(A) Offal (Heart, Liver, Kidney and Lung) and (B) Added Bovine/Ovine Or Porcine Serum 33

Defra project FA0122

Further validation of assays to monitor offal and added serum in meat products using commercial samples

Final report

Nottingham Trent University

Reviewed by the Authenticity Methods Working Group (AMWG): January 2016

Page 1 of 99

© Crown copyright 2018 You may re-use this information (excluding logos) free of charge in any format or medium, under the terms of the Open Government Licence v.3. To view this licence visit www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ or email [email protected]

This publication is available at www.gov.uk/government/publications

Any enquiries regarding this publication should be sent to us at: [email protected] www.gov.uk/defra

The views and opinions expressed in this report are those of the authors and may not in any circumstances be regarded as stating an official position of Defra.

Page 2 of 99

INDEX

Background Information 4

Chapter 1 Validation of detection of residual serum in raw meat 5

1A. Determination of threshold levels of residual serum in beef and pork meat 5 Sample details 5 Experimental details 5 Results 6 Residual bovine serum in different cuts/animals 6 Residual bovine serum in minced beef products 7 Residual porcine serum in different cuts/animals 8 Residual porcine serum in minced pork products 9 Residual serum in beef and pork mixtures 10 Conclusions 11

1B. Screening of frozen minced beef samples for added offal 12 Conclusions 13

1C. Determination of the effect of freezing on residual serum in pork and beef minced Meat 14 Effect of time of freezing on recovery of residual bovine serum 15 Effect of time of freezing on recovery of residual porcine serum 17 Effect of repeated freeze/thaw cycles on recovery of residual serum 19

Chapter 2 Establishing precision of Western blotting assays to determine addition of porcine/bovine serum to pork/beef 22

Intra- and inter-assay variation for assays of pork meat with added serum 23 Intra- and inter-assay variation for assays of beef meat with added serum 27 Summaries of intra- and inter-assay variation 31

Chapter 3 Defra pilot study of meat products for presence of (A) offal (heart, liver, kidney and lung) and (B) added bovine/ovine or porcine serum 33

3A. Sample collection 33

3B. Analysis of offal: detection and validation 35 Detection of offal 35 Validation of offal 37 Detailed summary of the products containing offal: offal type and amount 40 Summary tables of added offal by store and product types 43 Summary and overall conclusions of Chapter 3B. 45

3C. Analysis of added serum 46 Experimental details 46 Example data and detailed summaries 47 Summary tables of added serum by store and product type 52 Summary and overall conclusions of Chapter 3C. 54

3D. Summary of samples containing offal and/or serum at higher levels than expected 54

Chapter 4 Establish limit of detection (LOD) for offals via Western blot analysis using chromogenic reagents 56

Background 56 Methodology 56 Results 56 Conclusions 63

Appendices (separate file) 64 Page 3 of 99

Background Information

The EU definition of meat for labelling purposes is restricted to skeletal muscle with maximum limits for naturally adherent fat and connective tissue depending on the meat species.

The meat definition excludes offal (e.g. heart, liver) which, if included in a meat product must be listed separately in the ingredients list and cannot count towards the meat content. The species and type of offal must also be defined. Under general food labelling rules, all pre- packed foods must display an ingredients list. A quantitative ingredient declaration (QUID) of the amount of meat in a meat product must also be given. If blood or serum are used as ingredients, this should be accurately displayed on the label.

Previous anecdotal information has suggested that blood serum products or offal may be being added to some meat products and not labelled as such. These may be being added to a meat product to increase water holding capacity and extend the protein content, possibly leading to a false estimation of meat content as both blood products and offal cannot count towards the meat content. Offal and blood proteins are permitted food ingredients but should be accurately labelled on the package either at the retail or bulk catering level.

The work detailed in this report follows work previously conducted under contract to the Food Standards Agency (FSA)1 and Defra2 and contains a number of separate studies addressing gaps in knowledge relating to the previous projects. It also describes a local small scale pilot study to test the performance of previously developed methods across a broad range of sample types to ensure that the methods were fit for purpose and robust.

1 Q01092 Production and use of monoclonal anti-albumin antibodies to monitor the presence of bovine and porcine blood proteins in processed meat products.

2 FA0218 The proteomic detection of offal in meat products. FA0122 Validation of Western blot methods to monitor the presence of added bovine and porcine blood products in meat products.

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Chapter 1

Validation of detection of residual serum in raw meat

1A. Determination of threshold levels of residual serum in beef and pork meat

 Monitoring residual levels in raw meat (beef and pork).

 Monitoring residual levels of serum in samples of pork mince and beef mince from different retailers.

Levels of residual serum in different cuts of pork and beef meat have been monitored. In addition the levels of serum in retail minced pork and beef samples have also been checked.

Sample details

 Samples of different cuts of beef and pork were obtained from a local butcher with his own slaughtering facilities. Five animals (each cow and pig) were sourced and 5 individual cuts of meat were collected from each animal, giving a total of 25 samples of beef and 25 samples of pork. The cuts of beef originated from animals < 36 months old. Our butcher culls Charolais crosses and we believe that all the beef samples were from this cross; but we don’t have information about the female animal. In the case of pork, all animals were ‘commercial whites ‘.

 Retail minces were sourced from local retailers and included fresh and frozen products. 27 minced beef products (17 fresh and 10 frozen), 12 minced pork products (fresh only, since no frozen minced pork samples were available) were analysed. However 3 samples of frozen minced beef/pork mixtures (60 %, 65 %, 65% beef) were purchased and analysed for both porcine and bovine residual serum.

Samples were logged and given an individual code number.

Experimental details

Sample preparation Meat/mince sample (100 g) was homogenised in the coffee mill attachment of a Kenwood blender for 10 seconds to ensure a homogeneous sample.

A 5 g representative sample of this homogenate was weighed into a 50 ml tube containing 15 g SiLi beads, 30 ml cold phosphate buffered saline (PBS) were added and the sample homogenised using the MPBio FastPrep homogeniser (4 m/s for 30 seconds). The sample was then tumbled end over end for 30 minutes before centrifugation at 1500 x g for 15 minutes at 4°C. The supernatant was collected and frozen in aliquots for storage at -80 °C prior to analysis by Western blot.

SDS-PAGE and Western blotting Samples were analysed using SDS-PAGE and Western blotting. Standards, namely pure bovine serum albumin (BSA) and porcine serum albumin (PSA) were used to construct calibration graphs, from which the residual albumins in the test samples were calculated. All test samples were analysed in triplicate. These results were subsequently used to calculate Page 5 of 99 residual serum values, using published reference values for the levels of albumin in porcine and bovine serum (The Merck Veterinary Manual).

Results

Residual bovine serum in different cuts/animals

1 .4

1 .2

m u

r 1 .0

l 0 .8

a u

d 0 .6

s e

r 0 .4

% 0 .2

0 .0 0 1 2 3 4 5 A n im a l (B e e f)

C u t 1 C u t 2 C u t 3 C u t 4 C u t 5 M ean M e a n + /- S D

Figure 1.1 Residual bovine serum (%) in 5 different cuts of meat from 5 cows.

Table 1 .1 Mean % residual bovine serum in 5 different cows and 5 different cuts of meat.

Mean SD Animal A 0.509 0.070 B 0.494 0.108 C 0.536 0.102 D 0.272 0.154 E 0.385 0.101 Cut of meat 1 - stewing 0.426 0.166 2 - rib 0.523 0.058 3 - feather 0.414 0.180 4 - braising 0.435 0.191 5 - brisket 0.374 0.138

Overall mean 0.439 0.142

From our data there is no indication that differences occur between the 5 beef animals or the 5 cuts in one animal (confirmed via Mann Whitney analysis- since the population is not large enough to assume that it is normally distributed).

The collective data sets for all animals and all cuts were analysed using 3 different statistical tests (D’Agostino & Pearson, Shapiro-Wilk and Kolmogorov–Smirnov ) for normality and

Page 6 of 99 found to be normally distributed. It was therefore assumed that a range based on the error from the original 25 samples would be representative of the wider population.

The observed range for residual bovine serum in the 25 samples was estimated to be between 0.146 and 0.672 %. The combined data show a normal distribution, hence the statistically calculated range (mean ± 1.96 x SD) is between 0.161 and 0.717 % for all cuts/animals.

Residual bovine serum in minced beef products

1 .4

m 1 .2

u r

e 1 .0

a 0 .8

u d

i 0 .6

s e

r 0 .4

% 0 .2

0 .0

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 M in c e d B e e f s a m p le

F re s h m in c e d b e e f F ro z e n m in c e d b e e f  F ro z e n m in c e d b e e f o u tlie rs

M e a n b e e f c u ts M e a n b e e f c u ts + /- S D

Figure 1.2 Residual bovine serum (%) in retail minced beef products.

Table 1.2 Mean % residual bovine serum in retail minced beef products.

Mean SD Fresh 0.522 0.070 Frozen 0.769 0.286 Frozen + Fresh 0.614 0.215 Frozen, excluding outliers (>0.717 %) 0.568 0.103

All minced beef products (excluding 6 frozen outliers) contained residual serum within the statistically calculated higher threshold of 0.717 %.

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Residual porcine serum in different animals/cuts

1 .4

1 .2

m u

r 1 .0

l 0 .8

a u

d 0 .6

s e

r 0 .4

% 0 .2

0 .0 0 1 2 3 4 5 A n im a l (P o rk )

C u t 1 C u t 2 C u t 3 C u t 4 C u t 5

M ean M e a n + /- S D

Figure 1.3 Residual porcine serum (%) in 5 different cuts of meat from 5 pigs.

Table 1.3 Mean % residual porcine serum in 5 different pigs and 5 different cuts of meat.

