Study of physiological water content of poultry reared in the EU

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2 Study of physiological water content of poultry reared in the EU Contract number: 30-CE /00-25 Final Report LGC Contact Point: Selvarani Elahi Tel: +44 (0) Prepared by: Selvarani Elahi and Joanna Topping Report Number LGC CPFC/2012/492 2

3 This study was funded by the European Commission. The information and views set out in the report are those of the authors and do not necessarily reflect the official opinion of the Commission. The Commission does not guarantee the accuracy of the data included in this study. Neither the Commission nor any person acting on the Commission s behalf may be held responsible for the use which may be made of the information contained therein. This study was conducted by LGC Limited; the project team consisted of: Selvarani Elahi Joanna Topping Mark Woolfe Jesus Minguez Stephen Ellison. The European Commission Project Steering Group members were: Stefania Marrone Karlheinz Grobecker Marcin Zarzycki Gianluca Frinzi Gebhard Seiwald. 3

4 Contents 1. LGC Document Control Executive Summary Introduction Collection of Poultry Samples Sample Analysis Sample Homogenisation Determination of Water/Protein ratio Results Statistical Evaluation Comparison with study carried out in Calculation of limits Conclusions and Recommendations Conclusions Recommendations References Acknowledgements

5 List of Tables Table 1. Top 7 Poultry Producing Countries in the EU in Table 2. Full Factorial Balanced Design of the Key Variables Associated with Commercial Poultry Production in the EU 14 Table 3. Final sampling plan used for Italy 16 Table 4. Summary of Samples Collected 17 Table 5. Acceptance Criteria for Analytical Results 19 Table 6. Number of Repeat Analysis Performed by Laboratory 20 Table 7. Summary of methods used and sample weight taken by laboratory 21 Table 8. Water and Protein Summary Data 26 Table 9. The mean W/P ratios obtained in 2012 and Table 10. Significance of effects on W/P ratio. 27 Table 11. Significance of Countries 28 Table 12. Upper limit calculations: comparison of data published in 1993 report with data generated in Table Limits for 2, 4 and 6% extraneous water. 30 Table 14. Recalculated Upper Limits for 2, 4 and 6% extraneous water when a composite of 5 samples is used based on 2012 data. 31 Table 15. Current Upper limits compared with the proposed new upper limits. 33 List of Figures Figure 1. W/P ratio in chicken and turkey leg cuts as measured in EU Member States in 1993 and in Germany in Figure National broiler production for EU Member States Figure 3. Water and Nitrogen results by laboratory Figure 4. Water/Protein ratio by laboratory Figure 5. The W/P ratios obtained for gender, weight category and breed Figure 6. Water/Protein ratios in poultrymeat in the EU by country Figure 7. Water/Protein ratios per country comparing 1993, 2007 and 2012 studies Figure 8. Effect of recalculated and legislative limits on sample failure rate

6 1. LGC LGC Limited ( LGC ) is an international science based company and is Europe's leading independent provider of analytical, forensic and diagnostic services, and reference standards. LGC is the UK s designated National Measurement Institute for chemical and biochemical analysis and is also the host organisation for the UK s Government Chemist function. Separately, LGC also holds a range of National Reference Laboratory duties including the National Reference Laboratory for added water in poultrymeat. In 2012, LGC was appointed as one of the three National Reference Laboratory representatives on European Commission s Board of Experts in monitoring water content in poultrymeat. With headquarters in Teddington, South West London, the LGC Group employs 1,800 staff in 36 laboratories and centres across Europe and at sites in China, India, South Africa and the US. LGC was awarded this project following a competitive tender issued by the European Commission. 6

7 2. Document location The report is published on the European Commission website: 7

8 3. Executive Summary The aim of this study was to determine the physiological water content in chicken raised and slaughtered in the European Union (EU) in 2012 and to compare it to the results of a study conducted in 1993 to assess whether the limits for extraneous water in European legislation need to be revised. Seven Member States were chosen for participation in this study who accounted for more than 70% of total EU poultry production according to the figures published for Information was collected on current poultry rearing practices in the EU with respect to the most common breeds, weight classes, gender balance, flock/batch sizes and typical processing volumes. Using this information, a sampling plan was formulated that proposed collecting forty eight birds from each of the top EU poultry producing Member States. The sampling plan covered the key variables of breed, weight (light and heavy birds), gender, flock and cuts. Samples were collected from these seven Member States under the supervision of at least one LGC representative. The collected samples were frozen in the slaughterhouses or at the NRL in the Member State that samples were being sampled in and sent by overnight courier to LGC for sample homogenisation. Each sample was homogenised in accordance with ANNEX VIII of Commission Regulation (EC) 543/2008. Homogenised samples were stored frozen in aliquots until required for analysis. A stratified sampling plan was devised to distribute the samples to the participating eight NRLs, so that they each analysed a carefully selected set of samples from all seven Member States from which samples were taken. Thus the analyses were performed by eight NRLs. An analytical protocol stating acceptance criteria for the data was circulated to each laboratory. The laboratories were required to analyse a reference material (ERM - BB501a) with each batch of samples, which was supplied. The results were entered into a standard electronic reporting form and returned to LGC for statistical analysis. Although the statistical analysis showed differences between chicken samples collected from different poultry producing Member States and the gender of the birds, these differences were small compared to the spread of results observed, and hence were considered not to be of practical significance. The 2012 study has confirmed that younger birds do have slightly more water and slightly less protein when compared to the results from the 1993 study. Although the changes appear to be small, they are significant; an appreciable number of chicken breast and leg cuts on sale in the EU would be expected to fail the limits set in European legislation if they remain unchanged. This study provides strong evidence to support a decision to amend the limits in European legislation so that they reflect chicken reared in the EU in

