions can be used depending on local circumstances.)

Sterilisation
The removal or destruction of micro-organisms, including all relevant bacterial spores.

3 Materials

3.1 Indicator Micro-organisms

A heat resistant strain of Bacillus subtilis (Bac 1-12) is used as the test organism and was
chosen for its relatively high D-value, (0.71 minutes at 121°C) (ref. 5). It has a long
record of use in assessing the sterilisation of food.

The strain is cultivated overnight in trypticase soy broth (TSB; concentration 15g l-1
tripticase soy) at 37°C. Following incubation, 0.1ml volume of the broth is spread over
the surface of trypticase soy agar (TSA) plates. After incubation at 37°C for 3-5 days the
degree of sporulation is checked microscopically (e.g. observation under the microscope

© EHEDG Doc. 5 (Second edition) 3

by phase contrast or after staining with malachite green, crystal violet etc.). If greater
than 10%, the spores are harvested into physiological saline. This suspension is washed
twice in physiological saline and stored in 7ml portions in 20ml culture tubes at 5°C until
required. Spore suspensions may be kept for many months without losing relevant
characteristics. Immediately prior to use, spore portions are pasteurised at 80-82°C for 10
minutes and cooled at 15-25°C for 1h. The concentration of spores in the suspension is
determined by pour plating with TSA and incubating at 37°C for 3 days.

The heat resistance of the spore crop should be checked by immersing ampoules
containing a portion of the crop in either an oil bath or a steam chamber for an
appropriate time and temperature (e.g. 5 min at 121°C). The D-value can be calculated by
plotting log survivors versus time in minutes. Note: as this has to be undertaken for each
spore crop, it is suggested that large spore crops, sufficient to last for up to 12 months, be
prepared.

3.2 Tripticase Soy Broth

Trypticase soy broth (TSB) is pumped through the test circuit to provide a growth
medium for any indictor micro-organisms surviving in the test equipment after the
sterilisation test procedure.

3.3 Steam

The steam should be free from any contaminants (which may adversely affect the
germination and subsequent growth of Bacillus subtilis).

3.4 Test Equipment

Prior to testing, the equipment to be investigated is dismantled and thoroughly degreased

(using a solvent such as alcohol), cleaned by hand (using a neutral detergent solution)
and, if necessary, descaled (using a 1% w/w aqueous acetic acid solution). The
dismantled equipment (if the components are relatively small) should then be sterilised in
an autoclave at 121°C for 30 minutes. Alternatively, the equipment may be reassembled
and sterilised in-line by steam (121°C for 30 minutes).

Notes:

1. All construction materials, gaskets, etc. must be capable of withstanding the

cleaning and sterilisation procedures.
2. Equipment with shaft passages should be equipped with double seals and
provision made for flushing the space between the two seals with a sterilising
fluid.
3. Occasionally, gasket materials have antimicrobial properties, which may
influence the test results. Therefore, controls should be undertaken in which
gasket samples are soiled in the same conditions as the equipment in the test
procedure and just submerged in TSA (petri dishes). After solidification of the

© EHEDG Doc. 5 (Second edition) 4

 agar the petri dishes are incubated at 37°C for 24 hours. If after 24 hours no
growth is observed in the agar immediately surrounding the sample, the gasket
material must be regarded as unsuitable for this test method. If no growth is
observed on the petri dish at all, a second TSA plate, pre-inoculated with spore
suspension is incubated at 37°C for 24 hours to demonstrate positive growth of
the test strain.

4 Test Procedure

4.1 Equipment Soiling

The spore suspension is diluted with physiological saline to a concentration of

approximately 5 x 107 spores ml-1. A sample of the spore suspension is taken and the
concentration determined by pour-plating with TSA and incubating at 37°C for 3 days.
This diluted spore suspension is used to completely wet the inner surfaces of the
dismantled equipment, including all surfaces that are in contact with each other after re-
assembly (e.g. gaskets and gasket grooves). The equipment is allowed to dry at 20-25°C
(relative humidity, ≤65%) for ≥2 hours, after which it is re-assembled. If the temperature
is lower and/or the humidity is higher than specified, the time allowed for drying may
have to be extended. A visual assessment should be made of the coated surfaces, to
ensure sufficient drying, prior to re-assembly.

4.2 Sterilisation Procedure

An example of a test circuit used for conducting in-line steam sterilisability testing is
shown in Figure 1. An aseptic vessel fitted with two aseptic flow-through valves (Figure
2) and containing an appropriate volume of TSB, is sterilised in an autoclave at 121°C for
30 minutes.

Both side connections of the flow-through valves are short-circuited during autoclaving
(Figure 2) and the valves are left in the open position. After autoclaving the valves are
closed and the vessel is incorporated into the test circuit by means of the valves’ side
connections. Once the test circuit has been assembled, the item of equipment to be
evaluated is treated with steam at 121°C for 30 minutes. The steam must be saturated and
the required back pressure, 0.2Mpa (2 bar) absolute, controlled by means of the throttle
valve. Temperature and pressure within the system must be in agreement with those
expected for saturated steam. If this is not the case, the test is invalid (the steam may
contain gases, such as air). To ensure that no cold spots are formed in the system, care
must be taken to ensure that no condensate can accumulate during the steam treatment.
When the sterilisation procedure is completed, the two one-way valves either side of the
flow-through valves are closed. The flow-through valves are then opened, thus effecting
an aseptic connection with the TSB vessel. All test circuit components designated
‘aseptic’ must have been proven to be sterilisable and bacteria tight, otherwise they may
adversely influence the test results. In the case of small equipment, flexible tubing and

© EHEDG Doc. 5 (Second edition) 5

tube clamps may be used. All components (including any flexible tubing) must be
connected such that there are no places where solids or air can be trapped.

