Extracellular matrix affects mature biofilm and stress resistance of psychrotrophic spoilage Pseudomonas at cold temperature.

Psychrotrophic Pseudomonas as the dominant spoilage bacteria, have biofilm forming ability, increasing persistence and contamination in the chilled food. Biofilm formation of spoilage Pseudomonas at cold temperature was documented, however, role of extracellular matrix in mature biofilm and stress resistance of psychrotrophic Pseudomonas are much less abundant. The aim of this study was to investigate the biofilm forming characteristics of three spoilers P. fluorescens PF07, P. lundensis PL28, and P. psychrophile PP26 at 25 °C, 15 °C and 4 °C, and to explore their stress resistance to chemical and thermal treatments of mature biofilms. The results showed that biofilm biomass of three Pseudomonas at 4 °C was significantly higher than that at 15 °C and 25 °C. The secretion of extracellular polymeric substances (EPS) greatly increased in those Pseudomonas under low temperature, of which extracellular protein constituted about 71.03%-77.44%. Compared to 25 °C, the mature biofilms were observed to more aggregation and thicker spatial structure at 4 °C ranging from 42.7 to 54.6 µm, in contrast to 25.0-29.8 µm at 25 °C, especially strain PF07. These Pseudomonas biofilms switched into moderate hydrophobicity, and their swarming and swimming were significantly inhibited at low temperature. Furthermore, the resistance to NaClO and heating at 65 °C apparently enhanced for mature biofilm formed at 4 °C, indicating the difference in EPS matrix production influenced the stress resistance of biofilm. In addition, three strains contained alg and psl operons for exopolysaccharide biosynthesis, and biofilm related genes of algK, pslA, rpoS, and luxR were significantly up-regulated, while flgA gene was down-regulated at 4 °C compared to 25 °C, consistent with the above phenotype changes. Thus, the dramatic increase of mature biofilm and their stress resistance in psychrotrophic Pseudomonas were associated with large secretion and protection of extracellular matrix under low temperature, which provide a theoretical basis for subsequent biofilm control during cold chain.

Growth patterns of Pseudomonas aeruginosa in milk fortified with chitosan and selenium nanoparticles during refrigerated storage.

Pseudomonas spp are considered a common milk-associated psychotropic bacteria, leading to milk deterioration during storage; therefore, our study aimed to study the distribution of Pseudomonas aeruginosa in raw milk and its associated products then studying the growth behavior of P. aeruginosa in milk after employing chitosan nanoparticles (CsNPs 50, 25, and 15 mg/100ml) and selenium nanoparticles (SeNPs 0.5, 0.3 and 0.1 mg/100ml) as a trial to control the bacterial growth in milk during five days of cooling storage. Our study relies on the ion gelation method and green synthesis for the conversion of chitosan and selenium to nanosized particles respectively, we subsequently confirmed their shape using SEM and TEM. We employing Pseudomonas selective agar medium for monitoring the bacterial growth along the cooling storage. Our findings reported that high prevalence of Pseudomonas spp count in raw milk and kareish cheese and high incidence percent of P. aeruginosa in ice cream and yogurt respectively. Both synthesized nanoparticles exhibited antibacterial activity in a dose-dependent manner. Moreover, CsNPs50 could inhibit the P. aeruginosa survival growth to a mean average of 2.62 ± 1.18 log10cfu/ml in the fifth day of milk cooling storage; also, it was noted that the hexagonal particles SeNPs0.5 could inhibit 2.49 ± 11 log10cfu/ml in comparison to the control P. aeruginosa milk group exhibited growth survival rate 7.24 ± 2.57 log10cfu/ml under the same conditions. In conclusion, we suggest employing chitosan and selenium nanoparticles to improve milk safety and recommend future studies for the fate of nanoparticles in milk.

Mycosynthesis of silver nanoparticles using psychrotrophic strains of Tulasnella albida Bourdot & Galzin from the South Orkney Islands (Antarctica).