9 4. Introduction 4.1 Regulations A standard for marketing raw poultrymeat (whole, parts and offal of chicken, turkey, duck, goose, and guinea fowl) has been in place since The standard has now been incorporated into a consolidated marketing regulation for all agricultural products Council Regulation (EC) No 1234/2007 [1]. Detailed rules for the poultrymeat standard are given in Commission Regulation (EC) No 543/2008 [2]. This standard covers the quality classifications, description of cuts of poultrymeat, temperature requirements for storage of chilled, frozen and quick-frozen poultrymeat, as well as the limits of technically unavoidable water uptake (extraneous water) as a result of commercial preparation and cooling of poultrymeat. Water is an essential part of hygienic poultry preparation. It has been estimated that the process of removing feathers, evisceration and carcass washing even with air-chilling, chicken carcasses absorb around 1.8% water. The limits for extraneous water depend on the chilling method used either for poultrymeat sold chilled or pre-chilling of frozen poultrymeat. Commission Regulation (EC) No 543/2008 [2] gives the maximum limit of extraneous water for cuts/portions to be: 2% for air chilling 4% for air spray chilling 6% for immersion chilling, as determined by the chemical test specified in Annex VIII of the Regulation [2]. Enforcement of these limits ensures that poultrymeat is prepared according to good manufacturing and hygienic practice and that consumers are not being disadvantaged by excess added water in fresh poultrymeat. 4.2 Rationale for the Study Added water in food products, as an attempt to alter its quality parameters, is a fraudulent practice that has existed for over a century. However, in chilled/frozen poultrymeat, the presence of the so-called 'physiological water' (water naturally present in chicken) and some 'extraneous water', due to technically unavoidable water as a result of the chilling method used to cool poultrymeat, is acceptable. European legislation [2] sets limits for the amount of technically unavoidable water for the commonly used chilling methods so that the true extraneous water can be detected and consumers protected. The method for calculating extraneous water is based on the premise that the water/protein ratio (W/P) is a constant number for a given species of animal or bird and a specific cut. The W/P ratio will change in proportion to the amount of extraneous water present. Commission Regulation (EC) No 543/2008 [2] specifies that poultry cuts should be analysed in duplicate to determine the water and protein content in accordance with internationally recognised chemical methods [3,4]. W/P ratios are calculated for each sample and compared with upper limits for the W/P ratios specified in the Regulation [2]. Poultry production in the EU is an intensive agricultural activity. The industry is constantly striving for more efficient conversion of feed to carcass weight in the shortest possible time. The response to this requirement has been intensive breeding of chicken. Within the EU, the 9

10 three main broiler breeds used are Ross, Cobb and Hubbard. These breeds mature to give a market carcass weight around 1.5 kg within 5 weeks. In general, younger birds have slightly higher physiological water content, and lower protein contents; an extensive study [5] in the UK to reassess fat free nitrogen factors, (used to determine chicken content of composite chicken products), showed that the overall protein content of whole chicken meat without skin changed from 23% in 1963 (the last time this was studied in the UK) to 22% in 2000 and for skinless breast from 25% in 1963 to 24% in These differences reflect changes in rearing practices and breeds. An increasing trend in the number of failures observed by the National Reference Laboratories gave rise to a recent study on the initiative of the German Association of Poultry Processors (Bundesverband der Geflügelschlachtereien BVG) to determine the status of the physiological water content of poultrymeat produced in Germany. The German study [6] determined the water content of leg cuts from 280 chickens collected from different slaughterhouses which were representative of chicken reared in Germany in ,0 3,9 3,8 3,7 3,6 3,5 DE 2007 DE 1993 EU 1993 DK,UK,NL, FR,E S 3,4 3,3 Chicken Turkey Figure 1. W/P ratio in chicken and turkey leg cuts as measured in EU Member States in 1993 and in Germany in 2007 As shown in Figure 1 above, the German study revealed that the average W/P ratio, compared to 1993, the last study that assessed physiological water content in poultrymeat across Europe [7], had not changed for turkeys produced in Germany, but for chicken (broiler) cuts produced in Germany there had been an increase of around 7%. If this increase were applicable to all poultrymeat produced in the EU, then a significant number of poultrymeat cuts would exceed the limits specified in Regulation 543/2008/EC [2] for extraneous water content in poultrymeat. Since 1993, developments in respect of breeds and age/weight at slaughter have taken place across the EU, which may have caused a change in the physiological water content of 10