Mean SD Animal A 0.474 0.149 B 0.426 0.104 C 0.417 0.103 D 0.429 0.130 E 0.544 0.109 Cut of meat 1 - belly 0.436 0.108 2 - leg 0.386 0.098 3 - chine 0.422 0.083 4 - shoulder 0.509 0.145 5 - hock 0.580 0.061

Overall mean 0.458 0.120

From our data there is no indication that differences occur between the 5 pork animals or the 5 cuts in one animal (confirmed via Mann Whitney analysis- since the population is not large enough to assume that it is normally distributed) .

The collective data sets for all animals and all cuts were analysed using 3 different statistical tests (D’Agostino & Pearson, Shapiro-Wilk and Kolmogorov–Smirnov ) for normality and found to be normally distributed. It was therefore assumed that a range based on the error from the original 25 samples would be representative of the wider population.

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The observed range for residual porcine serum in the 25 samples was estimated to be between 0.269 and 0.687 %. The combined data show a normal distribution, hence the statistically calculated range (mean ± 1.96 x SD) is between 0.223 and 0.693 % for all cuts/animals.

Residual porcine serum in minced pork products

1 .4

1 .2

m u

r 1 .0

l 0 .8

a u

d 0 .6

s e

r 0 .4

% 0 .2

0 .0 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 M in c e d p o rk s a m p le

F re s h m in c e d p o rk

M e a n p o rk c u ts M e a n p o rk c u ts + /- S D

Figure 1.4 Residual porcine serum (%) in retail minced pork products.

Table 1.4 Mean % residual porcine serum in retail minced pork products.

Mean SD Fresh 0.618 0.097

Ten out of 12 fresh minced pork (83%) contained residual serum within the statistically calculated higher threshold of 0.693%; the rest were slightly above this threshold (0.705% and 0.737 %).

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Residual serum in beef and pork mixtures

Samples of frozen beef and pork mixtures were analysed for both bovine and porcine serum content.

Table 1.5 Estimated residual bovine and porcine serum in retail mixed minced beef and pork products.

Sample ID MB/P 1 MB/P 2 MB/P 3

% beef content (from ingredients list) 65 65 60

Measured residual bovine serum (%) 0.248 0.277 0.292

Expected residual bovine serum (%) 0.285 0.285 0.263

% pork content (from ingredients list) 35 35 40

Measured residual porcine serum (%) 0.023 0.216 0.255

Expected residual porcine serum (%) 0.160 0.160 0.183

In the majority of cases the measured residual bovine/porcine serum is very close to expected values and are generally within the statistically calculated ranges for beef and pork.

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Conclusions

Beef serum

 The measured range of residual serum in 5 cuts of meat from 5 cows is between 0.146 and 0.672 %. The mean (± SD) is 0.439 ± 0.142.

 The combined data are normally distributed so the range can be statistically calculated (mean ± 1.96 x SD) to be between 0.161 and 0.717 %.

 21 (78 %) retail minced beef products (fresh and frozen) fall within the calculated range.

 6 (22 %) retail minced beef products were above the calculated range; they were all frozen products and are suspected to contain added bovine serum.

Pork serum

 The measured range of residual serum in 5 cuts of meat from 5 pigs is between 0.269 and 0.687 %. The mean (± SD) is 0.458 ± 0.120.

 The combined data are normally distributed so the range can be statistically calculated (mean ± 1.96 x SD) to be between 0.223 and 0.693 %.

 10 (83 %) retail minced pork products fall within the calculated range and 2 (17 %) retail minced pork products were slightly above the calculated range.

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1B. Screening of frozen minced beef samples for added offal

This involved screening 5 retail frozen minced beef samples (MB1, MB4, MB5, MB7 and MB8) from section 1A, for added offal. These samples contained >0.717 % residual beef serum, and the purpose of the analyses was to determine if the increased level of serum was due to added offal.

Samples were cooked, extracted and analysed by Western blotting as described in the SOP. One negative (100% beef meat) and one positive control (beef meat containing 2 % (w/w) heart, liver, kidney or lung) were run in duplicate alongside triplicate lanes of the samples. Results are presented in Figure 1.5 and summarised in Table 1.6.

(A) Beef 2% BHe MB1 MB4 MB5 Beef 2% BHe MB7 MB8

(B) Beef 2% BLi MB1 MB4 MB5 Beef 2% BLi MB7 MB8

(D) Beef 2% BLu MB1 MB4 MB5 Beef 2% BLu MB7 MB8

Figure 1.5 Detection of offal in frozen minced beef samples by Western blot analysis. Extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blots probed for (A) heart marker protein (He), (B) liver marker protein (Li), (C) kidney marker protein (Ki) and (D) lung marker protein (Lu).

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Table 1.6 Summary of offal detected in retail frozen minced beef products.

Product ID % residual Heart Liver Kidney Lung serum* MB1 1.059 >2 % ND ~2 % ND

MB4 0.792 ND ND ~2 % ND

MB5 0.870 >2 % ND ND ND

MB7 0.842 Trace <2 % ~2 % >2 %

MB8 1.348 Trace <2 % >2 % >2 %

ND: Not detectable *Normal range of residual bovine serum (0.161 – 0.717 %)

Conclusions

 All minces contained offal, 2 samples (MB7 and 8) contained all four offals, and two samples contained single offals (MB4 and MB5).

 Liver was found at lower levels than the other offals.

 Kidney was the most prevalent offal found.

 The levels and type of offal did not correlate exactly with added serum.

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1C. Determination of the effect of freezing on residual serum in pork and beef minced meat

Previous analysis (Chapter 1A) of the residual serum content of several samples of frozen minced beef contained higher than expected levels of serum. It is therefore possible that freezing may have an influence on the recovery of residual serum. This possibility is addressed using minced beef and pork meat sourced from a butcher with slaughtering facilities and frozen using two freezing regimes. The effects of repeated freezing and thawing on the recovery of residual serum has also been determined.

Testing plan

Two freezing regimes were tested: #1 freeze directly at -20°C #2 freeze at -80°C for 6 hours to mimic an industrial blast freezer, then transfer to -20°C for long term storage.

Analysis was conducted on three replicates (a, b, c) of each sample.

For the freeze-thawing study, one sample (FrB3 & FrP3 in table below) was frozen and thawed 1x, 2x, 3x or 4x.

Sample plan

Sample ID Sample ID Freezing regime Time frozen (beef) (pork) FrB 1 FrP 1 None FrB 2 FrP 2 #1 24 hours FrB 3 FrP 3 #1 7 days (thaw 1) FrB 4 FrP 4 #1 6 weeks FrB 5 FrP 5 #2 24 hours FrB 6 FrP 6 #2 7 days FrB 7 FrP 7 #2 6 weeks

Freeze/Thaw study Time following freeze/thaw

FrB 8 FrP 8 #1 FrB/P3 freeze/thaw 2 7 days after thaw 1 FrB 9 FrP 9 #1 FrB/P3 freeze/thaw 3 7 days after thaw 2 FrB 10 FrP 10 #1 FrB/P3 freeze/thaw 4 21 days after thaw 3

Approximately 600 g each of minced beef and minced pork were purchased from a local butcher with its own slaughtering facilities. 75 g aliquots were taken into green screw lidded pots (100 ml capacity) and frozen according to the appropriate test freezing regime. After the required length of frozen storage the pots were removed from the freezer and allowed to thaw at room temperature. Following thawing the 75 g sample was homogenised ensuring any “drip” was incorporated back into the sample and 3 separate 5 g replicates were taken into 50 ml centrifuge tubes containing 15 g SiLi beads. 30 ml cold Phosphate Buffered Saline (PBS) were added and the sample homogenised using the MPBio FastPrep homogeniser Page 14 of 99 for 30 seconds (4m/s). Samples were tumbled end over end for 30 minutes prior to centrifugation at 1500 x g for 15 minutes at 4°C. Supernatants were collected and stored in aliquots at -80°C (3 x 1 ml and 5 x 30 µl) prior to analysis.

Western blot analysis

Extracts and standards (pure BSA or PSA) were diluted appropriately and loaded onto a 10 % gel, proteins were separated by electrophoresis (constant voltage 200 v) and transferred to nitrocellulose using the Invitrogen iblot equipment.

Results

Effect of time of freezing on recovery of residual bovine serum

Table 1.7 Effects of length of frozen storage of beef minced meat on % residual serum. #1 = freezing regime 1, #2 = freezing regime 2.

ID % residual serum Intra procedure variation (a,b,c)

Mean SD % CV Mean SD CV (n=3) (n=3) FrB1a Fresh 0.68 0.02 2.87 0.71 0.03 3.86 FrB1b 0.74 0.03 3.73 FrB1c 0.73 0.09 12.16

FrB2a #1 24 hr 0.70 0.02 2.77 0.71 0.05 6.52 FrB2b 0.76 0.03 3.48 FrB2c 0.66 0.02 3.47

FrB3a #1 7 days 0.67 0.02 3.24 0.64 0.03 4.62 FrB3b 0.63 0.02 2.91 FrB3c 0.62 0.01 1.42

FrB4a #1 6 wks 0.59 0.01 2.16 0.54 0.10 18.68 FrB4b 0.61 0.01 2.11 FrB4c 0.42 0.02 4.97

FrB5a #2 24 hr 0.71 0.01 1.50 0.71 0.02 2.06 FrB5b 0.72 0.03 4.02 FrB5c 0.70 0.04 5.97

FrB6a #2 7 days 0.56 0.01 2.30 0.54 0.03 4.77 FrB6b 0.54 0.01 2.39 FrB6c 0.51 0.06 11.41

FrB7a #2 6 wks 0.61 0.05 7.36 0.55 0.05 8.86 FrB7b 0.54 0.02 3.44 FrB7c 0.51 0.09 16.68

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Figure 1.6 Length of frozen storage of beef mince – effect on residual serum recovery.

