The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum‐based products
2021; Wiley; Volume: 19; Issue: 4 Linguagem: Inglês
10.2903/j.efsa.2021.6576
ISSN1831-4732
AutoresIngrid Clawin‐Rädecker, Jan De Block, Lotti Egger, C. Willis, Maria Teresa da Silva Felício, Winy Messens,
Tópico(s)Milk Quality and Mastitis in Dairy Cows
ResumoEFSA JournalVolume 19, Issue 4 e06576 Scientific ReportOpen Access The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum-based products European Food Safety Authority (EFSA), Corresponding Author biocontam@efsa.europa.eu Correspondence:biocontam@efsa.europa.euSearch for more papers by this authorIngrid Clawin-Rädecker, Search for more papers by this authorJan De Block, Search for more papers by this authorLotti Egger, Search for more papers by this authorCaroline Willis, Search for more papers by this authorMaria Teresa Da Silva Felicio, Search for more papers by this authorWiny Messens, Search for more papers by this author European Food Safety Authority (EFSA), Corresponding Author biocontam@efsa.europa.eu Correspondence:biocontam@efsa.europa.euSearch for more papers by this authorIngrid Clawin-Rädecker, Search for more papers by this authorJan De Block, Search for more papers by this authorLotti Egger, Search for more papers by this authorCaroline Willis, Search for more papers by this authorMaria Teresa Da Silva Felicio, Search for more papers by this authorWiny Messens, Search for more papers by this author First published: 30 April 2021 https://doi.org/10.2903/j.efsa.2021.6576 Requestor: European Commission Question number: EFSA-Q-2020-00331 Waiver: In accordance with Article 21 of the Decision of the Executive Director on Competing Interest Management, a waiver was granted to Charlotte (Lotti) Egger, an expert of the Working Group. Pursuant to Article 21(6) of the aforementioned Decision, the concerned expert was allowed to take part in the discussion and in the drafting phase of the EFSA Scientific Report on the use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum-based products, and was not allowed to be, or act as, a chairman, a vice chairman or rapporteur of the BIOHAZ Working Group on ALP milk pasteurisation (EFSA-Q-2020-00331). Any competing interests are recorded in the respective minutes of the meetings of that Working Group. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgements: EFSA wishes to acknowledge the contribution of Katrin Bote to this report for the scientific support. EFSA also wishes to thank the external reviewers Avelino Alvarez Ordóñez and Robert Davies. EFSA also wishes to acknowledge all European competent institutions, Member State bodies and other organisations that provided data for this scientific output. Approved: 6 April 2021 The Protocol is available under the Supporting Information section. AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Pasteurisation of raw milk, colostrum, dairy or colostrum-based products must be achieved using at least 72°C for 15 s, at least 63°C for 30 min or any equivalent combination, such that the alkaline phosphatase (ALP) test immediately after such treatment gives a negative result. For cows' milk, a negative result is when the measured activity is ≤ 350 milliunits of enzyme activity per litre (mU/L) using the ISO standard 11816-1. The use and limitations of an ALP test and possible alternative methods for verifying pasteurisation of those products from other animal species (in particular sheep and goats) were evaluated. The current limitations of ALP testing of bovine products also apply. ALP activity in raw ovine milk appears to be about three times higher and in caprine milk about five times lower than in bovine milk and is highly variable between breeds. It is influenced by season, lactation stage and fat content. Assuming a similar pathogen inactivation rate to cows' milk and based on the available data, there is 95–99% probability (extremely likely) that pasteurised goat milk and pasteurised sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively. The main alternative methods currently used are temperature monitoring using data loggers (which cannot detect other process failures such as cracked or leaking plates) and the enumeration of Enterobacteriaceae (which is not suitable for pasteurisation verification but is relevant for hygiene monitoring). The inactivation of certain enzymes other than ALP may be more suitable for the verification of pasteurisation but requires further study. Secondary products of heat treatment are not suitable as pasteurisation markers due to the high temperatures needed for their production. More research is needed to facilitate a definitive conclusion on the applicability of changes in native whey proteins as pasteurisation markers. Summary Pasteurisation of raw milk, colostrum, dairy or colostrum-based products must be achieved using heat treatment of at least 72°C for 15 s, at least 63°C for 30 min or any equivalent combination, such that the alkaline phosphatase (ALP) test immediately after such treatment gives a negative result. That is when the measured activity in cows' milk is ≤ 350 milliunits of enzyme activity per litre (mU/L) using the ISO reference method. Following a request from the European Commission, EFSA was asked to provide scientific and technical assistance on the use of ALP and possible alternative testing to verify thermal pasteurisation of milk, colostrum, dairy and colostrum-based products from sheep and goats. More specifically in Term of Reference 1 (ToR1), EFSA was requested to provide an overview of the scientific information available on the use and limitations of ALP testing