Genetic diversity of Gram-negative, proteolytic, psychrotrophic bacteria isolated from refrigerated raw milk

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Abstract

The random amplified polymorphic DNA (RAPD) fingerprinting technique was used to assess the genetic diversity of 70 isolates of Gram-negative proteolytic psychrotrophic bacteria that were isolated from refrigerated raw milk. Three oligonucleotides, which generated 87 fragments of polymorphic DNA, were used in the amplification reactions. The genetic distance values calculated using Jaccard's coefficient showed there was high genetic variability among the isolates. Cluster analysis procedures suggested that the genetic variability among isolates belonging to the same species was as high as the variability among different species. Clustering by the UPGMA hierarchical method and data graph dispersion indicated a tendency of the isolates to group according to whether they did or did not ferment glucose.

Introduction

Fresh bovine milk is usually regarded as sterile, but bacteria are introduced into the milk due to udder infections or from environmental sources during milking and processing (Phillips and Griffiths, 1990). Because of the high nutritional value, water content and near neutral pH of milk, many spoilage and pathogenic microorganisms can grow in it (Frank, 1997).

The conditions during storage and transport in refrigerated tanks cause the raw milk microbiota to change from predominantly Gram-positive to predominantly Gram-negative organisms as they grow. Gram-negative bacteria usually account for more than 90% of the microbial population in cold raw milk that has been stored. The Gram-negative flora is composed mainly of psychrotrophic species of Pseudomonas, Achromobacter, Aeromonas, Serratia, Alcaligenes, Chromobacterium, Flavobacterium and Enterobacter (García-Armesto and Sutherland, 1997, Sørhaug and Stepaniak, 1997, Ryser, 1999). Most of these bacteria produce extracellular proteolytic and lipolytic enzymes that are secreted into the milk. Many of these enzymes are not inactivated by pasteurizing at 72 °C for 15 s or by Ultra-High Temperature (UHT) treatment (Griffiths et al., 1981). The residual activities of these enzymes can reduce the organoleptic quality and shelf life of processed milk products (Fairbairn and Law, 1986).

Although many different conventional testing methods have been developed for the detection and enumeration of bacteria in food, these have relied almost exclusively on the use of specific culture media followed by a series of tests for confirmation of isolates. Conventional plate count methods are laborious, time-consuming and sometimes underestimate the numbers of bacteria. To overcome these limitations, molecular biological, biochemical and immunological techniques have been applied for the rapid and specific detection of microorganisms in food (Giraffa and Neviani, 2001).

Ribotyping has been widely adopted as a means of identifying Gram-negative bacteria isolated from milk or dairy products (Ralyea et al., 1998, Wiedmann et al., 2000, Dogan and Boor, 2003). Traditional classifications of bacteria based on phenotypic features do not always correlate well with molecular taxonomy (Woese, 1987). DNA-based techniques have provided new approaches to bacterial identification and taxonomy and are leading the way forward in bacterial genetic diversity studies (Rosselló-Mora and Amann, 2001). Dogan and Boor (2003) observed considerable ecological diversity among Pseudomonas spp. within the dairy-processing environment. They also found a strong association between the Pseudomonas spp. ribotype and spoilage capacity.

Among typing techniques based on the Polymerase Chain Reaction (PCR), the Random Amplified Polymorphic DNA (RAPD) technique is routinely used to assign bacteria to certain groups (Blixt et al., 2003, Martinez et al., 2003, Rückert et al., 2004). It can be used in environmental and food microbiology to detect and identify pathogens, for typing of isolates for epidemiological purposes and for the analysis of the clonality of populations (Giraffa and Neviani, 2001). Moreover, this technique has been used to trace the sources of spoilage bacteria within milk processing plants (Eneroth et al., 2000b). The objective of the present work was to study the genetic diversity of Gram-negative, proteolytic, psychrotrophic bacteria isolated from refrigerated raw milk using the RAPD technique in order to understand the taxonomic complexities of these bacteria.

Section snippets

Bacterial strains

The strains used in this study were obtained from the culture collection of the Laboratory of Food Microbiology, Department of Microbiology, Federal University of Viçosa. They were isolated from cooled raw milk stored at 4 °C, identified and characterized according to their ability to ferment glucose by Pinto (2004). The strains were stored in 20% glycerol at − 80 °C. A total of 70 strains of Gram-negative, proteolytic, psychrotrophic bacteria were analyzed by RAPD-typing.

Extraction of total DNA

For bacterial DNA

Results and discussion

The main psychrotrophic microflora encountered in raw milk are Gram-negative rods (Uraz and Çitak, 1998, Dogan and Boor, 2003) with Pseudomonas spp. comprising at least 50% of the total bacteria in milk (Champagne et al., 1994, Munsch-Alatossava and Alatossava, in press). The three primers used in this work yielded 87 polymorphic DNA fragments with sizes varying from 194 bp to 4361 bp with an average of 29 DNA fragments per primer. Monomorphic fragments were not observed (data not shown). The

Acknowledgments

This investigation was supported by FAPEMIG, Brazil and Maurilio L. Martins was supported by a fellowship from CNPq. We thank Professor Andrea Barros Ribon from Department of Biochemistry, Federal University of Viçosa, Brazil, for her comments and technical assistance.

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