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Felix Rwagasore, Frédéric Dehareng, Vincent Baeten, Georges Sinnaeve
Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium

 The aim of this work was to study the possibility of the gas chromatography (GC) to discriminate animal species from fat extracted from feeds. Indeed, according to the European regulation 1774/2002, the incorporation of some animal by-products in feed is allowed and others are not.  If banishment is based on animal species, it is capital to detect animal by-products according to their origin. We analysed fats because their addition in feed process is almost unavoidable. Beside the supplying energy, they improve the absorption of fat-soluble vitamins, diminish the pulverulence, and increase the efficiency of the consumed energy. Lubricant properties of fats are also necessary in concentrate processing technologies.

The fat is extracted according to a modified Folch procedure: The sample (4 g) is homogenized in 40 ml of Folch solution (Chloroform/Methanol 2:1) for 1 minute with an Ultraturax (IKA), then the flask is manually agitated and allowed to settle for 1 minute and the homogenisation is continued for 2 minutes more. The mixture is filtered and the filtrate is transferred to a 250ml separator funnel, one fourth of the total volume of 0.73 % sodium chloride in water is added, and the mixture is shaken thoroughly before being allowed to settle. The bottom layer is collected in a flask with funnel and a separator phase filter.
The aqueous (upper) layer is washed 2 times with a “wash solution” (Folch solution + 4th 0,73% NaCl) and each time, the bottom layer containing the purified lipid is filtered in the same flask. The bottom layer solvent is removed on a rotary film evaporator. The lipid extract is stored at -20°C prior analysis.

The fats are transesterified with a KOH/Methanol (2 M) solution (using C9 in hexane as Internal Standard)
Fatty Acids profiles have been acquired on a gas chromatograph (Agilent 5790N) equipped with a 100m CP-Sil 88 column (Varian) with “on column” injection mode and an FID detector.
This study was performed on a group of 20 pure animal meals (7 fish, 3 pork, and 7 bovine and 3 avian).
The Fatty Acid profiles were subjected to chemometrics techniques and showed a good separation of feed fats according to 12 main fatty acids (C14, C16, C16:1 c, C18, C18:1 c, C18:1 t, C18:2 c9 c12, C20:1, C22, C20:3(n-6), C20:4(n-6) and C20:5(n-3)).

Gas chromatography combined with chemometric techniques (PCA in our case) has shown its capability to discriminate feed fats from pure animal meals according to their species.
In this study we used pure feed fats, the next step will be the use of mixture of fats and from more sources like vegetable oils.

Keywords:
Meal; fish; beef; pork; avian; feed; fat; fatty acid profile; gas chromatography