for verifying pasteurisation in the above products derived from sheep and goats, compared to cattle. If information is available, the overview could be extended to products derived from other species such as solipeds and camelids, producing such products for human consumption. In ToR2, EFSA was requested to list the possible alternative methods to the determination of ALP activity, and their possible limitations for the verification of pasteurisation of the products immediately after such treatment in the processing plant, as well as on the end product placed on the market. The European Commission clarified that both ToR 1 and ToR 2 should be assessed considering the relevant products immediately after pasteurisation of milk or colostrum, in the processing plant or at farm level if the adequate equipment is in place, as well as in the end products placed on the market. The pasteurisation conditions of the assessment will consider those that have been legally defined. The overall approach to answer both ToRs was qualitative and based on using evidence extracted from the scientific literature, databases and expert knowledge. Also, a questionnaire was used to gather information about the current usage of ALP and possible alternatives to verify pasteurisation of relevant products in the EU. Regarding ToR1, the following assessment question 1 (AQ1) was formulated to address the ToR: What is the use and what are the limitations of ALP testing to verify thermal pasteurisation of milk or colostrum from sheep and goats (and other species such as solipeds and camelids, producing such products for human consumption), compared to cattle, both immediately after such treatment, as well as for the end products placed on the market (milk or colostrum for direct human consumption and milk or colostrum-based products such as yoghurt, cheese, ice cream, milk powder, cream, or fermented milk)? It was concluded that one-third of the 15 EU countries replying to the questionnaire reported using ALP testing for milk or milk products from non-bovine species, more specifically in goats' milk, sheep's milk, cheese from sheep's milk and cheese from goats' milk (in descending order). The limitations of ALP testing for verifying pasteurisation of milk and milk products from bovine species also apply to other species. It is recommended that the ALP test should be performed immediately after the heat treatment and that those factors that influence the residual ALP levels should be considered when interpreting the results. The ALP activity in raw sheep milk appears to be about three times higher, and in caprine milk about five times lower than in bovine milk. The level in raw milk from sheep and goats is highly variable between breeds and is influenced by season, lactation stage, fat content and udder health. Further variation of basal ALP levels among non-bovine species is expected due to greater variation in breeds of sheep, goats and equines compared to dairy cows. Combining the information on basal ALP levels and thermal inactivation behaviour of the enzyme in the respective species would facilitate an estimation of residual ALP after pasteurisation. However, only a few studies have investigated the thermal stability of ALP in milk derived from cows, sheep and goats, with conflicting evidence. Therefore, it is not possible to estimate residual ALP levels with certainty. Assuming that the inactivation of pathogens by heat would be the same in the milk of different species, and based on the available evidence from milk samples after pasteurisation, there is 95–99% probability (extremely likely) that pasteurised goat milk and pasteurised sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively. Nevertheless, it is recommended to collect further data in order to conclude whether the evidence now available is representative of all situations. For equine milk, the current test sensitivity does not allow the use of ALP testing as the basal ALP activity is very low. Camel milk also contains low basal levels and, additionally, a heat-stable ALP, and therefore, ALP testing is not appropriate either. The data available for cheese of non-bovine species do not allow limits to be evaluated. No data is available for colostrum or milk or colostrum-based dairy products such as yoghurt, ice cream, milk powder, cream or fermented milk. Regarding ToR2, the following AQ2 was formulated: What are the possible alternative methods to the determination of ALP activity, and their possible limitations, for the verification of thermal pasteurisation of milk or colostrum from sheep and goats, both immediately after such treatment, as well as for the end product placed on the market? The main alternative methods to verify pasteurisation of these products from non-bovine species are temperature monitoring over time during the heat treatment using data loggers and the enumeration of Enterobacteriaceae. The use of data loggers is standard practice to monitor the heat treatment applied over time but cannot detect other process failures or post-pasteurisation contamination. Enterobacteriaceae testing is relevant for monitoring the general hygiene of milk and milk products in accordance with the process hygiene criterion but is not suitable to verify that pasteurisation conditions have been properly applied. The assessment of different classes of heat treatment of milk can be performed by means of assaying other endogenous marker enzymes, secondary products of heat treatment or changes in whey proteins. The inactivation of some enzymes may be more suitable to verify pasteurisation conditions of milk from non-bovine