11 poultrymeat produced in the EU. Hence the European Commission decision to re-examine W/P ratios of chicken parts from chicken reared in the EU in 2012 to see whether they reflect changes in poultry rearing practices since

12 Production (kilotons) 5. Collection of Poultry Samples 5.1 Sampling plan Sampling Countries According to the Association of Poultry Processors and Poultry Trade in the EU countries (AVEC) report 2010, the total broiler production in 27 Member States of the EU in 2009 was 8802 kilotons (kt). In 2009 the top seven poultry producing Member States were the UK, Spain, France, Germany, Poland, Italy and the Netherlands. Data showing production in kilotons and percentage of the EU production are presented in Table 1 and Figure 2. Table 1. Top 7 Poultry Producing Countries in the EU in 2009 Country 2009 Annual Production (kt) % of EU production UK Spain France Germany Poland Italy Netherlands Total: Cumulative % of EU Production 0 0 UK Spain France Germany Poland Italy Netherlands Production (kilotons) Cumulative % Figure National broiler production for EU Member States. The line shows the cumulative percentage of total EU production (Data source BPA/AVEC). 12

13 These seven Member States account for just over 74% of the total EU production and so were considered to be representative of EU poultry production. Therefore the sampling plan was based on collecting chicken breast and leg cuts from each of the above Member States Design of Sampling Plan It was clear from the volumes of poultry produced in the EU on an annual basis that the sampling performed could not be representative on the basis of volume. However, it was considered essential that it must be representative of the processes being used across the EU. LGC consulted the British Poultry Council (BPC) / AVEC to establish the primary variables associated with commercial poultry production. The following parameters were identified as being key: Breed the most common breeds of poultry produced in the EU are Ross and Cobb. Although Hubbard is being increasingly used, it was not considered sufficiently widespread enough yet to warrant inclusion in this study. Weight there are two classes of weight, based on age; Light ( kg for week broilers) and Heavy ( kg for 7-8 weeks). Birds from each category were to be included. Although different Member States have different proportions of light and heavy birds in their markets, equal numbers were taken for this study from six of the seven Member States. Gender both males and females are important at both weights, although there was evidence to suggest that heavy females might not be available in some Member States e.g. UK and Italy as they mature more quickly than males and reach commercial carcass weights earlier. The above information was conveyed to NRLs in the other six Member States and further information was collected to help finalise the sampling plan, both in terms of what samples to take and whether it was feasible to take all required samples in one day. The 1993 EU study [7] sampled 10 birds (5 male and 5 female) from each of two flocks and if possible, from one breed. Since two specific breeds were planned to be sampled in the current study it was not possible to produce a balanced design with equal numbers of samples taken for the key variables if a sampling plan similar to the 1993 study was to be used. In order to balance the design, it was planned that six females (three light and three heavy) and six males (three light and three heavy) were to be taken per flock; resulting in twenty four birds in total for each breed. In order to maximize the number of samples taken from each Member State, it was decided that samples were to be taken from two distinct flocks for each breed. This would give a total of forty eight birds per Member State compared to the twenty birds sampled in Since the sampling protocol presented in Table 2 more than doubles the number of samples taken for the 1993 study, it gives increased confidence that the results will give a good picture of the physiological water content of the most common breeds of chicken reared in the EU in

14 Table 2. Full Factorial Balanced Design of the Key Variables Associated with Commercial Poultry Production in the EU Flock Number: 1 2 Breed: Ross Ross Weight: Light birds Heavy birds Female: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Male: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Flock Number: 3 4 Breed: Ross Ross Weight: Light birds Heavy birds Female: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Male: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Flock Number: 1 2 Breed: Cobb Cobb Weight: Light birds Heavy birds Female: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Male: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Flock Number: 3 4 Breed: Cobb Cobb Weight: Light birds Heavy birds Female: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Male: 3 3 Resultant Breast: 3 3 Resultant Leg: 3 3 Per Member State: Total number of birds: 48 Total number of breast samples for analysis: 48 Total number of leg samples for analysis: 48 From 7 Member States: Total number of birds: 336 Total number of breast samples for analysis: 336 Total number of leg samples for analysis:

