Fragi Strains Research Articles

Nanoemulsified clove essential oils-based edible coating controls Pseudomonas spp.-causing spoilage of tilapia (Oreochromis niloticus) fillets: Working mechanism and bacteria metabolic responses

Fish products suffer Pseudomonas-causing spoilage quickly during refrigeration storage, which could be solved by applying edible coating derived from nanoemulsified clove essential oils and fish gelatin (NCEO-FG). This study aimed to evaluate the effects and mechanism of NCEO-FG in preserving tilapia (Oreochromis niloticus) fillets that were inoculated with Pseudomonas spp. (Pseudomonas sp. strain ABa3, P. psychrophila strain ABe3, and P. fragi strain BBa3). NCEO caused remarkable leakage of proteins (198.5–252.8 μg/L) and nucleic acids (0.30–0.34 of OD260). After being incorporated into FG, NCEO-FG effectively delayed the deterioration of tilapia fillets because it significantly reduced the surviving bacteria populations (0.78 – 1.80 log CFU/g reductions) and inhibited the proteolysis and oxidation during cold storage. Further, the metabolic responses of NCEO-FG coated Pseudomonas spp. were revealed using NMR spectroscopy: the reducing levels of metabolites (e.g., pyruvate, amino acids, and betaine) suggested that the NCEO-FG disturbed energy and amino acid metabolisms of bacteria cells. However, the levels of metabolites (e.g., amino acids and osmoprotectants) were upregulated after 3 h and then back to normal concentration after 24 h, which indicated a defense system was built in bacterial cells to tolerate NCEO-FG. In short, this study confirmed that NCEO-FG could control the Pseudomonas-causing spoilage in fish fillets via elucidating the metabolisms.

In situ characterisation of biofilms formed by psychrotrophic meat spoilage pseudomonads

Psychrotrophic Pseudomonas species form biofilms on meat during refrigerated and temperature abuse conditions. Biofilm growth leads to slime formation on meat which is a key organoleptic degradation characteristic. Limited research has been undertaken characterising biofilms grown on meat during chilled aerobic storage. In this work, biofilms formed by two key meat spoilage organisms, Pseudomonas fragi and Pseudomonas lundensis were studied in situ using five strains from each species. Biofilm structures were studied using confocal microscope images, cellular arrangement, cell counts and biomass quantifications. This work demonstrated that highly dense, compact biofilms are a characteristic of P. fragi strains. P. lundensis formed biofilms with loosely arranged cells. The cells in P. fragi biofilm appear to be vertically oriented whereas this characteristic was absent in P. lundensis biofilms formed under identical conditions. Despite the continued access to nutrients, biofilms formed on meat by proteolytic Pseudomonas species dispersed after a population maximum was reached.

Characterization of Extracellular Polymeric Substances Produced by Pseudomonas fragi Under Air and Modified Atmosphere Packaging.

Extracellular polymeric substances (EPS) play an important role in bacterial biochemical properties. The characteristics of EPS from 2 strains of Pseudomonas fragi cultured in meat aerobically (control) and in modified atmosphere packaging (MAP) were studied. The amount and components of EPS, the surface properties, and the effect on biofilm formation of several spoilage organisms were evaluated. The results showed that MAP inhibited the growth of the P. fragi strains. Compared with the control, more loose and less bound EPS (containing protein and carbohydrate) were produced by P. fragi in MAP samples. MAP also caused increased cell autoaggregation and surface hydrophobicity. After the removal of the EPS, the surface property changes were strain-dependent, suggesting that membrane compositions were also changed. In addition, the EPS displayed significant antibiofilm activity on Pseudomonas fluorescens and Serratia liquefaciens. In conclusion, P. fragi strains not only modified the amount, components, and surface properties of EPS but also changed the cell membrane compositions to adapt to MAP stress. Moreover, EPS may play an important role in microbial community competitions.