This study is the first report on mycosynthesis of silver nanoparticles (NPs) using psychrotrophic Antarctic filamentous fungi, and the first report regarding Tulasnella (Basidiomycota). In this work, the ability to synthesize silver NPs from cell free filtrates of strains of Tulasnella albida isolated from Antarctica was assessed. All fungal filtrates were capable of synthesizing silver NPs with the addition of AgNO3. UV-vis spectroscopy, TEM and SEM microscopy analyses were performed to characterize the synthesized NPs. ATR-FTIR and Micro Raman spectroscopy analyses were conducted to find functional groups responsible for the reduction of AgNO3 and to detect the presence of silver oxide on the AgNPs. Theoretical calculations of optical absorption based on core-shell Ag-Ag2O were used to characterize the experimental absorption spectra of silver NPs colloids. Spherically shaped silver NPs, typically 2-3nm in diameter, were obtained. The largest ones showed a capping shell around them, which could be associated with the formation of small silver NPs. Functional groups corresponding to amides and alcohols were detected, confirming the presence of proteins as possible intermediates in the synthesis of AgNPs. On the other hand, the Micro Raman analysis confirms the presence of silver oxide on the surface of the AgNPs. This work presents a simple procedure for the synthesis of silver NPs using a psychrotrophic organism that could be interesting for the industry.

Study of the Ca2+-dependent gene expression of EuPrt, an extracellular metalloprotease produced by the psychro-tolerant bacterium Exiguobacterium undae Su-1.

The effect of a Ca2+ ion on the gene expression of an on-demand type of metalloprotease from psychrotrophic Exiguobacterium undae Su-1 (EuPrt) was studied. We first established a modified m m9 medium for strain Su-1 to examine its effect in more detail. Then, when the strain was cultured in m m9 medium and 1.0 m m CaCl2 was added, we detected the mature EuPrt and its precursor proteins via Western blotting analysis and found the relative protease activity and its transcription increased by 50-fold and 7-fold, respectively, at the peak. Furthermore, the intracellular concentration of Ca2+ ions was analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) with other metal ions along the growth of strain Su-1. The intracellular concentration of Ca2+ ion was found to increase as much as 3-fold in response to the addition of an extracellular Ca2+ ions, indicating that euPrt gene expression is regulated by sensing its intracellular concentration.

Lytic bacteriophages UFJF_PfDIW6 and UFJF_PfSW6 prevent Pseudomonas fluorescens growth in vitro and the proteolytic-caused spoilage of raw milk during chilled storage.

In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the proteolytic spoilage of raw milk during cold storage. The UFJF_PfDIW6 and UFJF_PfSW6 phages showed titers of 9.7 and 7.6 log PFU/ml; latent period of 115 and 25 min, and burst size of 145 and 25 PFU/infected cell, respectively. They also were highly specific to the host bacterium, morphologically classified as the Podoviridae family, stable at pH 5 to 11 and were not inactivated at 63 °C or 72 °C for 30 min. These phages found to be effective against P. fluorescens, reducing bacterial count throughout the entire exponential growth phase in broth formulated with milk at both 4 °C and 10 °C. This effect on bacteria growth led to inhibition by at least 2 days in proteases production, delaying the degradation of milk proteins. When applied together in raw milk stored at 4 °C, they reduced the total bacteria, psychrotrophic, and Pseudomonas by 3 log CFU/ml. This study's findings indicate that these phages have a great potential to prevent the growth of Pseudomonas and, consequently, to retard proteolytic spoilage of raw milk during chilled storage.

Soot biodegradation by psychrotolerant bacterial consortia.

