Q01101- Evaluation of a Simple Microscopy Protocol for Identifying Mechanically Separated Meat (MSM)inPork, Chicken and Turkey
Q01101- Evaluation of Simple Microscopy Protocol for Identifying Mechanically Separated Meat in Pork, Chicken and Turkey
Kathy Groves BSc (Hons) FRMS
FOREWORD
This research project was funded by the Food Standards Agency between October 2005 and March 2007.
EXECUTIVE SUMMARY
This project was contracted by the Food Standards Agency with the intention, if successful, to help local authorities and public analysts in the UK to assess whether a particular meat ingredient produced by mechanically removing residual meat from bones falls within the definition of mechanically separated meat (MSM), also known as mechanically recovered meat (MRM).
This definition in the Animal Products Hygiene Regulation (EC)853/2004[1]states that “mechanically separated meat is the product obtained by removing meat from flesh-bearing bones after boning or from poultry carcasses, using mechanical means resulting in the loss or modification of muscle fibre structure”.
If the ingredient is considered to be a mechanically separated product (i.e. MSM or MSM) then it falls outside the definition of “meat” and needs to be labelled separately. This definition came into force in 2006.
The project looked at whether a simple microscopy protocol could be used to differentiate between hand-deboned meat that has been mechanically treated (such as mincing/chopping), Baader and desinewed meats, and meat that has been mechanically separated from the carcass under high pressures.
The evaluation was based on the definition given above, and primarily considered the issue of loss or modification of muscle fibre structure. The evaluation also considered other structural aspects that became evident during the course of the project such as dispersed protein and connective tissue. Since the type of meat will affect the extent of disruption of the muscle fibres, the relatively softer chicken and turkey meats were included as well as tougher pork.
The work reported here compares the structures seen using a simple staining and light microscopy technique on a range of reference chicken, turkey and pork samples of known origin. The results showed clear differences in appearance and muscle integrity between minced and desinewed meat and MSM. These differences and the sample type were identified easily in a blind trial by scientists with minimal training in microscopy. An SOP with high resolution reference images has been prepared and produced separately on CD-ROM.
CONTENTS
INTRODUCTION
Food Hygiene and Labelling Requirements
MATERIALS
Reference Samples......
Stains and Reagents...... 8
METHODS...... 9
Examination of Samples and Preparation of Sections...... 9
Staining in Toluidine Blue...... 9
Staining in Picro-Sirius Red...... 9
Scoring system and test...... 10
Preparation of Cooked Samples...... 10
RESULTS...... 11
Sectioning and Staining Protocol - Comments...... 11
Observations on Chicken, Pork and Turkey Main Reference Samples...... 12
Appearance by Eye...... 12
Cryostat Sections Stained in Toluidine Blue...... 13
Observations on Chicken Reference Samples in Toluidine Blue...... 13
Observations on Pork Reference Samples in Toluidine Blue...... 14
Observations on chicken and pork samples derived from varying pressures on MPD60 in Toluidine Blue 14
Cryostat Sections Stained in Picro-Sirius Red
Observations on Chicken and Pork Additional Reference Samples using Toluidine Blue staining
Trial Scoring System
Observations on Cooked Samples
Chicken samples
Pork samples
DISCUSSION & CONCLUSIONS
REFERENCES
ABBREVIATIONS......
APPENDIX I– Images and sample conditions...... 32
APPENDIX II – Standard Operating Procedure...... 58
INTRODUCTION
This project was contracted by the Food Standards Agency with the aim to develop a method to assess whether a particular meat ingredient produced by mechanically removing residual meat from bones falls within the new definition of mechanically separated meat (MSM), also known as mechanically recovered meat (MRM).
Food Hygiene and Labelling Requirements
The definition of MSM is given in Annex I of Regulation (EC) 853/2004i,as “the product obtained by removing meat from flesh-bearing bones after boning or from poultry carcasses, using mechanical means resulting in the loss or modification of muscle fibre structure”. If the ingredient is considered to be mechanically separated product then it falls outside the definition of “meat” laid down in Commission Directive 2001/101/ECii, and needs to be labelled separately. This definition came into force in 2006.
The issue of mechanically removed meat (MRM/MSM), how to define it and determine whether it has been added to a product has existed for many years. Historically MRM was defined in Council Directive 64/433/EEC[2]on Hygiene requirements for fresh (red) meat as:
“(red) meat obtained by mechanical means from flesh bearing bones apart from bones of the head, extremities of limbs below carpal and tarsal joints, and for pigs the coccygeal vertebrae”. This was modified in 1995[3]to define MRM as coming from residual meat after boning, which had been obtained by mechanical means and passed through a fine mesh such that its cellular structure has been broken down and it flows in puree form.