species than ALP but studies would be required to evaluate this. Due to the high temperatures needed for the production of secondary products of heat treatment, methods based on their detection are not suitable as pasteurisation markers. Changes in native whey proteins depend on their levels in milk and their variability, making it difficult to set a meaningful limit for pasteurised milk currently. Recommendations for further studies were formulated relating to an in-depth thermal inactivation kinetics study of ALP inactivation in milk from the various animal species. More studies are also recommended to evaluate the use and limitations of ALP testing of colostrum and milk or colostrum-based products such as cheeses derived from goat and sheep milk and to evaluate the use of other endogenous enzyme markers for milk derived from other species such as solipeds and camelids. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor 1.1.1 Background When raw milk, colostrum, dairy or colostrum-based products from farmed animals undergo heat treatment, food business operators (FBOp) must ensure that the treatment complies with the conditions for pasteurisation or ultra-high temperature (UHT) treatment in accordance with Part II of Chapter II to Section IX of Annex III to Regulation (EC) No 853/2004 laying down specific hygiene rules for food of animal origin.11 Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin. OJ L 139, 30.4.2004, p. 55–205. Pasteurisation must be achieved by a treatment involving: a high temperature for a short time (at least 72°C for 15 s); a low temperature for a long time (at least 63°C for 30 min); or any other combination of time-temperature conditions to obtain an equivalent effect, such that the products show, where applicable, a negative reaction to an alkaline phosphatase (ALP) test immediately after such treatment. According to Chapter II in Annex III to Commission Implementing Regulation (EU) No 2019/62722 Commission Implementing Regulation (EU) 2019/627 of 15 March 2019 laying down uniform practical arrangements for the performance of official controls on products of animal origin intended for human consumption in accordance with Regulation (EU) 2017/625 of the European Parliament and of the Council and amending Commission Regulation (EC) 2074/2005 as regards official controls. OJ L 131, 17.5.2019, p. 51–100., such a test is considered to give a negative result if the measured activity in cows' milk is not higher than 350 milliunits of enzyme activity per litre (mU/L) using the ISO reference method 11816-1.33 ISO 11816-1 [IDF 155-1:2013]. Milk and milk products – Determination of alkaline phosphatase activity – Part 1: Fluorimetric method for milk and milk-based drinks. International Organization for Standardization, Geneva, Switzerland. While this verification method works well in products derived from cows' milk, difficulties have been encountered when applying it to products of sheep and goat origin and no cut off value has been laid down. This has been acknowledged by the wording "where applicable" in the legal provisions laid down in Regulation (EC) No 853/2004. A presentation made by the former European Union Reference Laboratory for milk and milk products (EURL-MMP) during its 14th Workshop of National Reference Laboratories (NRLs) in May 2011, representing the state of play at that moment on goat milk, is attached. 1.1.2 Terms of Reference (ToRs) In accordance with Article 31 of Regulation (EC) No 178/200244 Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–24., the Commission requests EFSA to provide scientific and technical assistance with an overview on the possible use of the ALP test for the above purpose in products derived from ewes and goats, and, on the availability of alternative methods. EFSA is requested to evaluate the use of ALP and possible alternative testing to verify thermal pasteurisation of milk, colostrum, dairy and colostrum-based products ('products') from sheep and goats. More specifically EFSA is requested: ToR 1: to provide an overview of the scientific information available on the use and limitations of ALP testing for verifying pasteurisation in the above products derived from sheep and goats, compared to cattle. If information is available, the overview could be extended to products derived from other species such as solipeds and camelids, producing such products for human consumption.ToR 2: to list the possible alternative methods to the determination of ALP activity, and their possible limitations for the verification of pasteurisation of the products immediately after such treatment in the processing plant, as well as on the end product placed on the market. 1.2 Interpretation of the ToRs The European Commission clarified that for both ToR 1 and ToR 2 the products to be assessed are milk, colostrum, dairy and colostrum-based products (referred to as 'relevant products' throughout this document) and these can be derived from sheep, goats and, if possible, from other animal species such as solipeds and camelids, producing such products for human consumption. Whenever reference is made in the report to milk, colostrum or dairy products, these are derived from cows' milk except when the animal species is specified. Cows' milk is the major type of milk produced in the EU. In the EU-28 in 2018, 172.2 million tonnes of milk was produced on farms, of which 96.8% was cows' milk, 1.6% ewes' milk, 1.3% goats' milk and 0.1% buffaloes' milk. In several MSs, milk other than cows' milk contributes significantly to milk production.55 https://ec.europa.eu/eurostat/statistics-explained/index.php/Milk_and_milk_product_statistics#Milk_production