Statistics - beef

Unpaired t test with Welch’s correction

Freezing method # 1 Freezing method # 2

Days Significant p = Significant p =

0 vs 1 No No 0 vs 7 Yes 0.0313 Yes 0.0012 0 vs 42 No Yes 0.0135

Conclusions – beef

There is no effect of freezing after day 1 with either freezing method. After 42 days the consequences of freezing are similar for both methods, i.e. a drop of around 25 % in the level of detectable bovine serum.

 Irrespective of the method of freezing, the long term effects are similar.

 There is no increase in serum levels extracted/detected after freezing; thus the higher levels of serum in the frozen outliers (chapter 1A) is not due to freezing resulting in improved access to albumin.

 The levels of serum detected are reduced by up to 25 % following 42 days freezing.

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Effect of time of freezing on recovery of residual porcine serum

Table 1.8 Effects of length of frozen storage of pork minced meat on residual serum. #1 = freezing regime 1, #2 = freezing regime 2.

ID % residual serum Intra procedure variation (a,b,c)

Mean SD % CV Mean SD CV (n=3) (n=3) FrP1a Fresh 0.66 0.03 4.24 0.66 0.05 7.40 FrP1b 0.60 0.02 3.10 FrP1c 0.70 0.03 4.77

FrP2a #1 24 hr 0.69 0.04 5.45 0.68 0.03 3.61 FrP2b 0.71 0.02 2.49 FrP2c 0.66 0.02 3.39

FrP3a #1 7 days 0.76 0.01 1.64 0.76 0.04 4.81 FrP3b 0.80 0.02 2.20 FrP3c 0.72 0.01 0.82

FrP4a #1 6 wks 0.68 0.04 6.32 0.66 0.03 3.78 FrP4b 0.65 0.02 3.60 FrP4c 0.64 0.06 9.37

FrP5a #2 24 hr 0.79 0.09 11.88 0.76 0.028 3.73 FrP5b 0.75 0.04 5.86 FrP5c 0.74 0.02 2.49

FrP6a #2 7 days 0.61 0.04 6.17 0.69 0.072 10.40 FrP6b 0.72 0.06 8.39 FrP6c 0.75 0.01 1.17

FrP7a #2 6 wks 0.63 0.02 3.69 0.70 0.072 10.19 FrP7b 0.77 0.07 8.38 FrP7c 0.71 0.01 1.59

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Figure 1.7 Length of frozen storage of pork mince – effect on residual serum recovery.

Statistics - pork

Unpaired t test with Welch’s correction

Freezing method # 1 Freezing method # 2

Days Significant p = Significant p =

0 vs 1 No Yes 0.0442 0 vs 7 Yes 0.0439 No 0 vs 42 No No

Conclusions - pork

 Overall there is no effect of freezing on recovery of residual pork serum irrespective of method.

 There is a slight increase in detectable pork serum after 7 days frozen storage, but this is not significant.

 Recovery of pork serum appears not to be affected by frozen storage for up to 6 weeks.

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Effect of repeated freeze/thaw cycles on recovery of residual serum

Table 1.9 Effects of repeated freeze/thaw cycles on recovery of residual serum from minced beef.

ID % residual serum Intra procedure variation (a,b,c)

Freeze/thaw Mean SD % CV Mean SD CV (n=3) (n=3) FrB3a 1x 0.67 0.02 3.24 0.64 0.03 4.62 FrB3b 0.63 0.02 2.91 FrB3c 0.62 0.01 1.42

FrB8a 2x 0.67 0.05 7.65 0.66 0.05 7.98 FrB8b 0.70 0.01 1.68 FrB8c 0.60 0.05 8.87

FrB9a 3x 0.65 0.07 10.68 0.69 0.04 5.97 FrB9b 0.73 0.07 10.11 FrB9c 0.70 0.02 3.16

FrB10a 4x 0.59 0.00 0.71 0.55 0.04 7.82 FrB10b 0.56 0.03 6.05 FrB10c 0.50 0.05 10.68

Table 1.10 Effects of repeated freeze/thaw cycles on recovery of residual serum from minced pork.

ID % residual serum Intra procedure variation (a,b,c)

Freeze/thaw Mean SD % CV Mean SD CV (n=3) (n=3) FrP3a 1x 0.76 0.01 1.64 0.76 0.04 4.81 FrP3b 0.80 0.02 2.20 FrP3c 0.72 0.01 0.82

FrP8a 2x 0.58 0.04 7.03 0.68 0.09 12.90 FrP8b 0.71 0.05 7.32 FrP8c 0.75 0.02 2.04

FrP9a 3x 0.68 0.08 11.60 0.69 0.07 10.81 FrP9b 0.76 0.06 7.88 FrP9c 0.62 0.02 2.62

FrP10a 4x 0.63 0.02 3.69 0.70 0.07 10.19 FrP10b 0.77 0.07 8.38 FrP10c 0.71 0.01 1.59

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Figure 1.8 Repeated freeze/thaw cycles on frozen minced beef and pork– effect on residual serum recovery.

Unpaired t test with Welch’s correction

Pork Beef

Days Significant p = Significant p =

1 vs 2 No No 2 vs 3 No No 3 vs 4 No Yes 0.0148 1 vs 4 No Yes 0.0496

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Conclusions

Pork  Recovery of residual serum from pork mince is unaffected by 4 freeze/thaw cycles.

Beef

 Four cycles of freeze/thawing of beef mince shows a decrease (14 %) in residual bovine serum detected.

 Freeze/thawing 2 -3 times is the same as freeze/thawing once.

 Freeze/thawing once does not affect detection of residual serum.

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Chapter 2

Establishing precision of Western blotting assays to determine addition of porcine/bovine serum to pork/beef.

This study involved the determination of inter and intra assay variation for a range of added sera (1%, 2%, 10%) in blind (home- produced) cooked samples. Standards (0, 1, 2, 5, 10, 15 % serum added to meat) and 3 blind unknowns with different levels of serum spiked into them (A, B, C) were prepared for the 4 following combinations (porcine serum in pork meat, porcine serum in beef meat, bovine serum in beef meat, bovine serum in pork meat). Cooked samples were extracted using SDS and analysed by Western blotting according to the updated SOP (see appendix). Each sample was extracted once at the same time as the standards for that particular combination. The blind sample was then assayed in triplicate alongside the appropriate standards on one SDS-PAGE gel. Estimations for added serum were calculated giving intra-assay variation. This was repeated on a further 2 occasions allowing the inter-assay variation to be calculated.

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Results

Intra- and inter-assay variation for assays of pork meat with added serum

Table 2.1 Intra assay variation for assays of pork meat containing added bovine serum (PMB)*

Date Unknown Intra assay variation ID % % mean % difference actual estimated mean SD % CV from actual (n=3) 17/2 PMB A rep 1 10.0 12.98 10.77 1.99 18.45 7.70 PMB A rep 2 10.0 10.24 PMB A rep 3 10.0 9.11

25/2 PMB A rep 1 10.0 9.89 11.46 1.47 12.78 14.60 PMB A rep 2 10.0 11.73 PMB A rep 3 10.0 12.78

26/2 PMB A rep 1 10.0 9.25 11.18 1.80 16.09 11.80 PMB A rep 2 10.0 12.80 PMB A rep 3 10.0 11.48

17/2 PMB B rep 1 1.0 1.03 0.93 0.14 15.42 7.00 PMB B rep 2 1.0 0.99 PMB B rep 3 1.0 0.77

25/2 PMB B rep 1 1.0 0.82 0.87 0.08 9.69 13.00 PMB B rep 2 1.0 0.82 PMB B rep 3 1.0 0.97

26/2 PMB B rep 1 1.0 0.78 0.92 0.12 13.30 8.00 PMB B rep 2 1.0 0.99 PMB B rep 3 1.0 1.00

17/2 PMB C rep 1 2.0 2.00 2.16 0.15 6.86 8.00 PMB C rep 2 2.0 2.19 PMB C rep 3 2.0 2.30

25/2 PMB C rep 1 2.0 2.43 2.17 0.22 10.16 8.50 PMB C rep 2 2.0 2.02 PMB C rep 3 2.0 2.08

26/2 PMB C rep 1 2.0 2.52 2.46 0.07 2.97 23.00 PMB C rep 2 2.0 2.47 PMB C rep 3 2.0 2.38

* PMB A = 10 %, PMB B = 1 %, PMB C = 2 % CV = coefficient of variation

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Table 2.2 Summary of inter assay variation for assays of pork meat containing added bovine serum (PMB)

Actual % added Mean estimated % added % of actual % difference

PMB A 10.00 10.77 107.70 7.70 10.00 11.46 114.60 14.60 10.00 11.18 111.80 11.80

Mean ± SD 11.14 ± 0.35 (CV = 3.11 %) 111.37 11.37 PMB B 1.00 0.93 93.00 7.00 1.00 0.87 87.00 13.00 1.00 0.92 92.00 8.00

Mean ± SD 0.91 ± 0.03 (CV = 3.66 %) 90.67 9.33 PMB C 2.00 2.16 108.00 8.00 2.00 2.17 108.50 8.50 2.00 2.46 123.00 23.00

Mean ± SD 2.26 ± 0.17 (CV = 7.33 %) 113.17 13.17 CV = coefficient of variation

Conclusions

 In 3 separate assays (on different days) estimating the addition of bovine serum to pork meat the intra- assay CV is < 20 % for all serum additions (1 %, 2 %, 10 % actual) (Table 2.1).

