Effects of Yucca schidigera extract on fermentation and degradation of steroidal saponins in the rumen simulation technique (RUSITEC)

A rumen simulation technique (RUSITEC) apparatus with eight 940 ml fermentation vessels was used to study the effects of the steroidal saponins in Yucca schidigera extract (YE) on ruminal microbial activity and saponin degradation. The YE contained approximately 4.4% (w/w) saponin, as smilagenin equivalents, and was included at 0 (control) or 0.5 mg ml⁻¹ (n=4) in the McDougall's buffer infused continuously into the vessels (dilution rate=0.75 day⁻¹). Each vessel received 5 g chopped alfalfa hay and 5 g concentrate (as-fed basis) daily for 22 days. Ammonia concentrations were lower (P<0.05) in effluent from vessels receiving YE than from controls for the first half of the study, but did not differ thereafter. Total amounts of VFA in effluent were not affected (P>0.05) by YE, but molar proportions of iso-butyric and iso-valeric acids were lower (P<0.05) in the YE vessels than in the controls in the first half of the experiment. Yucca extract at 0.5 mg ml⁻¹ did not affect (P>0.05) dry matter disappearance (DMD) from hay or from concentrate, nor did it affect total gas or methane production, or bacterial numbers (total or cellulolytic populations) in homogenates prepared from fermenter vessel liquid and feed particles. Protozoal numbers in the homogenates were substantially reduced (P<0.01) by YE (at 0.5 mg ml⁻¹), protease activity was increased (P<0.05), deaminase activity and activity against Ala₂ were unaffected (P>0.05) and activity against Ala₅ was reduced by 25% (P>0.05). When the homogenates from control and YE-supplemented (0.5 mg ml⁻¹) vessels were used to inoculate roll tubes containing 0 or 5 mg ml⁻¹ of YE, fewer colonies developed (P<0.01) in roll tubes containing YE than in those without YE, irrespective of the source of inoculum. Homogenates were also assayed for saponin degradation and for protease, peptidase and deaminase activities. Inoculum from the vessels receiving YE degraded saponin slightly during a 2 h incubation. Yucca extract at 0.5 mg ml⁻¹ altered proteolytic activity and reduced protozoal numbers, but did not affect DMD or bacterial activity, and did not induce resistance to YE at a concentration of 5 mg ml⁻¹.     

Production of a new exopolysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682 grown on glycerol

Pure glycerol and glycerol-rich product (GRP) obtained from the biodiesel industries were used as carbon source for the production of a new extracellular polysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682. The influence of temperature (20–40 °C) and pH (6.0–8.0) was studied. A temperature of 30 °C and pH control at 6.8 gave the maximum cell growth and EPS production. The culture attained a maximum cell dry weight (CDW) of 9.55 g l⁻¹ and an EPS concentration of 11.82 g l⁻¹ when cultivated with pure glycerol. GRP was a suitable carbon source, as shown by the slightly higher EPS concentration (12.18 g l⁻¹). The EPS productivity obtained with GRP (3.85 g l⁻¹ d⁻¹) was almost twice that obtained with pure glycerol (2.00 g l⁻¹ d⁻¹). Also, the yield on glycerol was higher for the cultivation with GRP (0.36 g g⁻¹) than for pure glycerol (0.28 g g⁻¹). The EPS was a high molecular weight heteropolysaccharide, composed by neutral sugars (37–80 wt% galactose, 2–30 wt% glucose, 0.5–25 wt% mannose and 0.5–20 wt% rhamnose) and containing acyl group substituents (pyruvil, acetyl and succinyl were identified). The EPS forms highly viscous aqueous dispersions with many potential commercial applications.     

