Milk and Milk Products Workshop Milk and Milk Products for the NRLs, Paris 2010 The European reference laboratory for Milk and Milk Products (EU-RL-MMP) held a workshop for national reference laboratories (NRLs) on 30 September and 1 October 2010. The Belgian national reference laboratory was represented by Hadewig Werbrouck (ILVO-T&V) and Véronique Ninane (CRA-W-DVP). Laurent Laloux (EU-RL-MMP) announced three changes regarding the EU-RL-MMP: the name “European reference laboratory Milk and Milk Products (EU-RL-MMP)” instead of “Community reference laboratory (CRL)”, the merger of the AFSSA and the AFSSET into ANSES (2) and the promotion of Véronique Deperrois who is now head of the Microbiology unit (3). Paolo Caricato (DG-SANCO) reported on the activities of DG-SANCO within the European Commission. A review of the activities of the European reference Laboratories, in particular an extension to “all fields” related to milk such as Staphylococcus aureus and Listeria spp.; an agreement to be reached with the United States on a limitation of border inspections of exported (or imported) milk products and the issue of inspections of pasteurised milk obtained from other species than cows. The development of methods to determine alkaline phosphatase (check if a product has been sufficiently pasteurized or not) in different species is now one of the priorities set by DGSANCO. This workshop was dedicated to only one subject : the total bacterial count in raw milk. Véronique Deperrois (EU-RL-MMP) gave an introduction to this subject by means of a presentation on the diversity of microflora in raw milk and on the influence of stress conditions on this diversity. Milk that leaves the udder is free of micro-organisms provided the cow does not show any signs of mastitis. The milk is contaminated only later by commensal microorganisms that are present in/on the teat of the cow, by man, by working tools, by the environment (e.g. litter and air), …. Maintenance, i.e. the practices adopted by the cattle farmer, of the “reservoirs” have an effect on the composition of raw milk; e.g. adding hay to the litter increases the number of lactobacilli in raw milk. Finally, one may say that the microflora in raw cow milk contains varying amounts of useful microorganisms (1) used e.g. in cheese making, such as leuconostocs, lactobacilli, lactococci, Propionibacteria, yeasts, fungi, streptococci, Enterococcus and “ripening” bacteria (cheese making); decay organisms (2) such as sporulated bacteria, psychrotrophic bacteria, coliforms, yeasts and fungi; and pathogenic microoganisms (3) such as staphylococci, Escherichia coli, Salmonella spp. and Listeria monocytogenes. This microbial diversity and the variability are the main factors that would possibly have to be taken into account when comparing methods used for determining the number of bacteria in raw milk since their targets may be different : bacteria are increasingly less capable of growing as a group in specific environmental conditions (some groups are inevitably excluded), sometimes “dead” bacteria, sometimes live bacteria, … For the implementation of Regulation (EC) No 853/2004 laying down specific hygiene rules for food of animal origin, the reference method for the plate count of raw milk is the method consisting of counting the colonies at 30°C, standardised as ISO 4833. The competence level of the European reference laboratories for this method was estimated by the EU-RL-MMP in ring tests carried out in 2007 and 2009. The results of these ring tests were presented by Alexandra Cauquil (EU-RL-MMP). She showed that the competence level of European laboratories was generally satisfactory. Moreover, that level had improved when compared to 2007: 88% of the participants

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obtained a good Z score against 82% in 2007. However, six European laboratories (not taken into account for assessing the results) did not use the reference method as yet. Paolo Caricato, the representative of DG-SANCO, was not pleased to hear this. Then, Rabid Miled (EU-RL-MMP) gave a complete view of the alternative methods for determining the bacterial count in raw milk. The alternative methods Bactoscan FC (Foss, Denmark) and Bactocount (Bentley, USA) were further discussed by representatives of the respective manufacturers: Berte Asmussen for Foss and Pierre Broutin for Bentley. The principle of these two methods is based upon the flow cytometry methodology with detection of bacteria by means of epifluorescence microscopy. In both cases the bacterial clusters are shattered into individual bacteria, the bacteria are coloured by means of a fluorescent dye and then the coloured bacteria are transferred to the reading system by means of a sheath liquid. The fluorescent dye of each individual bacterium is excited by a laser and detected by a detector. The reading system converts the light beams into electronic pulses. These pulses are then converted into the number of colony forming units by means of a conversion table. There was also a demonstration of the Bactocount (Bentley).

