Procedures guidelines guideline for microbiological evaluation of chilled dairy products

Procedures & Guidelines
Guideline for Microbiological Evaluation of
Chilled Dairy Products

FSQ-588003-0104


Table of contents

Preface ...................................................................................................... 2
Procedural Flow Chart................................................................................................3
Evaluation of customer requirements.......................................................................3
Evaluation of customer requirements.......................................................................4
Plant pre-requisites ....................................................................................................4
Commissioning ...........................................................................................................4
Filling test with product .............................................................................................5
Test procedure....................................................................................................................................................... 5

Microbiological evaluation of end product (trial) ................................... 6
Introduction .................................................................................................................6
Sampling procedure for the long term storage test.................................................7
Rapid screening test for detection of severe failure ...............................................8
Pre-incubation of packages.................................................................................................................................... 8
Incubation conditions ............................................................................................................................................ 8
Plating ................................................................................................................................................................... 9
Microbiological evaluation.................................................................................................................................. 10

Long term storage test for ESL products ...............................................................11
Pre-incubation of packages.................................................................................................................................. 11
Plating ................................................................................................................................................................. 11
Test evaluation .................................................................................................................................................... 11

Documentation..........................................................................................................12

Microbiological evaluation of end product (commercial production) 12
Sampling procedure .................................................................................................12
Microbiological evaluation.......................................................................................12
Filing of results .........................................................................................................12

Acknowledgement .................................................................................. 13

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Preface
This document is intended to support the supply and installation of processing and filling
equipment. The document is supplied as a guideline only and is not intended to be
prescriptive. It presumes that all operating manuals as well as generally accepted good
manufacturing practices are followed. It shall not imply or constitute any kind of
guarantee of the result of the testing nor shall it replace legally required methods
concerning product safety and quality.
The scope of the document is concerned with the verification of microbiological
performance only. The result does not necessarily reflect the long term performance of
the equipment, which is the cumulative result of many variable factors.
Prior to the verification of the microbiological performance the following events are a
suggested sequence to be followed:

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Procedural Flow Chart
Evaluation of customer
requirements

Verification of plant
pre-requisites

Commissioning of
equipment

Filling tests with
product – 3 trial runs

Sampling & product
incubation

Rapid screening test
100 samples minimum

Long term storage test
Sample size determined by

pre-agreed target failure
rate

Pre-incubate samples
24-28 h @ 23°C- 25°C

Incubation of samples at
reference temperature for
duration of shelf life

Incubation of plates
24-28 h @ 23°C- 25°C

Plating of samples,
Incubation as at left,
Evaluation of results of
plating

Evaluation of
microbiological results

Abort long-term storage test

Continue

Evaluation of test by
comparison of actual failure
rate with pre-agreed target
rate

Troubleshooting &
root cause analysis

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Evaluation of customer requirements
It is the responsibility of the local Tetra Pak Market Company to fully evaluate the
customer requirements, including appropriate legal requirements, for supply and
performance of equipment and services. This will form the basis for determining the
scope of supply.

Plant pre-requisites
Verification of the listed plant pre-requisites is recommended when:
• Equipment is installed in an existing plant
• A new product is developed
• Key components are changed
The following pre-requisites should be verified according to the generally accepted Good
Manufacturing Practice for liquid food production/manufacturers specifications:
• raw material quality
• thermal process
• CIP
• utilities
• environmental hygiene

Commissioning
Commissioning is the demonstration that the equipment, production line or plant
performs as specified or agreed upon. Commissioning should be carried out after
equipment installation according to specifications and methods supplied by the respective
Tetra Pak Business Unit.
The following tests are applicable:
• “Water Test”(verifies the technical parameters of equipment performance)
• Package Integrity Test
The procedure for testing package integrity supplied by the relevant Tetra Pak Business
Unit should be followed.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Filling test with product
The goal of the test is to evaluate the performance of the complete production line. The
expected target shelf life, storage temperature, detectable defect rate and method of
evaluation should be the subject of the agreement between customer and Tetra Pak
Market Company. The requirements shall be in accordance with applicable local
legislation.
Test procedure
A filling test should consist of at least three separate runs with a CIP and disinfection
between each run. The duration of each run shall be agreed between the customer and
Tetra Pak with a suggested minimum of at least 1 hour. Note that contamination related
to run duration such as condensation and environmental effects may not be detected with

a short run time.
Note: All test protocol shall be well documented for record purposes. Raw material
quality shall be verified prior to the test commencing to determine whether the raw
material is suitable for processing.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Microbiological evaluation of end product (trial)
Introduction
The following methods are recommended as Tetra Pak standard procedures, unless local
legal requirements provide differently.
Statistical evaluation will be according to the defect rate initially agreed upon with
reference to the table above.
The long term storage test is the reference test to determine the failure rate of the
production line during the defined shelf-life of the product.
In addition it is recommended to conduct a rapid screening test in order to detect any
severe recontamination by substandard cleaning or operational problems during
production.
The following methods are applicable to all categories of chilled low-acid dairy products
including:
-