15 5.2 Sampling Standard Operating Procedure In order to ensure that sample collection was as consistent as possible between the different collection sites in all seven Member States, a detailed standard operating procedure (SOP) was prepared. The sampling SOP detailed the samples that were to be collected within each Member State and described the method to be used for preparing the breast fillets and leg cuts, and the procedure for securely bagging the samples to maintain their integrity during transit to LGC. This SOP was discussed and agreed with the Commission project steering group before being circulated to all participating NRLs and selected slaughterhouses for comment. During the preparation of the SOP there was some discussion as how best to determine the gender of the poultry. The agreed approach was to compare the size of the wattle and comb; males have larger, more developed combs and wattles whereas females have smaller, paler combs and less obvious wattles. On some sampling occasions the carcasses were examined for the presence/absence of gonads which confirmed the accuracy of selection of gender based on the size of the head comb. A final version of the SOP, with photographs clearly demonstrating the procedure including the preparation of the breast and leg cuts, was circulated to all NRLs in the seven Member States where sampling was planned. 5.3 Amendments to Sampling Plans In order to provide the data that accurately reflected the range of birds available in the EU poultry market, some changes to the sampling plan presented in Table 2 were made to reflect local practices: In France, the Cobb breed is not used but Hubbard is reared instead and so Hubbard birds were sampled. In Italy less than 10% of the poultry production is Cobb, the majority being Ross; therefore only Ross birds were sampled in this Member State. In Italy, male and female birds are separated at hatching; the majority of female birds are used to produce small carcasses for roasting and not for production of portions therefore in Italy mostly male birds were sampled and these were from the medium and heavy weight ranges the final sampling plan used for Italy is presented in Table 3. The above changes were agreed with the respective NRLs and the Commission project steering group prior to sampling. 15

16 Table 3. Final sampling plan used for Italy Breed: Ross (308) Ross (708) Total Flock Number: Weight: Medium Heavy Medium Heavy Medium Heavy Medium Heavy Female: Resultant Breast: Resultant Leg: Male: Resultant Breast: Resultant Leg: Sample Collection Sampling was carried out in all seven Member State over a four month period (March June 2012) according to the final sampling plan presented in Table 2 (modified as described in section 5.3). In practice it was not possible to sample both light and heavy birds from the same flock as weight is related to the age of the birds and therefore light birds would not be available for slaughter on the same day as heavy birds from the same hatching. Thus, it was necessary to carry out sampling over two days in order to sample from the number of flocks required. On some occasions, due to the availability of flocks entering the slaughterhouse during the sampling period it was only possible to sample from one farm for a specific breed and weight category. In these instances, birds were sampled from two different sheds to obtain two different flocks/groups of birds. In some slaughterhouses it was not possible to trace birds to specific sheds; on these occasions birds were taken from one farm on the basis of information supplied that they were from mixed sheds. The samples were frozen at the slaughterhouse or NRL in the Member State where the samples were being collected for a minimum of 48 hours before being transported by overnight courier to LGC. A summary of all samples collected is presented in Table 4. 16

17 Table 4. Summary of Samples Collected Breed Cobb Hubbard Ross Gender Female Male Female Male Female Male Weight category H L M H L M H L M H L M H L M H L M France Germany Italy Netherlands Poland Spain UK L = Light weight birds as classified by each Member State. H= Heavy weight birds as classified by each Member State. M= Medium birds (Italy only) 17

18 6. Sample Analysis 6.1. Sample Homogenisation The homogenisation of the frozen portions was carried out in accordance with the method described in Annex VIII of Commission Regulation (EC) No. 543/2008. The samples were processed within 1 hour of removal from the freezer as specified in the Regulation. The frozen portions were cut into smaller strips using a saw and Delta clamp before being minced in a heavy duty mincer with a 4mm bore. Samples were re-minced, aliquoted and then stored frozen prior to analysis. It was noted that for leg portions, very small fragments of bone and parts of tendons/cartilage were still visible in the homogenised sample. Any further processing would, in our opinion, have potentially had an adverse effect on the integrity of the samples. As the homogenisation had been carried out in accordance with the requirements of Annex VIII of Commission Regulation (EC) No. 543/2008, the samples were deemed fit for purpose Determination of Water/Protein ratio Eight NRLs were recruited from across Europe to participate in the study; laboratories in Denmark, France, Germany, Ireland, Italy, the Netherlands, Spain and the UK participated. In order to ensure that each laboratory analysed an equal mix of cuts (breast/leg) from each country and breed, a stratified random sampling plan was developed by the LGC statistics team Quality Assurance To ensure that all laboratories analysed the samples in the same manner, a detailed SOP for analysis was prepared. Results were reported in a reporting spreadsheet which was provided to each laboratory. The analysis SOP gave instructions to analyse each sample in triplicate for: o water content (determined by the oven drying method as described in Regulation 543/2008/EC (ISO 1442, for the determination of water content or equivalent [3]). o nitrogen (determined by the Kjeldahl or equivalent method according to Regulation 543/2008/EC (ISO 937, for the determination of nitrogen content or equivalent [4]). In order to ensure that all the data reported by the different laboratories involved in the study was all of a high quality, specific performance criteria were specified: a quality control material (ERM -BB501a) was to be analysed in every batch and each sample was to be analysed in triplicate and the results were to be assessed against acceptance criteria which was specified in the analytical protocol. The acceptance criteria were calculated by the LGC statistics team based on: repeatability data quoted in the standard methods of 0.5g/100g water [3] or 0.1g/100g nitrogen [4] for the agreement between duplicate analyses. Horwitz equation [8] using 95% confidence limits for the quality control material. The resultant acceptance criteria are presented in Table 5. 18

