Mesophilic Strains Research Articles

Biochemical characterization of thermostable cellulase enzyme from mesophilic strains of actinomycete

A few mesophilic strains of actinomycete were used for detection, extraction and characterization of cellulase enzymes. These strains responded to produce all the three components of cellulase complex (endoglucanase, exoglucanase and β-glucosidase) in balanced quantities. Cellulase activity was determined on solid medium supplemented with 1% carboxy methyl cellulose (CMC). Production of cellulase was detected by the formation of clear or transparent zone around colonies. The greater size of transparent zone has been found proportional to the higher capabilities of the strains for enzymes. The extraction of cellulase enzyme was done in liquid basal medium. The assay of cellulase was observed by measuring the release of reducing sugar (RS) by DNS method. All the three components of cellulase viz. endoglucanase, exoglucanase and β -glucosidase were assayed in terms of CMCase, FPase and cellobiase, respectively and expressed in International units (IU). These strains were further tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well as on delignified cellulosics. The optimization for β-glucosidase enzyme was carried out by studying the various parameters viz. effect of pH, incubation period and nitrogen sources.

Structural data on a bacterial exopolysaccharide produced by a deep-sea Alteromonas macleodii strain

Some marine bacteria collected around deep-sea hydrothermal vents are able to produce, in laboratory conditions, complex and innovative exopolysaccharides. In a previous study, the mesophilic strain Alteromonas macleodii subsp. fijiensis biovar deepsane was collected on the East Pacific Rise at 2600m depth. It was isolated from a polychaete annelid Alvinella pompejana and is able to synthesise and excrete the exopolysaccharide deepsane. Biological activities have been screened and some protective properties have been established. Deepsane is commercially available in cosmetics under the name of Abyssine® for soothing and reducing irritation of sensitive skin against chemical, mechanical and UVB aggression. This study presents structural data for this original and complex bacterial exopolysaccharide and highlights some structural similarities with other known EPS produced by marine Alteromonas strains.

Excretome of the chitinolytic bacterium Clostridium paraputrificum J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.

Heat shock response as a cue for phenotypic variability: a study of psychrotrophic and mesophilic strains of Cellulosimicrobium cellulans

We report the expression pattern of heat shock proteins (HSPs) in an Antarctic psychrotrophic strain of Cellulosimicrobium cellulans (DSM 22151) by comparing with its mesophilic counterpart (DSM 43879T) at different temperatures. In both strains, the synthesis of HSPs declined with growth phase. The Antarctic isolate could upregulate 60 kDa, 52 kDa and 41.6 kDa HSPs at 60°C while for its mesophilic counterpart, regulation was restricted to a 64 kDa fraction. The highly unstable nature of this fraction could have led to the impaired thermotolerance exhibited by the mesophilic strain. The downshift of thermal shock from 60°C to 45°C resulted in further upregulation of HSPs in the psychrotrophic strain, while there was no marked variation in the mesophilic strain. This work highlights the significance of a 60 kDa protein in the Antarctic strain of C. cellulans and also indicates the role of a 52 kDa protein in the previously reported heat shock response. Contrary to expectations, the psychrotrophic strain was found to have better mechanisms to tolerate higher temperatures—a property that could give valuable insights into the evolution and adaptation of cold-adapted bacteria.

Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain

BackgroundAcetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination.ResultsThis work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography–mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. α-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C.ConclusionsGeobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.

A comprehensive and quantitative review of dark fermentative biohydrogen production.