Effect of high isostatic pressure on the peptidase activity and viability of Pseudomonas fragi isolated from a dairy processing plant

To produce safe and high quality processed milk, high pressure (HP) technology was tested to inactivate undesired microorganisms and their caseinolytic activity. A Pseudomonas fragi strain, isolated from the inner surface of a cheese-making machine from a dairy plant, was shown to harbour the aprX gene and cause casein proteolysis. Single-cycle HP processing of P. fragi-spiked milk at 450 MPa and 25 °C for 20 min decreased bacteria viability to lower levels and reduced peptidase activity by 14%. However when HP processing was performed at 50 °C, a synergistic effect on peptidase was observed, reaching 40% inactivation. Multiple HP treatment cycles at 450 MPa and 25 °C were less effective and reduced peptidase activity by only 23%. HP treatment could aid in the challenge to reduce AprX peptidase activity produced by microbial contaminants, but partial inactivation of peptidase was not effective in preventing UHT milk coagulation during storage at room temperature.

Antimicrobial activity of Myrtus communis L. water-ethanol extract against meat spoilage strains of Brochothrix thermosphacta and Pseudomonas fragi in vitro and in meat

The antimicrobial activity of hydro-alcoholic extract of Myrtus communis L. was investigate in vitro and in situ against different meat spoilage biotypes of Pseudomonas fragi and Brochothrix thermosphacta. Water-ethanol myrtle extract (WEME) was prepared from myrtle leaves and used to study the antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Naturally contaminated ground beef was used to evaluate in situ antibacterial activity of freeze-dried myrtle extract at different concentrations. In vitro antimicrobial activity of WEME was significantly more effective against B. thermosphacta than P. fragi strains. MIC and MLC values were in the range of 12.5–50 and 25–100 mg DM/ml, respectively, for B. thermosphacta and P. fragi strains. In situ results showed that the microbial population, except for Pseudomonas spp., decreased significantly in ground meat with added 5 % of freeze-dried myrtle extract. In conclusion, the findings demonstrate the effectiveness of myrtle extract to control or prevent the proliferation of meat spoilage bacteria.

Molecular analysis of poultry meat spoiling microbiota and heterogeneity of their proteolytic and lipolytic enzyme activities

Psychrotrophic Pseudomonas species P. fluorescens, P. fragi and P. lundensis were found as predominant bacteria of chicken meat stored at chill temperature, which showed high level of molecular diversity, while isolates of the psychrotrophic yeasts Candida zeylanoides, Metschnikowia pulcherrima, Rhodotorula glutinis and Rhodotorula mucilaginosa formed clusters of high level similarity within the different species as revealed by RAPD-PCR analysis.Combination of multiplex PCR and sequencing of the rpoB gene resulted correct identification of the Pseudomas isolates, while the routine diagnostic tests led to improper identification in case of half of the isolates, which indicated the extended biochemical and physiological heterogeneity of the food-borne pseudomonads. Majority of P. fluorescens and P. lundensis isolates were strong protease and lipase producers, while P. fragi strains were week or negative from this respect. Proteolytic and lipolytic activities of the isolated yeast strains were species specific and protease production was less frequent than lipolytic activities.

Pseudomonas fragi strains isolated from meat do not produce N-acyl homoserine lactones as signal molecules.

Quorum sensing (QS) is a signalling mechanism through which bacteria cellular functions are modified to promote access to nutrients and more favorable environmental niches. The frequent occurrence of Pseudomonas spp. in fresh and spoiled meat may involve enhanced gene expression regulated by QS. Several Pseudomonas spp. produce different N-acyl homoserine lactone (AHL) signal molecules. Meat spoilage during aerobic, refrigerated storage is often associated with the presence of Pseudomonas fragi. As with other Pseudomonas species in natural habitats, the dominance and activities of P. fragi in meat may be regulated by QS. In this study, five biosensor strains were used to detect AHL production on three different media by 72 different P. fragi strains isolated from fresh and spoiled meat. Positive and negative AHL-producing strains were used to verify the assays. None of the strains produced detectable quantities of AHLs, even when concentrated cell-free culture supernatants were assayed, nor did exogenous lactones increase biofilm formation in P. fragi strains. However, all isolates produced furanosyl borate diesters (type II autoinducers; AI-2) when tested using the bioluminescent biosensor strain of Vibrio harveyi (BB170). The production of AI-2 was presumed to be of metabolic origin even though Pseudomonas spp. have not been shown to harbor the luxS gene. Thus, the efficient development of P. fragi in fresh meat is not regulated by an AHL-mediated QS system. The mechanism of AI-2 production and its possible role in spoilage dynamics needs further study.

