Comparison of Real Time Polymerase Chain Reaction Quantification of Changes in hilA and rpoS Gene Expression of a Salmonella Typhimurium Poultry Isolate Grown at Fast Versus Slow Dilution Rates in an Anaerobic Continuous Culture System

The objective of this study was to determine the genetic responses of a Salmonella enterica Typhimurium poultry isolate during low and high dilution rates (D) in steady state continuous culture (CC) incubations. Samples for genetic analyses were taken from a previous study where S. Typhimurium cells had been grown in two chemostats operated concurrently, which were designated as Trials 1 and 2, with eight dilution rates sampled during steady state. Real-time polymerase chain reaction (PCR) on two target genes (rpoS and hilA) was analyzed as changes in expression of the target gene relative to the reference gene (16S rRNA). At the lowest D (0.0125 h^?1) in Trial 2, rpoS expression was more than twofold higher than the second highest relative expression. In Trial 1 hilA expression was 21.8-, 27.8-, and 21-fold higher in D 0.0125 h^?1, 0.025 h^?1, 0.05 h^?1, respectively, compared to D 0.1 h^?1, 0.54 h^?1, and 1.08 h^?1. In Trial 2, D 0.025 h^?1 and 0.27 h^?1 showed no difference in hilA expression but were significantly higher compared to other D. From these results, low glucose conditions may play an essential role in triggering rpoS induction, as well as contributing to potential virulence.     

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL⁻¹ for 0.15 h⁻¹ or 0.25 and 0.5 h⁻¹, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL⁻¹ for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h⁻¹ during FC cultures (460 IU mL⁻¹ h⁻¹ for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL⁻¹ h⁻¹ at 2.0 h⁻¹ without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.     

Heat transfer coefficients to canned green peas during end-over-end sterilisation

The effect of temperature (110, 120 and 130 °C), rotation speed (5, 10 and 15 r.p.m.) and headspace (4, 8 and 12 mm) on heat transfer coefficients to canned green peas during end‐over‐end sterilisation was studied using response surface methodology. The models developed for fluid‐to‐particle heat transfer coefficient, h_fp, and overall heat transfer coefficient, U, were adequate, showing no significant lack of fit and satisfactory correlation coefficients. For the two responses, temperature, rotation speed and headspace have a significant effect. U, ranged between 477 and 905 W m^?2 °C^?1, while h_fp, fluctuated between 480 and 1950 W m^?2 °C^?1. The highest h_fp and U values are obtained at high temperatures, rotation speeds and headspaces. The verification of the prediction models was satisfactory. Dimensionless correlations were developed for h_fp and U, with equations showing a good agreement with the experimental data. Heat transfer to liquids and particles was modelled using the Reynolds number, the Prandtl number and adimensional headspace.     

Xylitol bioproduction from wheat straw: hemicellulose hydrolysis and hydrolyzate fermentation

A 2² central composite design with five center points was performed to estimate the effects of temperature (120, 130 and 140 °C) and acid loading (100, 150 and 200 mg g^?1) on the yield of monomeric xylose recovery from wheat straw hemicellulose (Y_S/RM). Under the best hydrolysis condition (140 °C and 200 mg g^?1), a Y_S/RM of 0.26 g g^?1 was achieved. After vacuum concentration and detoxification by pH alteration and active charcoal adsorption, the hydrolyzate was used as source of xylose for xylitol bioproduction in a stirred tank reactor. A xylitol production of 30.8 g L^?1 was achieved after 54 h^?1 of fermentation, resulting in a productivity (Q_P) of 0.57 g L^?1 h^?1 and bioconversion yield (Y_P/S) of 0.88 g g^?1. The maximum specific rates of xylose consumption and xylitol production were 0.19 and 0.15 g g^?1 h^?1, respectively. Copyright © 2006 Society of Chemical Industry     