Biohydrogen production (BHP) can be achieved by direct or indirect biophotolysis, photo-fermentation and dark fermentation, whereof only the latter does not require the input of light energy. Our motivation to compile this review was to quantify and comprehensively report strains and process performance of dark fermentative BHP. This review summarizes the work done on pure and defined co-culture dark fermentative BHP since the year 1901. Qualitative growth characteristics and quantitative normalized results of H2 production for more than 2000 conditions are presented in a normalized and therefore comparable format to the scientific community.Statistically based evidence shows that thermophilic strains comprise high substrate conversion efficiency, but mesophilic strains achieve high volumetric productivity. Moreover, microbes of Thermoanaerobacterales (Family III) have to be preferred when aiming to achieve high substrate conversion efficiency in comparison to the families Clostridiaceae and Enterobacteriaceae.The limited number of results available on dark fermentative BHP from fed-batch cultivations indicates the yet underestimated potential of this bioprocessing application. A Design of Experiments strategy should be preferred for efficient bioprocess development and optimization of BHP aiming at improving medium, cultivation conditions and revealing inhibitory effects. This will enable comparing and optimizing strains and processes independent of initial conditions and scale.

A Thermophilic Cellulase Complex from Phialophora sp. G5 Showing High Capacity in Cellulose Hydrolysis

A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60-65°C and pH4.0-5.0, and had good long-term thermostability at 50°C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.

Global Analysis of the Genes Involved in the Thermotolerance Mechanism of ThermotolerantAcetobacter tropicalisSKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC-MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1.33 g l(-1), respectively. In addition, the surface tension (ST) decreased from 68 to 35.1 mN m(-1), suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon-contaminated sites.

Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.

Some quantitative data on bacterial attachment to pyrite

Bacterial attachment and biofilm formation are important for microbial bioleaching of metal sulfides, however, many details of the role played by bacteria are still unknown. Attachment as the first step in biofilm formation is critical. Our investigations are focused on these processes to control bacterial bioleaching processes. In moderately thermophilic pure cultures of Acidithiobacillus caldus and Leptospirillum ferriphilum, only the latter one showed significant attachment and biofilm formation to metal sulfides. In pure cultures of the mesophilic strains Acidithiobacillus spp., Leptospirillum ferrooxidans, Ferrimicrobium acidiphilum and a novel undescribed leaching organism designated SPIII/3, attachment was significant by L. ferrooxidans, At. thiooxidans and SPIII/3. In general, interaction of different species in mixed cultures resulted in increased attachment, increased production of extracellular polymeric substances (EPS) and leaching. Bacteria like Leptospirillum spp. enhanced the attachment of other species in mixed biofilms. Another finding is that large areas of the surface of minerals remain uncolonized, whereas at some places bacteria attach in clusters. Based on these findings we conclude that attachment and thereby bacterial leaching can be improved by the production of EPS by several strains especially in combination with Leptospirillum spp.

Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS-MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS-MS and 1-DE/MS-MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.

Characterization of bacteriocin from Lactococcus isolated from traditional Algerian dairy products

Mesophilic strains of lactic acid bacteria belonging to the genus Lactococcus were isolated from traditional dairy products and selected for their ability to produce antimicrobial substances such as bacteriocins. Six strains: Lactococcus lactis ssp. lactis (strains Lc l1, Lc l3, Lc l5), Lactococcus lactis ssp. cremoris (strains Lc c4, Lc c6) and Lactococcus lactis ssp. lactis biovar diacetylactis (strain Lc d2) were selected for their antagonistic activities against different groups of microorganisms. The most active strain, Lactococcus lactis ssp. lactis (Lc l5), produced its inhibitory substance (Bac5) in M17 broth presenting an important inhibition mainly against Listeria monocytogenes. It had no effect on its producer strain. These properties suggested that the inhibitory substance could be considered as a bacteriocin-like substance. SDS-PAGE analyses indicated that it was a homogeneous protein of higher molecular weight of 67 kDa. The bacteriocin was stable over a wide range of pH and temperature; it was active after autoclaving and was not affected by storage at −20°C for 6 months. Moreover, it was sensitive to proteolytic enzymes confirming the proteinaceous nature of the inhibitory substance.

Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5-9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K(m) values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

Cold Stress in Antarctic Fungi Targets Enzymes of the Glycolytic Pathway and Tricarboxylic Acid Cycle

ABSTRACTTo evaluate the concept of metabolic cold adaptation in Antarctic fungi, we compared the activities of several key enzymes of the glycolytic pathway and the TCA cycle (hexokinase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase) in psychrotolerant Penicillium sp. 161 and mesophilic Aspergillus glaucus 363 during both the stress exposure (6 h) and recovery phases. Mycelia of the Antarctic strains, grown until middle exponential phase at optimal temperature, were shifted to colder temperatures, i.e., 4 and 10°C. Our investigations showed a re-routing of carbon metabolism away from glycolysis into the pentose phosphate pathway (PPP), which serves as a cellular stress-resistance mechanism under cold stress conditions. Moreover, the data clearly suggest strain-dependent differences in cold stress response concerning TCA enzyme activities between both fungi. The psychrotolerant strain induces glyoxalate cycle activities and the mesophilic strain uses a reduction of respiratory activity. A recovery response after removal of the stress factor was observed.

MNNG-HOS osteosarcoma growth is enhanced by human fat and adipose-derived mesenchymal stem cells: Implication in reconstructive surgery following tumor treatment

patient survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. Here, we investigated the in vivo effects of a “heparin-like”, an oversulfated exopolysaccharide (OS-EPS) produced by a mesophilic strain found in deep-sea hydrothermal vents, on lung metastasis progression and animal survival. We compared this effect with that of a non-oversulfated exopolysaccharide (EPS) and unfractionated heparin. An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. Independent experiments (series treatedwith 6 mg/kg of EPS or OS-EPS or heparin) showed a significant increase of the survival rate in treated animals versus controls. The number ofmetastatic noduleswas strongly decreased by sulfated polysaccharide (OS-EPS and heparin) treated mice as revealed bymacroscopic analysis. In vitro, a 48-hour incubation with 25 μg/mL EPS or OS-EPS or heparin inhibited POS-1 cell line migration. Besides, the efficacyof polysaccharideswas testedonprimary bone tumor growth using a mouse-transplantable model of osteosarcoma. The preliminary results show that polysaccharides-treatment delayed local tumor growth, as compared to the untreated mice. In conclusion, we demonstrate that these sulfated polysaccharides significantly diminish osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals.

Methanogenic archaea isolated from Taiwan's Chelungpu fault.

Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification, and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, St545Mb(T) of newly proposed species Methanolobus chelungpuianus and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 m below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod-shaped organism capable of using H(2)-CO(2) and formate as substrates for methanogenesis. The morphological, biochemical, and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545Mb(T), isolated from a sandstone sample at 545 mbls, is a nonmotile, irregular, coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545Mb(T) was 99.0% similar to that of Methanolobus psychrophilus strain R15 and was 96 to 97.5% similar to the those of other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545Mb(T) were different from those of M. psychrophilus strain R15, and whole-genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545Mb(T) (DSM 19953(T); BCRC AR10030; JCM 15159) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.

Membrane fluidity and fatty acid comparisons in psychrotrophic and mesophilic strains of Acidithiobacillus ferrooxidans under cold growth temperatures

Psychrotrophic strains of Acidithiobacillus ferrooxidans have an important role in metal leaching and acid mine drainage (AMD) production in colder mining environments. We investigated cytoplasmic membrane fluidity and fatty acid alterations in response to low temperatures (5 and 15°C). Significant differences in membrane fluidity, measured by polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), were found where the psychrotrophic strains had a significantly more rigid membrane (P range = 0.41-0.45) and lower transition temperature midpoints (T (m) = 2.0°C) and broader transition range than the mesophilic strains (P range = 0.38-0.39; T (m) = 2.0-18°C) at cold temperatures. Membrane remodeling was evident in all strains with a common trend of increased unsaturated fatty acid component in response to lower growth temperatures. In psychrotrophic strains, decreases in 12:0 fatty acids distinguished the 5°C fatty acid profiles from those of the mesophilic strains that showed decreases in 16:0, 17:0, and cyclo-19:0 fatty acids. These changes were also correlated with the observed changes in membrane fluidity (R (2) = 63-97%). Psychrotrophic strains employ distinctive modulation of cytoplasmic membrane fluidity with uncommon membrane phase changes as part of their adaptation to the extreme AMD environment in colder climates.

