Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. METHODS AND RESULTS: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA. They were heat stable and retained activity even after treatment at 121 degrees C for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.     

Influence of storage conditions on the growth of Pseudomonas species in refrigerated raw milk

The refrigerated storage of raw milk throughout the dairy chain prior to heat treatment creates selective conditions for growth of psychrotolerant bacteria. These bacteria, mainly belonging to the genus Pseudomonas, are capable of producing thermoresistant extracellular proteases and lipases, which can cause spoilage and structural defects in pasteurized and ultra-high-temperature-treated milk (products). To map the influence of refrigerated storage on the growth of these pseudomonads, milk samples were taken after the first milking turn and incubated laboratory scale at temperatures simulating optimal and suboptimal preprocessing storage conditions. The outgrowth of Pseudomonas members was monitored over time by means of cultivation-independent denaturing gradient gel electrophoresis (DGGE). Isolates were identified by a polyphasic approach. These incubations revealed that outgrowth of Pseudomonas members occurred from the beginning of the dairy chain (farm tank) under both optimal and suboptimal storage conditions. An even greater risk for outgrowth, as indicated by a vast increase of about 2 log CFU per ml raw milk, existed downstream in the chain, especially when raw milk was stored under suboptimal conditions. This difference in Pseudomonas outgrowth between optimal and suboptimal storage was already statistically significant within the farm tank. The predominant taxa were identified as Pseudomonas gessardii, Pseudomonas gessardii-like, Pseudomonas fluorescens-like, Pseudomonas lundensis, Pseudomonas fragi, and Pseudomonas fragi-like. Those taxa show an important spoilage potential as determined on elective media for proteolysis and lipolysis.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Pseudomonas fluorescens Proteases in Ultrahigh-Temperature-Treated Milk

An inhibition enzyme-linked immunosorbent assay was developed to detect low levels of the proteases extracted from four strains of Pseudomonas fluorescens. The assay detected between 0.24 and 7.8 ng of protease per ml of ultrahigh-temperature-treated milk and could be completed within 6 h. It could be used as a framework for a test system for quantifying spoilage proteases in dairy products.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among Pseudomonas spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as P. fluorescens and P. putida by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk

Experiments were designed to elucidate a possible physiological mechanism for stimulated growth of Listeria monocytogenes by Pseudomonas fluorescens in milk. The ability of L. monocytogenes to grow in milk under aerobic and microaerophilic environments was compared. Neither environment favored the growth of the organism at 10 degrees C. Autoclaved whole milk was inoculated with P. fluorescens P26 and preincubated for 3 days at 10 degrees C followed by inoculation with L. monocytogenes Scott A and further incubation for 8 days at 10 degrees C. Changes in selected milk components were monitored over the 8-day period. The amount of lactose in the milk was determined, as well as the extent of proteolysis and lipolysis. Both L. monocytogenes and P. fluorescens were able to hydrolyze milk fat but were unable to use lactose. Milk protein was hydrolyzed by P. fluorescens but not by L. monocytogenes. Whole milk partially proteolyzed by treatment with purified protease was inoculated with L. monocytogenes. Results indicated that the growth of L. monocytogenes was stimulated in proteolyzed milk. This is the first report to provide evidence describing a likely mechanism for commensalism between L. monocytogenes and Pseudomonas spp.     

Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52.

In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.     

Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52.

In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.     

Exclusion of phospholipases (PLs)-producing bacteria in raw milk flushed with nitrogen gas (N(2))

