Low Acidic Conditions Research Articles

Abstract B32: A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors

Abstract The epidermal growth factor (EGF) signaling pathway relies on recognition by its receptor, EGFR, and subsequent downstream signaling by the KRAS and BRAF proteins to relay proper proliferative, migratory, and angiogenic functions. Cancers with activating KRAS or BRAF mutations are resistant to EGFR targeting agents and correspond to a significant unmet medical need. We hypothesized that an anti-EGFR antibody-drug conjugate (ADC) could be active against KRAS or BRAF mutated tumors, due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, we wished to engineer an antibody with enhanced specificity towards EGFR in the tumor microenvironment (TME) and attenuated binding to EGFR in normal tissue. This was achieved by screening a library of antibody variants (based on cetuximab) in a spatially addressed manner for binding to a recombinant version of the EGFR extracellular domain (EGFRECD) in two separate ELISA reaction conditions. High affinity binding to the EGFRECD was desired in the first condition, which approximated the physicochemical properties of the TME (acidic pH, high lactic acid concentration, 25% human serum). In the second assay condition, which approximated mAb binding to EGFRECD in normal tissue (neutral pH, low lactic acid concentration, 25% human serum), attenuated binding affinity was desired. We identified a lead mAb variant, cMab-1501, which possessed several fold reduced binding to EGFRECD in the neutral pH, low lactic acid condition, when compared to EGFRECD binding in the low-pH, high lactic acid, assay condition. To evaluate enhanced specificity for binding to EGFR in vivo, cMab-1501 was compared to cetuximab for binding to both human donor foreskin xenografts and human A431 tumor xenografts, using a DyLight 755 conjugated version of each antibody, and subsequent fluorescence detection with a Caliper IVIS system. cMab-1501 and cetuximab demonstrated relatively comparable binding towards human A431 tumor xenografts in vivo. In addition, cetuximab bound relatively equally between human tumor xenografts and human skin grafts. However, no binding to EGFR in the human skin graft was detected for cMab-1501 over all days measured; suggesting that cMab-1501 was highly specific for binding to EGFR in the TME. We next generated an cMab-1501 based ADC (antibody-drug conjugate), via maleimide chemistry carrying a protease cleavable valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E (vcPAB-MMAE) cytotoxic moiety, forming a cMab-1501-vcPAB-MMAE conjugate. Both the conjugated and un-conjugated versions of cMab-1501 were rapidly internalized by EGFR positive MDA-MB-231M tumor cells over several hours. In tumor xenograft models, the TME-specific anti-EGFR ADC demonstrated complete tumor regressions against two human EGFR overexpressing tumor types, MDA-MB-231M (TNBC, KRAS G13D) and HT-29 (CRC, BRAF V600E). In both in vivo models, tumors were resistant to treatment by cetuximab. These data suggest that it is possible to engineer a monoclonal antibody with enhanced specificity for its target within the TME and that an ADC-based approach could be utilized as potential treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Bob Veneziale, Lei Huang, Xiaoming Li, Qiping Zhao, Chunmei Zhao, Ryan Osgood, Jessica Cowell, Sanna Rosengren, Jason Parise, Ge Wei, Kim Phan, Robert Connor, Steve Rowe, Gilbert Keller, Gregory Frost, Dan Maneval, Curtis Thompson, Michael Shepard, Christopher Thanos. A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B32.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation.S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

Carotenoid transfer to oil upon high pressure homogenisation of tomato and carrot based matrices

The transfer of carotenoids into the oil phase during digestion is limited by matrix related factors that hamper carotenoid release and the conditions of low gastric acidity that limit carotenoid solubility. Therefore, the aim of this study was to investigate the use of high pressure homogenisation (HPH) as a driving force to favour carotenoid transfer to oil before digestion. The level of bio-encapsulation and carotenoid hydrophobicity were investigated as factors that may influence carotenoid transfer from tomato and carrot based matrices to oil upon HPH. The results indicated that the selective transfer of a particular carotenoid depended on its hydrophobicity. The cell wall in tomato cell clusters represents a limiting factor for carotenoid transfer. Overall, the findings indicate that HPH is efficient in transferring carotenoids to the oil phase and this can be crucial to improve the nutritional quality of carrot and tomato-based products.