Relative Oxidative Stability of Diacylglycerol and Triacylglycerol Oils

To compare the oxidative stability between diacylglycerol (DAG) oil and conventional triacylglycerol (TAG) oil (that is, soybean oil), the prepared stripped diacylglycerol oil (SDO) and soybean oil (SSBO) were stored at 60 °C in the dark for 144 h. During storage peroxide values (POVs), contents of aldehydes, unsaturated fatty acids were measured to evaluate the oxidative stabilities of the 2 oils. The results showed the content of C18:2, C18:3, and total unsaturated fatty acid decreased faster in DAG oil than in soybean oil, whereas the decreased rate of C18:1 was similar in 2 oils. Also, both rate constants (K₁ and K₂) obtained from POV (K₁) and total aldehydes (K₂) indicated that DAG oil (K₁ = 3.22 mmol/mol FA h^?1, K₂ = 0.023 h^?1) was oxidized more rapidly than soybean oil (K₁ = 2.56 mmol/mol FA h^?1, K₂ = 0.021 h^?1), which was mainly due to the difference of acylglycerol composition of the 2 oils along with higher C18:3 (9.6%) in SDO than SSBO (5.7%). It is concluded that DAG was more easily oxidized than soybean oil at 60 °C in the dark for 144 h.     

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

Evaluation of the conditions of carotenoids production in a synthetic medium by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor

This work studied the cultivation conditions for the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor. A Plackett–Burman design was used for the screening of the most important factors, followed by a complete second order design, to maximise the concentration of total carotenoids. The maximum concentration of 3425.9 μg L^?1 of total carotenoids was obtained in a medium containing 80 g L^?1 glucose, 15 g L^?1 peptone and 5 g L^?1 malt extract, with an aeration rate 1.5 vvm, 180 r.p.m., 25 °C and an initial pH of 4.0. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 90 h of fermentation. Carotenoid bio‐production was partially associated with cell growth. The specific carotenoid production (Y_P/X) was 238 μg carotenoids/g cells, whereas Y_P/S (substrate to product yield) was 41.3 μg g^?1. The specific growth rate (μ_x) was 0.045 h^?1. The highest cell and total carotenoid productivity were 0.19 g L^?1 h^?1 and 56.9 μg L^?1 h^?1, respectively.     

Development of active plastic crate and evaluation of phytopathogenic and pathogenic food spoilage micro‐organism inhibition

BACKGROUND: The plastic crates used in fruit and vegetable shipping can be vehicles of disease dissemination among production fields, since there is a chance of phytopathogenic micro‐organism adhesion on the crate surfaces when in contact with soil, contaminated produce or handling. The aim of this study was to develop an active plastic crate incorporated with a triclosan‐based antimicrobial agent and to evaluate its efficiency of micro‐organism inhibition. RESULTS: Staphylococcus aureus (a human pathogen), Clavibacter michiganensis ssp. michiganensis and Erwinia carotovora ssp. carotovora (phytopathogens) were the most sensitive micro‐organisms when in contact with samples of plastic crate incorporated with 30 g kg^?1 of antimicrobial agent. They presented diameters of approximately 5.0, 3.5 and 3.5 cm respectively in the halo test. Mean specific growth rates decreased in samples with 30 g kg^?1 of antimicrobial agent, compared with control samples, from 1.13 to 0 h^?1 for S. aureus, from 1.26 to 0.47 h^?1 for Escherichia coli and from 1.75 to 0.18 h^?1 for Listeria monocytogenes. The antimicrobial agent did not influence the mechanical properties of the crates. CONCLUSION: The active plastic crate has great potential to contribute to the safety of horticultural produce by restraining the proliferation of micro‐organisms among production fields. Copyright © 2008 Society of Chemical Industry     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