Prolonged cold storage of raw milks favors the growth of psychrotrophs, which produce heat-resistant exoenzymes of considerable spoilage potential; the bacterial proteases and lipases affect raw milk quality; among them phospholipases (PLs) may target the milk fat globule. More importantly, bacterial PLs are key virulence factors for numerous species. Two studies examined the use of nitrogen (N(2)) gas and examined its effect on psychrotrophs, proteases and lipase producers when the milk was stored in closed vessels; however, the effect on PLs producers is unknown. Here we show that by considering an open system the PLs producers were sooner or later excluded in raw milk (whereas the PLs producers in the non-treated controls culminated at 10(8)CFU/ml), by effective gas treatments that bring oxygen (O(2)) levels in milk lower than 0.1ppm. No increase of the PLs producers among the anaerobes was noticed during the course of the experiments. In the experiments performed at 6.0 degrees C, the delay after which the PLs producers were no longer detectable seemed independent of the initial level of PLs producers in raw milk (lower than 10(3)CFU/ml). We anticipate that flushing pure N(2) gas in raw milk tanks, considered as open systems, along the cold chain of raw milk storage and transportation, may be an additional technique to control psychrotrophs, and may also constitute an interesting perspective for limiting their spoilage and pathogenic potential in food materials in general.     

Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits

During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora, and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of dairy products. The diversity, dynamics, and enzymatic traits of culturable psychrotrophs in raw milk from four farms were investigated over a 10-month period. About 20% of the isolates were found to be novel species, indicating that there is still much to be learned about culturable psychrotrophs in raw milk. The psychrotrophic isolates were identified and classified in seven classes. Three classes were predominant, with high species richness (18 to 21 species per class) in different seasons of the year: Gammaproteobacteria in spring and winter, Bacilli in summer, and Actinobacteria in autumn. The four minor classes were Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria. The dominant classes were found in all four dairies, although every dairy had its own unique "bacterial profile." Most but not all bacterial isolates had either lipolytic or both lipolytic and proteolytic activities. Only a few isolates showed proteolytic activity alone. The dominant genera, Pseudomonas and Acinetobacter (Gammaproteobacteria), showed mainly lipolytic activity, Microbacterium (Actinobacteria) was highly lipolytic and proteolytic, and the lactic acid bacteria (Lactococcus and Leuconostoc) displayed very minor enzymatic ability. Hence, the composition of psychrotrophic bacterial flora in raw milk has an important role in the determination of milk quality. Monitoring the dominant psychrotrophic species responsible for the production of heat-stable proteolytic and lipolytic enzymes offers a sensitive and efficient tool for maintaining better milk quality in the milk industry.     

Characterization of contaminants from a sanitized milk processing plant

Milk processing lines offer a wide variety of microenvironments where a diversity of microorganisms can proliferate. We sampled crevices and junctions where, due to deficient reach by typical sanitizing procedures, bacteria can survive and establish biofilms. The sampling sites were the holding cell, cold storage tank, pasteurizer and storage tank--transfer pump junction. The culturable bacteria that were isolated after the sanitation procedure were predominantly Pseudomonas spp., Serratia spp, Staphylococcus sciuri and Stenotrophomonas maltophilia. We assayed several phenotypic characteristics such as the ability to secrete enzymes and siderophores, as well as the capacity of the strains to form biofilms that might contribute to their survival in a mixed species environment. The Pseudomonas spp. isolates were found to either produce proteases or lecithinases at high levels. Interestingly, protease production showed an inverse correlation with siderophore production. Furthermore, all of the Serratia spp. isolates were strong biofilm formers and spoilage enzymes producers. The organisms identified were not mere contaminants, but also producers of proteins with the potential to lower the quality and shelf-life of milk. In addition, we found that a considerable number of the Serratia and Pseudomonas spp. isolated from the pasteurizer were capable of secreting compounds with antimicrobial properties.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties.

A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.     

Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties

A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.     

Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods

The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Growth of psychrotrophic bacteria in raw and UHT-treated goats' milk

The growth of six strains of Pseudomonas fluorescens, two of Ps. fragi, and one of Serratia liquefaciens was followed in raw and UHT-treated goats' milk, held at 4 degrees C. Generation times for Ps. fluorescens in UHT milk ranged from 5.19 to 5.81 h, increasing markedly in raw milk (8.34-21.49 h). Growth of Ps. fragi did not differ significantly between raw (4.56, 4.65 h) and UHT (5.04, 7.24 h) milk. Generation times for S. liquefaciens were 6.63 and 14.07 h, for UHT and raw milk respectively.