 The majority of estimated added serum values (8 out of 9) are within 15 % of the actual amount of added serum (Table 2.1).

 For inter-assay variation all the CVs are < 10 % (Table 2.2).

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Table 2.3 Intra assay variation for assays of pork meat containing added porcine serum (PMP)

Date Unknown Intra assay variation ID % % mean % difference actual estimated mean SD % CV from actual (n=3) 21/1 PMP A rep 1 2.0 2.01 2.26 0.26 11.53 13.00 PMP A rep 2 2.0 2.25 PMP A rep 3 2.0 2.53

4/3 PMP A rep 1 2.0 2.34 2.50 0.14 5.74 25.00 PMP A rep 2 2.0 2.56 PMP A rep 3 2.0 2.61

17/3 PMP A rep 1 2.0 2.54 2.79 0.59 21.02 39.50 PMP A rep 2 2.0 3.46 PMP A rep 3 2.0 2.37

21/1 PMP B rep 1 1.0 1.03 0.84 0.17 20.03 16.00 PMP B rep 2 1.0 0.71 PMP B rep 3 1.0 0.78

4/3 PMP B rep 1 1.0 1.30 1.49 0.19 12.79 49.00 PMP B rep 2 1.0 1.68 PMP B rep 3 1.0 1.48

17/3 PMP B rep 1 1.0 1.16 1.11 0.12 10.78 11.00 PMP B rep 2 1.0 1.19 PMP B rep 3 1.0 0.97

3/2 PMP C rep 1 10.0 8.37 10.28 3.48 33.83 2.80 PMP C rep 2 10.0 14.29 PMP C rep 3 10.0 8.17

10/2 PMP C rep 1 10.0 13.20 10.98 2.22 20.17 9.80 PMP C rep 2 10.0 10.98 PMP C rep 3 10.0 8.77

11/3 PMP C rep 1 10.0 9.87 10.81 0.82 7.63 8.10 PMP C rep 2 10.0 11.42 PMP C rep 3 10.0 11.13

*PMP A = 2 %, PMP B = 1 %, PMP C = 10 % CV = coefficient of variation

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Table 2.4 Summary of inter assay variation for assays of pork meat containing added porcine serum (PMP)

Actual % added Mean estimated % added CV % of actual % difference

PMP A 2.00 2.26 11.53 113.00 13.00 2.00 2.50 5.74 125.00 25.00 2.00 2.79 21.02 139.50 39.50

Mean ± 2.52 ± 0.27 (CV = 10.54 %) 125.83 25.83 SD PMP B 1.00 0.84 20.03 84.00 16.00 1.00 1.49 12.79 149.00 49.00 1.00 1.11 13.30 111.00 11.00

Mean ± 1.15 ± 0.33 (CV = 28.43 %) 114.67 25.33 SD PMP C 10.00 10.28 33.83 102.80 2.80 10.00 10.98 20.17 109.80 9.80 10.00 10.81 7.63 108.10 8.10

Mean ± 10.69 ± 0.37 (CV = 3.45 %) 106.90 6.90 SD CV = coefficient of variation

Conclusions

 The intra-assay variation of the addition of porcine serum to pork meat has a maximum CV of 33.83 % with the majority (8 out of 9) being <25 % (Table 2.3).

 The majority (7 out of 9) of estimated added serum values are ≤ 25 % of the actual amounts added (Table 2.3).

 Inter-assay variation is < 30 % (Table 2.4).

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Intra- and inter-assay variation for assays of beef meat with added serum

Table 2.5 Intra assay variation for assays of beef meat containing added porcine serum (BMP)*

Date Unknown Intra assay variation ID % % mean % difference actual estimated mean SD % CV from actual (n=3) 12/2 BMP A rep 1 2.0 2.17 2.28 0.26 11.30 14.00 BMP A rep 2 2.0 2.57 BMP A rep 3 2.0 2.09

11/3 BMP A rep 1 2.0 1.94 2.02 0.14 6.86 1.00 BMP A rep 2 2.0 1.94 BMP A rep 3 2.0 2.18

17/3 BMP A rep 1 2.0 2.46 2.42 0.13 5.56 21.00 BMP A rep 2 2.0 2.53 BMP A rep 3 2.0 2.27

12/2 BMP B rep 1 10.0 10.03 10.39 0.31 3.00 3.90 BMP B rep 2 10.0 10.57 BMP B rep 3 10.0 10.57

17/3 BMP B rep 1 10.0 7.97 9.16 1.20 13.10 8.40 BMP B rep 2 10.0 9.15 BMP B rep 3 10.0 10.37

24/3 BMP B rep 1 10.0 12.11 12.24 0.51 4.18 22.40 BMP B rep 2 10.0 11.80 BMP B rep 3 10.0 12.80

10/2 BMP C rep 1 1.0 0.67 0.53 0.18 33.66 47.00 BMP C rep 2 1.0 0.60 BMP C rep 3 1.0 0.33

12/2 BMP C rep 1 1.0 0.56 0.52 0.04 7.82 48.00 BMP C rep 2 1.0 0.51 BMP C rep 3 1.0 0.48

17/3 BMP C rep 1 1.0 0.72 0.71 0.09 12.03 29.00 BMP C rep 2 1.0 0.79 BMP C rep 3 1.0 0.62

*BMP A = 2 %, BMP B = 10 %, BMP C = 1 % CV = coefficient of variation

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Table 2.6 Summary of inter assay variation for assays of beef meat containing added porcine serum (BMP)

Actual % added Mean estimated % added CV % of actual % difference

BMP A 2.00 2.28 11.30 114.00 14.00 2.00 2.02 6.86 101.00 1.00 2.00 2.42 5.56 121.00 21.00

Mean ± 2.24 ± 0.20 (CV = 9.15 %) 112.00 12.00 SD BMP B 10.00 10.39 3.00 103.90 3.90 10.00 9.16 13.10 91.60 8.40 10.00 12.24 4.18 122.40 22.40

Mean ± 10.60 ± 1.55 (CV = 14.61 %) 105.97 11.57 SD BMP C 1.00 0.53 33.66 53.00 47.00 1.00 0.52 7.82 52.00 48.00 1.00 0.71 12.03 71.00 29.00

Mean ± 0.59 ± 0.11 (CV = 18.58 %) 58.67 41.33 SD CV = coefficient of variation

Conclusions

 The intra-assay variation for the addition of porcine serum to beef meat shows the majority of CVs < 15 % with one outlier at around 33 % (Table 2.5).

 Values of estimated added serum are within 25 % of the actual added serum for additions of 2 and 10 % porcine serum, but for 1 %, added serum values have been estimated at up to 50 % different from the actual (Table 2.5).

 Inter-assay variation is <20 % (Table 2.6).

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Table 2.7 Intra assay variation for assays of beef meat containing added beef serum (BMB)

Date Unknown Intra assay variation ID % % mean % difference actual estimated mean SD % CV from actual (n=3) 26/2 BMB A rep 1 1.0 0.71 0.92 0.19 20.13 8.00 BMB A rep 2 1.0 1.05 BMB A rep 3 1.0 1.01

17/3 BMB A rep 1 1.0 1.81 1.81 0.03 1.39 81.00 BMB A rep 2 1.0 1.84 BMB A rep 3 1.0 1.79

31/3 BMB A rep 1 1.0 1.18 1.48 0.26 17.48 48.00 BMB A rep 2 1.0 1.65 BMB A rep 3 1.0 1.60

26/2 BMB B rep 1 2.0 1.76 1.83 0.35 19.42 8.50 BMB B rep 2 2.0 2.21 BMB B rep 3 2.0 1.51

17/3 BMB B rep 1 2.0 2.38 2.15 0.34 15.79 7.50 BMB B rep 2 2.0 2.31 BMB B rep 3 2.0 1.76

31/3 BMB B rep 1 2.0 3.02 3.52 0.47 13.43 76.00 BMB B rep 2 2.0 3.57 BMB B rep 3 2.0 3.96

10/2 BMB C rep 1 10.0 5.12 5.12 0.15 2.93 48.80 BMB C rep 2 10.0 4.97 BMB C rep 3 10.0 5.27

4/3 BMB C rep 1 10.0 11.98 10.51 1.90 18.04 5.10 BMB C rep 2 10.0 11.18 BMB C rep 3 10.0 8.37

17/3 BMB C rep 1 10.0 9.52 8.87 1.89 21.35 11.30 BMB C rep 2 10.0 10.36 BMB C rep 3 10.0 6.74

BMB A = 1 %, BMB B = 2 %, BMB C = 10 % CV = coefficient of variation

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Table 2.8 Summary of inter assay variation for assays of beef meat containing added bovine serum (BMB)

Actual % added Mean estimated % added CV % of actual % difference

BMB A 1.00 0.92 20.13 92.00 8.00 1.00 1.81 1.39 181.00 81.00 1.00 1.48 17.48 148.00 48.00

Mean ± 1.40 ± 0.45 (CV = 31.9 %) 140.33 45.67 SD BMB B 2.00 1.83 19.42 91.50 8.50 2.00 2.15 15.79 107.50 7.50 2.00 3.52 13.43 176.00 76.00

Mean ± 2.50 ± 0.90 (CV = 35.9 %) 125.00 30.67 SD BMB C 10.00 5.12 2.93 51.20 48.80 10.00 10.51 18.04 105.10 5.10 10.00 8.87 21.35 88.70 11.30

Mean ± 8.17 ± 2.76 (CV = 33.8 %) 81.67 21.73 SD CV = coefficient of variation

Conclusions

 The intra-assay variation for the addition of bovine serum to beef meat shows CVs of <25 % (Table 2.7).

