HPLC quantification of biogenic amines in cheeses : correlation with PCR-detection of tyramine-producing microorganisms Marı ´a Ferna ´ndez, Daniel M Linares, Beatriz del Rı ´o, Victor Ladero and Miguel A Alvarez* Instituto de Productos La ´cteos de Asturias (IPLA-CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Spain Received 24 July 2006 and accepted for publication 20 November 2006 The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese. Keywords : Biogenic amines, tyramine, cheese, HPLC, PCR. Biogenic amines (BAs) are low molecular weight organic bases sometimes present in foods. They are mainly pro- duced by the microbial decarboxylation of certain amino acids. BA formation in foods should be controlled since these compounds are associated with respiratory distress, headache, hyper- and hypotension, and allergies. These problems are particularly severe in people with low levels of monoamine and diamino-oxidase (enzymes belonging to the BA detoxification system) ; such deficiencies are as- sociated with genetic background and certain medical treatments (Joosten & Northolt, 1987, Hala ´sz et al. 1994). Although there are no regulations governing the BA con- tent in most foodstuffs, the European legislation (Directive 91/493/EEC) set a limit for the histamine levels in fishery products of 100–200 mg Kg –1 for fresh fish and up to 400 mg Kg –1 for cured products. The US Food and Drug Administration set a limit for histamine in canned tuna of 500 ppm (http.//www.fda.gov). Some authors have suggested a general limit for histamine levels in food of 100 mg Kg –1 (Ten Brick et al. 1990). Histamine, tyramine, putrescine and cadaverine are the most common BAs in fermented foods. They are mainly produced by lactic acid bacteria (LAB ; Lonvaud-Funel, 2001) ; their appearance and accumulation are therefore influenced by the environmental factors (temperature, pH, availability of substrate, etc.) that affect the growth and/or decarboxylase activity of these organisms (Valsamaki et al. 2000). Methods that can rapidly detect BA-producing strains in foodstuffs are required if food quality and safety is to be assured. Such a capability would also help the dairy industry inspect raw materials destined for use in food production. Cheese is one of the foods most commonly associated with BA poisoning; indeed, the term ‘cheese reaction’ has been coined to refer to it (Ten Brick et al. 1990). The microbiota present in cheese has different origins (milk, starters, and contaminating microorganisms) and its de- velopment is affected by factors such as the treatment the milk receives and the ripening conditions. Several authors have shown the BA content of cheese made from raw milk to be higher than that of cheese made from pasteurized milk (Joosten & Northolt, 1987; Ordon ˜ez et al. 1997; Novella-Rodrı ´guez et al. 2004). Since BAs accumulate in food products, the duration of ripening is a critical factor affecting the final BA content (Ordon ˜ez et al. 1997; Novella-Rodrı ´guez et al. 2003a). In addition, the high level of proteolysis that occurs in cheeses (which provides the amino acid substrates for the production of BAs), com- bined with the acidic environment of this food, favors the synthesis and activity of decarboxylation enzymes (Joosten & Northolt, 1987). *For correspondence ; e-mail : maag@ipla.csic.es Journal of Dairy Research (2007) 74 276–282. f Proprietors of Journal of Dairy Research 2007 276 doi:10.1017/S0022029907002488 First published online 30 April 2007 Printed in the United Kingdom