Tài liệu 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 FSQ – 588003-0104 i 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: FSQ – 588003-0104 2 (13) 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 FSQ – 588003-0104 3 (13) 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. FSQ – 588003-0104 4 (13) 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. FSQ – 588003-0104 5 (13) 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. FSQ – 588003-0104 6 (13) 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 FSQ – 588003-0104 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 7 (13) 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. FSQ – 588003-0104 8 (13) 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 [email protected] or [email protected] 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. FSQ – 588003-0104 9 (13) 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. FSQ – 588003-0104 10 (13) 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. FSQ – 588003-0104 11 (13) 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. FSQ – 588003-0104 12 (13) 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. FSQ – 588003-0104 13 (13)