To probe the bioavailability of soot released into the atmosphere is pivotal to understanding their environmental impacts. Soot aerosol absorbs organic matter, creating a hot spot for biogeochemical transformation and the global carbon cycle. Soot primarily contains condensed aromatics chemically recalcitrant; however, oligotrophic microorganisms might use it as a nutritional source. This study investigated the influence of psychrotolerant bacterial consortia on soot. Significant increase in the bacterial biomass, reduction in water-insoluble organic carbon (OC) and elemental carbon (EC) in soot residues and increase in water-soluble OC in the filtrate signifies the use of soot as a carbon and nutritional source. The influence on morphology and composition of soot was reported using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy, and Energy Dispersive X-Ray analysis (EDX). The FTIR analysis showed significant variations in the pattern of soot spectra, suggesting degradation. Elemental analysis and EDX showed a reduction in carbon percentage. Besides, the reduction of optical density with incubation time signifies the OC and EC consumption. This study shows that soot can be a substrate and pivotal factor in the microbial food web. Nowadays, soot emission to the environment is growing; therefore, soot involvement in microbe-mediated processes should be closely focused.

Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China.

The quality of raw milk is a key factor influencing the whole dairy processing chain. The richness and diversity of bacteria in raw milk affect its quality and safety. However, traditional microbial detection methods mainly depend on the known microbe culture and are often time consuming. Thus, the development of efficient ways for supervising any possible microbiological contamination is desiderated. In the current work, single-molecule real-time (SMRT) sequencing, developed by Pacific Biosciences (PacBio), was applied to acquire long reads and applied for discrimination of bacteria at species level. Forty samples of raw milk obtained from Beijing, Hebei, Inner Mongolia, Shanghai, and Guangdong in China during summer, autumn, and winter were investigated. Among 35 bacteria species identified in these samples, Acinetobacter albensis, Pseudomonas gessardii, Pseudomonas weihenstephanensis, and Rahnella inusitata were the bacteria with the highest relative abundance in the overall sample, whereas the bacteria with the highest relative abundance in raw milk samples of different origins and seasons are different. Significant differences in bacterial richness and bacterial community diversity in raw milk grouped according to different production areas and different sampling seasons were confirmed by Welch's t-test. Interestingly, the transport distance and transport time positively correlated with the relative abundance of Pseudomonas weihenstephanensis, suggesting that the content of this bacteria was expected to be a standard for evaluating the freshness of raw milk. Pathogens Bacillus cereus and Klebsiella pneumoniae were detected in most samples, indicating that the raw milk was at risk of contamination by pathogenic bacteria. Moreover, the findings of this study provide important evidence for quality and safety monitoring and biological control of raw milk.

Pseudomonas fluorescens and Pseudomonas putida from refrigerated raw milk: genetic diversity and lipoproteolytic activity.

In this research communication the genetic diversity of Pseudomonas fluorescens (n = 67) and Pseudomonas putida (n = 44) isolated from refrigerated raw milk from bulk tank trucks were verified. The relationship between the genetic profile of the isolates and their lipoproteolytic potential was evaluated using skim milk agar and tributyrin agar (21°C/72 h). The lipoproteolytic potential (low or high), evaluated by the diameter of the halos (cm), was correlated with the number of milk producing properties that contributed to each sample (one sample = one bulk tank truck; 8-80 producers/sample) and the distance between the dairy properties and the processing plant (21-370 km). P. fluorescens was confirmed in all samples, while P. putida in 60% samples. For both species, two clusters (I and II) were observed, and the first one showed lower genotypic diversity and the presence of isolates with 100% similarity. P. fluorescens isolates presenting at least 70% similarity were 83.9% in Cluster I (n = 31) and 44.4% in Cluster II. In both clusters (I and II) observed in the P. fluorescens dendrogram, the occurrence of high proteolytic and lipolytic potential were equivalent. The higher the number of farms per milk sample, the greater the lipoproteolytic intensity of P. fluorescens isolates. In relation to P. putida isolates, 74% presented at least 50% similarity in Cluster I (n = 27) and only 35% in Cluster II (n = 17). The occurrence of high proteolysis linked to P. putida was proportional between both Clusters, but the occurrence of high lipolysis was greater in Cluster II. No significant association was detected between P. putida isolates and the variables studied. The results indicate the circulation of P. putida and P. fluorescens with 100% similarity in different milk producing regions. The level of genetic diversity was related only to the lipolytic capacity of P. putida.