 Values of estimated beef serum additions are variable ranging from 5.1 to 81 % difference from the actual added (Table 2.7).

 Inter-assay variation is around 35 % (Table 2.8).

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Summaries of intra- and inter-assay variation

Table 2.9 Summary table of inter-assay variation

BM = beef meat PM = pork meat

Actual % Estimated % SD CV (%) % difference added added serum from actual serum Mean (n=3) PM + bovine 1% 0.91 0.03 3.66 6.70 serum 2% 2.26 0.17 7.33 13.17 10% 11.14 0.35 3.11 13.70

PM + porcine 1% 1.15 0.33 28.43 14.67 serum 2% 2.52 0.27 10.54 25.83 10% 10.69 0.37 3.45 6.90

BM + porcine 1% 0.59 0.11 18.58 41.33 serum 2% 2.24 0.20 9.15 12.00 10% 10.60 1.55 14.61 5.97

BM + bovine 1% 1.40 0.45 31.90 40.33 serum 2% 2.50 0.90 35.91 25.00 10% 8.17 2.76 33.83 18.33

Figure 2.1 Correlation between actual and estimated percentage of added serum for four data sets (PMB, PMP, BMB, BMP). R2 values are shown in the figure legend for the linear correlation or “lines of best fit”.

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Table 2.10 Summary of intra and inter assay repeatability

PM+Beef PM+Pork BM+Pork BM+Beef serum serum serum serum

Precision Most within Majority Within 25 % of Variable, 15 % of actual ≤ 25 % of actual for 2 % ranging from actual and 10 % 5 – 81 % added serum. different from Up to 50 % actual different for 1 % added

Intra-assay CV= <20 % for Max. CV = Majority CVs All CVs repeatability all serum 33.8 %. <15 % <25 % additions Majority < 25 %

Inter-assay All CVs <10 % CVs <11 % for All CVs < 20 % All CVs up to repeatability 2 and 10% (<15 % for 2 % 35 % added and 10 % <30 % for 1 % added serum) added

To summarise these data, BMB is shown to be the most variable showing deviation from the rest of the data sets (figure 2.1). In a background of beef meat there is more variation in the detection of added bovine serum (Inter assay, CVs are around 35 % for all levels of added beef serum) than added porcine serum ( CV<20 %) (Table 2.10). The data sets PMB, PMP and BMP show similar profiles. In a background of pork meat detecting the addition of pork serum is the more variable (PMP, CV< 30 %) than the detection of beef serum (PMB, CV< 10 %) (Table 2.10).

Conclusions

 Assays provide semi-quantitative analysis for the addition of 2 % and 10 % added serum.

 Levels of 1 % added serum can always be detected, but quantification is more variable, especially in the case of the addition of added serum of the same species.

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Chapter 3

Defra pilot study of meat products for presence of (A) offal (heart, liver, kidney and lung) and (B) added bovine/ovine or porcine serum

3A. Sample Collection

One hundred retail and food service samples were purchased from various outlets in the East Midlands area and included products containing beef, pork and lamb as ingredients. They were analysed for the presence of (A) offal (heart, liver, kidney and lung) and (B) additional bovine/ovine or porcine serum. The sample size provided a reasonable selection of product types from several sources but is not intended to be statistically representative of the market.

Products were collected, packaging photographed and products logged by Nottingham Trent University (NTU). The products were collected from retailers (major, small and independent retail stores [including butchers]), fast food takeaways and burger vans. Retail product types included fresh, frozen and canned products and ready meals (e.g Lasagne), and included value, standard and premium products. This diverse range of products included a wide range of different matrices in order to test the robustness of the assays; the selection of products was approved by both Defra and the FSA. A summary of the types of stores visited and the products collected is summarised in Tables 3.1, 3.2 and 3.3. Details of all product packaging are listed in Appendix 1.

Table 3.1 Summary of store types visited and the number of products collected

Stores Products Store Type visited collected

National retailer 12 63

Small national 5 9 retailer

Independent retailer 6 8

Food service 5 5 provider (National) Food service provider 15 15 (Independent)

TOTAL 43 100

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Table 3.2 Summary of the types of retail products collected

Raw Cooked Product type products products TOTAL collected collected 17 Minced meat 0 17

Burger/ kebab/ 34 26 8 meatball

Sausage 7 1 8

Ready meal 0 10 10

Canned/ Jar 0 11 11

TOTAL 50 30 80

Table 3.3 Summary of the types of food service products collected

Product type (all Products cooked) collected Burger/ meatball 15

Kebab 4

Sausage 1

TOTAL 20

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3B. Analysis of offal: detection and validation (for detailed summary refer to Appendix 2)

Detection of offal

Samples for offal analysis were extracted and analysed by Western blotting as described in the SOP FA0122(3) v1.2 (Standard operating procedure for the extraction of offal marker proteins in raw and processed meat products, and their subsequent detection and semi- quantification). Samples were assayed in triplicate alongside relevant positive (meat containing 2 % (w/w) added heart, liver, kidney or lung) and negative controls (100 % meat). Examples of Western blots showing the detection of heart, liver, kidney and lung are presented in Figures 3.1 – 3.4.

The protein used as the marker for kidney is not kidney-specific, we have detected it at high levels in kidney and at much lower levels in liver and lung, but not in skeletal muscle or heart (see Q01105 final report). According to the literature, the protein has been detected in liver, pancreas, prostate, spleen, stomach and large intestine with highest levels in kidney, small intestine and placenta, however, it is not expressed in skeletal muscle or heart (UniProt Knowledgebase). For the purposes of this report we will refer to it as a ‘kidney’ marker protein as it is not kidney-specific.

Beef 2 % BHe DPS 31 DPS 53 DPS 59

Beef 2 % BHe DPS 82 DPS 85 DPS 86

Beef 2 % BHe DPS 92 DPS 93 DPS 95

Figure 3.1 Examples of HEART detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a heart-specific protein. Beef = beef skeletal muscle; BHe = beef heart in beef skeletal muscle

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Beef 2 % BLi DPS 5 DPS 8 DPS 9

Lamb 2 % LLi DPS 13 DPS 20 DPS 74

Beef 2 % BLi DPS 58 DPS 69 DPS 79

Figure 3.2 Examples of LIVER detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a liver-specific protein. Beef/Lamb = beef/lamb skeletal muscle; BLi = beef liver in beef skeletal muscle; LLi = lamb liver in lamb skeletal muscle.

Beef 2 % BKi DPS 6 DPS 12 DPS 19

Beef 2 % BKi DPS 22 DPS 24 DPS 33

Beef 2 % BKi DPS 41 DPS 54 DPS 67

Figure 3.3 Examples of ‘KIDNEY’ detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a ‘kidney’ marker protein. Beef = beef skeletal muscle; BKi = beef kidney in beef skeletal muscle

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Beef 2 % BLu DPS 6 DPS 12 DPS 19

Beef 2 % BLu DPS 22 DPS 24 DPS 33

Beef 2 % BLu DPS 41 DPS 54 DPS 67

Figure 3.4 Examples of LUNG detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a lung-specific protein. Beef = beef skeletal muscle; BLu = beef lung in beef skeletal muscle

Conclusions

 A total of 19 products tested postive for one or more offals, 9 were retail samples and 10 were food service samples.

 9 products contained heart, 1 contained liver, 3 contained ‘kidney’, 4 contained heart and ‘kidney’ and 2 contained liver and ‘kidney’.

 No lung was detected.

Validation of offal

Offal in positive pilot study samples was quantified by Western blotting. In brief, pilot study samples were assayed in triplicate alongside a range of reference standards. Densitometry analysis using AIDA image analysis software was used to produce standard curves and estimate the offal content of the pilot study samples. Examples of Western blots for heart, liver and kidney quantification are shown in Figures 3.5, 3.6 and 3.7 respectively.

Where heart or liver were detected at a level of 1 % or above, the species origin and offal type were confirmed by mass spectrometry (MS) using SOP Q01105; (SOP for the extraction of offal marker proteins in raw and processed meat products, and their subsequent detection and quantification).

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% (w/w) beef heart in beef meat DPS 22 DPS 85 DPS 93 0 1 2 5 10 15

% (w/w) beef heart in beef meat DPS 86 DPS 88 DPS 90 0 5 10 15 20 25

Figure 3.5 Quantification of beef heart in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a heart-specific marker protein.

Thirteen samples contained heart at a level of 2 % (w/w) or above. In all the products the heart was confirmed to be beef by mass spectrometry.

% (w/w) lamb liver in meat DPS 13 DPS 20 DPS 40 0 0 1 2 4

Figure 3.6 Quantification of lamb liver in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a liver-specific marker protein.

The three samples which tested positive for liver were all lamb based products, only DPS 20 contained a level above 1 % (w/w) and this was confirmed to be lamb liver by mass spectrometry. Mass spectrometry confirmed the presence of the liver marker protein in DPS 40 despite the level being only ~0.5 % (w/w), but due to the low levels it wasn’t possible to establish if this was beef or lamb liver.

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% (w/w) beef kidney in beef meat DPS 33 0 0 2 2 5 5 10 10 15 15

% (w/w) beef kidney in meat DPS 90 DPS 92 DPS 95 0 1 2 5

Figure 3.7 Quantification of ‘kidney’ in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a ‘kidney’ marker protein.

The protein used as a marker for ‘kidney’ was detected in 9 products, it was found at a level of >1 % (w/w) in 5 products.

To confirm the presence of kidney in these products, a second antibody to a kidney-specific protein was used. The limit of detection for kidney when using the alternative marker protein is currently ~ 5 % (w/w) and therefore the presence of kidney could not be confirmed in the products containing less than 5 % (w/w) ‘kidney’. Only DPS 33 contained > 5 % ‘kidney’, see Figure 3.8 for the detection of kidney in DPS 33.

