Detection of cows' milk in ewes' milk and cheese by an immunodotting method

Abstract

Adulteration of ewes' milk with cows' milk is a wellknown fraud. It can become a serious problem in cheese manufacture as the composition of the coagulated milk influences the organoleptic characteristics of the final product. Analytical methods for detecting cows' milk in ewes' milk or cheese are based on the composition of milk fat or milk protein (Ramos & Juarez, 1984). Gas liquid chromatography has been used to analyse the fatty acid composition of fat from milk mixture and cheese (Benassi, 1963), but this method is useless when adulteration is with skim milk. Moreover, the sensitivity of the method is low, only allowing detection of the addition of > 10% cows' milk (Ramos etal. 1977). Methods of milk protein analysis include mainly immunological and electrophoretic techniques. The latter are based on the different electrophoretic mobility of ewes' and cows' milk caseins (Assenat, 1967). However, adulteration in aged cheese is difficult to detect because of the complexity of the electropherograms, due to proteolysis occurring during the ripening process (Grappin et al. 1985). Most of the immunological methods currently used in these analyses are based on precipitation reactions between whey proteins, mainly immunoglobulins, and specific antibodies (Bernhauer et al. 1983; Elbertzhageng & Wenzel, 1982). While these methods are suitable for distinguishing milk from different animal species, the immunochemical reactivity of whey proteins is affected by heat treatment (Ramos & Juarez, 1984), while the antigenicity of the heat stable caseins is poor. Antisera against caseins have low titres, so precipitation techniques are often unsuitable for detecting cows' milk in ewes' milk or cheese. This paper describes a method based on immunodotting techniques, which does not require high titre antisera, and its application to the detection of cows' milk in ewes' milk cheese. MATERIALS AND METHODS Preparation of whole casein Bovine or ovine milk was defatted by centrifugation at 2000 g for 15 min at 4 °C. Skim milk was acidified to pH 4-6 with 1 M-HC1. Whole casein was recovered by centrifugation for 10 min at 2000 g, washed and neutralized with 0-5 M-NaOH to pH 70. The above operations were repeated twice to eliminate any remaining whey proteins.

Current research on goat's milk in Spain

“…Calvo's group has developed 2 enzyme immunoassays based on immunodot techniques using antibodies against caseins (Aranda et al, 1988) or immunoglobulins (Calvo et al, 1989) to detect cow's milk in ewe's milk. Recently the same authors (Aranda et al, 1993) have designed an lmmunodot assay to detect the presence of goat's and/or cow's milk in ewe's milk and ewe's milk cheeses.…”

Authentication of dairy products by immunochemical methods: a review

“…Two similar antisera, one raised against whole bovine casein and the other against whole caprine casein, after immunoaffinity purification, were successfully employed in ELISA determi-nation of bovine and caprine casein in ovine milk and cheeses (Rodríguez et al 1993(Rodríguez et al , 1994. As expected, the results were not affected by the heat treatments applied to milk (Aranda et al 1988), but immunochemical analysis of the cheese samples based on such antibody preparations was not fully reliable. Indeed, the molecular species actually targeted by these antibodies have not been identified and consequently no information regarding the preservation of the antibody-recognized epitopes, depending on protein hydrolysis during cheese ripening, was available.…”

“…Nevertheless, immunoadsorption of an antiserum developed against whole bovine casein on insolubilized whole ovine casein allowed polyclonal antibodies which specifically recognized bovine casein in mixtures of bovine and ovine milk by an immunodotting method to be obtained (Aranda et al 1988). Two similar antisera, one raised against whole bovine casein and the other against whole caprine casein, after immunoaffinity purification, were successfully employed in ELISA determi-nation of bovine and caprine casein in ovine milk and cheeses (Rodríguez et al 1993(Rodríguez et al , 1994.…”

“…(Unpublished results) preparations, the anti-β-casein (106-110) antiserum, cross-reacted with the native and plasmin-generated γ 2 -casein from bovine and water buffalo casein but did not recognize the majority of peptides resulting from the water buffalo β-casein plasminolysis and, in particular, the bovine γ 2 -casein-like band in the electrophoretic profile of pure water buffalo casein. Based on the use of the anti-β-casein (106-110) antiserum, immunoblotting analysis of experimental Mozzarella cheese samples from Aranda et al (1988) Polyclonal antibodies Caprine IgG 0.5% of caprine or bovine milk in ovine milk Aranda et al (1993) Polyclonal antibodies Bovine whey proteins 1% of bovine milk in caprine milk Castro et al (1992) Polyclonal antibodies Caprine whey proteins 0.5% of caprine milk in ovine milk García et al (1993) Polyclonal antibodies Bovine whey proteins 3% of bovine milk in ovine milk García et al (1989) Polyclonal antibodies Bovine whey proteins 1% of bovine milk in ovine milk García et al (1990) Polyclonal antibodies Bovine γ-casein 0.1% of bovine milk in ovine and caprine milk and cheese Richter et al (1997) Polyclonal antibodies Whole bovine casein 1% of bovine milk in ovine milk and cheese Rodríguez et al (1993) Polyclonal antibodies Whole caprine casein 1% of caprine milk in ovine milk and cheese Rodríguez et al (1994) Monoclonal antibody Bovine β-casein 0.5% of bovine milk in ovine and caprine milk and cheese Anguita et al (1997a) Monoclonal antibody Caprine α s2 -casein 0.25% of caprine milk in ovine milk Haza et al (1997) Monoclonal antibody Caprine α s2 -casein 0.5% of caprine milk in ovine cheese Haza et al (1999) Monoclonal antibody Bovine IgG 0.1% of bovine milk in ovine, caprine, and buffalo milk …”

Kishk - a dried fermented milk / cereal mixture. 2 Assessment of a variety of protein analytical techniques for determining adulteration and proteolysis

“…Adulteration can be detected at low levels, and an example of such a technique is given in the European Union (EU) reference methods [20]. The immunoassay methods rely on using specifie mono-or polyclonal specifie antibodies reactive to: (a) cow's~-Lg [16,21,32,41]; (b) cow's IgG [10,Il,37]; (c) cow's x-casein [12]; (d) asl-casein [43]; (e) goat's whey protein [22,23]; (f) Y2-and Y3-caseins [2]; (g) goat casein [24,39,40,42]; and (h) cow's -casein [6,7]. The sensitivity of such methods is also :::: 0.5 %.…”