Homogeneous liquid–liquid extraction of U(VI) from HNO3 aqueous solution to betainium bis(trifluoromethylsulfonyl)imide ionic liquid and recovery of extracted U(VI)

Abstract In order to examine the applicability of a thermomorphic ionic liquid, betainium bis(trifluoromethylsulfonyl)imide ([Hbet][Tf 2 N]), for the decontamination of solid wastes contaminated with uranium, we have studied fundamental extraction behavior of U(VI) from HNO 3 aqueous solutions to [Hbet][Tf 2 N] phases using a homogeneous liquid–liquid extraction method. As a result, it was found that the U(VI) species are extracted with relatively high distribution ratios ( D > 1) in low acidic condition without any additional extractant, and proposed that the U(VI) species are extracted through the exchange reaction between [Hbet] + and [UO 2 (bet)] 2+ . The complex formation of [UO 2 (bet)] 2+ was found to play an important role in the extraction, because the U(VI) species were not extracted with trimethylpropylammonium bis(trifluoromethylsulfonyl)imide ([TMPA][Tf 2 N]) without any functional groups. We also examined the recovery method of U(VI) species from the [Hbet][Tf 2 N] phase. As a result, it was found that the precipitation of uranyl peroxide with the addition of H 2 O 2 aqueous solution is the useful method for recovering U(VI) species.

Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions

Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in <1, 2, 5, and 21% O2 at pH 7.2 or 6.2 in the absence or presence of the proinflammatory cytokine, interleukin-1β (10ng/ml).In addition, chondrocytes were cultured with resveratrol (10µM) or N-acetylcysteine (NAC) (2mM).Cell viability, glycosaminoglycan (GAG) release, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), GSH:GSSG ratio, and SOD1 and SOD2 protein expression were measured. Very low levels of oxygen (<1%), acidosis (pH 6.2), and exposure to IL-1β led to reductions in cell viability, increased GAG release, alterations in ΔΨm and ROS levels, and reduced GSH:GSSG ratio. In addition, SOD1 and SOD2 protein expressions were reduced. Both resveratrol and NAC partially restored ΔΨm and ROS levels and prevented GAG release and cell loss and normalized SOD1 and SOD2 protein expression. In particular NAC was highly effective at restoring the GSH:GSSG ratio.These results show that the antioxidants resveratrol and N-acetylcysteine can counteract the redox imbalance in articular chondrocytes induced by low oxygen and acidic conditions.

Improved dissolution and absorption of ketoconazole in the presence of organic acids as pH-modifiers

Formulation development of poorly water-soluble compounds can be challenging because of incomplete dissolution that causes low and variable bioavailability. Enhancing compound solubility is important and many techniques have been investigated to that end, but they require specific materials and machinery. This study investigates the incorporation of a pH-modifier as a method to increase compound solubility and uses ketoconazole (KZ), which is weakly basic (pKa: 6.5), as a model compound. Organic acids are effective pH-modifiers and are generally used in pharmaceutical industries. We successfully obtained granules containing variable organic acids (KZ/acid granule) using a high-shear mixer. Dissolution tests of the KZ/acid granule resulted in highly enhanced solubility under non-sink conditions. Adding water-soluble acids, such as citric acid (CA) and tartaric acid, resulted in more than 8-fold higher dissolution at pH 6.0 compared to that of KZ only. The granules containing citric acid (KZ/CA granule) improved the dissolution of KZ after oral administration to rats under low gastric acid conditions, where the bioavailability of the KZ/CA granules at elevated gastric pH was comparable with that of KZ only at gastric acidic pH. The incorporation of organic acids would result in effective therapeutic outcomes independent of gastric pH in patients. In addition, higher bioavailability of KZ was observed after oral administration of KZ/CA granules under gastric acidic pH conditions than that of KZ alone. Thus, CA improved the dissolution and absorption rate of KZ after oral administration.

