Characteristics of a novel bacterial polysaccharide consisted of glucose and mannose as major components

A bacterial strain, designated KMBL 5781, producing a high level of extracellular biopolymer was isolated from soil and identified as Mitsuaria chitosanitabida based on the 16S rRNA gene sequences. The biopolymer was purified by the sequential precipitation using ethanol, cethyl trimethyl ammonium bromide and then ethanol again. Its molecular weight was estimated to be about 54.7 kDa by the MALDI-TOF analysis. GC–MS analysis revealed that it is a polysaccharide consisting of glucose, mannose and galactose with approximate molar ratio of 18:6:1. In lower concentrations such as 0.5% and 1% viscosities of the EPS solution was higher than that of the xanthan gum used as a control. At the final range of shear rate the viscosity of 0.5% EPS was 34 cP while 0.5% xanthan showed 19.8 cP. The 1% EPS solution at the same rate has shown 59 cP and the viscosity of 1% Xanthan was 35 cP. When heated from 20 to 92 °C, the EPS solution (2%) remain stable from room temperature until 60 °C and showed significantly higher viscosity at 92 °C than 2% xanthan. The EPS hydrogel (2%) was strongly stable, which released no water during the incubation at 4 °C for 20 days and even after the tubes containing the gel was centrifuged at 4000× g for 10 min. Addition of the EPS into the starch solution resulted in the increase the weight and volume of the gel.     

Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.     

Antimicrobial activity against foodborne pathogens of chitosan biopolymer films of different molecular weights

Antimicrobial activity against Listeria monocytogenes, Escherichia coli 0157:H7 and Samonella typhimurium of chitosan biopolymer films (CBFs) prepared with four different viscosities of chitosans (10, 40, 100 and 200 mPa s) were investigated by agar diffusion assay. The films were also characterized with measurements of color, tensile strength (TS), % elongation (EL), water vapor permeability and oxygen permeability. CBFs prepared with 100 mPa s chitosan showed an antimicrobial effect only on 10⁴ cfu/mL inoculation of L. monocytogenes while other viscosities showed an antilisterial effect on all concentrations (10⁴-10⁶ cfu/mL) of L. monocytogenes. CBFs prepared with 10 mPa s (CBF-10) and 40 mPa s (CBF-40) chitosans showed an inhibitory effect against E. coli 0157:H7 and S. typhimurium only at the 10⁴ cfu/mL concentration. CBFs prepared with the two higher viscosity chitosans did not show any effect regardless of bacterial level. TS and EL of the CBFs increased with increasing viscosity up to 100 mPa s. Molecular weight distribution was found to be positively correlated with viscosity. The oxygen permeability of the CBFs increased with increasing viscosity of chitosans, but water vapor transmission rate was not similarly affected. In conclusion, CBFs were more effective at inhibition of L. monocytogenes than S. typhimurium and E. Coli O157:H7. Molecular weight of chitosan must be chosen selectively to control the target foodborne pathogens.     

Kinetics of inulinase production by solid-state fermentation in a packed-bed bioreactor

In this work the cell growth and inulinase production by Kluyveromyces marxianus NRRL Y-7571 were investigated in a packed-bed bioreactor. For this purpose, seven experimental runs were carried out in order to verify the influence of the inlet air temperature and volumetric air flow rate on the process dynamics. The results showed that the manipulated variables affected significantly the process performance. The best condition for enzyme production was an inlet air temperature and volumetric flow rate of 30 °C and 3 m³ h⁻¹, respectively, reaching an activity of around 463 U gds⁻¹. The results obtained here can be useful in the scale-up and optimisation of packed-bed bioreactors configuration for inulinase production.     

Preparation of microcapsules containing antimicrobial lipopeptide from Bacillus amyloliquefaciens ES-2 by spray drying

Using a fermentation broth produced by Bacillus amyloliquefaciens ES-2 as a raw material, a preparation method for antimicrobial lipopeptide powders was developed. Antimicrobial lipopeptide microcapsules were obtained by a spray-drying process using maltodextrin and porous starch as composite wall materials. Optimal encapsulation conditions comprised a ratio of wall materials to the core material of 5%, a ratio of porous starch to maltodextrin of 1/9, a drying air flow of 70 m³/h and an inlet gas temperature of 220 °C, producing a high yield of microcapsules of 63.9 ± 0.5%. The lipopeptides retained their desired bioactivity and had a spherical form after spray drying, with an antimicrobial potency of 25711.3 ± 34.5 IU/g evaluated by the cylinder-plate method, and their preservation percent was about 81.5%. Because of its low cost, high efficiency, simplicity and easy of scale-up, the technology is expected to be efficient for producing antimicrobial lipopeptide powders and to be helpful in the application of antimicrobial lipopeptides as food additives.     

