News from the European Reference Laboratory for Milk and Milk Products (EU-RL MMP), Paris, 2011

Transcription

1 News from the European Reference Laboratory for Milk and Milk Products (EU-RL MMP), Paris, 2011 Véronique Ninane* & Koen De Reu** *CRA-W, Département Valorisation des Productions, 24 Chaussée de Namur, B-5030 Gembloux **ILVO-T&V, Instituut voor Landbouw en Visserijonderzoek Eenheid Technologie en Voeding, Brusselsesteenweg 370, B-9090 Melle Introduction Each year, the EU-RL MMP holds a workshop for the national reference laboratories (NRL). The latest session, that took place on 2 and 3 May 2011, was dedicated to the entire field of analyses that is covered by the EU-RL MMP. The state of progress, the needs with respect to analysis and legislation concerning total flora, somatic cells and alkaline phosphatase were discussed by means of presentations and debates. Performance of the European Reference Laboratories (EU-RLs) In his introduction to the workshop, Klaus Kostenzer, representing DG SANCO of the European Commission, gave a detailed overview of the recent evaluation of the activities conducted by the different EU-RLs. The quality of the performances of the EU-RL MMP was judged good to very good. Conducting inter-laboratory tests The heart of the matter was approached through an item that includes all analysis parameters, i.e. the carrying out of comparative inter-laboratory tests. Soraya Amarouche of the French Laboratoire national de métrologie et d essai (LNE) described the broad outline of the standards ISO (2010): General requirements for proficiency testing and ISO (2005): Statistical methods for use in proficiency testing by interlaboratory comparison. The latter standard is a supplement to ISO Guide 43. It gives a detailed description of the statistical methods that must be used by those who set up proficiency tests and makes recommendations with respect to the use of these methods. The standard, among other things, mentions an indicative threshold value for judging the homogeneity of samples ; but Soraya Amarouche emphasized that this value is not always appropriate and may therefore be adjusted. The practical organization of inter-laboratory tests was discussed by the representatives of two NRLs, Karin Knappstein of the German NRL and Koen De Reu (ILVO-T&V) of the Belgian NRL, and by Thomas Berger of Agroscope (Switzerland). The aim of inter-laboratory tests was different for each of these speakers: comparison between routine devices and comparison between routine devices and with the reference method or determination of the value of reference material. In addition, Koen De Reu (ILVO-T&V) explained the process used for evaluating the 30

2 performance of the Belgian NRL by means of performance indicators as well as the financial penalty system linked to that process. It appeared from these accounts that the Belgian system is up to date when compared to the European developments in the field of analytical guidance. The representatives of all Member States were impressed by the completeness of the inter-laboratory tests presented and by the fact that part of these tests are done under accreditation: at the ILVO-T&V, some inter-laboratory tests are prepared under accreditation. It is therefore not surprising that many questions were asked to Koen De Reu when he had finished his presentation. Furthermore, the setting up of inter-laboratory tests was also at the centre of the presentations concerning the progress of EU-RL MMP studies and study projects on stability. As it is, the EU-RL MMP develops projects with a view to defining protocols on the treatment and the management of samples that are suitable for distributing tests among geographically distant laboratories. In that respect, the stability of raw goat s milk samples, to which preservatives had or had not been added, was tested at 12 C over a 10 days period (Rabed Miled). This study showed that adding a mixture of boric acid, glycerol and potassium sorbate makes raw goat s milk stable with respect to total flora. A project has been planned that deals with the formulation of raw cow milk samples that are naturally contaminated with somatic cells and are stable for that parameter (Alexandra Cauquil). The project was discussed and completed, more particularly on the basis of the information given by Koen De Reu on the method of preparation applied by the Belgian NRL to raw cow milk samples intended for inter-laboratory tests on somatic cell counting. Finally, more details were given on the protocol for checking the homogeneity and the stability of milk samples distributed for the purpose of the inter-laboratory test set up by the EU-RL MMP with a view to the determination of alkaline phosphatase (Caroline Vignaud, ANSES, Paris). Total flora As for the criterion «total flora», the validation of alternative methods and the establishment of their conversion relation are the main problems that need to be solved. The total flora of raw milk can be counted by means of an alternative method if that method has been validated against the reference method and when the rules of the ISO standard have been complied with. These rules have been established for foodstuffs and are not very appropriate and not very clear for the matrix in question: raw milk. In order to make these rules more explicit, the EU-RL MMP completed them with specific standards for milk, the FIL/IDF 161 standard that is being reviewed ( ISO 16297) and 128, and prepared a practical synthesis of the references to be consulted when implementing each of the validation points mentioned in standard ISO (Véronique Deperrois, ANSES, Paris). As for the conversion relation for alternative methods, the total flora of raw milk is determined by means of instrumental methods and specific conversion relations for each of the countries involved. At the previous meeting was made a suggestion to establish a working group that would have to examine whether these conversion relations can be harmonised at EU level (Ninane & Werbrouck, 2010). The EU-RL MMP initiated the feasibility study and suggested three statistical approaches that may be used to establish the incidence of the regional factor (geographic origin) on the conversion relation (Laurent Guillier, ANSES, Paris). The members who committed themselves to participate in the working group, including Véronique Ninane (CRA-W) for the Belgian NRL, will be invited to contribute to the further development of the study. Finally, the EU-RL MMP set up a study with a view to identify the factors that affect the conversion relation of the Bactocount (Bentley, USA) for counting the total flora in raw goat s milk (Rabed Miled). 31