Raw milk is defined in Regulation No (EC) 853/20041 as 'milk produced by the secretion of the mammary gland of farmed animals that has not been heated to more than 40°C or undergone any treatment that has an equivalent effect'. Dairy products are defined as 'processed products resulting from the processing of raw milk or from the further processing of such processed products'. Colostrum is defined as 'the fluid secreted by the mammary glands of milk-producing animals up to 3–5 days post parturition that is rich in antibodies and minerals and precedes the production of raw milk'. Colostrum-based products are defined as 'processed products resulting from the processing of colostrum or from the further processing of such processed products'. It was also clarified that both ToR 1 and ToR 2 should be assessed considering the relevant products immediately after thermal pasteurisation of milk or colostrum, in the processing plant or at farm level if the adequate equipment is in place, as well as in the end products placed on the market. The end products are the milk or colostrum for direct human consumption and any products based on those such as yoghurt, cheese, ice cream, milk powder, cream or fermented milk. 'End products placed on the market' should be understood as to be supplied 'at retail' or by 'direct supply to the final consumer'. Thermal pasteurisation will be referred to as 'pasteurisation' in the remainder of this document and will consider the legally defined treatment conditions. According to the Codex code for milk and milk products (CAC, 2004), 'pasteurisation is the application of heat to milk and liquid milk products aimed at reducing the number of any pathogenic microorganisms to a level at which they do not constitute a significant health hazard'. It results in the elimination of the most heat-resistant, non-spore-forming pathogenic bacteria and contributes to the extension of the shelf-life. The description of pasteurisation given by the International Dairy Federation (IDF, 1986) remains very appropriate: 'a process applied with the aim of avoiding public health hazards arising from pathogenic microorganisms associated with milk, by heat treatment which is consistent with minimal chemical, physical and organoleptic changes in the product'. This can be achieved by heating at high temperature for a short time (HTST; at least 72°C for 15 s) or at low temperature for a longer time (LTLT; at least 63°C for 30 min) or at any other combination of time–temperature (t/T) to obtain an equivalent effect, such that the ALP activity in milk is reduced to an activity not higher than 350 mU/L. The lactoperoxidase (LPO) enzyme is still active after pasteurisation; in some countries such as Switzerland (Eberhard and Gallmann, 1994), LPO negative milk is referred to as 'highly pasteurised' milk, but this is not a clearly defined term in the EU. This mandate concerns the evaluation of the potential use, and limitations, of ALP activity for the verification of pasteurisation of the relevant products from sheep, goats and, if possible, from other species such as solipeds and camelids, as it is currently used for the verification of the application of pasteurisation to bovine milk. Other possible uses of this test, e.g. to assess colostrum quality or immunoglobulin G (IgG) concentration, are excluded from this mandate. Alternative testing to verify thermal pasteurisation of the relevant products should include alternative methods to the ISO 11816-1:2013 standard3 for the determination of ALP activity, as well as possible alternatives to the determination of ALP activity. Based on the interpretations described above, the following assessment questions (AQs) were formulated in order to address the ToR: AQ1: What is the use and what are the limitations of ALP testing to verify thermal pasteurisation of milk or colostrum from sheep and goats (and other species such as solipeds and camelids, producing such products for human consumption), compared to cattle, both immediately after such treatment, as well as on the end products placed on the market (milk or colostrum for direct human consumption and milk or colostrum-based products such as yoghurt, cheese, ice cream, milk powder, cream, or fermented milk)? AQ2: What are the possible alternative methods to the determination of ALP activity, and their possible limitations, for the verification of thermal pasteurisation of milk or colostrum from sheep and goats, both immediately after such treatment, as well as on the end product placed on the market (as above)? 1.3 Additional information 1.3.1 Study from the European Union Reference Laboratory for milk and milk products on ALP limits in goat milk A study was carried out by the former EURL-MMP66 According to the Commission Regulation (EU) 2017/2460 of October 2017, the European Union reference laboratory for milk and milk products (EURL-MMP) has stopped its activities on 31 December 2017. on ALP testing and limits in goats' milk from different Member States (MS). The results of this study ('Fixation of ALP limits in goat milk, EU study') were presented during the 14th Workshop of the NRLs in May 2011 and provided to EFSA as an addendum to this mandate. Preliminary data collected from nine countries showed that most countries complied with the legal ALP limit defined for cows' milk of 350 mU/L, but two countries had goats' milk samples with values higher than 350 mU/L. During the autumn of 2009, additional data were collected from another eight countries. One country partly complied with the legal ALP limit defined for cows' milk of 350 mU/L, whereas the other seven countries fully complied with the legal limit. The fact that, overall, two countries did not comply with the legal ALP limit defined for cows' milk triggered discussions at European Commission level regarding whether a derogation would be pertinent for certain countries, or if a higher limit should be allowed for all countries. The European Commission decided to refer this Article 31 mandate to EFSA before taking any management decisions on this topic. 