In the UK a BMPA (formerly BMMA) code of practice was agreed in 1987 to restrict the raw materials and types of bones used, and to ensure that prohibited offals listed in the meat product regulations were not present. The BMPA agreed with LACORS that MSM could be defined as residual meat mechanically removed off bones after deboning that flows in puree form and whose cellular structure has been broken down.
Originally MSM was considered as meat and could be labelled as such when incorporated into food products. However in a court case in 19881 it was laid down that MSM should be declared separately, and in 1997 the Standing Committee for Foodstuffs was of the opinion that it should be declared separately in the ingredients list.
To complicate the issue further, an EU definition update was discussed to satisfy three different measures:
a)a definition of meatii for labelling and QUID[4]
b)a consolidated Hygiene Regulation on Animal Products[5]
c)TSE Regulations for controlling BSE.[6]
These three measures had different requirements in themselves. The labelling and quantitative ingredient declaration (QUID) needed very specific definitions on MSM since it would not count as meat. The Hygiene regulation needed a wide definition as there was a microbiological risk in mechanical processing, and finally the TSE Regulation needed a specific definition to remove specified risk material.
The end result after these discussions was that in the UK MSM could not count towards the minimum meat content of a product, and had to be labelled separately from the meat in the ingredients list. As well as the current definition of MSM given above, there are regulations and restrictions depending on the calcium content and also the age of the material after slaughter and temperature of transport of the material. This meant that if the material was classed as MSM it was subject to close regulation on these issues and needed to be treated differently from meat in terms of inspection, transport and use in cooked products.vii With so many requirements and restrictions placed on the definition of MSM, difficulty arises in the enforcement of these regulations. A method is therefore needed to assist in the determination of MSM, for example by finding a marker specific to MSM.
Attempts have been made to identify a marker specific to mechanically separated meat using several different techniques including microscopy2,3,4,5,6. Previously, researchers have used different staining techniques to highlight bone, connective tissue, and hyaline cartilage and muscle differences. The approach in each case was to identify a marker for MSM so that its presence in products, especially comminuted products, could be detected, but to date no clear marker has been found.
Production and Manufacture of MSM and desinewed meat
Conventionally meat was separated from flesh bearing bones, using machines run at very high pressures (up to 200 bar), and the products obtained were low in connective tissue, with a very fine consistency and a puree-like texture and appearance. Machines used to remove all bone using high pressure were developed from a patent filed in 1976, and became the industry standards under the name ‘Stork Protecon’ for the production of what became known as mechanically removed meat (now commonly termed as mechanically separated meat). There were four different sizes of machine, with the largest, the MPD60, being used for red meat, which is tougher than poultry. The pressures used in these machines can be varied by the operator, and a number of different high pressures were used in this project as detailed later.
The exclusion of MSM from the definition of meat has forced the meat industry to look at alternative ways to recover the large quantity of meat from the bones. Machines have been developed which remove meat from bones at lower pressures and which purport to retain the fibre structure of the muscle. The products obtained from these machines have been commercially termed “desinewed meat”. Desinewed meat, from which sinews, tendons and bone fragments have been removed, is considered a meat preparation as defined in the hygiene regulations, i.e. it has gone through a process insufficient to modify the internal muscle fibre structure of the meat. Although no definition of desinewed meat exists in legislation, further guidance on the production of desinewed meat is available on the Food Standards Agency’s website.7
There are different machines on the market to carry out the deboning and/or desinewing operation, and the resulting material depends on the type of meaty carcass going into the machine and the conditions used. Recently there have been modifications to the standard Stork Protecon machines so that they run at much lower pressures (2-50 bar), and can be combined with other machines like the Baader and Sepamatic for a two-stage deboning and desinewing process.
The Beehive machine is a single stage deboning and desinewing machine, most suitable for producing desinewed meat from poultry. The operation of the machine involves a rotating drum and a sideways mincer, with a valve to control rate of bone waste ejected. Another very popular deboning and desinewing machine, particularly for pork, is the Townsend DMM 50 system. This is essentially a low pressure deboning machine that uses a hydraulic ram and coarse filter to remove bone and large pieces of connective tissue followed by a desinewed step, to produce what is referred to as ‘3mm pork’, or “trim”.