Quantification of the Effect of Culturing Temperature on Salt-Induced Heat Resistance of Bacillus Species

Short- and long-term exposure to mild stress conditions can activate stress adaptation mechanisms in pathogens, resulting in a protective effect toward otherwise lethal stresses. The mesophilic strains Bacillus cereus ATCC 14579 and ATCC 10987 and the psychrotolerant strain B. weihenstephanensis KBAB4 were cultured at 12 degrees C and 30 degrees C until the exponential growth phase (i) in the absence of salt, (ii) in the presence of salt, and (iii) with salt shock after they reached the exponential growth phase and subsequently heat inactivated. Both the first-order model and the Weibull model were fitted to the inactivation kinetics, and statistical indices were calculated to select for each condition the most appropriate model to describe the inactivation data. The third-decimal reduction times (which reflected the times needed to reduce the initial number of microorganisms by three decimal powers) were determined for quantitative comparison. The heat resistance of both mesophilic strains increased when cells were salt cultured and salt shocked at 30 degrees C, whereas these salt-induced effects were not significant for the psychrotolerant strain. In contrast, only the psychrotolerant strain showed salt-induced heat resistance when cells were cultured at 12 degrees C. Therefore, culturing temperature and strain diversity are important aspects to address when adaptive stress responses are quantified. The activated adaptive stress response had an even larger impact on the number of surviving microorganisms when the stress factor (i.e., salt) was still present during inactivation. These factors should be considered when stress-integrated predictive models are developed that can be used in the food industry to balance and optimize processing conditions of minimally processed foods.

Role of Nbr1 in bone remodeling

The growth and differentiation of bone cells is controlled by various factors which can be modulated by heparan sulfates. Here, we investigated the effects of a low-molecular-weight “heparin-like”, an oversulfated exopolysaccharide (OS-EPS) produced by a mesophilic strain found in deep-sea hydrothermal vents, on bone cells in regard to their differentiation, proliferation, and function in vitro. We compared this effect with that of a non-oversulfated exopolysaccharide (EPS). Cell viability was assessed by trypan blue exclusion, and alive and dead cells were manually counted. Besides, by time-lapse experiments, the cell divisions were manually scored. Mesenchymal stem cells (MSCs) isolated and propagated previously from the rat bone marrow were tested for their capabilities to differentiate into osteoblasts in vitro. We showed that exogenous application of OS-EPS to cultures of primary MSCs during in vitro differentiation inhibited MSC mineralization. In addition, OS-EPS strongly reduces the proliferation of differentiating osteoblasts MSCs as we observed for the rat bone marrow cells. This proliferation inhibition was not due to an induction of apoptosis. These results suggest that OS-EPS reduces the differentiated cells proliferation, leading to strong inhibition of mineralized nodule formation. In contrast, OS-EPS enhanced no differenciated MSCs proliferation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfatation in the proliferation and differentiation effects. Bone remodelling is regulated by osteogenic cells which act individually through cellular and molecular interaction. Osteoclasts are bone-resorptive cells that differentiate from hematopoietic precursors upon receptor activator of nuclear factor kappaB ligand (RANKL) activation. To investigate the OS-EPS effect in osteoclastic differentiation, we used several models: murine RAW264.7 cells and human CD14+ monocytes. In the two models used, the addition of OS-EPS inhibited RANKL-induced osteoclastogenesis. In contrast to EPS or other glycosaminoglycan, surface plasmon resonance experiments revealed that RANKL and OS-EPS are able to form a complex. In addition, a strong decrease of the osteoclastic precursor cells adhesion was observed. These results suggest that OS-EPS is a strongly inhibitor of osteoclastogenesis. It also underlines the importance of OS-EPS as a co-receptor for the signaling of RANKL. Conflict of interest: None declared.