Selection of pH buffers for use in conductimetric microbiological assays

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5° to 30°C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

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.     

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.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1-4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.     

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Volatile compounds produced by meat pseudomonads and relate reference strains during growth on beef stored in air at chill temperatures

Volatile compounds produced by 31 strains of pseudomonads and by reference strains of Pseudomonas fragi and Ps. fluorescens biotype 1 during growth on beef stored at 6 degrees C in air were analysed by gas chromatography-mass spectrometry of headspace gases. Compounds of major sensory significance were ethyl and methyl esters of C2-C8 fatty acids and sulphur-containing compounds which included methane- and isopropanethiols and their related sulphides and thioesters but not hydrogen sulphide. Ester production was mainly associated with growth of some, but not all, Ps. fragi and related meat strains but sulphur-containing compounds were produced by all but a single meat strain. A minority of other meat strains produced greater amounts of methyl ketones, secondary alcohols and unsaturated hydrocarbons believed to be of lipid origin.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida, 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0 degrees-10 degrees C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

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°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.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida , 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0°–10°C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Volatile compounds produced by meat pseudomonads and related reference strains during growth on beef stored in air at chill temperatures

Volatile compounds produced by 31 strains of pseudomonads and by reference strains of Pseudomonas fragi and Ps. fluorescens biotype 1 during growth on beef stored at 6°C in air were analysed by gas chromatography-mass spectrometry of headspace gases. Compounds of major sensory significance were ethyl and methyl esters of C₂–C₈ fatty acids and sulphur-containing compounds which included methane- and isopropanethiols and their related sulphides and thioesters but not hydrogen sulphide. Ester production was mainly associated with growth of some, but not all, Ps. fragi and related meat strains but sulphur-containing compounds were produced by all but a single meat strain. A minority of other meat strains produced greater amounts of methyl ketones, secondary alcohols and unsaturated hydrocarbons believed to be of lipid origin.     

A numerical taxonomic study of the Pseudomonas flora isolated from poultry meat

Pseudomonas strains were isolated from both fresh and cold-stored broiler skin. Phenotypically-based numerical taxonomic techniques were used to characterize the isolates and 36 reference strains. For this purpose, Biolog GN Microplates, API 20NE and a number of other biochemical tests were used. Jaccard clustering revealed the predominance of four major Pseudomonas groups: Ps. fragi, Ps. lundensis, strains belonging to Ps. fluorescens biovars and an unidentified group of strains displaying a high degree of similarity to Ps. fluorescens biovars. Within Ps. fluorescens, biovar A was best represented. The marked proteolytic character of members of Ps. fluorescens biovars A, B and C, as well as of members of the unidentified cluster, supports their possible role in the origin of organoleptic defects. In the Ps. lundensis cluster, a distinct group of Ps. lundensis-like species was found. Further genotypic studies should be carried out to clarify the taxonomic status of the Ps. lundensis-like strains and that of the unidentified group resembling Ps. fluorescens biovars A and B.     

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb

E.H. DROSINOS AND R.G. BOARD. 1994. Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere ( Ps. fragi only) enriched with carbon dioxide at 4C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.     

Cross-reactivity of antibodies raised to Pseudomonas fluorescens protease with extracellular proteins produced by meat-spoiling pseudomonads

The cross-reactivity patterns of antibodies to Pseudomonas fluorescens protease with the extracellular proteins produced by a number of meat-spoiling pseudomonads were studied. Immunoblotting studies showed that purified IgG to Ps. fluorescens protease cross-reacted with extracellular proteins in the cell culture supernatant fluids of Pseudomonas spp., including Ps. fragi and Ps. lundensis. In the case of Ps. lundensis and Pseudomonas spp. 11390, the cross-reactive moieties were of similar molecular weight to the Ps. fluorescens protease (46 kDa). However, in Ps. fragi the cross-reactive moiety was a lower molecular weight protein (8 kDa). This may represent a fragment of the active enzyme. These results indicate the presence of common antigenic determinants among the proteases of meat spoiling pseudomonads.     

Antibacterial activity among Pseudomonas strains of meat origin

Almost 750 Pseudomonas strains from meat, fish, milk or plant were screened for inhibitory potential towards 10 selected Pseudomonas fragi isolated from meat. Only strains of fish origin and a reference Ps. putida strain produced an inhibitory substance which was either a siderophore or a bacteriocin-like substance. The two systems were efficient in meat extract medium suggesting potential use for controlling meat quality.     

A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk

Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats' milk, which had been stored at 5 degrees C for 7 d. The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests. The results yielded 195 characters suitable for taxonomic analysis. Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S. The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples. Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1–4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.     

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.     

Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86%. SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g. Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.