This project looked at whether a simple microscopy protocol could be used to differentiate between hand-deboned meat (HDM) that has been minced and chopped, Baader and other desinewed meats, and finally meat that has been mechanically separated under high pressures. The various structures in the samples were analysed, and a subjective assessment made as to whether they can be considered as MSM, based on the requirement in the official definition relating to the loss or modification of muscle fibre structure. Since the type of meat will affect the extent of disruption of the muscle fibres, the relatively softer chicken and turkey meats were included as well as tougher pork. The results with other meats such as beef and lamb would need to be studied separately.
The machines described have been used to produce the samples evaluated in this project, and the project does not encompass all of the machines available for producing MSM or desinewed meat. Generally, the machines can be adapted to run at different pressures to produce samples with different levels of muscle fibre disruption. Specific conditions and settings for production of the different samples used for this project are listed in Appendix 1. Changing these conditions will alter the state of the final product (and hence the assessment and interpretation of the results).
Aims of the project
At the outset there were two objectives for this project. The first was to use as simple a preparation and staining procedure as possible to evaluate the differences between reference samples of known origin. If the differences were clear then it would provide a guide for enforcement authorities to come to a reasonable decision as to the nature of the meat ingredient.
The second objective was to look more closely at a technique for staining the muscle structure and connective tissue since unpublished work carried out at Leatherhead Food Research using the stain Picro-Sirius Red indicated that there was a difference in staining of mechanically separated meat and control meat. Although the protocol used was not simple, it was included to see whether there was a visible difference in the membranes around the muscle fibres after high pressure separation.
MATERIALS
Reference Samples
Tables 1 and 2 list the samples examined in this project. The main reference chicken and pork samples (Table 1) were produced by one company specifically for this project and also for a separate FSA projectat Royal Holloway College, University of London using chemical markers to identify MSM (Q01102). Further technical and production details of these main reference samples are included in Appendix I. The first set of chicken and pork samples were produced early on in the project. The MPD60 “pressure” samples were produced much later in the project. They were produced to see whether there was a difference in structure with samples of MSM produced at slightly lower high pressures.
The turkey samples were produced by a company specialising in turkey-based products. Additional reference samples were supplied by other companies (Table 2). All of the reference images of cooked and uncooked meat samples, produced using the different manufacture conditions described in the tables below, are supplied in this report. Samples provided in this project are referred to by the machine they were produced from, but as the conditions and settings for each machine can be altered, it is important to note that the machine used does not necessarily define the product type (MSM or desinewed). A standard operating procedure (SOP) document has been produced to accompany the report, and provides extra replicate reference images, in addition to the images coded in the table.
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Table 1: Main Reference Samples Examined
SAMPLE / CONDITIONS (see Appendix 1) / PLATE NUMBERS / SOP plate numberMinced chicken (A1) / 3mm plate, 98% yield (Table 3) / 6a, 12a, 16a / 5a, 10a, x
Beehive Chicken (B1) / Setting U, 43.2% yield (Table 4) / 6b, 12d, 16c / 5b, 13a, x
DMM50 Chicken (C1) / 50 Bar, 28.4% yield (Table 5) / 6c, 12c, 16b / 5c, 12a, x
MPD60 Chicken (D1) / 200, Bar 62% yield (Table 6) / 6d, 12e, 16d / 5d, 14a, x
LIMA Chicken (E1) / 15% yield (Table 7) / 6e, 12f, 16e / 5e, 15a, x
Minced and chopped chicken (F1) / As in Table 3 followed by extra chopping in a blender / 12b / 11a