Pilot study on novel skin care method by augmentation with Staphylococcus epidermidis, an autologous skin microbe – A blinded randomized clinical trial

Background and objectiveStaphylococcus epidermidis is an autologous bacterium that is beneficial to skin health. Our goal was to develop a novel, personalized basic cosmetic that exploits this characteristic. MethodsWe conducted a double-blinded, randomized clinical trial on augmentation with S. epidermidis as a pilot study, in which S. epidermidis was collected from the subject, cultured for proliferation, and then continuously applied to the subject's own face before sleep twice per week for four weeks in order to increase colonization levels. ResultsThe results showed that this treatment increased the lipid content of the skin and suppressed water evaporation, thereby markedly improving skin moisture retention. Moreover, augmentation with S. epidermidis maintained a low acidic condition on the skin surface. The low risk of undesirable effects induced by augmentation with S. epidermidis was also confirmed by measuring erythema and melanin levels. ConclusionsThese results may serve as a driving force to accelerate the development of novel, personalized basic cosmetics.

Understanding the Meat Color Attributes by Direct Electrochemistry of Myoglobin Films on Electrodes

Protein film electrochemistry (PFE) is a unique method with high simplicity to obtain mechanistic information about complex biological electron transfer and catalytic processes. The emerging applications of PFE are in the development of selective biosensors, biofuel production, renewable energy processes, green biocatalytic systems, and biomedical tools.[i],[ii],[iii] Along this line, we are exploring the novel utilization of heme-protein electrochemistry to understand the meat color qualities. Myoglobin (Mb) is a muscle protein predominantly responsible for meat color. Mb can exist in three different forms namely deoxy-, oxy-, and metmyoglobin. The extent of oxymyoglobin (Mb-FeII-O2) state mainly influences the characteristic red color of meat. On the other hand, the oxidation of oxy- or deoxymyoglobin leads to discoloration of meat or metmyoglobin (Mb-FeIII) accumulation.Our objective was to investigate the kinetics of metmyoglobin reduction and oxymyoglobin formation using myoglobin model and examine its relevance to meat color properties. For our initial electrochemical analysis, we have chosen three meat related pH conditions such as pH 7.4 (physiological muscle), pH 6.4 (stress-out animal muscle), and pH 5.6 (typical postmortem muscle) and investigated the rates of Mb-FeIII reduction and Mb-FeII-O2 formation using Mb-films adsorbed on graphite electrodes. We observed that under the physiological muscle pH condition, the rates of Mb-FeIII reduction and the subsequent formation of Mb-FeII-O2 were highly favored in comparison to the low acidic pH conditions. Our electrochemical results correlated well with the prior biochemical and redox-titration studies that reported the faster discoloration of meat with lowering pH conditions.[iv],[v] Our investigation suggests that myoglobin-PFE is simple, inexpensive, rapid, and powerful in probing atomic level redox-properties, and thus a valuable tool to predict meat color. Acknowledgements. This work was supported by the Oklahoma State University.[i]. (a) M. Meredith and S.D. Minteer, Annu. Rev. Anal. Chem., 2012, 5, 157-179; (b) S. Xu and S.D. Minteer, ACS Catalysis, 2013, 3, 1756-1763.[ii]. L. Halamkova, J. Halamek, V. Bocharova, A. Szczupak, L. Alfonta and E. Katz, J . Am . Chem . Soc ., 2012,4 , 5040.[iii]. (a) S. Krishnan and F. A. Armstrong, Chem . Sci ., 2012, 3, 1015-1023; (b) S. Krishnan and J. F. Rusling, Thin Iron Heme Enzyme Films on Electrodes and Nanoparticles for Biocatalysis, Chapter 5, p.125, in New and Future Developments in Catalysis, S. Suib (Ed.), Elsevier Publishers, 2013; (c) C. Walgama and S. Krishnan,J. Electrochem. Soc., 2014, 161, H1-H6; (d) S. Krishnan and C. Walgama, Anal. Chem., 2013, 85, 11420−11426.[iv]. A. L. Alderton, C. Faustman, D. C. Liebler, D. W. Hill, Biochemistry 2003 , 42, 4398.[v]. R. Ramanathan, R. A. Mancini, G. A. Dady, C. B. Van Buiten, Meat Science 2013, 93, 888.