3 Somatic cells The discussions on somatic cell count focused on the capacity of the reference laboratories to use the microscopic method that is described in the ISO standard, which is the reference method for that parameter. In order to evaluate the capacity of the European analysis network to use that method, the EU-RL MMP set up an inter-laboratory test at the end of 2010 (Alexandra Cauquil). That test revealed that 85 % of the participating laboratories, among which the Belgian NRL, use the method in an appropriate way. An accurate identification of the difficulties met by other laboratories, carried out with the aid of the EU-RL MMP, should lead to an increase of that percentage. The results of the test also reveal that the overall performance of the European network in terms of repeatability and reproducibility have improved when compared to the previous test (2008). The EU-RL MMP intends to further improve these performances by means of a practical training. In addition, the development of a structure for producing reference material for the somatic cell count should lead to an improvement of both the performance of the reference method and the mutual consistency of routine methods. The International Dairy Federation (IDF) and the International Committee for Animal Recording (ICAR, Italy) have joined their efforts to develop a suitable organisation structure, a so-called reference system (Thomas Berger, Ascroscope, Liebefeld-Posieux, Switzerland). The Belgian NRL is involved in the development of this system: Véronique Ninane (CRA-W) takes part in the activities of this working group of the IDF and the ICAR. 32

4 Alkaline phosphatase Several analytical aspects have been examined with regard to alkaline phosphatase the activity of which is used to detect pasteurization. One of these aspects is the validation of the fluorometric method for use in goat s milk that is described in the ISO standard (2006). In 2010, the EU-RL MMP set up an inter-laboratory test for the purpose of determining the repeatability and the reproducibility of the method in goat s milk. 22 laboratories were involved in that test, including the ILVO-T&V for the Belgian NRL. The test related to 6 alkaline phosphatase activity rates (Anne-Cécile Boitelle, ANSES, Paris). The quality of the results of the inter-laboratory test is such that the test may be used for validation, which was the purpose for which it was set up. Figure 1: Fluorophos instrument with incubator for the determination of the alkaline phosphatase activity Figure 2: Positive sample (left) and negative sample (right) analyzing the alkaline phosphatase activity 33

