Genetic diversity of Gram-negative, proteolytic, psychrotrophic bacteria isolated from refrigerated raw milk
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.
References (28)
- et al.
Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria
International Journal of Food Microbiology
(2003) - et al.
A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads
Journal of Microbiological Methods
(2002) - et al.
Contamination of milk with Gram-negative spoilage bacteria during filling of retail containers
International Journal of Food Microbiology
(2000) - et al.
Contamination routes of Gram-negative spoilage bacteria in the production of pasteurized milk, evaluated by randomly amplified polymorphic DNA (RAPD)
International Dairy Journal
(2000) - et al.
DNA-based, culture independent strategies for evaluating microbial communities in food-associated ecosystems
International Journal of Food Microbiology
(2001) - et al.
Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing
International Journal of Food Microbiology
(2003) - et al.
Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk
International Journal of Food Microbiology
(2005) - et al.
Bacterial tracking in a dairy production system using phenotypic and ribotyping methods
Journal of Food Protection
(1998) - et al.
The species concept for prokaryotes
FEMS Microbiology Review
(2001) - et al.
A RAPD-based survey of thermophilic bacilli in milk powders from different countries
International Journal of Food Microbiology
(2004)
Psychrotrophs in dairy products: their effects and their control
Critical Reviews in Food Science and Nutrition
Programa Genes — versão Windows
Modelos Biométricos Aplicados ao Melhoramento Genético volume 2, 1th
Análises multidimensionais
Cited by (66)
Pathogenic microorganisms in milk: Their source, hazardous role and identification
2022, Advances in Dairy Microbial ProductsStrategies for raw sheep milk storage in smallholdings: Effect of freezing or long-term refrigerated storage on microbial growth
2019, Journal of Dairy ScienceCitation Excerpt :Finally, no changes in PPC were observed. We expected the highest growth of psychrotolerant bacteria because low temperatures favor growth of this group of microorganisms compared with mesophilic microorganisms (Martins et al., 2006). Despite the low thermoresistance, psychrotrophic microorganisms (included in the psychrotolerant group) can be especially damaging to dairy products because they produce proteolytic and lipolytic enzymes, which are resistant to pasteurization and can cause defects in dairy products throughout storage, such as the development of a bitter taste and rancidity (Pinto et al., 2006).
Safety assessment of Gram-negative bacteria associated with traditional French cheeses
2019, Food MicrobiologyCitation Excerpt :The safety status as well as contribution of most GNB to the cheese process is poorly documented. In previous years, GNB were classically considered as indicators of hygienic problems (Bockelmann et al., 2005; Tornadijo et al., 1993) and responsible for defects in cheese texture and flavor due to the production of extracellular proteolytic and lipolytic enzymes (Amato et al., 2012; Martins et al., 2006). For example, dairy related Pseudomonas spp. strains have been shown to produce volatile compounds such as ethyl esters and alcohols that may negatively affect cheese sensory characteristics (Arslan et al., 2011; Morales et al., 2005).
Hazards of a ‘healthy’ trend? An appraisal of the risks of raw milk consumption and the potential of novel treatment technologies to serve as alternatives to pasteurization
2018, Trends in Food Science and Technology