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Proposed study on the determination of the species origin of gelatine in commercially available foods, including a supplementary validation study. - FA0126

Description
Gelatine is manufactured from the collagen in skin and bone material of cow, pig, poultry and fish carcasses and is used in a wide range of foods, cosmetics, and pharmaceutical products. It is used as a thickener, gelling agent and to enhance ‘mouth feel’ in foods and in beverages as a clarifying agent. Furthermore, gelatine has been used as a water binding agent in meat and can thus be used to increase the apparent meat content. There are religious, ethical and labelling issues surrounding the inclusion of a product derived from cow or pig material in confectionery and other foods.

There are significant mis-recordings relating to the species provenance of gelatines on the paperwork used in the food industry. Fera holds a library of gelatines from the EU food industry. Each of these gelatines arrived with paperwork declaring the species origin. However, testing in-house revealed that around 15% of this paperwork was inaccurate. Further, during the course of a Defra-funded project (Q01132), 20 gelatines of known origin were analysed by a specialist German PCR firm and by specialist ELISA kits. 60% of the samples were reported incorrectly. A publication has been prepared for submission to a peer-reviewed journal (Food Chemistry, Grundy et al. 2012) for this work.

Work was undertaken by Fera in collaboration with the University of York with funding from the Food Standards Agency (FSA), and Interact to determine the species origin of gelatine in foods (2007). The method is based on the identification of a suite of species and tissue (skin / bone) determining collagen peptides by high sensitivity, high resolution LC-MS/MS mass spectrometry. Work for the FSA to determine the species origin of gelatine in chicken fillets sourced by Trading Standards gained much media interest when bovine gelatine was detected in chicken.

An inter-lab trial (Defra Project Q01132) has recently been managed by Fera using SOPs developed here. The trial participants (TEAGASC, IRMM, Rikilt, Cafia, LGC and US FDA) showed that the method could be successfully transferred to other laboratories to discriminate between gelatine species origins. This, as far as we are aware, is the first proteomics-based inter-lab trial. There is a strong legislative driver to enforce labelling in this area and Defra have made Fera aware that there is a high level of interest from industry and consumer groups. Regular trial updates have thus been given to the EU Commission at Defra’s suggestion.

The proposed project involves a validation study of the performance of the methods on commercially available products to determine the species origin of gelatine. The products could, for example, include ice creams, sweet flans, thickened sauces and beverages However, the products will be selected in agreement with Defra, using the latest relevant intelligence from the Food Standards Agency. In the past, we have used an LC-MS/MS instrument off-site to screen the gelatine. This would carry considerable cost to this project. An equivalent high resolution, high sensitivity instrument is now available at Fera and some time has been allowed to transfer the method to this instrument. Twenty samples will undergo an initial screening for species origin of gelatine. A validation study will then be carried out in which the method will be validated on five of the previously screened samples. Again, these samples will be agreed with Defra. Since we have been able to transfer our gelatine extraction to a range of food matrices, we are confident that it will be possible to transfer the method to a great variety of foods and other products as required.
Objective
The project aims to (1) transfer the method onto an equivalent high resolution, high sensitivity LC-MS/MS at Fera, (2) perform a small amount of method extension to develop methods to extract gelatine from other food matrices, and (3) perform an initial screen of twenty commercially available samples to determine the species provenance of the gelatine (4) perform a validation study of the method using commercial samples.

Objective 1 Method transfer, duration 5 months
Task 1.1
Materials will be sourced for method transfer.
Task 1.2
The existing method will be transferred on to Fera’s new high sensitivity, high resolution LC-MS/MS in order to make savings on the costs of hiring such an instrument.
Task 1.3
The method will be adapted to cover a larger range of food and beverage matrices, the details of which will be agreed with Defra, taking into account FSA and other intelligence to target products. These matrices, for example, could include commercially available desserts, ice creams, sweet flans, tart toppings, sauces and beverages. Task 1.4
An interim report will be prepared and submitted and a teleconference will be held with Defra to agree, amongst other details, on the types of samples to be included in the next stage.

Milestone 1. Submit interim report.

Objective 1 must be completed prior to commencement of Objective 2 in order to have the necessary LC-MS/MS method in place and to determine/agree the types of food matrix to be screened.

Objective 2 Pre-Screening of commercial samples, duration 4 months

Task 2.1
Twenty commercial samples will be sourced in close consultation with Defra, taking into account FSA and other intelligence to target products. The samples will be purchased in duplicate and logged (LIMS) in detail. The packaging will also be photographed for archiving purposes.
Task 2.2
The samples will be homogenized and gelatine will be extracted from one of each of the duplicate samples. Screening of the samples will be conducted by LC-MS/MS to determine species of origin. Results will be reported to Defra in an email and a teleconference will be organized to agree on the samples to be used for Objective 3.

Milestone 2. Teleconference with Defra to agree samples for use in Objective 3

Objective 3 Intra-laboratory validation of method on commercial samples, duration 3 months

Task 3.1
A method validation study will be carried out. Five of the previously screened samples will be selected, in consultation with Defra and taking into account FSA and other intelligence to target products. An aliquot of each of the five samples will be extracted and analysed on six different occasions to generate data regarding intra-laboratory reproducibility and robustness.
Concerning Quality Assurance, additional food products found to contain no gelatine will also be included as blanks during the analysis detailed above in order to check the method as follows. Following sample preparation, samples and blanks will be transferred to clean tubes and assigned a unique identifier number by an analyst not involved in extraction process so that all samples are analysed ‘blind’ and in random order and, following analysis, it will be checked that the expected results were obtained.
Task 3.2
The final report will be prepared for Defra and a teleconference will be held. The work will be presented at the Authenticity Methods Working Group meeting as appropriate.

Milestone 3 Submit final report.
Project Documents
• EVID4 - Final project report : FA0126 EVID4   (5385k)
Time-Scale and Cost
From: 2013

To: 2014

Cost: £62,392
Contractor / Funded Organisations
F E R A (FERA)
Keywords
Food