Procedures guidelines guideline for microbiological evaluation of chilled dairy products

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

Procedures & Guidelines

Guideline for Microbiological Evaluation of

Chilled Dairy Products

FSQ-588003-0104

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

FSQ – 588003-0104 2 (13)

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:

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 plates

24-28 h @ 23°C- 25°C

Evaluation of

microbiological results

Abort long-term storage test

Troubleshooting &

root cause analysis

Continue

Incubation of samples at reference temperature for duration of shelf life

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

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

FSQ – 588003-0104 4 (13)

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

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

FSQ – 588003-0104 6 (13)

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:

- 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

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)

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

1.5 2.37 3.15 3.83 5,25 8.48 200 1.00 1.58 2.0 2.56 3.50 5.65 300 0.75 1.18 1.57 1.93 2.67 4,24 400 0.60 0.95 1.26 1.54 2.10 3.39 500 0.50 0.79 1.05 1.29 1.75 2.83 600 0.43 0.68 0.90 1.10 1.50 2.42 700 0.37 0.59 0.79 0.966 1.31 2.12 800 0.33 0.53 0.70 0.859 1.16 1.89 900 0.30 0.47 0.63 0.774 1.05 1.70 1000 0.15 0.24 0.32 0.385 0.53 0.85 2000 0.10 0.16 0.21 0.258 0.35 0.56 3000

0.060 0.095 0.13 0.155 0.21 0.34 5000 0.050 0.079 0.10 0.129 0.18 0.28 6000 0.043 0.068 0.090 0.111 0.15 0.24 7000 0.037 0.059 0.079 0.097 0.13 0.21 8000

FSQ – 588003-0104 8 (13)

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

Plating

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

Fritz.lembke@tetrapak.com or Kristina.Petersson@tetrapak.com 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

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 pre-incubation 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

Same procedure applied as described above for high-heat treated milk

FSQ – 588003-0104 10 (13)

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

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

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

10 µl loop