Evaluation of the effects of sodium chloride, potassium sorbate, nisin and lysozyme on the probability of growth of Clostridium sporogenes

SummaryThe aim of this study was to evaluate the combined effect of salt (sodium chloride, 0–8% w/v), sorbate (potassium sorbate, 0–4.5% w/v), nisin (0–500 ppm) and lysozyme (0–500 ppm) on the survival of Clostridium sporogenes as a non‐toxigenic surrogate of Clostridium botulinum in terms of the probability of growth by using a central composite rotatable design. The results indicated that salt and sorbate were the most effective factors in preventing the growth of Cl. sporogenes in high‐moisture (&gt;95%) and low‐acid conditions. The probability of growth of Cl. sporogenes in broth was reduced by combinations of salt and sorbate. Nisin and lysozyme had insignificant effects on the probability of growth of Cl. sporogenes (P &gt; 0.05). Lysozyme individually and in combination with nisin had no inhibitory effect on Cl. sporogenes. Overall, the addition of sorbate and lysozyme may allow the salt concentration to be reduced while preventing growth.

Sensitivity ofMycobacterium aviumsubspparatuberculosis,Escherichia coliandSalmonella entericaserotype Typhimurium to low pH, high organic acids and ensiling

To evaluate the persistence of Mycobacterium avium subsp paratuberculosis (Myco. paratuberculosis), Salmonella enterica serotype Typhimurium (Salm.Typhimurium) and a commensal Escherichia coli (E. coli) isolate under the low pH and high organic acid (OA) conditions of ensiling of forages. Decay rates and the time required to obtain a 90% reduction in cell concentration were calculated following (i) exposure to buffered OA (pH 4.0, 5.0, 6.0 or 7.0) (ii) exposure to silage exudates and (iii) survival through ensiling of forage materials. Salm. Typhimurium had higher decay rates in silage exudates (-0.5601 day(-1) ) than did E. coli (-0.1265 day(-1) ), but both exhibited lower decay rates in silage than in OA or silage exudates. Myco. paratuberculosis showed no decrease in silage and decay rates in silage exudates were significantly lower (2-12 times) than for the other two organisms. Escherichia coli, Salm. Typhimurium and Myco. paratuberculosis exhibit marked differences in response to acidity. All three organisms show acid resistance, but Myco. paratuberculosis in particular, if present in manure and applied to forage grasses, may survive the low pH and high OA of the ensilaging process; silage may therefore be a potential route of infection if ingested by a susceptible animal. This information contributes to the understanding of potential risks associated with silage preservation and contamination of livestock feed with manure-borne pathogens.

Kinetics and Thermodynamics of Atmospherically Relevant Aqueous Phase Reactions of α-Pinene Oxide

Recent work has demonstrated that isoprene-derived epoxide intermediates are responsible for a wide variety of chemical species found in ambient secondary organic aerosol (SOA). Since the second most abundant biogenic hydrocarbon, α-pinene, is also known to form an epoxide intermediate, nuclear magnetic resonance techniques were used to study products, kinetics, and equilibria of the aqueous phase reactions of that epoxide, α-pinene oxide. The present results indicate that α-pinene oxide will react very quickly with aqueous atmospheric particles, even under low acidity conditions. Depending on the acid concentration of the aqueous solutions, a number of new products are observed from the reaction of α-pinene oxide, some of which are expected to partition back to the gas phase. In contrast to some previous results, no long-lived organosulfate or organonitrate species are observed, and no species which retain the α-pinene bicyclic carbon backbone are observed. Rather, the overall product distribution can be explained by various rearrangements of the initial carbocation intermediate formed in the ring opening of α-pinene oxide, all of which can be rationalized by the thermodynamically driven relief of the bicyclic ring strain in the α-pinene carbon backbone.