19 Analyte Table 5. Acceptance Criteria for Analytical Results Acceptance criteria for quality control material (95% confidence) Acceptance criteria for standard deviation of triplicate sample results (99% confidence) Water 61.8 ± 2.7 g /100g <0.40 g /100g Nitrogen 2.30 ± 0.16 g /100g <0.08 g /100g Each participating laboratory was sent: 84 homogenised samples which had been allocated to each laboratory based on the stratified random sampling plan. A certified reference material (ERM -BB501a). An Analytical Protocol with: A reporting spreadsheet which automatically calculated the standard deviation for each set of triplicate results and indicated whether the results were within the acceptable range or not. Instructions to repeat the analysis of a sample if it failed the acceptance criteria for a triplicate analysis specified in Table 5. Instructions to repeat the entire batch if the quality control material in a batch failed the acceptance criteria specified in Table 5. All participating laboratories returned complete sets of data for water and nitrogen content for their samples in accordance with the requirements of the analytical protocol supplied. None of the laboratories reported any failures for the quality control material. Six of the eight laboratories for water and four of the eight laboratories for nitrogen reported that some samples did not pass the acceptance criteria and so had to be repeated. In most cases, but not all, the repeat analysis resulted in acceptable data. After consultation with the LGC statistics team, it was decided not to analyse samples more than twice as it was thought that it would not further improve the information on these samples. The number of samples requiring repeat analyses is presented in Table 6. 19

20 Table 6. Number of Repeat Analysis Performed by Laboratory Water Nitrogen Lab no. no. tested no. repeats % repeats no. tested no. repeats % repeats (12 leg samples) (3 leg samples) (3 leg samples) (5 leg samples) (1 leg sample) (3 leg samples) (0 leg samples) (9 leg samples) NA* 8 84 (11 leg samples) NA All labs 672 (43 leg samples) (4 leg samples) 1.3 * Repeat data was not available as the laboratory reported that instrumental failure required a large number of samples to be repeated. Three laboratories noted that the samples requiring repeat analyses contained small pieces of bone and cartilage. LGC made a record of these sample numbers so that the results could be assessed during statistical analysis. None of the individual results were identified as outliers. Analysis of Table 6 shows that although the nitrogen repeats (only 9 out of 672 samples) were split almost equally between the breast and leg samples, the samples which needed repeat analysis for water were exclusively the leg cuts. This is likely to reflect the more heterogeneous nature of the leg samples which contained bone, skin and cartilage as well as muscle and were more difficult to homogenise to a completely smooth paste. A total of forty three leg samples needed repeat analysis for water out of a total of three hundred and thirty six water analyses conducted, which constitutes a repeat rate of 13% compared to <1% for breast samples. By setting acceptance criteria, the impact of the heterogeneity of leg samples, homogenised in accordance with the requirements of Annex VIII of Commission Regulation (EC) No. 543/2008, was minimised in this study. However, it is recommended that the fitness for purpose of the homogenisation procedure specified in Annex VIII of Commission Regulation (EC) No. 543/2008 for leg samples, for the purposes of control analyses undertaken by NRL, should be discussed at the annual meeting of NRLs. Not all laboratories reported the need for repeat analysis, so the initial sample weight taken for the analysis in each laboratory was compared and this information is presented in Table 7. 20

21 Table 7. Summary of methods used and sample weight taken by laboratory Lab no. 1 2 Water Sample weight (g) 4 5 Method Manual by weighing Manual by weighing Nitrogen Sample weight (g) Method 0.8 to 1 Kjeldahl 0.2 to 0.4 Leco model No FP323 (Dumas) to 7.8 (mean 5.2) 5 5 to to Manual by weighing Manual by weighing Manual by weighing Manual by weighing Manual by weighing Manual by weighing 1.1 to 1.6 (mean 1.4) Kjedahl 1 Kjeldahl 2 ± 0.2 Kjeldahl 1.4 to 1.5 Kjeldahl 2 to 2.1 Kjeldahl 1 Leco model No CNS2000 (Dumas) If sample homogeneity was a significant issue, a smaller sample size would be expected to be more likely to give rise to more repeats. However, the smallest sample weights were taken for the determination of nitrogen and yet very few samples required repeat analyses for nitrogen. All NRLs took similar sample weights for the determination of water yet saw very differing rates of repeat analyses. From the data available, there does not appear to be a significant correlation between sample weight and repeat analyses. Samples which failed on repeat analysis were not included in the statistical evaluation (n=1 breast sample, and n=24 leg samples); additional statistical analysis confirmed that omitting the technically invalid sample results had no material effect on the study conclusions. 21