Standards

% (w/w) beef kidney in beef meat 0 0 5 5 10 10 15 15 DPS 33 Non-specific band

Full length kidney- specific protein

Truncated kidney- specific protein

Figure 3.8 Quantification of kidney in DPS 33 using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blots probed with antibodies to a highly specific kidney protein.

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The alternative kidney-specific marker protein antibody did not detect kidney in DPS33, suggesting something other than kidney is present in this product. Further research and development is needed to determine what other tissue(s) are present in this product.

Detailed summary of the products containing offal: offal type and amount

Table 3.4 provides a detailed summary of the products containing offal. Following the horsemeat incident a 1 % (w/w) level was established as a reporting threshold for horsemeat in beef and was generally accepted as representing deliberate contamination3, but a recent report (FA0137) established that cross contamination in meat plants can lead to >1 % levels of accidental contamination. For the detection of heart and liver (in beef, lamb, pork) and for the additional offal marker protein present in kidney the limit of detection of the methods is 1 % (w/w). Quantitation of the exact level present is challenging and this needs to be set against the measurement uncertainty of the methods. For this reason the Authenticity Methods Working Group (AWMG) decided that a level of above 4 % (w/w) was considered to be the level above which it can be confidently said that the true level is above1 % (w/w) in the samples tested. Taking into account the accepted threshold for deliberate contamination and set against the method’s performance characteristics, AMWG agreed that the results should be reported using the bandings shown below, rather than individual values:

< 4 % offal Viewed as likely trace or adventitious contamination unless declared 4 – 12 % offal Deliberate addition, unless declared > 12 % offal Deliberate addition, unless declared

3 2014 FSA Board paper http://www.food.gov.uk/sites/default/files/multimedia/pdfs/board/fsa-140104v1.pdf; LGC study http://www.food.gov.uk/news-updates/news/2014/6071/mincemeat

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Table 3.4 Offal type and amount detected in pilot study samples (For full details of estimated offal amounts, refer to Appendix 2)

# For catering establishments it is not a legal requirement to give the full ingredients list for a food, however the caterer will have this information and will provide it to a customer on request.

% meat Condition Offal Offal detection/ quantification Sample Product Additional (selected on declared Offal detected Mass Spec. ID type Conclusion – presence of offal comments ingredients) purchase on label? type / level Validation Beef (69%), Beef heart DPS 5 burger cooked No > 12 % heart Yes beef fat confirmed Yes minced Unable to *Offal type not yet known, but DPS 6 Beef (100%) raw No < 4 % ‘kidney’* meat determine not liver, heart or lung. Level too low to establish if it is kidney. 1Unable to Yes < 4 % liver1 minced Lamb determine (level * Offal type not yet known, but DPS 13 raw No meat (100%) too low) not liver, heart or lung. Level too < 4 % ‘kidney’* low to establish if it is kidney. sandwich Beef heart DPS 15 Beef (?%) cooked No 4 – 12 % heart Yes steaks confirmed Yes Pork (46%), Unable to * Offal type not yet known, but DPS 16 sausage pork rind, cooked No < 4 % ‘kidney’* determine not liver, heart or lung. Level too pork fat low to establish if it is kidney minced Lamb < 4 % liver Lamb liver DPS 20 raw No Yes meat (100%) confirmed minced Beef heart DPS 22 Beef (100%) raw No < 4 % heart Yes meat confirmed Yes minced 4 – 12 % Unable to DPS 33 Beef (100%) raw No *Offal type not yet known, but meat ‘kidney’* determine NOT kidney, liver, heart or lung. Lamb Yes < 4 % liver (45%), * Offal type not yet known, but DPS 40 burger raw No Liver confirmed Mutton not liver, heart or lung. Level too < 4 % ‘kidney’* (42%) low to establish if it is kidney Soya bean Assumed to Not Beef heart DPS 85 burger cooked 4 – 12 % heart Yes proteins be beef known# confirmed detected by MS Assumed to Not Beef heart DPS 86 burger cooked >12 % heart Yes be beef known# confirmed Yes >12 % heart Assumed to Not Beef heart * Offal type not yet known, but DPS 88 burger cooked be beef known# confirmed not liver, heart or lung. Level too < 4 % ‘kidney* low to establish if it is kidney. Yes 4 – 12 % heart Not Beef heart * Offal type not yet known, but DPS 90 burger Beef cooked known# confirmed not liver, heart or lung. Level too < 4 % ‘kidney’* low to establish if it is kidney. Not Beef heart DPS 91 burger Beef cooked 4 – 12 % heart Yes known# confirmed Yes >12 % heart Not Beef heart * Offal type not yet known, but DPS 92 burger Beef cooked known# confirmed not liver, heart or lung. Level too < 4 % ‘kidney’* low to establish if it is kidney Assumed to Not Beef heart DPS 93 burger cooked 4 – 12 % heart Yes be beef known# confirmed Yes >12 % heart Assumed to Not Beef heart * Offal type not yet known, but DPS 95 burger cooked be beef known# confirmed not liver, heart or lung. Level too < 4 % ‘kidney’* low to establish if it is kidney. Not Beef heart DPS 96 burger Beef cooked >12 % heart Yes known# confirmed Soya bean Assumed to Not Beef heart DPS 99 burger cooked 4 – 12 % heart Yes proteins be beef known# confirmed detected by MS.

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Additional comments and observations

In addition to the products which tested positive for one or more offals, one of the lamb kebabs (DPS 100) from a fast food takeaway had a unexpected protein profile on a 1D SDS polyacrylamide gel and therefore the species origin of meat in this product was analysed by mass spectrometry. Beef, lamb and chicken were detected in this product (results not shown).

Many products in the pilot study had a 1D SDS polyacrylamide gel protein profile that was quite different to 100 % skeletal muscle (meat) suggesting that products could contain something other than skeletal muscle (but not heart/liver/kidney/lung). Further work is required to investigate what other ingredients manufacturers may be using as a substitute for skeletal muscle.

Two food service burgers contained soya bean proteins (presence confirmed by mass spectrometry in DPS 85, DPS 99). As packaging was unavailable at the point of purchase, consumers will not be aware of the presence of this potential allergen.

Conclusions

 12 products contained heart at a band level of 4 % (w/w) or above. In all products the heart was confirmed to be beef by mass spectrometry.

 3 products contained lambs liver, the presence of liver was confirmed in 2 of the products by mass spectrometry.

 1 product contained high levels of a protein expressed in kidney, and reported to be expressed in small intestine, placenta, liver, pancreas, prostate, spleen, stomach and large intestine. Assays confirmed it was not kidney or liver. A further 8 products contained lower levels of this protein, but we are unable to determine if kidney is present in these products.

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Summary tables of added offal by store and product types

Tables 3.5 to 3.7 summarise the offal types found in the different product and store types.

Table 3.5 Summary of offal type by store type

Store Type Products Tested/ Offal type collected validated for offal National retailer 63 6 1 contained heart 1 contained liver 2 contained ‘kidney’ 2 contained liver & ‘kidney’ Small national 9 2 2 contained heart retailer Independent retailer 8 1 1 contained ‘kidney’

Food service 5 0 provider (National) Food service 15 10 6 contained heart provider 4 contained heart & ‘kidney’ (Independent) TOTAL 100 19

Table 3.6 Summary of offal type by retail product

Product type Products Tested/ Offal type collected validated for offal Minced meat 17 5 1 contained heart 1 contained liver 2 contained ‘kidney’ 1 contained liver & ‘kidney’ Burger/ kebab/ 34 3 2 contained heart meatball 1 contained liver & ‘kidney’ Sausage 8 1 1 contained ‘kidney’

Ready meal 10 0

Canned/ Jar 11 0

TOTAL 80 9

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Table 3.7 Summary of offal type by food service product

Product type Products Tested/ Offal type collected validated for offal Burger/ meatball 15 10 6 contained heart 4 contained heart & ‘kidney’ Kebab 4 0

Sausage 1 0

TOTAL 20 10

Conclusions

 11 %, (9 out of 80), samples from retail outlets were found to contain undeclared offal, 5 raw frozen minced meat; 2 frozen burgers (1 cooked, 1 raw); 1 cooked frozen sandwich steak and 1 cooked frozen sausage.

 6 were purchased from large national retailers, 2 from small national retailers and 1 from an independent retailer.

 50 %, (10 out of 20), cooked food service samples (all burgers) contained offal.

Follow up studies conducted by Local Authorities (LA)

All results were reported to Defra and the FSA; enforcement officers were informed and follow up investigations have taken place. For food service products, offal was declared as an ingredient on the bulk catering pack for 8 products; 2 products are being further investigated. This is evidence that the methods can robustly detect offal in these meat products.

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Summary and overall conclusions of Chapter 3B

 19 products (19 %) tested positive for one or more offals, with 13 of these containing offal at a level of 4 % (w/w) or above.

 ~ 10 % of retail products contained undeclared offal.

 50 % of the food service products (10 out of 20) tested positive for offal, all contained heart and 8 of these had beef heart declared on the catering packaging.

 ~ 30 % of retail minced meat tested contained undeclared offal.

 Heart was the most prevalent offal with >4 % (w/w) beef heart confirmed in 12 products.

 One product (DPS 33) contained high levels of a non-skeletal muscle protein, it was confirmed that the protein is not of kidney, liver, heart or lung origin. It is not yet known what ‘offal’ tissue is present in this product.

 One doner kebab was found to contain a mixture of 3 species: beef, chicken and lamb.

