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The identification of meat species in vegetarian foods by QRT-PCR
Project Code: Q01049
Wiseman, G ; Bowler, P; Ohara, K
The analysis of vegetarian foods to detect contaminating materials of animal origin appears not to have been specifically addressed by analysts. It is accepted that inadvertent contamination is the most likely source of any adulteration, having originated from shared production lines rather than the deliberate act of adding a high cost ingredient i.e. meat, when compared to the cost of vegetables and common food ingredients derived from plants. It is accepted that food contaminated with animal by-products is unacceptable to vegetarians as well as many ethnic groups. It is also recognised that the unacceptable adulterating species varies with ethnic groups.
2 Rationale and Objectives
There are a large number of established methods for the detection of meat species and products in foods. These use a variety of analytical approaches, many of these methods however, are applicable only to raw or undegraded meat; this particular disadvantage has been addressed using DNA methods. In many cases the limitations associated with the method are that of limited specificity, sensitivity or the number of samples that can be analysed in parallel. Most DNA methods use the Polymerase Chain Reaction (PCR) to amplify specific DNA sequences; normally the products of the reaction are assessed following fractionation on the basis of size using an agarose gel. For many applications and in particular where sensitivity or cross-contamination are issues, this technique has been superseded by a closedtube methods that allows the reaction to be monitored without the need for the fractionation step. This technique is called real-time PCR, in which a fluorescent signal generated during the amplification of the specific DNA is used to follow the reaction. Real-time PCR has been used successfully to analyse very short target sequences present in processed foods and hence was chosen to address the detection of meat in vegetarian foods. The same technique has already been applied effectively to the qualitative and quantitative detection of genetically modified organisms (GMOs) in both ingredients and final products and to the detection and quantification of common wheat in high quality durum pastas. Hence, it was hoped that this sensitive technique would be equally appropriate for detecting small fragments of DNA in processed foods.
The present study has used DNA sequences that are common to all animals as a means of detection. During the project a series of model vegetarian foods were prepared containing known levels of adulterating meats. These have been analysed and it has been shown that in general, contaminating meat can be detected at about 0.05%, however, the exact level that can be detected depends upon the actual food itself. The presence of milk and cheese, which are common components of vegetarian food, creates a particular analytical problem due to the fact that they contain bovine/ovine DNA. This has been solved using DNA sequences that are found in skeletal muscle and are absent from dairy products allowing the detection of adulterating meat in the presence of bovine/ovine milk or cheese. Studies aimed at quantifying the adulterating meat were also carried out.
4 Outcome/Key Results Obtained
1. A general qualitative method, which is independent of species has been used to detect contaminating meat in vegetarian products.
2. Contaminating beef when present as a homogenised blend of meats from animals of differing gender can be detected at about 0.05%, however, the exact level that can be detected depends upon the actual food itself.
3. The presence of bovine and ovine milk and cheese within vegetarian products complicates the analysis of adulterating meat. However, this can be resolved using DNA sequences that are not present in dairy produce. It should be noted that the presence of meat from only female animals remains undetectable in these instances. In practice, this situation is very unlikely to occur, as most contamination will involve finely divided meat originating from a number of different animals.
4. The presence of egg in the vegetarian products did not cause analytical problems.
5. Limited trials have shown the method to be applicable to cooked samples.
6. The absolute amount of DNA present in a particular materials varies with individual, tissue, and growth conditions; a situation that makes precise quantification difficult.
7. Studies aimed at quantifying the adulteration of vegetarian foods by skeletal meat have shown that reasonable quantification can be achieved in the absence of dairy produce.
8. Progress has been made in addressing the issue of quantification using externally added DNA. This provides a benchmark that can be used subsequently to assess the levels of contaminating meat.
9. Despite the use of added reference DNA, products containing milk and cheese continue to generate anomalous results.
10. The analysis of commercial products showed evidence of very occasional low-level contamination consistent with ineffective manufacturers cleaning procedures.
5 What it means and why it’s important
The work carried out during the project has provided the FSA with a qualitative method that will identify the presence of skeletal meat in vegetarian products at about the 0.05% level, even in the presence of bovine milk and cheese. The analysis can be performed on processed products obtained from supermarket shelves allowing enforcement agencies to determine if skeletal meat has been deliberately included in a vegetarian food or if adulteration has occurred due to ineffective cleaning regimes in the factory. It will also allow retailers to check their suppliers and manufacturers to ensure they perform their duties of due diligence. It will further allow checks to be devised that will benefit the various ethnic groups in the UK whose religious beliefs forbid consumption of particular species. The project has also demonstrated that despite the use of carefully controlled reference materials the quantification of the adulterating meat content in products containing milk or cheese is very difficult indeed. Hence, further investigations should be carried out if quantification remains a target for the FSA.
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