EFFECT OF PHYSICAL FACTORS ON ACETIC ACID PRODUCTION IN BRETTANOMYCES STRAINS

Four species of Brettanomyces (intermedius, bruxellensis, custersianus, clausenii) were examined to ascertain their acetic acid production capacity. The results showed that B. bruxellensis was the strain with the best volumetric productivity and specific production rate (P = 0.065 gL^?1 h^?1; V_p = 0.43 gg^?1h^?1). The best kinetic parameters were reached (P = 0.133 gL^?1 h^?1; Y_p/s = 0.23; P_max = 11.64 gL^?1) at an airflow of 288 Lh^?1 (0.6 vvm, OTR = 124 mgO₂L^?1 h^?1), and substrate inhibition was not observed. The influence of temperature and agitation on acetic acid production by B. bruxellensis in a glucose medium was investigated at different levels, 26, 30, 34C and 250, 350, 450 rpm, respectively. Temperature and agitation were shown to be deci‐sive factors (P < 0.05) in acetic acid production at 288 Lh^?1(0.6 vvm, OTR = 124 mgO₂L^?1 h). The optimal conditions for a high volumetric productivity were 30C and 250 rpm, respectively.     

The Influence of Oxygen Volumetric Mass Transfer Rates on Cyclodextrin Glycosyltransferase Production by Alkaliphilic Bacillus circulans in Batch and Fed-Batch Cultivations

In this work, the influence of oxygen mass transfer rates on the production of cyclodextrin glycosyltransferase (CGTase) by the alkaliphilic bacterium Bacillus circulans ATCC 21783 was investigated. Experimental design and response surface methodology were applied to optimize agitation speed and air flow rate in batch cultivations, in order to identify their significant effects and interactions with the synthesis of CGTase. Results were expressed as the volumetric mass transfer rates of oxygen (k_la, [per hour]). The maximal CGTase productivity of 155 U mL⁻¹ h⁻¹ was achieved with k_la of 48 h⁻¹. CGTase production was also studied in fed-batch cultures using the optimized parameters obtained in the batch experiments. The maximal CGTase productivity on fed-batch cultivations was 137 U mL⁻¹ h⁻¹ with feeding rates of starch at 0.17 g L⁻¹ h⁻¹.     

The effect of oxygen and redox potential on growth of Clostridium botulinum type E from a spore inoculum

Small volumes of oxygen introduced into vials of medium at pH 7 prepared under anaerobic conditions reacted with the medium during sterilization by heating, and also raised the redox potential. The partial pressure of oxygen (pO₂) in the medium, and the redox potential (E_h) were measured and their effect on the number of spores of Clostridium botulinum type E required to produce growth, and hence on the probability of growth from single spore inocula, was determined.In the presence of a pO₂ of up to 4·5 × 10⁻³ atm resulting in an E_h of c. + 217 mV, the probability of growth from single spores within 5 days at 20°C was equal to that in strictly anaerobic conditions at an E_h of − 400 mV. At pO₂ values of between 5·3 × 10⁻³ atm and 8·4 × 10⁻³ atm corresponding to E_h values of between + 226 mV and + 254 mV an inoculum of between 10 and 1000 spores was required to produce growth and at pO₂ values of between 1·12 × 10⁻² atm,and 1·6 × 10⁻² atm, corresponding to redox potentials of between + 271 mV and + 294 mV, the number of spores required to produce growth was between 2 × 10⁴ and > 2 × 10⁵. The relationship between pO₂ and E_h depends on the chemical nature of a culture medium or food, and in order to assess the probable influence of these parameters on growth of C. botulinum in a medium or a food it is necessary to determine both the redox potential and the partial pressure of oxygen.     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect in Saccharomyces cerevisiae