 Many products in the pilot survey had a 1D protein profile that was quite different to 100 % skeletal muscle (meat) suggesting that products could contain something other than skeletal muscle (but not heart/liver/kidney/lung).

 Two food service burgers contained soya bean proteins (presence confirmed by mass spectrometry). As there is no packaging consumers will not be aware of the presence of this potential allergen.

Limitations of the method

 Quantification may be affected by the addition of non-meat matrices.  The kidney marker is not specific to kidney, but is not found in skeletal muscle. We have detected it at high levels in kidney and much lower levels in liver and lung. According to the literature, the protein has also been found in: pancreas, prostate, spleen, stomach, small and large intestine and placenta.  The limit of detection using the second kidney specific marker is approximately 5 % (w/w).

Further work

 Identify an alternative kidney specific marker.  Establish the identity of the “non-skeletal muscle component” in DPS 33.  Further work to determine the method performance characteristics and better understand method uncertainty.

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3C. Analysis of added serum (for detailed summary refer to Appendix 3)

Experimental details

All 100 samples for analysis were analysed raw unless already cooked. Samples were extracted and analysed by probing Western blots for the presence of both porcine and bovine serum (2 gels) as described in the SOPs [SOP FA0122(1) v1.2; SOP for the extraction of processed meat products prior to the analysis of added serum”; SOP FA0122(2) v1.2; SOP for Immunoblot analysis of bovine or porcine serum in extracts of processed meat products].

Each extracted sample was analysed in triplicate alongside standards of appropriate meat content.

Initial analysis of the results was based on data generated previously, as described in Chapter 1A (determination of the threshold levels of residual serum in beef and pork meat). The purpose of this study was to determine the variation in residual serum across the types of meat cuts likely to be used in processed meat products and across different animals to obtain a representative serum value for each species. Limited resources meant that this study was based on five different animals and five cuts of meat from each animal giving a total data set of 25 samples each for pork and beef.

For the analysis of the 100 pilot study samples, Western blots were analysed using densitometry software to determine the intensity of each band on the blot allowing the comparison of standard and sample data.

Using this densitometry as a measure of band intensity and the previously calculated error value for each species a top threshold value for serum was extrapolated for each species. This top threshold value for each standard on an individual gel was used to compare against the signal for the pilot study samples. Since it is recognised that the data produced are not linear (i.e. signals are not directly proportional to analyte concentration), appropriate percentage meat standards with/without 2 % (w/w) added serum were also included on many gels.

It became clear that occasionally the calculated top threshold value overlapped with the value for 2 % added serum, so for further validation of some samples, 100 % meat and 100 % meat with 2 % added serum were used as standards regardless of the meat content of the individual samples. This way if the sample was displaying a signal much higher than the 2 % added serum we could be more confident that the sample contained serum at a level higher than expected.

For raw sausage samples containing rusk as a significant ingredient, rusk was added to standards of appropriate meat content, i.e. sausage samples of 32 to 46 % meat were analysed alongside 45 % meat standards containing 15 % rusk, with/without 2 % added serum.

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Results

Example data and detailed summaries

Examples of data demonstrating the presence of added serum are shown in Figures 3.9 and 3.10.

100 % RB RB % 100

100% RB 2% BS BS 2% RB 100%

DPS34

DPS 3 DPS

100 % RB RB % 100

100%RB 2%BS 2%BS 100%RB DPS6 DPS 33 DPS

Figure 3.9 Examples of added beef serum detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for beef serum. (RB= raw beef, BS = beef serum)

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100 % CP CP % 100

DPS 16

100% CP 2% 2% CP 100%

45 % CP 5%Ru 50% CC 50% 5%Ru CP % 45

45 % RP 15 % Ru % 15 RP % 45

DPS 21 16

DPS 27 16 45 % RP 15 % Ru 2%PS 2%PS Ru % 15 RP % 45

Figure 3.10 Examples of added pork serum detection in pilot study samples using Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for beef serum. (RP= raw pork, PS = pork serum, Ru = rusk)

Table 3.8 provides an overall summary of samples containing higher than expected levels of added serum. Tables 3.9 & 3.10 respectively summarise results of samples showing equivocal results and unusual observations.

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Table 3.8 Summary of samples containing serum at levels higher than expected based on in-house standards

Sample Product % meat Condition on Conditi Added serum detection Additional ID type (selected purchase on of Higher Higher Conclusion Information ingredients) sample than than 2% - Higher at threshold added serum than analysi serum expected? s Retail DPS 6 minced Beef (100 %) raw/frozen raw Yes Yes Yes below meat threshold for cooked assay DPS 8 burger Beef (69 %), cooked/frozen cooked Yes Yes Yes beef fat DPS 13 minced Lamb (100 raw/frozen raw n/a Yes Yes below 2% meat %)* added for cooked assay DPS 16 sausage Pork (46 %), cooked/frozen cooked Yes Yes Yes pork rind, pork fat DPS 27 sausage Pork (32 %), raw/frozen raw Yes Yes Yes below pork rind, pork threshold for fat cooked assay DPS 31 burger Beef (85 %), cooked/ cooked Yes Yes Yes Beef Fat refrigerated DPS 33 minced Beef (100 %) raw/frozen raw Yes Yes Yes below meat threshold for cooked assay DPS 70 kebab Beef (67 %) raw/frozen raw Yes Yes Yes below threshold for cooked assay Food Service DPS 81 burger Beef cooked cooked Yes Yes Yes

DPS 84 burger Beef cooked cooked Yes Yes Yes

DPS 88 burger Species not cooked cooked Yes Yes Yes declared but assumed to be beef DPS 90 burger Beef cooked cooked Yes Yes Yes

DPS 96 burger Beef cooked cooked Yes Yes Yes

DPS 99 burger Species not cooked cooked Yes Yes Yes declared but assumed to be beef

* It should be noted that the level of residual serum has not been determined in lamb or mutton. The choice of standard is a key factor and was considered to be the most difficult aspect of the work. For example, the addition of other food matrices (rusk for example) is known to affect the serum signal and the level of rusk in processed meat products is variable, so the use of 100 % meat as a standard may lead to false negatives.

Conclusions

 14 % of the samples contained higher than expected levels of serum; 5 were raw and 9 were cooked.

 Higher than expected levels of serum were found in 11 beef samples, 1 lamb sample and 2 pork samples.

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Table 3.9 Summary of samples showing equivocal results.

Added serum detection Condition of sample Sample Product % meat Condition on Higher Higher Conclusion Additional ID type (selected purchase at Information analysis than than 2% - Higher ingredients) threshold added serum than serum expected?

DPS2 sausage Pork (42%), raw/frozen raw Yes No Uncertain pork rind

DPS10 sausage Pork (42%), raw/frozen raw Yes No Uncertain pork rind

DPS12 minced Beef (100%) raw/frozen raw Yes No? Uncertain Very close to meat 2% added serum value

DPS21 sausage Pork (44%), raw/frozen raw Yes No Uncertain Pork Rind

DPS63 sausage Pork (42%), raw/frozen raw Yes No Uncertain pork fat (10%)

DPS64 meatballs Beef (59%), cooked/frozen cooked Yes No Uncertain beef tendon (8%)

Conclusions

 6 samples showed equivocal results.

 Most of these samples (4) were raw sausages, these gave levels of serum higher than the calculated threshold for pork meat, but came well below the signal when assayed against appropriate standards containing 2 % added serum.

 DPS 12 (frozen minced beef) showed a borderline result when compared against standards with 2 % added serum.

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Table 3.10 Summary of samples showing unusual observations.

Sample Product % meat Condition Added serum detection Additional ID type (selected on Information ingredients) purchase Higher Higher Conclusion - Higher than than 2% serum than expected? threshold added serum

DPS82 meatballs Species not cooked - - Bovine/Ovine or Porcine known serum not detected

DPS85 burger Species not cooked No No Bovine serum low Soyabean declared but compared to 100 % proteins assumed to be cooked beef standard. detected by MS beef Trace of pork serum present

DPS93 burger Species not cooked - - Bovine/Ovine or Porcine Presence of declared but serum not detected beef meat assumed to be confirmed by beef MS

DPS100 kebab Doner kebab, cooked - - Bovine/Ovine or Porcine Meat species therefore expect serum not detected. detected by MS: lamb lamb, beef and chicken

Conclusions

 Unable to detect either beef/lamb or pork serum in 3 samples. Presence of meat species was confirmed by mass spectrometry in 2 samples.

 One sample, assumed to be beef, but ingredients are not known, contained a trace of pork serum.

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Summary tables of added serum by store and product types

Tables 3.11 to 3.13 summarise the added serum detected by product and store types.

Table 3.11 Summary of samples containing serum at levels higher than expected by store type

Store Type Products Serum at level higher collected than expected National retailer 63 5

Small national 9 1 retailer Independent retailer 8 2

Food service 5 2 provider (National) Food service 15 4 provider (Independent) TOTAL 100 14

Table 3.12 Summary of samples that contained higher than expected by levels of serum by product type

Product type Products Serum at level higher collected than expected Minced meat 17 3

Burger/ kebab/ 34 3 meatball Sausage 8 2

Ready meal 10 0

Canned/ Jar 11 0

TOTAL 80 8

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Table 3.13 Summary of food service product types that contained serum at levels higher than expected

Product type Products Serum at level higher collected than expected

Burger/ meatball 15 6

Kebab 4 0

Sausage 1 0

TOTAL 20 6

Conclusions

 10 %, (8 out of 80), samples from retail outlets were found to contain serum at levels higher than expected, 3 raw frozen minced meat; 2 cooked burgers (1 frozen, 1 refrigerated); 1 raw frozen kebab, 1 raw frozen sausage and 1 cooked frozen sausage.

