Detection of heat stable proteins from meat of ruminant

Details Detection of heat stable proteins from meat of ruminant Detection of heat stable proteins from meat of ruminant

1996-02-15

1999-06-08

Caputa, Anthony C.

Chemistry: analytical and immunological testing

Food or dairy products

Meat or eggs

436 8, 436 17, 436 18, 436 20, 436543, 436825, 436177, 436 71, 436 792, 436 793, 436 794, 436 795, G01N 3312, G01N 3368, G01N 3353

Patent

active

059104465

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a method for the detection of proteins in rendered animal material, and particularly a method for detecting heat stable ruminant proteins in such material.
A ban on rendered ruminant protein in ruminant feedstuffs was instituted by the United Kingdom Ministry of Agriculture, Fisheries and Food in July 1988 in response to outbreak of Bovine Spongiform Encephalopathy--the Bovine Spongiform Encephalopathy Order 1988 (Statutory Instrument 1988/1039). This, and the serious nature of this disease, provides a need to detect these proteins in feedstuffs.
Animal material that passes through a rendering process is subjected to temperatures ranging from 102.degree. C. to 145.degree. C. with a typical exposure time of up to two hours. Current immunological tests aimed at determining the species of origin of resulting meat and bonemeal have limited capability, with little or no sensitivity at below 1% contamination when speciating material cooked above 130.degree. C. for more than one and a half hours; these temperatures being confirmed by differential scanning calorimetry.
Meat speciation is commonly carried out using ELISA systems based on antibodies against muscle heat stable proteins. Berger et al, (1988) J. Association of Official Analytical Chemists, 7, (2), 406-409, have used antisera raised to raw skeletal muscle to detect pork and chicken heated to 120.degree. C. for 15 minutes, while Kang'ethe and Gathuma (1987) Meat Science 19, 265-270, used antisera against cooked muscle proteins, mainly from ruminants, with similar effect. Heat stable proteins have also been demonstrated as present in adrenal glands, brain, testes, heart and kidney.
Material for rendering is composed mainly of bone, fat and viscera, with skeletal muscle being a lesser component. Collagen, which is present in all tissues, converts to a soluble form when heated to become the main constituent of gelatin. Water soluble extracts of meat and bonemeal frequently solidify due to the high gelatin content and this gelatin is capable of depressing the immunoassay sensitivity (e.g. see results in Kang'ethe and Linqvist (1987) in Journal of Science of Food & Agriculture, 39, 179-184.
Thus the immunoassay of heat stable protein in rendered materials such as meat and bonemeal is problematical. Commercial kits currently available can detect bovine and ovine species in these materials at a dilution no greater than one part in 100, and this level of sensitivity decreases in materials that have been treated under high temperature rendering conditions, ie. at about 135.degree. C.-155.degree. C. for up to one and a half hours.
The heat stable sample preparation regimes of Berger et al and Kang'ethe and others can be summarised as follows:
(i) The method of Berger et al (1988) mixes cooked or canned meat with distilled water over a short periods e.g. 1 minutes leaves the mixture to stand for about one hour at room temperature then centrifuges it, the supernatant being used for the assay step.
(ii) The method of Kang'ethe et al (1986) Journal of Science of Food & Agriculture, 37, 157-164 homogenises the meat products in saline in a ratio of about 1:1 (w/v) and then sonicates the mixture at 300 W for 10 minutes, the sonicate is centrifuged at about 2000 g for 15 minutes and resultant supernatant dialysed against saline for 3 days before use in the assay step.
(iii) The method of Kang'ethe and Lindqvist (1987) is initially similar to that of Kang'ethe et al (1986) but uses fresh meat, and after the centrifugation step the supernatant is filtered, centrifuged at 86000.times.g for 30 minutes, and the resulting supernatant autoclaved at 121.degree. C. for 30 minutes. The autoclaved material is again centrifuged at 2000 g for 15 minutes and the antigens precipitated from the supernatant by addition of 3 vols absolute ethanol; the mixture being left overnight and the precipitate being recovered by centrifugation. The precipitate is then dissolved in saline, concentrated by ultrafiltration (30 kDa cut-off) and fractionated by gel filtrat

REFERENCES:
England et al (1990) Methods in Enzymology. vol. 158, 157-166.

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