Mating pheromones stimulate Saccharomyces cerevisiae yeast cells to form a pointed projection that becomes the site of cell fusion during conjugation. To investigate the role of mating projections, we screened for mutations that enhanced the weak mating defect of MAT a ste2‐T326 cells that are defective in forming pointed projections. These cells are also 10‐fold more sensitive to α‐factor pheromone because ste2‐T326 encodes truncated α‐factor receptors that are not regulated properly. Mutations in AXL1, STE6 and FUS3 were identified in the screen. AXL1 was studied further because it is required for efficient a ‐factor pheromone production and for selecting the site for bud morphogenesis. Mutation of AXL1 did not enhance the morphogenesis or pheromone sensitivity defects of ste2‐T326. Instead, the synergistic mating defect was apparently due to decreased a ‐factor production because the axl1Δ ste2‐T326 cells mated well with a sst2 α mating partner that is supersensitive to a ‐factor. When combined with a wild‐type mating partner, the ste2‐T326 axl1Δ cells failed to mate because they did not lock cell walls, one of the earliest steps in conjugation. Analysis of axl1Δ in combination with other mutations that cause defects in morphogenesis or pheromone sensitivity (e.g. bar1, sst2, afr1) indicated that both phenotypes of ste2‐T326 cells, supersensitivity to α‐factor and the defect in forming pointed projections, contributed to the synergistic mating defect. We suggest a model that the synergistic mating defect is caused by the combined effects of ste2‐T326 and axl1Δ on the presentation of a ‐factor to partner cells. Altogether, these results demonstrate an important linkage between the incoming and outgoing pheromone signals during the intercellular communication that promotes yeast mating. Copyright © 1999 John Wiley & Sons, Ltd.     

Mechanical flower thinning improves the fruit quality of apples

BACKGROUND: Apple ‘Golden Delicious Reinders’ and ‘Gala Mondial’ trees were mechanically blossom‐thinned with 30–77 × g (300–480 rpm rotation) and 5 or 7.5 km h^?1 vehicle speed to improve fruit quality, minimise leaf damage, reduce hand and chemical thinning and to prevent or overcome alternate bearing; adjacent untreated or manually thinned apple trees served as controls. RESULTS: Mechanical thinning (43 × g, 360 rpm, 5–7.5 km h^?1) had a positive effect on fruit size (15% larger), firmness (8.4 in Gala vs. 7.6 kg cm^?2 in the unthinned control), sweetness (124 vs. 117 g kg^?1 sugar in the control), contained the largest malic acid content (4 g kg^?1 vs. 3.4 g kg^?1 in the control) and 17% more anthocyanin (normalised anthocyanin index = 0.8 in Gala vs. 0.7 in the control); fruit of Golden and Gala showed additionally advanced starch breakdown and ripened earlier. CONCLUSIONS: Since increases in rotor speed, viz. centrifugal force, versus increases in the vehicle speed resulted in opposing effects, an integrated coefficient of thinning (ICT) was devised with optimum values of 10–40 (at 43 × g, 5–7.5 km h^?1), where an ICT > 50 led to tree damage and ICT < 8 led to sub‐optimum thinning efficacy. Copyright © 2010 Society of Chemical Industry     

Integral utilisation of barley husk for the production of food additives

A new and effective chemical–biotechnological process for the global utilisation of barley husk (obtained from the spent grains in the brewing process) is reported. With the proposed process the three main components of the lignocellulosic residue (cellulose, hemicellulose and lignin) are utilised. A first treatment with sulfuric acid (pre‐hydrolysis) allowed the solubilisation of hemicelluloses to give xylose and glucose‐containing liquors (suitable to make fermentation media for the continuous lactic acid (LA) production with L. pentosus) and a solid phase containing cellulose and lignin. In this set of experiments, a maximum volumetric productivity (Q_P) = 2.077 g L^?1 h^?1 and product yield (Y_P/S) = 0.62 g g^?1 were obtained for a dilution rate of 0.01 h^?1. The solid residues from pre‐hydrolysis were treated with NaOH in order to increase their cellulase digestibility, and dissolve the lignin content. The cellulose residue was used as substrates for lactic acid production by simultaneous saccharification and fermentation (SSF) in media containing Trichoderma reesei cellulases and Lactobacillus rhamnosus cells using the complete MRS broth or a cheaper medium. In both cases similar LA concentrations and volumetric productivities were achieved (P = 73.4–71.0 g L^?1 and Q_P = 1.28–1.25 g L^?1 h^?1, respectively), where P is LA concentration. The lignin solution obtained after the alkaline treatment was extracted with ethyl acetate in order to obtain the phenolic components. The extract obtained at pH 3 showed three times more antioxidant activity than the one extracted at pH 12.8, with an EC₅₀ of 1.396 g L^?1 for pH 3 and 4.604 g L^?1 for pH 12.8. The best extracts showed twice antioxidant activity than BHT. Copyright © 2007 Society of Chemical Industry     