 5 were purchased from large national retailers, 1 from a small national retailer, 2 from independent retailers.

 30 %, (6 out of 20), cooked food service samples (burgers) contained serum at levels higher than expected.

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Summary and overall conclusions of Chapter 3C

 14 products contained higher than expected levels of added serum.

 10 % of retail products (8 out of 80) contained higher than expected levels of added serum.

 30 % of food service products (6 out of 20) contained higher than expected levels of added serum.

 We were unable to detect any residual serum (bovine/ovine/porcine) in canned products, possibly due to the highly processed nature of the material.

 It is difficult to make conclusions regarding the presence of added serum in the food service products as we do not have access to an ingredients list.

Limitations of the method

 At present analysis of raw material is considered to be more reliable than analysis of cooked material. Most validation has been undertaken with raw samples to date.  Appropriate standards will be different for every sample according to its ingredients and meat content. Therefore it is difficult to define suitable reference material, this is a key factor and the most difficult aspect of the work.  Addition of other food matrices (rusk, for example) is known to reduce the serum signal and the level of rusk in many processed meat products is variable, so the use of 100 % meat as a standard may lead to false negatives.  Residual serum was not detected in canned products, possibly due to the highly processed nature of the material.

Further work

 Further identification and validation of suitable sets of reference material, customised for different types of samples, to allow detection of additional serum above an agreed amount.  Determination of threshold levels of residual serum in lamb.  Further investigation of the performance of the assays on cooked material.  Effect of age, sex and breed of animal on “normal” residual serum levels.

3D. Summary of samples containing offal and/or serum at levels higher than expected

Table 3.14 provides a summary of both offal and added serum in the pilot study.

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Table 3.14 Summary of pilot study samples containing offal and/or serum at levels higher than expected, collected from retail and food service outlets in the East Midlands

Offal detected Added serum detected Offal + added serum detected

Sample Product % meat (selected Condition Offal Higher serum than ID type ingredients) on detected? expected? purchase DPS 5 burger Beef (69 %) cooked Yes No DPS 6 minced meat Beef (100 %) raw Yes Yes DPS 8 burger Beef (69 %) cooked No Yes DPS 13 minced meat Lamb (100%) raw Yes Yes DPS 15 sandwich Beef (?%) cooked Yes No steaks DPS 16 sausages Pork (46 %), pork cooked Yes Yes rind, pork fat DPS 20 minced meat Lamb (100 %) raw Yes No DPS 22 minced meat Beef (100 %) raw Yes No DPS 27 sausages Pork (32 %), pork raw No Yes rind, pork fat DPS 31 burger Beef (85 %) cooked No Yes DPS 33 minced meat Beef (100 %) raw Yes Yes DPS 40 burger Lamb (45 %), raw Yes No Mutton (42 %) DPS 70 kebab Beef (67 %) raw No Yes DPS 81 burger Beef cooked No Yes DPS 84 burger Beef cooked No Yes DPS 85 burger Species assumed cooked Yes Bovine serum low to be beef compared to standard. Trace pork serum DPS 86 burger Species assumed cooked Yes No to be beef DPS 88 burger Species assumed cooked Yes Yes to be beef DPS 90 burger Beef cooked Yes Yes DPS 91 burger Beef cooked Yes No DPS 92 burger Beef cooked Yes No DPS 93 burger Species assumed cooked Yes Bovine/Ovine or Porcine to be beef serum not detected DPS 95 burger Species assumed cooked Yes No to be beef DPS 96 burger Beef cooked Yes Yes DPS 99 burger Species assumed cooked Yes Yes to be beef

Conclusions

 25 % of meat products collected from retail and food service outlets contained offal and/or serum at levels higher than expected; 12 % were from food service and 13 % were from retail.

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Chapter 4

Establish limit of detection (LOD) for offals via Western blot analysis using chromogenic reagents

Background

The current Western blot SOP for offal detection uses an enhanced chemiluminescence (ECL) reagent and a CCD camera system to capture and quantify the results; these systems are expensive and not currently available in Public Analyst laboratories. To facilitate transfer of the offal detection assays to Public Analysts it is necessary to find an alternative, less technical and potentially more economical way to visualise Western blots; possibly achievable using chromogenic substrates. These could offer a simple and cost-effective method of detection without the need for special processing or visualising equipment. However, this approach is likely to be much less sensitive than our current method.

Initial trials comparing the two most sensitive commercially available chromogenic reagents revealed that a 1-step ultra TMB-blotting substrate was the most sensitive (data not shown). The latter reagent is reported to have a detection limit of 20 pg, this compares to femtogram levels for the ECL reagent used in our laboratory and therefore we were not expecting the detection of offal to be as sensitive.

Methodology

Reference material for determining the limit of detection were prepared by spiking beef, lamb and pork meat with 0 – 10 % (w/w) offal (heart, liver, kidney and lung). The reference samples were analysed on 3 separate occasions using the chromogenic substrate (1-step ultra TMB-blotting substrate; Thermoscientific 37574) to establish the limit of detection for each offal from the 3 species. The coloured product/band formed was visualised by eye and the blots were scanned using a Fujifilm LAS-4000 CCD imaging system and a flatbed scanner so the results could be viewed electronically. Images generated using the flatbed scanner could not be analysed using existing software (AIDA image analysis software) to provide quantitative data.

Results

Figures 4.1 – 4.11 show the results of the chromogenic detection of offals from cows, lambs and pigs.

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% (w/w) beef heart in beef meat 0 1 2 5 10

Figure 4.1 Determination of experimental LOD for BEEF HEART detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a heart-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

% (w/w) lamb heart in lamb meat 0 1 2 5 10

Figure 4.2 Determination of experimental LOD for LAMB HEART detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a heart-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

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% (w/w) pork heart in pork meat 0 1 2 5 10

Figure 4.3 Determination of experimental LOD for PORK HEART detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a heart-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

% (w/w) beef liver in beef meat 0 1 2 5 10

Figure 4.4 Determination of experimental LOD for BEEF LIVER detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a liver-specific protein. Bands were visualised using a chromogenic reagent Data from assays conducted on 3 separate occasions are shown. .

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% (w/w) lamb liver in lamb meat 0 1 2 5 10

Figure 4.5 Determination of experimental LOD for LAMB LIVER detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a liver-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

% (w/w) pork liver in pork meat 0 1 2 5 10

Figure 4.6 Determination of experimental LOD for PORK LIVER detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a liver-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 2 separate occasions are shown.

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% (w/w) beef kidney in beef meat 0 1 2 5 10

Figure 4.7 Determination of experimental LOD for BEEF KIDNEY detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a kidney marker protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

% (w/w) lamb kidney in lamb meat 0 1 2 5 10

Figure 4.8 Determination of experimental LOD for LAMB KIDNEY detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a kidney marker protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

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% (w/w) pork kidney in pork meat 0 1 2 5 10

Figure 4.9 Determination of experimental LOD for PORK KIDNEY detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a kidney marker protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

% (w/w) beef lung in beef meat 0 1 2 5 10

Figure 4.10 Determination of experimental LOD for BEEF LUNG detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a lung-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

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% (w/w) lamb lung in lamb meat 0 1 2 5 10

Figure 4.11 Determination of experimental LOD for LAMB LUNG detection by Western blot analysis. Protein extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the resultant Western blots probed for a lung-specific protein. Bands were visualised using a chromogenic reagent. Data from assays conducted on 3 separate occasions are shown.

The levels of offals detected by eye are summarised in Table 4.1 along with a comparison of the experimental LOD for offals using the ECL detection method. It should be noted that the experimental LOD for heart and kidney using ECL is likely to be lower than 1 % (w/w); we did not check levels lower than 1 % (w/w).

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Table 4.1 Summary of limit of detection for offals by Western blotting using chromogenic detection

Chromogenic detection LOD

using Offal Overall LOD by LOD by LOD by ECL conclusion for eye gel 1 eye gel 2 eye gel 2 detection LOD Beef 1 % 1 % 1 % 1 % 1 % HEART Lamb 1 % 1 % 1 % 1 % 1 % Pork 1 % 1 % 1 % 1 % 1 % Beef 1 % 2 % 2 % 2 % 1 % Lamb 5 % 5 % 5 % 5 % 1 % LIVER Unable to Pork determine, 10 % 10 % 10 % 1 % but > 10 % Beef 1 % 1 % 1 % 1 % 1 % KIDNEY Lamb 1 % 2 % 2 % 2 % 1 % Pork 1 % 1 % 1 % 1 % 1 % Beef 1 % 1-2 % 1 % >1 % 1 % Lamb 2 % 2 % 2 % 2 % 1 % LUNG Unable to Unable to Unable to Pork determine, determine, determine, >10 % 10 % but > 10 % but > 10 % but > 10 %

Conclusions

 The LOD for offals was generally higher using the chromogenic detection reagent than the ECL reagent.

 However, 1 % (w/w) heart (all species) and 1 % (w/w) beef and pork kidney could be detected using the chromogenic substrate.

 The increase in the experimental LOD for liver was greatest for pig liver, at around 10 % (w/w); and was respectively 5 % (w/w) and 2 % (w/w) for lamb and beef liver.

 LOD for lamb kidney was increased from 1 % (w/w) with ECL to 2 % (w/w) using a chromogenic substrate.

 LOD for beef and lamb lung was increased from 1 % (w/w) with ECL to 2 % (w/w) with a chromogenic substrate.

To conclude, when using ECL 1 % (w/w) offal could always be detected (except for pork lung). Using the chromogenic substrate, 1 % (w/w) level could be consistently detected for heart. For other offals, using the chromogenic substrate the LOD was 2 – 10 % (w/w).

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