Influence of corn starch based bio-active edible coating containing fumaric acid on the lipid quality and microbial shelf life of silver pomfret fish steaks stored at 4 °C.

The present study aimed at assessing the impact of addition of fumaric acid (0.5%), as an active agent, in a corn starch (2%) based edible coating, on the lipid quality and microbial shelf life of silver pomfret (Pampus argenteus) fish steaks stored at 4 °C. Treating fish steaks with FA resulted in a bacteriostatic effect leading to reduced counts of total mesophilic and psychrotrophic bacteria, H2S producing bacteria and Pseudomonas spp. The total mesophilic bacterial count of uncoated control sample exceeded the permissible limit of 7 log cfu g-1 on 6th day and had the lowest microbial shelf life. FA incorporation in the CS coating improved the microbial stability of fish steaks resulting in a shelf life of 15 days. The outcomes of the study suggest that CS based coating is beneficial in delaying lipid oxidation as displayed by the lower TBA and PV values while FA is an effective agent for further increasing the preservative action of CS coating by significantly inhibiting microbial growth as well as lipid quality deterioration, which could be exploited by the seafood industry as an active packaging component.

Transcriptomic time-series analysis of cold- and heat-shock response in psychrotrophic lactic acid bacteria.

BACKGROUND: Psychrotrophic lactic acid bacteria (LAB) species are the dominant species in the microbiota of cold-stored modified-atmosphere-packaged food products and are the main cause of food spoilage. Despite the importance of psychrotrophic LAB, their response to cold or heat has not been studied. Here, we studied the transcriptome-level cold- and heat-shock response of spoilage lactic acid bacteria with time-series RNA-seq for Le. gelidum, Lc. piscium, and P. oligofermentans at 0 °C, 4 °C, 14 °C, 25 °C, and 28 °C. RESULTS: We observed that the cold-shock protein A (cspA) gene was the main cold-shock protein gene in all three species. Our results indicated that DEAD-box RNA helicase genes (cshA, cshB) also play a critical role in cold-shock response in psychrotrophic LAB. In addition, several RNase genes were involved in cold-shock response in Lc. piscium and P. oligofermentans. Moreover, gene network inference analysis provided candidate genes involved in cold-shock response. Ribosomal proteins, tRNA modification, rRNA modification, and ABC and efflux MFS transporter genes clustered with cold-shock response genes in all three species, indicating that these genes could be part of the cold-shock response machinery. Heat-shock treatment caused upregulation of Clp protease and chaperone genes in all three species. We identified transcription binding site motifs for heat-shock response genes in Le. gelidum and Lc. piscium. Finally, we showed that food spoilage-related genes were upregulated at cold temperatures. CONCLUSIONS: The results of this study provide new insights on the cold- and heat-shock response of psychrotrophic LAB. In addition, candidate genes involved in cold- and heat-shock response predicted using gene network inference analysis could be used as targets for future studies.

NADP+ -dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A.

The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH-D and 13AIDH-M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH-D and 13AIDH-M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH-D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH-M activity was completely lost under the same condition. In addition, 13AIDH-D showed much higher specific activity than 13AIDH-M. From northern and western blot analyses, the 13AIDH-D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH-M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold-living bacteria to their growth at low temperatures was confirmed in strain 13A.

Wickerhamomyces psychrolipolyticus f.a., sp. nov., a novel yeast species producing two kinds of lipases with activity at different temperatures.