Invertase activity in fresh and processed honeys

The enzymatic activities of 147 samples of commercially produced Spanish polyfloral and monofloral honeys are reported. Important variations in invertase activity and peroxide accumulation were determined to obtain objective information related to honey quality. Variations from 4.04 (Robinia pseudoacacia) to 25.61 g sucrose hydrolysed per 100 g h⁻¹ (SN) (Castanea sativa) and from 11.31 (Erica vagans) to 45.25 µg H₂O₂ g⁻¹ h⁻¹ (Hedysarum coronarium) were detected, with a range from 10.57 to 46.2 SN in polyfloral honeys. Honeys from Erica spp had different invertase activities—high for E cinerea (18.3 SN) and low for E vagans (8.36 SN). The different heating processes of honey have been evaluated to determine the best treatment for preserving invertase activity. The hydroxymethylfurfural content is not a sufficiently good indicator of the degree of deterioration of honey. Another objective was to assess the use of invertase activity in honey quality legislation. © 2000 Society of Chemical Industry     

Pharmacokinetics of xanthohumol and metabolites in rats after oral and intravenous administration

Scope Xanthohumol (XN), a dietary flavonoid found in hops, may have health‐protective actions against cardiovascular disease and type 2 diabetes. Yet, there are limited data on the pharmacokinetics (PK) of XN. This study provides PK parameters for XN and its major metabolites in rats. Methods and results A PK study was conducted in male jugular vein‐cannulated Sprague‐Dawley rats. Rats (n = 12/group) received an intravenous (IV) injection (1.86 mg/kg BW) or an oral gavage of a low (1.86 mg/kg BW), medium (5.64 mg/kg BW), or high (16.9 mg/kg BW) dose of XN. Plasma samples were analyzed for XN and its metabolites using LC‐MS/MS. The maximum concentration (C_max) and area under the curve (AUC_{0‐96 h}) of total XN (free and conjugated) were 2.9±0.1 mg/L and 2.5±0.3 h^* mg/L in IV group, 0.019±0.002 mg/L and 0.84±0.17 h^* mg/L in the oral low group, 0.043±0.002 mg/L and 1.03±0.12 h^* mg/L in the oral medium group, and 0.15±0.01 mg/L and 2.49±0.10 h^* mg/L in the oral high group. Conclusion The bioavailability of XN is dose‐dependent and approximately 0.33, 0.13, and 0.11 in rats, for the low‐, medium‐, and high‐dose groups, respectively.     

Co-production of Lactic Acid and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> Cells from Whey Permeate with Nutrient Supplements

Lactic acid and cell production from whey permeate by Lactobacillus rhamnosus with different nutrient supplements were investigated in this study. Yeast extract was identified as the most effective nutrient affecting lactic acid production. Increase in inoculum size from 0.05% to 1% (v/v) resulted in a substantial increase in lactic acid productivity from 0.66 to 0.83 g L⁻¹ h⁻¹ (P < 0.001). The optimal temperature for lactic acid production was 37 °C, while the highest cell production was obtained at 42 °C. When whey permeate and yeast extract concentrations were 6.8% (w/v) and 3 g L⁻¹, respectively, lactic acid productivity reached 0.85 g L⁻¹ h⁻¹ after 48-h cultivation, which is 3.40 times of those without nutrient supplements.