Production of Volatile Compounds in Cheese by Pseudomonas fragi Strains of Dairy Origin

Volatile compounds produced in cheese by five Pseudomonas fragi strains isolated from 1-day-old raw milk cheeses were investigated. Each strain was representative of a different biochemical group of isolates of identical phenotypic characteristics, according to identification with API 20 NE strips. The five strains were ascribed to the species P. fragi after 16S rRNA sequencing because of their high degree of coincidence with P. fragi ATCC 4973. In each of two experiments, carried out on different days, five cheeses were made at laboratory scale from pasteurized milk separately inoculated with approximately 105 CFU/ml of each P. fragi strain. After 12 days at 10°C, mean counts of P. fragi strains were close to 1010 CFU/g in the outer part of cheeses and close to 108 CFU/g in the inner part. A total of 131 volatile compounds, 49 of which were further characterized, were identified in cheeses by gas chromatography–mass spectrometry after extraction with a purge and trap apparatus. Abundances of compounds were generally higher in the outer part of cheeses. Production of volatile compounds was clearly strain dependent. Only two strains produced ethyl esters, and three produced nonethyl esters. Ethyl acetate, ethyl butyrate, ethyl caproate, methyl acetate, isopropyl acetate, and propyl tiglate were the major esters, and ethanol, 2-propanol, and 3-methyl butanol were the major alcohols. Undecene was the major hydrocarbon, dimethyl sulfide and methyl thiocyanate the major sulfur compounds, and 2-pentanone the major ketone. Two aromatic compounds, styrene and o-dichlorobenzene, were present in all cheeses.

The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.

The Proteolytic Activity of Pseudomonas fragi, Pseudomonas taetrolens and Related Species at Low Temperatures

The proteolytic activity of Pseudomonas strains including P. fragi, P. taetrolens, P. aeruginosa, P. chlororaphis, P. fluorescens, P. putida, and P. synxantha was determined at various temperatures from 4°C to 37°C. Out of 43 strains tested, 33 strains grew well at 10°C within 3 days, and all strains except for a P. aeruginosa strain grew at 4°C within 7days. Four P. fragi strains, one P. taetrolens strain, one P. chlororaphis strain, two P. fluorescens strains, and one P. synxantha strain decomposed skimmilk at 10°C within 7 days. Ten P. fragi strains, one P. taetrolens strain, one P. chlororaphis strain, three P. fluorescens strains, and one P. synxantha strain did so at 4°C within 3 weeks. Strains of P. putida did not decompose skimmilk within 3 weeks. The proteolytic activity of P. fragi, P. taetrolens and their related species at low temperatures was discussed from the viewpoint of food hygiene

Enhancement of fruity aroma production of Pseudomonas fragi grown on skim milk, whey and whey permeate supplemented with C3-C7 fatty acids

Pseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C3-C7 fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C3-C7 fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.

Antagonism of Foodborne Bacteria by Pseudomonas spp.: A Possible Role for Iron

Antagonistic action of Pseudomonas spp. against foodborne spoilage and pathogenic bacteria was studied to determine mechanisms involved in the establishment of dominance by these organisms in food systems. Thirteen Pseudomonas strains from plant and food origin were tested for the ability to inhibit other Pseudomonas spp. on brain heart infusion agar using a bacteriocin screening assay. P. aeruginosa AA was the most active, inhibiting P. phaseolicola, P. pisi, P. putida, P. fluorescens, and P. fragi strains. When testing for antagonism against non-pseudomonads, four Pseudomonas spp. showed wide spectrum activity against a variety of gram positive and gram negative bacteria. Growth of most Pseudomonas spp. on an iron-deficient medium increased antagonism. In assays conducted in liquid media, inhibitory activity varied greatly as a result of the composition of the media. A minimal media extract from a P. aeruginosa AA culture significantly inhibited the growth of P. fluorescens and Staphylococcus aureus in milk.

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk.

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 degrees to 30 degrees 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.

Effects of Homologous Bacteriophage on Growth of Pseudomonas fragi WY in Milk1

Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml.

Identification of the Volatile Compounds Produced in Sterile Fish Muscle (Sebastes melanops) by Pseudomonas fragi1

Volatile compounds produced by Pseudomonas fragi strain 18 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. The fruity odor that developed in fish muscle during incipient spoilage was attributed to a synergistic flavor interaction involving the ethyl esters of acetate, butyrate, and hexanoate.