Favorable effect of very low initial KLa value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii

Xylitol production from xylose by a self-isolated furfural and 5-hydroxymethyl furfural assimilating Pichia guilliermondii was studied under oxygen limitation. An extremely low initial volumetric oxygen transfer coefficient (0.075 h^− 1) was found most favorable to the xylitol production with yield of 0.61 g g^− 1. Related enzymes activities were also investigated and discussed.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

Relationships among selected variables affecting the resistance of Salmonella enterica, serovar Enteritidis to thermosonication

In order to find the relationships among variables affecting the resistance of salmonella to the thermosonication treatment, the combined effect of simultaneously applied ultrasonic waves and heat treatment on the survival of a strain of Salmonella enterica serovar Enteritidis was studied in several volumes (100, 200, 500, 750 and 1200 mL) of distilled water. The higher the liquid volume, the greater the decimal reduction times (D_R). The joint confidence regions for D_R and z were calculated showing significant (p < 0.05) differences for the different treated volumes. The influence of the applied ultrasonic power on the kinetic constants was modelled using a logarithmic equation. The sonication of whole eggs provided kinetic constants in agreement with the logarithmic model found. A model was developed to integrate all the variables (temperature, time and ultrasonic power), attaining a close fit of the experimental data (R² = 0.95). This model allows the effect of sonication to be predicted under different process conditions and, therefore, is useful for the scale-up of the system.     

Fate of biofilm cells of Cronobacter sakazakii under modified atmosphere conditions

Survival of biofilm cells of Cronobacter sakazakii formed on stainless steel and polyvinyl chloride (PVC) on exposure to different atmosphere conditions was studied. Biofilms were formed on stainless steel and PVC coupons by using three strains of C. sakazakii. Six day old biofilms on stainless steel and PVC coupons were stored under N₂ gas, CO₂ gas, and air for up to 20 days. N₂ and CO₂ gases resulted in significant (p < 0.05) further reductions of 1.79 and 2.47 log CFU/cm² after 20 days of storage, respectively, compared to air storage. N₂ and CO₂ gases led to less reduction of biofilm cells on PVC compared to those on stainless steel. N₂ and CO₂ gases resulted in significant (p < 0.05) further reductions of 0.98 and 1.20 log CFU/cm² after 20 days of storage, respectively, compared to air storage.     

Response surface methodology applied to supercritical carbon dioxide extraction of Piper nigrum L. essential oil

This study was conducted to optimize the supercritical carbon dioxide (SC-CO₂) extraction of Piper nigrum L. essential oil using response surface methodology (RSM). In order to obtain the maximum yield of the essential oil, experiments were carried out using a three-factor central composite design (CCD) under following conditions: pressure of 15–30 MPa, temperature of 40–50 °C and dynamic extraction time of 40–80 min. A second-order polynomial regression model expressing the total extraction yield as a function of main SC-CO₂ variables was significantly (p < 0.05) fitted, with high coefficient of determination value (R² > 0.985). The results showed that the best extraction yield (2.16%) was obtained at 30 MPa, 50 °C and 80 min. Pressure showed the most significant (p < 0.05) effect on the yield variation. The chemical composition of the essential oil was determined using GC-flame ionization detection (FID) and gas chromatography–mass spectrometry (GC–MS) analysis. The main constituents (concentration > 3.0%, calculated as % peak area) in the P. nigrum L. essential oil obtained through SC-CO₂ extraction were determined to be β-caryophyllene (24.34%), limonene (15.84%), sabinene (15.04%), 3-carene (9.44%), β-pinene (9.27%) and copaene (4.52%).     

Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples

Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica are the most common foodborne bacterial pathogens and are responsible for many outbreaks. Therefore, multiplex detection of these three using a single assay platform is highly desirable. The objective was to develop and optimize a fiber optic sensor for simultaneous detection of these three from food. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. Pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Ready-to-eat beef, chicken and turkey meats were inoculated with each pathogen (∼100 cfu/25 g), enriched in SEL (Salmonella, E. coli, Listeria), a multipathogen selective enrichment broth for 18 h and tested with the biosensor. The biosensor was able to detect each pathogen, individually or in a mixture with very little cross-reactivity. The limit of detection for the sensor was ∼10³ cfu/ml for all three pathogens. Furthermore, the biosensor successfully detected each pathogen, grown in a mixture from enriched meat samples under 24 h. The pathogen presence was further verified by PCR and immunofluorescence assay. The multiplex fiber optic sensor shows promise for detection of the three pathogens if present in the same sample eliminating the use of multiple single pathogen detection platforms.     

Effect of hot acidic fructose solution on caramelisation intermediates including colour,hydroxymethylfurfural and antioxidative activity changes

Fructose model solutions of various concentrations were adjusted to pH 3.0, then incubated at 75, 85, and 95 °C, respectively, for studying the reaction kinetics of colour development, hydroxymethylfurfural (HMF) production, and antioxidative activities change in the caramelisation reaction. Results showed that colour development, HMF production, DPPH radical scavenging activity and ABTS^·+ radical scavenging activity in the system increased linearly with a first-ordered kinetics on fructose concentration. Their values of Q₁₀ were 1.87, 4.48, 2.06, and 2.24, and activation energies were 66, 160, 77, and 86 kJ/mol, respectively.     

Syngas fermentation in a 100-L pilot scale fermentor: Design and process considerations

Fermentation of syngas offers several advantages compared to chemical catalysts such as higher specificity of biocatalysts, lower energy costs, and higher carbon efficiency. Scale-up of syngas fermentation from a bench scale to a pilot scale fermentor is a critical step leading to commercialization. The primary objective of this research was to install and commission a pilot scale fermentor, and subsequently scale-up the Clostridium strain P11 fermentation from a 7.5-L fermentor to a pilot scale 100-L fermentor. Initial preparation and fermentations were conducted in strictly anaerobic conditions. The fermentation system was maintained in a batch mode with continuous syngas supply. The effect of anaerobic fermentation in a pilot scale fermentor was evaluated. In addition, the impact of improving the syngas mass transfer coefficient on the utilization and product formation was studied. Results indicate a six fold improvement in ethanol concentration compared to serum bottle fermentation, and formation of other compounds such as isopropyl alcohol, acetic acid and butanol, which are of commercial importance.     

Physicochemical and functional properties of flours from three Black gram (Phaseolus mungo L.) cultivars

Flours processed from three black gram cultivars (Mash1‐1, PU‐19 and T‐9) were studied for proximate composition, physicochemical and functional properties. Protein, fat, ash and crude fibre content varied significantly (P ≤ 0.05) from 24.5 – 28.4%, 1.1–1.4%, 2.7–3.3% and 2.7–3.4%, respectively, among the cultivars. Significant differences were also observed in other physicochemical properties. Pasting properties like peak viscosity, breakdown viscosity and setback viscosity were in the range of 2078.7–2473.0 cP, 644.0–863.7 cP and 588.3–804.0 cP, respectively. Textural profile analysis of flour gels displayed significant differences in hardness (13.0–18.9 g), gumminess (2.8–5.9 g), chewiness (2.0–5.7 g) and adhesiveness (21.0–40.9 g). Differential scanning calorimetry revealed starch gelatinisation from 63.5 to 75.6 °C and amylose lipid complex melting from 105.0 to 136.1 °C. Protein solubility profile in the pH range of 2–9 varied from 6.3 to 97.3%. Emulsifying activity index and Emulsifying stability index at pH 3, 5 and 7 varied correspondingly from 6.0 to 14.5 m² g^?1 and 18.8 to 64.9 min. Foaming capacity of 2% flour suspensions at pH 2, 4, 6, 8 and 10 varied significantly from 67.3 to 130% among the cultivars. Foam stability varied at different pH and was observed between 0 and 70.8% among the cultivars.     