Minced pork (A2) / 3mm plate, 98% yield (Table 8) / 7a, 13a, 17a / 6a, 19a, x
Beehive pork (B2) / Setting U, 30.5% yield (Table 9) / 7b, 13d, 17c / 6b, 22a, x
DMM50 pork (C2) / 60 Bar, 55.9% yield (Table 10) / 7c, 13c, 17b / 6c, 21a, x
MPD60 pork (D2) / 200 Bar, 36.9% yield (Table 11) / 7d, 13e, 17d / 6d, 23a, x
LIMA pork (E2) / 12% yield (Table 12) / 7e, 13f, 17e / 6e, 24a, x
Minced and chopped pork (F2) / As in Table 8 followed by extra chopping in a blender / 13b / 20a
Turkey drumstick pieces (G) / Tsd 03100 ex Systemates / 10a, 15a / 9a, 28a
Desinewed Turkey drumstick (H) / Tsd43100 ex Sepamatic std yield / 10b, 15b / 9b, 29a
Deboned Turkey necks (B3) / Ex Beehive std yield / 10c, 15c / 9c, 30a
FMT Turkey necks (B4) / Ex Beehive 40% yield / 10d, 15d, 15e / 9d, 31a, 31b
Unprocessed Turkey necks / No processing at all / 15f / x
Chicken on MPD60 “low pressure” (D3)
Still high pressure MSM
Still high pressure / MPD60 150 Bar dwell 3sec/6sec / 8a, 14a / 7a, 16a
Chicken on MPD60 “medium pressure” (D4)
Still high pressure MSM / MPD60 180 Bar dwell 3sec/6sec / 8b, 14c / 7b, 17a
Chicken on MPD60 “high pressure” (D5)
Still high pressure MSM / MPD60 220 Bar dwell 3sec/6sec / 8c, 14e / 7c, 18a
Pork on MPD60 “low pressure” (D6)
Still high pressure MSM
ow pressure” / MPD60 150 Bar dwell 3sec/6sec / 9a, 14b / 8a, 25a
Pork on MPD60 “medium pressure” (D7)
Still high pressure MSM / MPD60 180 Bar dwell 3sec/6sec / 9b, 14d / 8b, 26a
Pork on MPD60 “high pressure” (D7)
Still high pressure MSM / MPD60 220 Bar dwell 3sec/6sec / 9c, 14f / 8c, 27b
x – slide or sample not included in Standard Operating Procedure document
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Table 2: Additional Reference Samples Examined
Colour coding indicates manufactured at the same site and time
SAMPLE / CONDITIONS / PLATE NUMBERSPork MSM ex Belgium / No details available. Commercial sample / 19f, 20a
Desinewed Ground Pork / Protecon MRS 60 modified to run at a lower pressure of 70-110 Bar followed by a Sepamatic / Baader with a 3mm drum. / 19e, 20b
Desinewed Chicken Necks DMP45 / DMP45 65Bar/ 2mm Baader / 18a, 23e
Desinewed Chicken 3 part wings DMP45 / DMP45 75Bar/3mm Baader / 23b
Desinewed Chicken Keel Bone
Carcase DMP45 / DMP45 30Bar/3mm Baader / 18b, 23d
Desinewed Chicken Brk/Drums DMP45 / DMP45 75 Bar/3mm Baader / 18c, 21b, 22a, 22b
Desinewed Chicken 3 part wings Baader / Baader set at 18 Bar / 18f, 23a
Desinewed Chicken Keel Bone Carcase Baader / Baader set at 18 Bar / 18e, 23c
Desinewed Chicken Brk/Drums Baader / Baader set at 18 Bar / 18d, 21a
Minced Spent hen breast / Hand mincer at LFI – 5mm plate / 19c, 24a
Spent hen breast / Minced through a Sepamatic with 3mm drum / 19a, 24b
Spent hen breast / Baader with 2mm drum / 19b, 24c
Desinewed French Chicken / Baader 3mm drum less than 4 Bar / 19d, 25
Stains and Reagents
Tissue Tek (Agar Scientific66a Cambridge Road,Stansted, Essex, CM24 8DA United Kingdom); hydrochloric acid; Histoclear (Taab Laboratories Equipment Ltd3 Minerva House, Calleva Park, Aldermaston, Berks, RG7 8NA, England); Hystomount (Agar Scientific Ltd); 0.1% toluidine blue in water prepared according to Flint and Firth7(1988); picric Acid; sirius red; formalin; absolute ethanol; acetone.
METHODS
Examination of Samples and Preparation of Sections
A portion of each sample was thawed completely and photographed. For sectioning, small amounts (approximately 1cm3) of each sample were placed on a metal stub using the mounting medium (Tissue-Tek) and the stub frozen in liquid nitrogen (plate 1). The stub and sample were allowed to equilibrate at -20oC in a cryostat (plate 2a) for at least 30 mins before sections were cut (approximately 10µ thick) and collected on glass microscope slides at room temperature. Five samples were taken from different areas of each main reference sample. Each sample was frozen and sectioned, and at least six sections collected from each sample. Samples for sections were taken from three areas of the additional reference samples and cooked samples (plates 18a-25). Two alternative ways to prepare the samples were assessedin order to evaluate how useful they could be for those without a cryostat. In the first method the sample was thawed and the meat smeared directly onto the slide. In the second method a razor blade or scalpel was used to hand-cut frozen sections from samples directly out of the -20oC freezer.