Two yeast strains isolated from soil collected in Hokkaido, Japan, were found to secrete two extracellular lipases that exhibited activities at both 25 and 4 °C. Both strains could utilize olive oil, rapeseed oil, lard and fish oil as sole carbon sources. The similarity of the D1/D2 domain of the large subunit ribosomal RNA (LSU rRNA) sequence of these yeast strains to that of other yeasts in the GenBank database was very low (<96 %). The phylogenetic trees based on the LSU rRNA sequences and translation elongation factor-1-α (tef1-α) sequences indicated that both strains represented a member of the Wickerhamomyces /Candida clade. Sexual reproduction was not observed. The name Wickerhamomyces psychrolipolyticus f.a., sp. nov is proposed for this newly described yeast species producing cold-active lipases. This novel species is distinguishable from the type strains of other related species, Wickerhamomyces alni, Candida ulmi and Candida quercuum due to their abilities to grow at 4 to 30 °C, to produce lipase that is active also at 4 °C and to assimilate soluble starch.

Effect of thermoresistant protease of Pseudomonas fluorescens on rennet coagulation properties and proteolysis of milk.

This study aimed to investigate the effect of different activity levels of a thermoresistant protease, produced by Pseudomonas fluorescens (ATCC 17556), on the cheesemaking properties of milk and proteolysis levels. Sterilized reconstituted skim milk powder was inoculated with the bacteria, and after incubation, centrifuged to obtain a supernatant-containing protease. Raw milk was collected and inoculated to obtain a protease activity of 0.15, 0.60, and 1.5 U/L of milk (treatments P1, P4, and P10, respectively). One sample was not inoculated (control) and noninoculated supernatant was added to a fifth sample to be used as a negative control. Samples were stored at 4°C for 72 h. After 0, 48, and 72 h, the rennet coagulation properties and proteolysis levels were assessed. The protease produced was thermoresistant, as no significant differences were observed in the activity in the pasteurized (72°C for 15 s) and nonpasteurized supernatants. The chromatograms and electrophoretograms indicated that the protease preferably hydrolyzed κ-casein and ß-casein, and levels of proteolysis increased with added protease activity over storage time. The hydrolysis of αS-caseins and major whey proteins increased considerably in P10 milk samples. At 0 h, the increase in the level of protease activity decreased the rennet coagulation time (RCT, min) of the samples, possibly due to synergistic proteolysis of κ-casein into para-κ-casein. However, over prolonged storage, hydrolysis of ß-casein and αS-casein increased in P4 and P10 samples. The RCT of P4 samples increased over time and the coagulum became softer, whereas P10 samples did not coagulate after 48 h of storage. In contrast, the RCT of P1 samples decreased over time and a firmer coagulum was obtained, possibly due to a lower rate of hydrolysis of ß-casein and αS-casein. Increased levels of protease could result in further hydrolysis of caseins, affecting the processability of milk over storage time.

Effect of Pseudomonas fluorescens proteases on the quality of Cheddar cheese.

The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk on the manufacture and quality of Cheddar cheese. Fresh raw milk was collected, standardized, and pasteurized at 72°C for 15 s, and the enzyme was added to give a protease activity of 0.15 or 0.60 U/L (treatments P1 and P4, respectively), while one sample had no enzyme added (control). Milk was stored at 4°C for 48 h and Cheddar cheese was manufactured after 0 and 48 h of storage. Results indicated that the protease was active in milk during 48 h of storage; however, its effect on milk composition was minimal. The protein that was preferentially hydrolyzed by the protease over storage was ß-casein, followed by κ-casein. The mean cheese yield and recovery of fat and protein obtained for all cheeses were not affected by protease activity. The protease showed low activity during cheese manufacture, possibly because of unfavorable conditions, including low pH. One of the factors that might have influenced protease activity was the pH of the curd (approximately 6.55 after acidification and 5.35 at milling), which was lower than that at which the enzyme would have optimum activity (pH 7 to 9). Consequently, the composition, pH, patterns of proteolysis, and hardness of all cheeses produced were similar and in accordance with values expected for that type of cheese, independently of the protease activity level. However, slight increases in proteolysis were observed in P4 cheeses and produced using milk stored for 48 h. Both the P1 and P4 cheeses had higher concentrations of free amino acids (FAA) compared with the control, whereas urea-PAGE electrophoretograms indicated a greater breakdown of caseins in the P4 cheese samples, which may be related to possible increases in numbers of proteolytic bacteria in milk during storage. Therefore, the thermoresistant psychrotrophic bacterial protease(s) tested in this study may affect the manufacture or quality of Cheddar cheese during ripening to a relatively limited extent. However, controlling initial levels of proteolytic bacteria in raw milk remains essential, because proteolysis affects the development of flavor and texture in cheese.

