Sophie Marchand

Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples.

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.

The Biodiversity of the Microbiota Producing Heat-Resistant Enzymes Responsible for Spoilage in Processed Bovine Milk and Dairy Products

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.

Cyclic Lipodepsipeptides Produced by Pseudomonas spp. Naturally Present in Raw Milk Induce Inhibitory Effects on Microbiological Inhibitor Assays for Antibiotic Residue Screening

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Understanding and preventing consumer milk microbial spoilage and chemical deterioration.

Abstract: This chapter gives a broad overview of the possible spoilage defects in consumer milk, being either of a microbiological nature (e.g. spore formers and Pseudomonas enzymes) or of a chemical nature (e.g. light-induced oxidation). As these defects can be quite specific for, e.g., pasteurised versus ultra high temperature (UHT) treated milk, a detailed description of the mechanisms of spoilage, the main factors (on farm or industry level) influencing this spoilage, and the methods (including emerging methods on farm or industry level) to prevent spoilage, are discussed for different types of consumer milk. Finally, some future trends and further reading advice are given.

Destabilization and off-flavors generated by Pseudomonas proteases during or after UHT-processing of milk

BackgroundPseudomonads play a major role in the spoilage of UHT processed dairy products, due to their growth-related protease production in raw milk.ResultsTo assess the off-flavor generating capacity of these AprX proteases in milk after UHT-processing, six major milk spoiling Pseudomonas groups were investigated. Sensory evaluation of the different processed milk samples showed large differences in the degree of proteolysis related to onset of off-flavors. Nevertheless, it was illustrated that P. fragi has the greatest spoilage potential within the tested Pseudomonas groups, when it comes to generating off-flavors.ConclusionsNo clear correlation could be obtained between protein hydrolysis and the presence of off-flavors in UHT milk.