Lethal effects of sulfuryl fluoride on eggs of different ages and other life stages of the warehouse moth Ephestia elutella (Hübner)

Sulfuryl fluoride (SF) is a fumigant used for the control of stored-product pest insects and is a feasible alternative to some uses of methyl bromide. SF was first registered in Germany in 2004 for disinfestation of emptied flourmills, emptied food storages and dried fruit. One- to 4-day-old eggs, larvae and pupae of the warehouse moth, Ephestia elutella (Hübner) (Lepidoptera) were investigated for their susceptibility to SF under different conditions. Each life stage was exposed for 18 h, 24 h or 48 h, to 11.6 g/m³ or 21.3 g/m³ at 15 °C, 20 °C or 25 °C and 65% relative humidity. A Fourier transform infrared (FTIR) spectrometer was used to determine the concentrations of SF. Within 18 h of exposure, all larvae and pupae of E. elutella died at 11.6 g/m³ at all three temperatures. The 1 and 2- day-old eggs were generally more susceptible, whereas the 3 and 4-day-old eggs were more tolerant to the SF treatment. All eggs of all ages were controlled within 48 h of exposure to the concentration of 21.3 ± 1.3 g/m³ at temperatures of 20 °C and 25 °C.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Helvellisin,a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa

A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 °C, with about 40% activity remaining at 87 °C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14 600 U/mg toward casein. The K_m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 °C. The V_max was 5.35 × 10^− 2 mg ml^− 1 min^− 1. It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg²⁺, Fe²⁺ and Mn²⁺, but was curtailed by Cu²⁺, Hg²⁺ and Fe³⁺. It was devoid of antifungal and ribonuclease activities.     

Evidence for a role of biosurfactants produced by Pseudomonas fluorescens in the spoilage of fresh aerobically stored chicken meat

Fresh chicken meat is a fat-rich environment and we therefore hypothesised that production of biosurfactants to increase bioavailability of fats may represent one way in which spoilage bacteria might enhance the availability of nutrients. Numbers of Pseudomonas were determined on a total of 20 fresh and 20 spoiled chicken thighs with skin. A total of 400 randomly isolated Pseudomonas colonies from fresh (200) and spoiled (200) chicken were screened for the presence of biosurfactant production. Biosurfactant producing strains represented 5% and 72% of the Pseudomonas spp. isolates from fresh (mean count 2.3 log₁₀ cfu g⁻¹) and spoiled (mean count 7.4 log₁₀ cfu g⁻¹) chicken skin, respectively. Partially-purified biosurfactants derived from a subgroup of four Pseudomonasfluorescens strains obtained through the screening process were subsequently used to investigate the role that the addition of these compounds plays in the spoilage of aerobically stored chicken. Emulsification potential of the four selected biosurfactants was measured against a range of hydrocarbons and oils. All four biosurfactants displayed a greater ability to emulsify rendered chicken fat than hydrocarbons (paraffin liquid, toluene and hexane) and oils (canola, olive, sunflower and vegetable). Storage trials (4 °C) of chicken meat treated with the four selected biosurfactants revealed a significantly greater (P < 0.05) total aerobic count in biosurfactant treated samples, as compared to untreated samples on each day (0, 1, 2, 3) of storage. For biosurfactant treated samples the greatest increase in total aerobic count (1.3-1.7 log₁₀ cfu g⁻¹) occurred following one day of incubation. These results indicate that biosurfactants produced by Pseudomonas spp. may play an important role in the spoilage of aerobically stored chicken meat by making nutrients more freely available and providing strains producing them with a competitive advantage.     

A novel nanosensor based on Pt:Co nanoalloy ionic liquid carbon paste electrode for voltammetric determination of vitamin B9 in food samples

In this work we describe a novel nanoalloy (Pt:Co) room temperature ionic liquid (RTIL) modified carbon paste electrode as a high sensitive sensor for voltammetric determination of vitamin B₉ in food samples. The sensor exhibits an enhanced effectiveness for the electro-oxidation of vitamin B₉ in aqueous solution. The oxidation peak potential for this matter at a surface of the ionic liquid (n-hexyl-3-methylimidazolium hexafluoro phosphate) Pt:Co carbon paste electrode (Pt:Co/IL/CPE) appeared at 685 mV that was about 110 mV lower than the oxidation peak potential at the surface of the traditional carbon paste electrode (CPE) under similar condition. The mechanism of the electro-oxidation process on the surface of the modified electrode was analyzed. Square wave voltammetry (SWV) was applied as a very sensitive electrochemical method for the determination of sub-micro-molar amounts of vitamin B₉. The linear response range and detection limit were found to be 1.0 × 10⁻⁷ to 5.0 × 10⁻⁴ M and 4.0 × 10⁻⁸ M, respectively. The prepared modified electrode shows several advantages such as simple preparation method, high stability, high sensitivity, and excellent catalytic activity, long-term stability and remarkable voltammetric reproducibility for eletrooxidation of vitamin B₉. The proposed sensor was successfully applied for the determination of vitamin B₉ in food samples.