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.

Multispecies biofilm formation by the contaminating microbiota in raw milk.

Biofilms can be formed on the surfaces of dairy processing equipment and are a potential source of product contamination. This study evaluated the diversity of multispecies biofilms formed on stainless steel (SS) due to the contaminating microbiota in raw milk. Samples of raw milk were used: one was fresh milk and the other maintained in refrigerated bulk tanks for up to 48 h. The mesophilic aerobic contamination was ∼104 CFU ml-1 in fresh milk and 106 CFU ml-1 in bulk milk. SS coupons were kept immersed in the milk at 7 ±2 °C for 10 days, and every two days, the raw milk was changed for samples of the same origin collected on the current day. After incubation for 10 days, sessile cells in the biofilm reached 105 CFU cm-2 in the presence of fresh milk, and 106 CFU cm-2 in the presence of bulk milk. The genetic diversity analysis showed that Gammaproteobacteria and Bacilli predominated in the biofilms throughout the incubation of both milk samples and these biofilms showed a reduction in diversity over time. The main classes of bacteria found in these biofilms have representatives of great importance since many of them have spoilage potential.

Risk presented to minimally processed chilled foods by psychrotrophic Bacillus cereus.

BACKGROUND: Spores of psychrotrophic Bacillus cereus may survive the mild heat treatments given to minimally processed chilled foods. Subsequent germination and cell multiplication during refrigerated storage may lead to bacterial concentrations that are hazardous to health. SCOPE AND APPROACH: This review is concerned with the characterisation of factors that prevent psychrotrophic B. cereus reaching hazardous concentrations in minimally processed chilled foods and associated foodborne illness. A risk assessment framework is used to quantify the risk associated with B. cereus and minimally processed chilled foods. KEY FINDINGS AND CONCLUSIONS: Bacillus cereus is responsible for two types of food poisoning, diarrhoeal (an infection) and emetic (an intoxication); however, no reported outbreaks of food poisoning have been associated with B. cereus and correctly stored commercially-produced minimally processed chilled foods. In the UK alone, more than 1010 packs of these foods have been sold in recent years without reported illness, thus the risk presented is very low. Further quantification of the risk is merited, and this requires additional data. The lack of association between diarrhoeal food poisoning and correctly stored commercially-produced minimally processed chilled foods indicates that an infectious dose has not been reached. This may reflect low pathogenicity of psychrotrophic strains. The lack of reported association of psychrotrophic B. cereus with emetic illness and correctly stored commercially-produced minimally processed chilled foods indicates that a toxic dose of the emetic toxin has not been formed. Laboratory studies show that strains form very small quantities of emetic toxin at chilled temperatures.

High pressure sensitization of heat-resistant and pathogenic foodborne spores to nisin.

