Multiplex qPCR for the detection and quantification of putrescine-producing lactic acid bacteria in dairy products

Putrescine is one of the most abundant biogenic amines (BA) in dairy products, in which it is mainly produced through the deamination of agmatine. Agmatine deaminase activity has been detected in a variety of lactic acid bacteria (LAB) mostly belonging to the genera Enterococcus and Lactobacillus, but also in Lactococcus lactis. Current PCR methods for detecting putrescine producers are based on the amplification of part of the agmatine deaminase gene (aguA). However, the existence of L. lactis strains in which this gene is inactivated by an insertion sequence (IS) invalidates such methods for use with dairy products. This paper describes a culture-independent multiplex qPCR method based on the specific amplification of the region of the agmatine deaminase gene cluster (AGDIc) – in which an interfering IS may be inserted – for detecting, quantifying and identifying LAB truly capable of producing putrescine from agmatine. The proposed method was found to be specific and to have a wide dynamic range. This allowed for the quantification of putrescine-producing bacterial groups in a survey of commercial cheeses. The different cheeses had different numbers and types of putrescine-producing LAB. The relationships between putrescine concentration, numbers of putrescine-producing LAB and cheesemaking technological factors are discussed.