22 Nitrogen (g/100g) Moisture (g/100g) 7. Results The nitrogen data was converted to crude protein content at LGC by multiplying the mean nitrogen result by 6.25 [9]. The water and protein content was used to calculate the water/protein ratio for each sample as described in Annex VIII of Regulation (EC) No. 543/2008. The results by laboratory are presented in: Figure 3 water and protein contents Figure 4 W/P ratios Figure 5 W/P ratios obtained for gender, weight category and breed. The figures show that there was little difference between the results from different laboratories. Moisture Water Breast Leg Lab ID Nitrogen 4.0 Breast Leg Lab ID Figure 3. Water and Nitrogen results by laboratory 22

23 W/P Water Protein Ratio Breast Leg Lab ID Figure 4. Water/Protein ratio by laboratory 23

24 W/P W/P W/P W/P - Gender Breast Leg F: F Female M M: Male Labs W/P - Wcat Breast Leg H: Heavy H L M L: Light M: Medium Labs W/P - Breed Breast Leg C: Cobb H: CHubbard Hu R: Ross R Labs Figure 5. The W/P ratios obtained for gender, weight category and breed. 24

25 W/P Both country of origin and gender showed statistically significant effects (refer to section 8) on the W/P ratio though the effects were small compared to the dispersion observed for individual samples as demonstrated in Figure 6. W/P Breast Leg Fr Ge It Ne Po Sp UK Country Fr Ge It Ne Po Sp UK Figure 6. Water/Protein ratios in poultrymeat in the EU by country. The results are presented as median and spread of the results for individual countries. 25

26 8. Statistical Evaluation The results were analysed by LGC s statistics team to detect differences between: Breed Age/Weight Gender Country Cut Laboratory effects. This involved the following treatment of the data: a) Outlier identification methods in line with ISO b) Multi-way analysis of variance to test for the significance of the above effects compared to measurement variance. c) Variance component extraction to establish the individual variance contributions d) Estimation of the mean water content, protein content and W/P ratio. e) Calculation of confidence limits appropriate for the sample size used in current regulations. f) Comparison of results with the results of the 1993 study. g) Evaluate whether the limits Regulation (EC) No. 543/2008 are still relevant Comparison with study carried out in 1993 The mean water and protein results for chicken breast and leg cuts are presented in Table 8. Table 8. Water and Protein Summary Data Analyte Cut Mean * (g/100g) SD * (g/100g) Breast (73.94) 0.74 (1.05) Water Leg (66.19) 1.68 (1.93) Protein Breast (23.19) 0.15 (0.12) Leg (17.56) 0.10 (0.13) The results are mean values for n = 335 breast and n = 312 leg. Numbers in blue are the results for the 1993 where n= 120 for both breast and leg. The results show that the average physiological water content of EU produced chicken has increased since the previous study carried out in The mean water content has increased by 1.4% in breast samples and 1.8% in leg samples. There are corresponding decreases of 0.95% and 2.85% protein in breast and leg cuts respectively. The W/P ratio was calculated for each sample and the results compared with the previous studies; the EU-wide 1993 study and a study carried out in Germany in 2007 (Figure 7). 26

27 Water Protein Ratio Breast Leg DK Fr Ge It Ne Po Sp UK Country Figure 7. Water/Protein ratios per country comparing 1993, 2007 and 2012 studies. The dotted lines are the overall mean for the individual studies (Red: 1993; Blue: 2012). The water protein ratios (W/P) observed in the 1993, 2007 and 2012 studies are compared in Figure 7. The distribution of data in each study is consistent with a normal distribution; none of the observations are detected as outliers by Grubb s test (p = 0.4). The mean W/P ratios observed are presented in Table 9. DK Fr Ge It Ne Po Sp UK Table 9. The mean W/P ratios obtained in 2012 and 1993 Cut 2012 Mean 2012 SD 1993 Mean 1993 SD Breast Leg The data were analysed for significant differences between samples from different groups; the result of this analysis is presented in Table 10. Table 10. Significance of effects on W/P ratio. Factor Breast Leg Lab Breed Country Gender Wcat Breed:Gender Breed:Country Breed:Wcat p-values < 0.05 p -values > 0.05 Empty cells: factor was removed from the linear model. Full models output can be seen in Annex 3. 27