Today, there is no effective non-thermal method to inactivate unwanted bacterial spores in foods. High-Pressure (HP) process has been shown to act synergistically with moderate heating and the bacteriocin nisin to inactivate spores but the mechanisms have not been elucidated. The purpose of the present work was to investigate in depth the synergy of HP and nisin on various foodborne spore species and to bring new elements of understandings. For this purpose, spores of Bacillus pumilus, B. sporothermodurans, B. licheniformis, B. weihenstephanensis, and Clostridium sp. were suspended in MES buffer, in skim milk or in a liquid medium simulating cooked ham brine and treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Nisin (20 or 50 IU/mL) was added at three different points during treatment: during HP, during and or in the plating medium of enumeration. In the latter two cases, a high synergy was observed with the inhibition of the spores of Bacillus spp. The evaluation of the germinated fraction of Bacillus spp. spores after HP revealed that this synergy was likely due to the action of nisin on HP-sensitized spores, rather than on HP-germinated spores. Thus, the combination of nisin and HP can lead to Bacillus spp. spore inhibition at 20 °C. And Nisin can act on HP-treated spores, even if they are not germinated. This paper provides new information about the inhibition of spores by the combination of HP and nisin. The high synergy observed at low temperature has not been reported yet and could allow food preservation without the use of any thermal process.

The contribution of fast growing, psychrotrophic microorganisms on biodiversity of refrigerated raw cow's milk with high bacterial counts and their food spoilage potential.

Storage of raw milk in the bulk tank creates an environment which selects for psychrotrophic bacteria. Results from earlier studies suggested that the microbiota of bulk tank milk with high bacterial counts is dominated by single, cold-adapted species with high growth rates at low temperatures. We checked this assumption in more detail and analyzed the microbial diversity of 48 samples from bulk tank raw cow's milk with bacterial counts >100,000 cfu/mL from different geographic regions by culture-dependent and -independent methods. Contrary to presumptions from earlier studies, only the minority (24%) of samples was dominated by a single bacterial species and diversity was not correlated with bacterial counts. The dominating species in this group of samples were identified as psychrotrophic Acinetobacter and Pseudomonas species, related to poor hygiene and spoilage, or mesophilic, mastitis-related Streptococcus species and Escherichia coli. This shows that storage of raw milk under refrigeration does not always lead to a selection of cold-adapted bacteria. Approximately half of the raw milk isolates showed either lipolytic or proteolytic activity at 10 °C or 4 °C. Consistent or increased enzymatic activity at cold temperatures was detected for Acinetobacter spp. and Pseudomonas spp., but also for genera with minor abundance, e.g. Carnobacterium and Arthrobacter.

Bacterial spoilage profiles in the gills of Pacific oysters (Crassostrea gigas) and Eastern oysters (C. virginica) during refrigerated storage.

Microorganisms harbored in oyster gills are potentially related to the spoilage and safety of oyster during storage. In this study, the microbial activities and pH changes of the gills of the two species, Crassostrea gigas and C. virginica, harvested from three different sites were determined and sensory evaluation was conducted during refrigerated storage. The bacteria in gills with an initial aerobic plate count (APC) of 3.1-4.5 log CFU/g rose remarkably to 7.8-8.8 log CFU/g after 8-days of storage. The APC of Enterobacteriaceae increased from 2.5 to 3.6 log CFU/g to 4.5-4.8 log CFU/g, and that of lactic acid bacteria (LAB) fluctuated in the range of 1.4-3.0 log CFU/g during the whole storage period. The results of sensory analysis indicated that the oysters had 8-days of shelf-life and that the gill presented the fastest deterioration rate. The pH of all samples showed a decrease in the early stages followed by an increased after 4-days of storage. The dynamic changes in microbial profiles were depicted to characterize gill spoilage by Illumina Miseq sequencing to characterize gill spoilage. The results revealed that oysters harvested at different sites showed common bacterial profiles containing Arcobacter, Spirochaeta, Pseudoalteromonas, Marinomonas, Fusobacterium, Psychrobacter, Psychromonas, and Oceanisphaera when spoiled, especially, among which Psychrobacter and Psychromonas (psychrotrophic genus) were represented as the most important gill spoiled bacteria during refrigerated storage, and Arcobacter with pathogenic potential was the dominated bacteria in all spoiled oysters. The consumption quality and safety of refrigerated oysters could be greatly improved by targeted control of bacteria in oyster gills according to the results the present study provided.