Characterization of the biofilm matrix composition of psychrotrophic, meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.

Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.

The lactoperoxidase system (LS), an antimicrobial system naturally present in milk that is activated by H2O2, has been used to inhibit microbial outgrowth in raw milk in areas where refrigeration is not viable. This study evaluated lactose oxidase (LO) as a novel activator of the LS. Lactose oxidase oxidizes lactose and produces H2O2 needed for the activation of the LS. The antimicrobial effect of different concentrations of LO with and without components of the LS, thiocyanate (TCN) and lactoperoxidase (LP), was evaluated in model systems and then applied in pasteurized milk and raw milk. In general, an increase in LO caused greater reductions of Pseudomonas fragi in the model systems and treatments were more effective at 6°C than at 21°C. At 6°C, the LO solution at 0.12 and 1.2 g/L showed significantly higher microbial reduction than the control when both added alone and combined with LS components. At 21°C, treatments with 1.2 g/L of LO solution achieved a reduction of >2.93 log cfu/mL in 24 h, but at lower levels there was not a significant reduction from the control. Higher concentrations of TCN led to a greater P. fragi reduction at both temperatures when LO was added alone but not when combined with LP. In pasteurized milk, the LO solution at 0.12 g/L caused a reduction of approximately 1.4 log of P. fragi within 24 h when added alone and a reduction of approximately 2.7 log when combined with LP and TCN. Bacterial counts remained at significantly lower levels than the control during storage, and the TCN-supplemented milk exhibited an approximately 6-log difference from the control by d 7. In raw milk, the total bacterial growth curve showed a longer lag phase when the LS was activated by LO (11.3 ± 1.4 h) compared with the control (4.0 ± 1.0 h), but it was not different from the recommended method (9.4 ± 1.0 h). However, the total bacterial count after 24 h for the sample treated with LO and TCN (5.3 log cfu/mL) was significantly lower compared with the control (7.2 log cfu/mL) and the recommended method (6.1 log cfu/mL). Results from this study suggest that LO is an alternative source of H2O2 that enhances the microbial inhibition achieved by the LS. Lactose oxidase could be used to develop enzyme-based preservation technologies for applications where cold chain access is limited. This enzymatic approach to improving the shelf life of dairy products also represents a novel option for clean label spoilage control.

Modified atmosphere packaging decreased Pseudomonas fragi cell metabolism and extracellular proteolytic activities on meat.

Modified atmosphere packaging (MAP) is considered an effective method for extending the shelf life of meat. The use of optimal mixture of gases (CO2 and N2) in food packaging containers has been proved to effectively inhibit the growth of microorganisms in poultry meat. In general, a minimum CO2 concentration range of 20%-30% is required for the inhibitory effect. The aim of this study was to investigate the mechanism by which MAP (CO2/N2 30%/70%) inhibits Pseudomonas fragi, a dominant spoilage microorganism in aerobically stored chilled meat. The cell physiological changes were determined by measuring membrane integrity, membrane potential, ATP level, and extracellular proteolytic activity. The results showed that samples stored under MA retained cell membrane integrity, but lost significant membrane potential and ATP synthesis activity. Furthermore, the peptides issued from 2 structural proteins (myosin and actin) were mainly identified in air samples, indicating that these fragments result from bacterial proteolytic activity while MAP inhibited this activity. Overall, the study found that cell metabolism and extracellular protease activity decreased under MAP conditions. This study showed that MAP is an effective food preservation strategy and revealed mechanisms by which MAP inhibits spoilage.

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field.

BACKGROUND: Moderate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties-rather than chemical additives-to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear. RESULTS: The spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular ß-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days. CONCLUSION: Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.

Cold stress promoting a psychrotolerant bacterium Pseudomonas fragi P121 producing trehaloase.