Invertebrate shredder as a factor controlling the fixation potential for metals/metalloids in organic matter during decay

Wetlands are well known for water treatment and as a sink for elements (e.g. metals/metalloids). The elements are mainly fixed by sediments, microbial biofilms and plants, whereas the organic part of the sediment has a potential important role. These organic sediments underlie the process of organic matter decomposition, which proceeds in the three distinct steps of leaching, microbial conditioning and fragmentation by aquatic fauna. The effect of organic matter fragmentation on elemental fixation was shown previously only under neutral pH conditions. Hence, a batch experiment was conducted to assess the effect of invertebrate shredder on elemental accumulation during litter decay under low acidic conditions. Here it is shown that this fragmentation of the organic material by aquatic fauna enhances the elemental fixation potential of the organic sediments significantly, also under low acidic conditions. During the process of fragmentation the surface area of the organic matter increases by increasing amount of smaller particles. This in turn results in higher adsorption capacity of a larger amount of growing biofilm enhancing the surface even more (heterotrophic microorganisms). The growing biofilm (periphyton) with its larger surface area and hence more functional groups result in higher elemental fixation. Therefore, invertebrate shredders as ecosystem engineers enhance the capability for elemental fixation by fragmentation of organic matter, which is an important process in wetlands ecosystems usable in a broad pH range.

Uranyl ion interaction at the water/NiO(100) interface: A predictive investigation by first-principles molecular dynamic simulations

The behavior of the UO(2)(2+) uranyl ion at the water/NiO(100) interface was investigated for the first time using Born-Oppenheimer molecular dynamic simulations with the spin polarized DFT + U extension. A water/NiO(100) interface model was first optimized on a defect-free five layers slab thickness, proposed as a reliable surface model, with an explicit treatment of the solvent. Water molecules are adsorbed with a well-defined structure in a thickness of about 4 Å above the surface. The first layer, adsorbed on nickel atoms, remains mainly in molecular form but can partly dissociate at 293 K. Considering low acidic conditions, a bidentate uranyl ion complex was characterized on two surface oxygen species (arising from water molecules adsorption on nickel atoms) with d(U-O(adsorption))=2.39 Å. This complex is stable at 293 K due to iono-covalent bonds with an estimated charge transfer of 0.58 electron from the surface to the uranyl ion.

Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain That Co-Ferments Glucose and Xylose: II. Influence of pH and Acetic Acid on Ethanol Production

The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0-6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150-200mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.

Structural changes of bovine milk fat globules during in vitro digestion

An in vitro digestion model that simulated gastric and intestinal fasting conditions was used to monitor the physical, chemical, and structural changes of fat globules from raw bovine milk. During in vitro gastric digestion, the fat globules were stable under low-acidic conditions. Some peptides and β-lactoglobulin were resistant to proteolysis by pepsin. Phospholipids, proteins, and peptides stabilized the globules in the stomach model. During in vitro intestinal digestion, most of the β-lactoglobulin and residual peptides were hydrolyzed by trypsin and chymotrypsin, and the lipolytic products, released from the hydrolysis of the triglyceride core of the globules, led to destabilization and coalescence of the globules. By accumulating at the surface of the fat globules, the lipolytic products formed a lamellar phase and their solubilization by bile salts resulted in the formation of disk-shaped micelles. This study brings new interesting insights on the digestion of bovine milk.