4.3 Detection of Surviving Spores

The TSB is introduced into the test equipment by means of a peristaltic pump. To avoid
anaerobic conditions the broth is circulated for 2 hours every day. The flow rate at which
the broth is circulated will depend on the volume contained within the system and should
be set to give two volume changes within the vessel during the 2-hour circulation each
day. The test circuit is kept at ambient temperature (approximately 20-25°C) for at least 5
days and, if applicable, the equipment is operated carefully 10 times during this period
(e.g. valves may be actuated and pumps operated manually). If the ambient temperature
fluctuates outside the stated limits, care should be taken that the growth of any spores of
B. subtilis Bac 1-12 is not adversely affected.

4.4 Interpretation of Results

If the broth remains clear after 5 days the equipment is classified as in-line steam
sterilisable. If the broth becomes turbid, a sample is taken and examined microscopically
for the presence of B. subtilis Bac 1-12. If there is any doubt as to the identity of the
micro-organisms, a further sample of the broth is pour plated with TSA and incubated at
37°C for 3-5 days. Colonies of sporulated B. subtilis Bac 1-12 are greyish/green.

4.5 Positive Controls

When, after sterilisability testing, the TSB is still clear (no growth) a small volume of the
spore suspension used to inoculate the test item is aseptically inoculated into the TSB of
the test circuit. Growth of the spores in the broth, within 3 days at ambient temperature, is
indicative of both the viability of the inoculating culture and the capacity of the TSB to
allow maximum growth (productivity of TSB).

5 Discussion

Whilst it is well recognised that ‘no visible growth’ is suggestive of a population of <106
cells ml-1, the test strain grows readily in TSB at 25°C (turbidity within 24 hours) and a
relatively long incubation time has been chosen (5 days). The reason for this is that
surviving spores may be heat-damaged and would need a longer recovery time. In
addition, surviving spores may be trapped or hidden between equipment parts, in which
case they have to grow out by multiplication before they reach the circulating broth.
Sterilisation at 121°C for 30 minutes (a process equivalent to >33 decimal reductions
with a D-value of 0.71 min at 121°C) is sufficient to kill the spores of B. subtilis Bac 1-
12 in the concentrations applied (approximately 5x107 ml-1), provided there are no cold
spots in the system. In summary, therefore, a visually clear broth is indicative of freedom
of spores within the test item, whilst turbidity of the broth is indicative of the presence of
cold spots, and hence spore survival, within the equipment.

© EHEDG Doc. 5 (Second edition) 6

Tests should be conducted a minimum of three times, if the same results are obtained
each time. If varying results are still obtained after a maximum of five tests, a thorough
examination should be conducted to ascertain whether the tests have been adversely
influenced by faults in either the item of test equipment, the test circuit or the testing
conditions and/or analysis. If any faults are discovered, these should be rectified and the
tests repeated. If no obvious faults are discovered and the results still remain variable, it
can be concluded that the item of test equipment is unlikely to be suitable for in-line
steam sterilisation unless modifications to the equipment are made or special measures
are taken to avoid problems in practice.

Equipment that proves not to be sterilisable by the described method, may be sterilisable
at higher temperatures and/or longer sterilisation times. If supported by experimental
evidence, such equipment may be classified as ‘steam sterilisable in-line only with
extended sterilisation times and/or temperatures higher than 121°C’, specifying the
applicable times and temperatures.

6 References

1. EHEDG Document No.2 (2004). A method for the assessment of in-place

cleanability of food processing equipment.
2. EHEDG Document No.4. (1993). A method for the assessment of in-line
pasteurisation of food processing equipment. Also as an extended abstract in
Trends in Food Science and Technology, February 1993, 4, 52-55.
3. EHEDG Document No.7. (2004). A method for the assessment of bacteria
tightness of food processing equipment. E
4. Lelieveld, H.L.M. (1985). Hygienic design and test methods. Journal of the
Society of Dairy Technology, 38, No. 1, 14-16.
5. Put, H.M.C., and Aalbersberg, W.I.J. (1967). Occurrence of Bacillus subtilis with
High Heat Resistance. Journal of Applied Bacteriology, 30, 411-419.

Order information on EHEDG documents may be obtained from www.ehedg.org.

© EHEDG Doc. 5 (Second edition) 7

 Figure 1. Test circuit for testing the bacteria tightness of equipment.

© EHEDG Doc. 5 (Second edition) 8

 1 = TSB vessel
3 = Aseptic flow-through valves
10 = Bacteria/air filter

Figure 2. TSB vessel with aseptic flow-through valves during sterilisation in an autoclave.

© EHEDG Doc. 5 (Second edition) 9

The production of EHEDG Guidelines is supported by the European Commission
under the Quality of Life Programme, project HYFOMA (QLK1-CT-2000-01359),
co-ordinated by TNO Nutrition and Food Research, Zeist, the Netherlands.