Bactofugated milk

-

Micro-filtrated milk

-

High heat-treated milk (135 °C / <1 second or higher than 71,7°C for 15 seconds,
respectively, according to the Council Directive 92/46/EEC. Max 127 °C in
Germany)

-

Ultra-pasteurised milk (138 °C- 2 seconds according to FDA regulations in the USA)

Note: The rapid screening test is not intended to replace the long-term storage test, as
it will not necessarily detect all recontaminations.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Sampling procedure for the long term storage test
The number of packages that must be tested from each run depends on the acceptable
defect rate, which Tetra Pak shall have negotiated with the customer before testing starts.
The number of packages will be based on statistics. The results of the samples taken in
each run should be added together for purposes of statistical evaluation. Some examples

are given in the table below. The figures are based on a confidence level of 95%. The
table below shows the maximum percentage defect rate based on the number of defective
packs found in the total tested (sum of three trials).

Maximum defect rates, %
Confidence level 95 %

Note: Besides the determination of defect rates the legal requirements must be fulfilled
including pathogens (e.g. EU, PMO or National directives). The samples should be taken
randomly over the whole production run.
Number of defects
0
1
2.99
4.75
1.5
2.37
1.00
1.58
0.75
1.18
0.60
0.95
0.50
0.79
0.43
0.68
0.37
0.59
0.33

0.53
0.30
0.47
0.15
0.24
0.10
0.16
0.075
0.12
0.060
0.095
0.050
0.079
0.043
0.068
0.037
0.059

2
6.3
3.15
2.0
1.57
1.26
1.05
0.90
0.79
0.70
0.63
0.32

0.21
0.16
0.13
0.10
0.090
0.079

3
7.57
3.83
2.56
1.93
1.54
1.29
1.10
0.966
0.859
0.774
0.385
0.258
0.194
0.155
0.129
0.111
0.097

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5
N/A

5,25
3.50
2.67
2.10
1.75
1.50
1.31
1.16
1.05
0.53
0.35
0.26
0.21
0.18
0.15
0.13

10
N/A
8.48
5.65
4,24
3.39
2.83
2.42
2.12
1.89
1.70
0.85
0.56

0.42
0.34
0.28
0.24
0.21

Sample size
100
200
300
400
500
600
700
800
900
1000
2000
3000
4000
5000
6000
7000
8000

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Rapid screening test for detection of severe failure
A minimum of 100 samples should be taken randomly over the production (see also table
above).
Pre-incubation of packages
The packages should be incubated for 24 - 28 hours at 23 °C - 25°C.
This does not include the time for temperature equilibration, which has to be determined
before or during the incubation as described under “Incubation conditions” (usually 3 – 4
hours).
Note: This time temperature combination has to be seen as a compromise between getting
false negative and false positive growth. This compromise can be accepted as the objective
of the rapid screening test is not to determine the failure rate in the production line but only
to detect severe contaminations in a test run (e.g. cleaning problems) and to be able to
repeat a test without waiting till the end of the long shelf life test.
The incubation conditions given here also are in accordance with the literature data
including the IDF documentation on recontamination tests.
Incubation conditions
Experiences have shown that in practice inadequate incubation conditions are usually the
main reason for getting false (negative or positive) or unexplainable results from incubation
tests. Therefore we would strongly make the following recommendations to ensure proper
incubation conditions:
• Make sure that there is a homogeneous temperature profile in all areas of the incubator
where you are storing the samples by conducting temperature measurements.
Temperature differences between coldest and warmest spot should not exceed +/- 2 °C.
Often simple means as e.g. additional radiators or ventilators can significantly improve
the situation.
• When incubating the packages make sure that the distance between single packages is
equal to the width of the single package to ensure air circulation and shorter temperature
equilibration between room and product temperature (e.g. 10 cm for Tetra Brik 1000 cc,
or 7 cm for Tetra Rex 1000 cc packages).

• During incubation of packages monitor the temperature profile in at least 3 to 4 packages
at different positions with temperature loggers and protocol the time until the product in
the packages has reached the incubator room temperature.
• Consider the ratio of total incubation room volume and product volume to be incubated.
The necessary equilibration time for the temperature might differ significantly when
incubating too many samples in a small incubation facility. If the incubator volume vs.
product volume is small increase the incubation room temperature a few centigrade to
compensate the initial temperature drop created by the cold samples.

Note: Incubation of packages at higher temperatures is not recommended due to the risk
of growth of microorganisms, which will not affect product under defined conditions of
chilled storage.
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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products
Plating
• Standard pasteurized, bactofugated and micro-filtrated milk.
The recommended medium is: 50% PCA+50% VRB agar. This media ensures inhibition
of most Gram-positive bacteria and does not inhibit Pseudomonas.
Other media with similar inhibition characteristics are also acceptable (please contact
or for further details).
The recommended method for evaluation of the rapid screening test is streaking a volume
of 10 µl of pre-incubated samples on the recommended agar. The method of application
is a straight-line inoculation, beginning approximately 5 mm from the edge of the plate
and ending about 5 mm from the opposite edge of the plate.
Plates are incubated for 24 to 28 hours at 23 °C - 25 °C.