28 Both country of origin and gender showed statistically significant effects on the W/P ratio though the effects were small compared to the dispersion observed for individual samples. Further analysis (Table 11) revealed that: Spain shows a difference from other countries for the breast cut higher values Poland differs from Germany for the leg cut - higher values. Table 11. Significance of Countries Pairs Breast Leg Po- Ge Sp- Ge Sp- Ne Sp- Po Sp - UK Male - Female p-values < 0.05 Spain shows differences from other countries for breast meat, while only Ge-Po differ for leg meat. The gender is only significant for breast meat. Once again the differences were small compared to the dispersion of results and so would not be considered to be of practical significance Calculation of limits The values for the W/P ratios obtained in 1993 were used to calculate limits within which 95% of the poultrymeat cuts should fall if they were compliant with the legislation i.e. if they did not contain more than 2%, 4% or 6% extraneous water depending on the method of chilling. The limits were calculated according to Equation 1 shown in Box 1. The tolerance () value in the equation takes into account any extraneous water in the sample that may have been taken up during the chilling process. The tolerance value is calculated using Equation 2 in Box 1. 28

29 Equation 1 y x t v * 1 1 * n n2 ( n 2 1) * s1 ( n ( n 1) ( n ) * s 1) 2 2 Where: n 1 & s 1 = number of observation and standard deviation in the study n 2 & s 2 = future number of observations and standard deviation Equation 2 t v = students t value with n1+n2-2 degrees of freedom = tolerance ẋ = overall mean in the current study Where: E W * P 100 E phys E w = Extraneous water P phys = Physiological protein W 100* P phys Box 1. Equations from 1993 study used to calculate upper limits for water in poultrymeat. In the 1993 study, a tolerance value of 0.3 was used for the limit calculation. This was stated to be equivalent to a mean water uptake of about 7%. In order to directly compare the two studies, the same calculation was performed using the 2012 data and a tolerance value of 0.3. Since there has been a decrease in the amount of protein in the chicken cuts between 1993 and 2012 a tolerance value of 0.3 is equivalent to 6.5% water in 2012 breast cuts and 4.9% extraneous water in 2012 leg cuts. The data was calculated to give upper limits for extraneous water which would be appropriate for using three, five, seven and ten future samples as was done using the 1993 study. The limits obtained using the data from both studies are presented in Table

30 Table 12. Upper limit calculations: comparison of data published in 1993 report with data generated in 2012 Cut Mean W/P ratio Std Dev Number of observations in study Number of future samples Upper limit Breast 1993 Breast 2012 Leg 1993 Leg The tolerance value was fixed at (=0.3) as was used in the 1996 report. In order to determine the upper limits for 2, 4 and 6% extraneous water, the upper limit calculation was repeated by calculating the appropriate tolerance value (and then carrying out the limit calculation using equation 1. This data is not shown in the 1993 report but the numbers generated using this process with the data from the 1993 study are presented in Table 13. Table Limits for 2, 4 and 6% extraneous water. Cut Breast Leg Extraneous Water (%) Calculated Upper Limit *Upper Limits in (EC) 543/ It was assumed that the number of future samples to be tested on each occasion was five. * The upper limits calculated using data from the 1993 study were rounded to the nearest 0.05 and used in Commission Regulation (EC) 543/2008. In this regulation, the limit for chicken breast without skin is set at 3.40 for all chilling methods; it is not clear why a different limit was not applied for spray and immersion chilling for this particular cut of meat. Since the 2012 data shows that there has been an increase in the mean water content and water/protein ratio compared to the results of the 1993 study, the upper limits were 30

31 recalculated using the 2012 data. Specifically, the tolerance values were calculated using the 2012 protein data for 2%, 4% and 6% extraneous water on the basis of taking samples (cuts) for analysis. Additionally, unlike the 1993 study, allowances were made to account for the fact that: the 5 samples (cuts) taken for analysis would be from the same country of origin, and the analysis of the sample(s) would be performed in a single laboratory. The recalculated limits are presented in Table 14. Table 14. Recalculated Upper Limits for 2, 4 and 6% extraneous water when a composite of 5 samples is used based on 2012 data. Cut Extraneous Water (%) Recalculated Upper Limit Breast Leg As would be expected based on the observed increase in physiological water, the upper limits have increased. In order to demonstrate the appropriateness of the new limits, the current legislative limits and the new recalculated limits for 2% extraneous water (air chilling) were applied to the data in this study and are presented in Figure 8. Figure 8 shows that the proportion of: breast samples from the 2012 study that would be expected to fail the current legislative limit for air chilling (2% extraneous water) is 7.9% compared with only 0.13% expected failures for the new recalculated limits. leg samples from the 2012 study that would be expected to fail the current legislative limit for air chilling (2% extraneous water) is 13% compared with only 0.02% expected failures at the new recalculated limits. 31

