Atividade proteolítica de bactérias psicrotróficas isoladas de leite cru e implicações nos testes de adição fraudulenta de soro de queijo.

Abstract

Fraud in milk by whey addition is a recurring issue in Brazil, while the contamination and spoilage of milk by psychrotrophic bacteria is one of the main problems faced by the dairy industry. Among these bacteria, the genus Pseudomonas has been highlighted in numerous microbiological studies as the predominant group in the microbiota of cold stored milk. Recently, this predominance has been confirmed via the application of molecular techniques for microbial identification. Yet, molecular analyses have served to raise questions regarding the accuracy of some earlier phenotypic identifications, and concerning the role of previously undetected and/or underestimated genera/species which may be found as major components of the microbiota of cold stored milk. Problems related to the quality of milk and dairy products due to the growth and enzymatic activities of psychrotrophs are compounded by the fact that the production of proteases by the bacteria may cause false-positive results in tests employed for detection of milk fraud by whey addition. This is a common practice in some countries, including Brazil, and usually detected by analyzing the CMP index. In the current study, a total of 12 strains of psychrotrophic bacteria isolated from milk and phenotypically identified in a previous study (ARCURI et al., 2008) were reidentified using genotypic methods based upon analysis of the sequence (16S rDNA) encoding the small subunit of ribossomal RNA. This approach provided identification to the species level for four strains received as Acinetobacter spp. as A. guillouiae (3) and A. johnsonii (1), and identified as Pseudomonas rhodesiae an isolate received as P. fluorescens. In addition, three isolates received as P. putida were reclassified as P. psychrophila and an isolate phenotypically identified as Aeromonas hydrophila was identified as A. punctata. The growth of these bacteria at refrigeration temperatures was examined by inoculation of milk (ca 103 CFU mL-1) with incubation at different times/temperatures (refrigeration), in accordance with a factorial design (22) with three repetitions at the central point. After each incubation period, the production of CMP was assessed by high performance liquid chromatography (HPLC-GF) and the results of a subgroup of samples were compared with those obtained by immunoassay and liquid chromatography coupled with electrospray ionization mass spectrometry in tandem mode (HPLC-ESI-MS/MS), which sensitivity makes possible the identification of false-positive results. The production of CMP, as assessed by HPLC-GF was maximal at 10°C/4d for all strains, with the exception of the acinetobacters. Two strains of P. rhodesiae showed the highest levels, followed by a strain of A. hydrophila (P07). Linear models were constructed for bacterial growth; however, for CMP production 66.6% of the samples showed a quadratic trend. The HPLC-ESI-MS/MS analysis showed that the percentage of pseudo CMP in some samples (condition 10°C/4d) was in excess of 50% compared to true CMP for all samples, except for the milk inoculated with an isolate identified as A. punctata (P65). Despite the fact that the temperature (10°C) is not permitted under Brazilian legislation for x milk storage, the data demonstrated the potential of these strains to generate false-positive results (via HPLC-GF) for the fraudulent addition of whey.