A newly isolated Pseudomonas fragi P121 strain in a soil sample taken from the Arctic Circle is able to produce trehalose. The P121 strain was able to grow at temperatures ranging from 4 to 25 °C, had an optimum pH of 6.5, and an optimum salt concentration of 2 %. The P121 strain had a survival rate of 29.1 % after being repeatedly frozen and thawed five times, and a survival rate of 78.9 % when placed in physiological saline for 15 days at 20 °C after cold shock, which is far higher than the type strain Pseudomonas fragi ATCC 4973. The P121 strain could produce 2.89 g/L trehalose, which was 18.6 % of dry cell weight within 52 h in a 25 L fermention tank using the malt extract prepared from barley as medium at 15 °C, while only 11.8 % of dry cell weight at 20 °C. These results suggested that cold stress promoted the strain producing trehalose. It is the first reported cold-tolerant bacterium that produces trehalose, which may protect cells against the cold environment.

The effect of reduced sodium chloride content on the microbiological and biochemical properties of a soft surface-ripened cheese.

Many health authorities have targeted salt reduction in food products as a means to reduce dietary sodium intake due to the harmful effects associated with its excessive consumption. In the present work, we evaluated the effect of reducing sodium chloride (NaCl) content on the microbiological and biochemical characteristics of an experimental surface-ripened cheese. A control cheese (1.8% NaCl) and a cheese with a reduced NaCl content (1.3% NaCl) were sampled weekly over a period of 27d. Reducing NaCl content induced microbial perturbations such as the lesser development of the yeast Debaryomyces hansenii and the greater development of the gram-negative bacterium Hafnia alvei. This was accompanied by changes in proteolytic kinetics and in profiles of volatile aroma compounds and biogenic amine production. Finally, the development of the spoilage microorganism Pseudomonas fragi was significantly higher in the cheese with a reduced salt content.

Pseudomonas spp. convert metmyoglobin into deoxymyoglobin.

Meat 'reddening' by bacteria was observed in chilled beef. To identify the reddening bacteria, isolates were inoculated onto beef and the changes in CIE L*a*b* values monitored. As a result, two Pseudomonas spp., including Pseudomonas fragi which is commonly observed in raw meat, were selected and identified as reddening bacteria. The reddening was coincidentally occurred with the appearance of slime, and the increase in thiobarbituric acid-reactive substances (TBARS) was simultaneously suppressed. In myoglobin-containing nutrient broth, it is shown spectroscopically that P. fragi converted metmyoglobin into deoxymyoglobin. It was concluded that the meat reddening was due to the formation of deoxymyoglobin, induced by the very-low-oxygen tension brought about by Pseudomonad's oxygen consumption: This oxygen depletion simultaneously suppressed TBARS increase.

Effect of industrial and natural biocides on spoilage, pathogenic and technological strains grown in biofilm.

This study aimed at investigating bactericide solutions effective on spoilage and pathogenic bacteria while preserving technological bacteria. Two compounds of essential oil (thymol and eugenol), one essential oil of Satureja thymbra and two industrial biocides (PE 270-30, Brillo) were tested on technological strains (Staphylococcus equorum, Staphylococcus succinus and Lactobacillus sakei) grown in monoculture biofilm and on a mixed biofilm of pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes) and spoilage bacteria (Pseudomonas fragi, Escherichia coli). Biofilm cultures were performed in glass fibre filters for 24h at 20 degrees C before application of biocides. Thymol and eugenol had no effect on the mixed biofilm. S. thymbra (2%) was highly effective on spoilage strains (5 log reduction), and S. equorum (4 log reduction) was moderately effective on pathogens (2.3 log reduction) and not effective on S. succinus and L. sakei (0.5 log reduction). PE-270-30 with 10% Na((2))SO((4)) decreased spoilage bacteria (5.1 log reduction), maintained the technological bacteria, but did not reduce the pathogens. The disinfectant Brillo (3%) killed all the strains. These results showed the difficulty in obtaining a biocide that is effective in destroying spoilage and pathogenic bacteria while preserving technological bacteria. Essential oils could be a good alternative for eradicating spoilage bacteria in the food environment where they are often found at high levels.

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 10(5) CFU/ml of each P. fragi strain. After 12 days at 10 degrees C, mean counts of P. fragi strains were close to 10(10) CFU/g in the outer part of cheeses and close to 10(8) 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.