Fig 1: Bactoscan FC and Bactocount IBC. These methods may be used within the context of Regulation 853/2004 provided that they have been validated in accordance with the ISO 16140 standard and that their conversion relation has been established in accordance with the ISO 21187 standard. Betrand Lombard (EU-RL-MMP) gave a short review of the main rules. It was said, in concrete, that none of the alternative methods have already been validated but that a provisional approval had been issued for Bactoscan FC given the fact that part of the validation assessment is documented and to the fact that the Bactoscan FC equipment has been used for more than 10 years in most European countries. Yet, the manufacturer (Foss) will have to have the equipment validated in accordance with the ISO 16140 standard; if not, it will not be allowed to keep on using the equipment for the purpose of Regulation 853/2004. The EU-RL-MMP did not set a period of time but it is expected that the validation of the BactoscanFC will take up some two years. With a view to validation within the context of a ring test, EU-RL-MMP asked the manufacturers, including those of the Bactoscan FC, to call on the help of a specialised organisation. That aspect does not come under the authority

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of the NRLs. Three European certified organisations specialised in that type of work were mentioned: Microval, AFNOR certification and Nord Val. As for the evaluation intralaboratory tests have been performed for both the bacterial count and the somatic cells count by the EU-RL-MMP with a view to validating the equipment. The tests have been completed and will be published shortly. Bertand Lombard (EU-RL-MMP) mentioned some changes brought on by the review of the ISO 16140 standard (standardisation). These changes relate to the statistical processing, the introduction of acceptation criteria, the improved workability (praticability) and precision of the scope. As it is, the standard does not apply to an alternative method that is specific for the laboratory (in this case an interlaboratory test is useless) but it does apply to commercial methods. The issue of standardization, with respect to the ISO 16297/IDF 161 standard, was concluded by Harrie Van den Bijgaart (Qlip, Nederland). This is a specific standard and an addition to the ISO 16140 standard that may be applied to the evaluation part, i.e. the intralaboratory tests of the alternative methods for the bacterial count in milk. Given the complexity of standardisation with respect to the evaluation of alternative methods for determining the total flora in milk, the EU-RL-MMP suggested to write a practical guide with a detailed description of the procedures (both standards ISO 16140 and 13297 combined). The responsibility of the NRLs with regard to the appropriateness of the conversion relation for the alternative methods (preparation and control according to ISO 21187 standard) was pointed out. The “checklist” drawn up by the EU-RL-MMP, mainly with the help of Koen De Reu (ILVO-T&V), was distributed among the NRLs to help them judge the quality of the work done by routine laboratories. Jolanta Rola (PIWET, Poland) who used the “checklist” also made some suggestions for the improvement of the list and for focusing the list on the final purpose. In addition was set up a working group that is to re-examine the list taking into account the remarks made. Summing up, one might say that the general rule is that one conversion relation may be established per type of equipment and per country. Exceptionally, several conversion relations are possible but a motivation will be required in that case (e.g. significantly different climates). In practice, the establishment of a conversion relation is in fact hampered by the difficulty to distribute the same milk samples among different laboratories when the distance between laboratories is too great. An interesting approach that makes it possible to sidestep this problem was illustrated by Giusseppe Bolzoni (IZLER, Italy). His approach (details of statistical processing are available on www.izler.it ß reference material) suggests that there is a possibility to have one single conversion relation in Europe. A working group was set up that must examine the feasibility of that ambition. Véronique Ninane (CRAW-DVP) is a member of this working group.