32 a) Breast cuts Recalculated (2012) 0.13 % Current limit 7.9 % W/P ratio a) Leg cuts Recalculated (2012) 0.02 % Current limit 13 % W/P ratio The fraction of a) breast cut samples and b) leg cut samples from the 2012 study population that would fail at the current legislative limit for 2% extraneous compared with the recalculated limit (based on an assumed normal distribution with mean and standard deviation as estimated for the present data set). Figure 8. Effect of recalculated and legislative limits on sample failure rate. 32

33 9. Conclusions and Recommendations 9.1. Conclusions The data obtained from the 2012 study shows:- Physiological water content of both breast and leg cuts have increased by 1-2%. Physiological protein content of both breast and leg cuts has decreased by 1-3%. For chicken breast cuts, the mean W/P ratio and standard deviation are slightly higher than those obtained in the 1993 study. For chicken leg cuts, the mean W/P ratio is slightly higher with a lower standard deviation than found in the 1993 study. The upper limits (95% confidence interval) for extraneous water in chicken breast and leg cuts increase appreciably on recalculation using the W/P ratio data obtained in this study Recommendations The data obtained in this study supports the recommendation to amend the limits for extraneous water given in Commission Regulation (EC) 543/2008 to take into account the physiological water content of chicken currently produced in the EU in The proposed new limits for 2% (air chilling), 4% (air spray chilling) and 6% (immersion chilling) extraneous water content are shown in Table 15. Table 15. Current Upper limits compared with the proposed new upper limits. Cut Skinless chicken breast Leg cuts with skin Extraneous Water (%) Upper Limit Regulation (EC) 543/2008 Proposed new Upper Limits * * The current upper limit for skinless chicken breast of 3.40 [2] is given for all three chilling methods (air chilling, air spray chilling and immersion chilling). This is considered to be an anomaly in the current legislation [2] as the legislation clearly states that chicken absorbs different amounts of technically unavoidable extraneous water when cooled using the three chilling methods; 2% for air chilling, 4% air spray chilling and 6% for immersion chilling. Thus, as it is for other cuts, the upper limits for skinless chicken breast for: air spray chilling should have been > than the upper limit for air chilling immersion chilling should have been > than the upper limit for air spray chilling. 33

34 It is recommended that the upper limits for skinless chicken breast are amended to reflect the extra extraneous water associated with air spray chilling (4%) and immersion chilling (6%) as they are for all other cuts in the current legislation [2]. The limits presented in Table 15 have been derived from: the results of the 2012 study using the equation used in the 1993 study making an allowance for the fact that the five samples (cuts) taken for analysis will originate from one country and will be analysed in one laboratory. This study found a higher repeat analysis rate for leg cuts compared to breast cuts (13% of leg cuts required repeat analysis compared to <1% for breast cuts) due to poor repeatability; it is recommended that the fitness for purpose of the homogenisation procedure specified in Annex VIII of Commission Regulation (EC) No. 543/2008 for leg samples, for the purposes of control analyses undertaken by NRL, should be discussed at the annual meeting of NRLs. 34

35 10. References 1. Council Regulation (EC) No 1234/2007 of 22 October 2007 establishing a common organisation of agricultural markets and on specific provisions for certain agricultural products (Single CMO Regulation). OJ L299, , p Commission Regulation (EC) No 543/2008 of 16 June 2008 laying down detailed rules for the application of Council Regulation (EC) No 1234/2007 as regards the marketing standards for poultrymeat. OJ L157, , p ISO 1442:1997. Meat and meat products - Determination of moisture content (Reference method). 4. ISO 937:1978. Meat and meat products - Determination of nitrogen content (Reference method). 5. Food Standards Agency UK - Survey of Added Water in Chickens and Chicken Products (Number 08/00). Published 1 Nov Effects of sample preparation on the water/protein ratio of poultry cuts in relation to the identification of extraneous water. MRI (Max Rubner Institute), 2009: MMY&zeilenzahl_zaehler=47&NextRow=10 7. Commission of the European Communities - Physiological Water Content of Frozen and Quick Frozen Chicken and Turkey Parts Control methods for extraneous water ISBN Luxembourg: Office for Official Publications of the European Communities, 1993: 8. Horwitz W: 1982 Evaluation of analytical methods used for regulation of foods and drugs. Anal. Chem. 54, 1982, 67A-76A. 9. FAO/WHO: 1973 Energy and protein requirements. Technical report No. 522 Ad Hoc Expert Committee. Rome: FAO/WHO 35

36 11. Acknowledgements The authors would like to acknowledge the contribution of the following: Consumer Protection team at LGC for assistance with sampling and undertaking the determination of moisture and nitrogen in samples. The Statistics team at LGC for undertaking the statistical analysis for the project. The participating slaughterhouses and their staff The participating NRLs for assistance with sampling and undertaking the determination of moisture and nitrogen in samples. The European Commission for funding the work. The European Commission project steering group. 36