Bioluminescence test methodology could be used as an alternative with the proviso that
each positive test sample be verified by conventional streak plating as described above.
Note: Pour - or surface plating of 0.01 to 0.1 ml can also be used but is not
recommended due to higher risk if recontamination and sometimes delayed growth in
pour plate agar.
•

High heat-treated milk (< 135 °C / 1 seconds)
Samples should be streaked or plated on Standard PC-Agar or TGA. A selective agar
as recommended for pasteurized products is not needed because the heat process
conditions will eliminate all bacteria which might be able to grow during the preincubation of the packages and might lead to false-positive growth. Another
advantage of using a non-selective media is that sporeformer originating from a
downstream contamination can also be detected.
Incubation conditions and alternative methods apply as above.

•

Ultra pasteurised milk (138 °C- 2 seconds FDA)
Same procedure applied as described above for high-heat treated milk.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products
Microbiological evaluation
The plates are evaluated by growth / no growth where less than 10 colonies on the streaks
or plate are considered as negative.

If there are 3 or more defective packages (> 7% defect level) in a sample size of 100
packages, it is recommended that the long-term storage test be discontinued immediately.
Note: If a volume of 10 µl is used the dilution factor will be 100 in order to express the
result in ml. After adequate pre incubation usually the count will be > log 5 to log7 / ml
this corresponds to > log 3(1,000) to log 5 (100,000) colonies per streak. Less than 10
colonies in the streak should be regarded as negative.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Long term storage test for ESL products
Pre-incubation of packages
The packages should be stored until the expiry date that has been defined as the target
shelf life. The storage temperature is the reference temperature that has been defined by
legislation or by agreement. The difference in temperature between layers of packages
should not be more than +/-1 °C. Packages should be stacked in a way that allows free
circulation of air between packages. Means to check and record the temperature during
the storage must be used.
The decision to continue further depends on the result of the rapid screening test.
Plating
The recommended method for evaluation of the target shelf life is streaking a volume of
10 µl on Standard Plate Count agar. The method of application is a straight-line
inoculation, beginning approximately 5 mm from the edge of the plate and ending about
5 mm from the opposite edge of the plate (see diagram below)

Streak line
10 µl loop

This is not a strict quantitative method but will show whether the sample is within the
legal demands or not (e.g. > 100 colonies on streak corresponds to > log 4 cfu / ml which
is normally the range of legal requirements (PMO, EC directive, Milk Hygiene
Ordinance). If, however there is a strict demand on a quantitative result by the customer,
either pour plating or the spiral plating technique is options.
The plates should be incubated at 23 °C - 25 °C for 72 hours for checking slow growing
micro-organisms (coryneform, moulds etc.). It is recommended to check plates for
growth also after 24 and 48 hours.
Microbiological evaluation of plates is carried out as previously described.
Test evaluation
A defect in terms of a microbial failure must be defined, e.g.:
• A total plate count greater than 20.000 to 50.000 cfu /ml depending on applicable
legislation.
• A positive streak showing > 10 cfu’s is regarded as a failure.
• Where no legislation exists, a target failure rate should be the subject of agreement
between customer and Tetra Pak.
The number of packages to be tested in total depends upon the pre-agreed acceptable
percentage failure rate and should be statistically determined. Refer to the table above.
Note: In case of failure, the test should only be repeated when troubleshooting has been
performed, and the problem root cause identified and rectified. Preferably the result
from the rapid screening test from the first test run should be available before the second
test run starts.

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Procedures & Guidelines
Guideline for Microbiological Evaluation of Chilled Dairy Products

Documentation
All testing procedures need to be carefully documented for reference purposes.
The results need to be filed and interpreted by a microbiological specialist and the
"commissioning team"(pre-agreed combination of joint group customer/TP) will decide
if the production line is acceptable for commercial production.

Microbiological
production)

evaluation

of

end

product

(commercial

This procedure could be applied in addition to the regular microbiological quality control
carried out by the customer.

Sampling procedure
When the results from the line performance test are acceptable and the commercial
production starts, the sampling procedure will be switched to regular quality control.
The following sampling scheme could be implemented:

Samples are taken every 30 to 60 minutes from each filler and the minimum number of
samples to be drawn per hour as follows:
Minimum 2 samples / hour

1 sample for rapid
detection for
severe failures

1 sample for long
term storage at
reference temperature

Microbiological evaluation
The samples should be analysed according to the appropriate methodology described as
described earlier.

Filing of results
It is recommended that the results obtained in regular quality control be filed in a
computer based data system, e.g. spreadsheet software such as Microsoft Excel or
compatible.
Should the failure rate deviate from the applicable internal standard level, a
troubleshooting programme shall be considered including production step control until
the failure rate is on the standard level again.

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Procedures & Guidelines

Guideline for Microbiological Evaluation of Chilled Dairy Products

Acknowledgement
This document is has been created as a joint effort between the former Tetra Pak ESLAC
group, Tetra Pak Research & Development and the Tetra Pak FiSQA-Group.
Grateful thanks are due to:
Lennart Olbjer
for his invaluable assistance with the statistics in this document. The inclusion of relevant
and user-friendly statistics plays a major role in the sampling and evaluation procedures.

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