1.3.2 Legal background Part II of Chapter II of Section IX of Annex III to Regulation (EC) No 853/20041 laying down specific hygiene rules for food of animal origin describes the specific requirements for heat treatment of raw milk, colostrum and dairy or colostrum-based products. FBOp must ensure that the treatment satisfies the requirements laid down in Chapter XI of Annex II to Regulation (EC) No 852/200477 Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. OJ L 139, 30.4.2004, p. 1–54.. In particular, they shall ensure, when using the following processes, that they comply with the specifications mentioned: Pasteurisation is achieved by a treatment involving: a HTST (at least 72°C for 15 s); a LTLT (at least 63°C for 30 min); or any other combination of t/T conditions to obtain an equivalent effect, such that the products show, where applicable, a negative reaction to an ALP test immediately after such treatment. UHT treatment is achieved by a treatment: involving a continuous flow of heat at a high temperature for a short time (not less than 135°C in combination with a suitable holding time) such that there are no viable microorganisms or spores capable of growing in the treated product when kept in an aseptic closed container at ambient temperature, and sufficient to ensure that the products remain microbiologically stable after incubating for 15 days at 30°C in closed containers or for 7 days at 55°C in closed containers or after any other method demonstrating that the appropriate heat treatment has been applied. When considering whether to subject raw milk and colostrum to heat treatment, FBOp must: have regard to the procedures developed in accordance with the HACCP principles pursuant to Regulation (EC) No 852/20047; and comply with any requirements that the competent authority (CA) may impose in this regard when approving establishments or carrying out checks in accordance with Regulation (EC) No 854/200488 Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. OJ L 139, 30.4.2004, p. 206–320.. Chapter II in Annex III to Commission Implementing Regulation (EU) No 2019/6272 lays down the conditions to determine the ALP activity in pasteurised cow's milk as follows: To determine the ALP activity in pasteurised cow's milk, the ISO standard 11816-13 must be applied as the reference method. The ALP activity is expressed as mU/L. One unit of ALP activity is the amount of ALP enzyme that catalyses the transformation of 1 micromole of substrate per minute. An ALP test is considered to give a negative result if the measured activity in cows' milk is not higher than 350 mU/L. The use of alternative analytical methods is acceptable when they are validated against the reference method mentioned in point A in accordance with internationally accepted protocols and rules of good laboratory practice. The Codex code for milk and milk products (CAC, 2004) established performance and process criteria for pasteurised milk and liquid milk products. 'As C. burnettii is the most heat-resistant non-sporulating pathogen likely to be present in milk, pasteurisation is designed to achieve at least a 5 log reduction of C. burnettii in whole milk (4% milkfat)' (performance criteria). In relation to the process criteria, 'according to validations carried out on whole milk, the minimum pasteurisation conditions are those having bactericidal effects equivalent to heating every particle of the milk to 72°C for 15 s (continuous flow pasteurisation) or 63°C for 30 min (batch pasteurisation). Similar conditions can be obtained by joining the line connecting these points on a log time versus temperature graph. Processing times necessary rapidly decrease with minimal increase in temperature. Extrapolation to temperatures outside the range of 63–72°C, in particular, processing at temperatures above 72°C must be treated with the utmost caution as the ability for them to be scientifically [validated] is beyond current experimental techniques. When changes in the composition, processing and use of the product are proposed, the necessary changes to the scheduled heat treatment should be established and a qualified person should evaluate the efficiency of the heat treatment. For instance, the fat content of cream makes it necessary to apply minimum conditions greater than for milk, minimum 75°C for 15 s. Formulated liquid milk products with high sugar content or high viscosity also require pasteurisation conditions in excess of the minimum conditions defined for milk'. Part III of Chapter I of Section IX of Annex III to Regulation (EC) No 853/20041 specifies that FBOp producing or, as appropriate, collecting raw milk and colostrum must ensure compliance with the following requirements before heat treatment; raw cows' milk must have a plate count at 30°C of less than 100,000 CFU per mL. raw milk from other species must have a plate count at 30°C of less than 1,500,000 CFU per mL. Part III of Chapter II of Section IX of Annex III to Regulation (EC) No 853/20041 lays down for FBOp manufacturing dairy products the criteria for raw cows' milk immediately before being
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