Facile synthesis and morphology control of graphene oxide/polyaniline nanocomposites via in-situ polymerization process

This study reports the synthesis of graphene oxide (GO)/polyaniline (PANI) nanocomposites with controllable morphologies through in-situ polymerization of aniline monomers in the presence of GO sheets. Specific reaction parameters including solution acidity, aniline concentration, and reaction temperature are used to control the final shape of the composite product. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images are used to explore the morphology of the composite. Thermogravimetric analysis (TGA), X-ray diffraction (XRD), FT–IR and UV–vis spectrophotometers are utilized to characterize the intermediates and the final products of the GO/PANI composites. Experiment results reveal that the polymerization operated in low acidity and low temperature conditions inclines to form GO/PANI nanotubes. On the other hand, the polymerization operated in high acidity inclines to form either nanospheres or aligned nanofiber arrays. These different morphologies are resulted from different polymerization routes and the formation mechanisms of these different shapes of nanocomposites are explored. Among the various nanocomposites, the GO/PANI nanospheres exhibit a highest electrochemical surface area. This study provides a facile and effective strategy to control the morphology of GO/PANI nanocomposites with characteristic electrochemical property.

Dilute Acid Hydrolysis of Sugar Cane Bagasse at High Temperatures: A Kinetic Study of Cellulose Saccharification and Glucose Decomposition. Part I: Sulfuric Acid as the Catalyst

The kinetics of sugar cane bagasse cellulose saccharification and the decomposition of glucose under extremely low acid (ELA) conditions, (0.07%), 0.14%, and 0.28% H2SO4, and at high temperatures were investigated using batch reactors. The first-order rate constants were obtained by weight loss, remaining glucose, and fitting glucose concentration profiles determined with HPLC using the Saeman model. The maximum glucose yields reached 67.6% (200 °C, 0.07% H2SO4, 30 min), 69.8% (210 °C, 0.14% H2SO4, 10 min), and 67.3% (210 °C, 0.28% H2SO4, 6 min). ELA conditions produced remarkable glucose yields when applied to bagasse cellulose. The first-order rate constants were used to calculate activation energies and extrathermodynamic parameters to elucidate the reaction mechanism under ELA conditions. The effect of acid concentration on cellulose hydrolysis and glucose decomposition was also investigated. The observed activation energies and reaction orders with respect to hydronium ion for cellulose hydrolysis an...

EFFECT OF ACID ADAPTATION AND DIFFERENT SALT CONCENTRATIONS ON SURVIVAL OF LISTERIA MONOCYTOGENES IN TURKISH WHITE CHEESE

Survival of acid-adapted and nonacid adapted Listeria monocytogenes in Turkish white cheese with different salt concentrations (3.8 and 6.7% NaCl) was investigated. Cheese blocks were inoculated with L. monocytogenes and stored at 4C for 15 days. Samples were taken on different days and analyzed for the numbers of the pathogen, and the samples were challenged with simulated gastric fluid (SGF) for determining acid tolerance. There was no significant differences between the numbers of acid-adapted and nonadapted L. monocytogenes cells regardless of the salt concentration on sampling days (P > 0.05). Challenge in SGF revealed that there was no significant difference between acid-adapted and nonadapted bacteria in cheese with 3.8% NaCl throughout the storage period, while significant (P < 0.05) differences between those cells in cheese with 6.7% NaCl were found after day 5. These results suggest that acid-adapted L. monocytogenes exposed to high salt concentration such as 6.7% may have an advantage in SGF. PRACTICAL APPLICATIONS Evaluation of potential stress responses of pathogens in food is important to understand the behavior of pathogens that can survive under stresses. As an environmental contaminant, Listeria monocytogenes may acquire adaptation to low acid conditions in cheese plants. Our results suggested that although there was no significant difference between acid-adapted and nonadapted cells both in industrial (3.8% salt) and traditional (6.7% salt) white cheese, salt concentration significantly affected the survival of acid-adapted cells in simulated gastric fluid (pH 2.3). These findings may indicate that L. monocytogenes, which is adapted to acid before contaminating high salt cheeses, may survive in stomach and cause infection. It can be said that increased salt concentration in cheese may not provide a barrier against acid-adapted L. monocytogenes.

Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

BackgroundCold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.ResultsA gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures.ConclusionBy taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.

Syntrophic acetate oxidation in two-phase (acid–methane) anaerobic digesters

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.