5 Another aspect that was discussed during the session is that of the residual alkaline phosphatase activity in pasteurized goat s milk, and more in particular, the legally acceptable limit value of alkaline phosphatase activity. In a concrete manner, the EU-RL MMP examined the possibility to use for goat s milk the limit value of 350 mu/l that was set by the EU for cow s milk. This study showed that the alkaline phosphatase activity following pasteurization of goat s milk is generally below the limit value set for cow s milk but that this value is exceeded in goat s milk coming from two Member States (Marina Nicolas, ANSES, Paris). DG SANCO should now take the initiative for legislation since it has enough information to make a decision. The determination of an activity limit value for cheese made from pasteurized milk was also on the agenda of the workshop. The adoption of a limit value is based upon the inventory of values found in practice and, hence, on the capacity of the laboratories to determine the alkaline phosphatase activity in cheese. In 2009, the EU-RL MMP set up a practical workshop on standard ISO (which was then being reviewed) that includes a description of the reference method for that parameter, with a view to improving the testing capacity of the NRL network. Following that workshop, the capacity of the NRLs to use the method was evaluated by means of an inter-laboratory test. Four out of the 19 participating laboratories did not have a sufficient capacity and were re-briefed and then subjected to an evaluation test. After these two briefing and evaluation sessions, 17 laboratories, among which the ILVO-T&V for the Belgian NRL, were found capable of determining the alkaline phosphatase activity in cheese in a correct way. The list of the residual alkaline phosphatase activity values found in practice may now be completed in order to be more representative of the real situation for cheese in Europe (Marina Nicolas, ANSES, Paris). In the meantime, the EU-RL MMP suggested to adopt a working limit value of 10 mu/g which may later be adjusted if necessary. That value has already been tested on some cheese types. So, the Swiss Agroscope laboratory tested the limit value on a certain number of Swiss cheeses made of cow s milk, using the reference method very accurately (Charlotte Egger, Agroscope,Liebefeld-Posiux, Switzerland). All cheeses of raw or thermised milk that were examined, showed an activity rate of more than 10 mu/g whereas most of the cheeses made from pasteurized milk had a residual alkaline phosphatase activity of less than 10 mu/g. One soft cheese, Limburger, showed a residual alkaline phosphatase activity of more than 10 mu/g although it was made from pasteurized milk. In that particular case, the superficial flora contributed to the alkaline phosphatase activity that was measured: when the activity was measured after removal of a thicker rind (exceeding the standard thickness for that cheese, i.e.0.5 cm instead of as thin as possible ), it was indeed less than 10 mu/g. The importance of the place,where the sample was taken, was then shown by means of large wheels ( cm diameter) of cheese made from thermised milk: the wheels are so big that the cheese at the centre of the wheel stays warm long enough to deactivate alkaline phosphatase in the same way as in the case of pasteurisation. Whereas for Swiss cheeses, an adjustment of the standard seems at first sight sufficient to make the proposed limit value acceptable, this is not so for other cheese types. As a matter of tact, the EU-RL MMP showed that the manufacturing process of some cheeses may be at the origin of a change in alkaline phosphatase activity and may lead to a misinterpretation of results (Marina Nicolas, ANSES, Paris). During the manufacturing process of mozzarella type cheese e.g., alkaline phospatase is sometimes deactivated, leading to an activity of less than 10 mu/g, although this cheese is made from raw milk. The participants attention was also drawn to the opposite phenomenon: cheese made from pasteurized milk that showed an alkaline phosphatase activity of more than 10 mu/g. In the latter case, an activity rate of more than 10 mu/g was measured at the centre of the cheese and not only on the surface, such as in the case of Limburger cheese; so, removing a thicker rind does not solve the problem. It is very likely that the microflora is at the origin of this abnormally high alkaline phosphatase activity. As for the analysis, the ISO method for determining alkaline phosphatase in cheese still needs to be validated. In that respect, the EU-RL MMP intends to carry out a preliminary study on the characterization of the method in 2011 and to deal with the inter-laboratory aspects of validation in In order to check pasteurization of milk of other animal species (such as camels), the use of other alkaline phosphatase markers, such as lactoperoxidase of γ-gt, is being considered. 34

6 Reference Ninane V. & Werbrouck H. (2010). Workshop of the NRLs for Milk and Milk Products, Paris, Labinfo AFSCA, 5,