Hadewig Werbrouck (ILVO-T&V, Melle) and Véronique Ninane (CRA-W-DVP, Gembloux) [email protected] and [email protected]

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Developments in the field of standards and legislation New IDF- FIL (International Dairy Federation – Fédération International de Laiterie) standards in 2010 (from 2 November 2009 up to 8 October 2010): Standards: ISO 1211|IDF 001:2010 - Milk - Determination of fat content - Gravimetric method (Reference method) ISO 5536|IDF 023:2009 - Milkfat products - Determination of Water content - Karl Fischer method ISO 12081|IDF 036:2010 - Milk - Determination of calcium content - Titrimetric method ISO 6091|IDF 086:2010 - Dried milk - Determination of titratable acidity (Reference method ISO 6732|IDF 103:2010 - Milk and milk products - Determination of iron content - Spectrometric method (Reference method) ISO 5546|IDF 115:2010 - Caseins and caseinates - Determination of pH (Reference method) ISO 13366-1|IDF 148-1:2008 - Milk - Enumeration of somatic cells - Part 1: Microscopic method (Reference method) + Technical Corrigendum 1 (2009) ISO 27205|IDF 149:2010 - Fermented milk products – Bacterial starter cultures - Standard of identity ISO 11813|IDF 156:2010 - Milk and milk products – Determination of zinc content - Flame atomic absorption spectrometric method ISO 17678|IDF 202:2010 - Milk and milk products – Determination of milk fat purity by gas chromatographic analysis of triglycerides (Reference method) ISO 26462|IDF 214:2010 - Milk - Determination of lactose content - Enzymatic method using difference in pH ISO/TS 27105|IDF/RM 216:2009 - Milk and milk products – Determination of hen’s egg white lysozyme by HPLC ISO/TS 27106|IDF/RM 217:2009 - Cheese - Determination of nisin A content by LC-MS and LC-MS-MS ISO 29981|IDF 220:2010 - Milk products - Enumeration of presumptive bifidobacteria - Colony count technique at 37 degrees C ISO 10932|IDF 223:2010 - Milk and milk products – Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria Other interesting IDF publications in 2009-2010: Bulletin of the IDF No. 440/2009 - Interlaboratory Collaborative Study on the Kjeldahl Reference Method for Nitrogen Determination in Sheep and Goat Milk Bulletin of the IDF No. 441/2009 - Monitoring success of paratuberculosis programs Proceedings of 2nd Paratuberculosis Forum, Minneapolis, August 2009 Bulletin of the IDF No. 442/2010 - Current situation & compilation of commercially available screening methods for the detection of inhibitors/antibiotic residues in milk - E-Form Bulletin of the IDF No. 443/2010 - Environmental issues at dairy farm level - E-Form Bulletin of the IDF No. 444/2010 - Feed-associated Mycotoxins in the Dairy Chain: Occurrence and Control - EForm IDF congresses: 2011_IDF Regional Conference on Domestic Milk Supply and Demand Systems : Lessons from Experience”, 26 - 29 April 2011, Seoul, Korea 2011_IDF International Symposium on Sheep, Goat and other non-Cow Milk, 16 - 18 May 2011, Athens, Greece 2011_IDF/ISO Analytical Week, 23-27 May 2011, Lyon, France 2011_IDF World Dairy Summit, 15-19 October 2011, Parma, Italy 2012_IDF International Symposium on Cheese Ripening and Technology, 20-24 May 2012, Madison, Wisconsin, USA

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2012_IDF/INRA International Symposium on Spray Dried Dairy Products, 19-21 June 2012, St. Malo, France 2012_IDF World Dairy Summit, 03-09 November 2012, Cape Town, South Africa 2013_IDF World Dairy Summit, October 2013, Yokohama, Japan

Koen De Reu (ILVO-T&V) Jessy Claeys (ILVO-T&V)

[email protected] [email protected]

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