Comparison of two matrices for selective recovery of C595 diabody fragment (dbFv) from Escherichia coli lysates

A comparative study of the performance of two types of adsorbent (Streamline Quartz Base and Upfront Matrices), derivatized with the same affinity ligand (RPAP) to recover C595 diabody fragment (dbFv) from Escherichia coli lysates has been undertaken. Both streamline and Upfront Matrices are characterized by a particle size range of 100–300 μm. Streamline has a density of 1.20 g cm⁻³ and ligand concentration of 0.85 μmol ml⁻¹. Upfront has a density of 1.35 g cm⁻³ and ligand concentration of 0.83 μmol ml⁻¹. The release of C595 dbFv from E. coli cells was achieved by a chemical lysis method. The recovery performance of both adsorbents was evaluated in terms of operational productivity and elution yield of C595 dbFv in packed bed (clarified feedstock) and expanded bed (unclarified and clarified feedstock) chromatography systems. Streamline and Upfront adsorbents exhibited diabody operational productivities of 131 and 202 mg l⁻¹, respectively, with an elution yield of 92 and 94%, respectively, in packed bed operation. Streamline and Upfront adsorbents exhibited diabody operational productivities of 54.5 and 123.7 mg l⁻¹, respectively, with an elution yield of 89 and 92%, respectively, in expanded bed operation.     

Mathematical analysis of trickling filter response to pentachlorophenol shock load

The response of a laboratory trickling filter to a step increase in pentachlorophenol (PCP) feed concentration was analyzed using continuous stirred tank (CSTR) and plug flow reactor (PFR) models. The CSTR model provided a slightly better fit to experimental data than the plug flow model when specific growth rate, μ, and PCP-degrading biomass concentration before the shock load, X₀, were variable parameters but was clearly superior when the mean residence time, τ, was added as a third parameter. The three-parameter CSTR model accurately represented six of seven concentration response curves corresponding to step increases in PCP feed concentration of 12–165 mg l⁻¹ and 20–150 mg l⁻¹. The continuing improvement in system response to repetitive 20–150 mg l⁻¹ shock loads was reflected by a monotonic increase in the optimal estimates of initial rate of biomass production.     

Effect of soybean oil and Tween 80 on the production of botryosphaeran by Botryosphaeria rhodina MAMB-05

The addition of soybean oil and Tween 80 was evaluated with the objective of increasing the production of botryosphaeran, an exopolysaccharide (EPS) of the (1 → 3;1 → 6)-β-d-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. Factorial design and analysis by response surface methodology was developed to select the main factors that would affect and enhance EPS production. The optimized culture conditions were: 40 g l⁻¹ glucose with 10 ml l⁻¹ soybean oil, and 4.5 ml l⁻¹ Tween 80, during 72 h cultivation at 28 °C (180 rpm) and initial pH 5.7. The predicted result for botryosphaeran production was 8.22 ± 1.36 g l⁻¹, and compared with the experimental value of 7.74 ± 0.13 g l⁻¹. Partial characterization of the botryosphaeran produced under the optimized conditions showed one type of polysaccharide with β-glycosidic linkages containing glucose as monosaccharide.     

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli. Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg⁻¹, was towards polygalacturonic acid. Significant ramie (Boehmeria nivea) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml⁻¹ with a productivity of 48.3 U ml⁻¹ h⁻¹ under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml⁻¹ with a productivity of 57.5 U ml⁻¹ h⁻¹ after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l⁻¹ in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Selenium uptake,speciation and stressed response of Nicotiana tabacum L.

The research on the function and mechanism of selenium (Se) is of great significance for the development of Se-enriched agricultural products. In this paper, uptake, speciation distribution, the effects on the flue-cured tobacco growth and antioxidant system of Se at different levels (0–22.2 mg Se kg⁻¹) were studied through a pot experiment, aiming to clarify flue-cured tobacco's response to Se stress and the relationship between Se speciation and antioxidant system. The results showed that the leaf area and number, the biomass and the chlorophyll content reached the maximum at 4.4 mg kg⁻¹ of Se treatment. Selenium at low levels (≤4.4 mg kg⁻¹) stimulated the growth of flue-cured tobacco by elevating the capability of antioxidant stress and reducing the malondialdehyde (MDA) content to 0.6–0.8 times of that of the control. However, high Se levels (≥11.1 mg kg⁻¹) depressed the capability of antioxidant stress and raised the MDA content to 1.5-fold of that of the control, and meanwhile the biomass of the aboveground parts and underground parts declined notably. The Se content in different parts of flue-cured tobacco significantly increased with the growth of Se levels. The range of Se content in roots, leaves and stems at 2.2–22.2 mg kg⁻¹ of Se treatment were 16.7–58.6 mg kg⁻¹, 2.6–37.3 mg kg⁻¹ and 2.2–10.3 mg kg⁻¹, respectively. According to the detection of different Se speciation, only selenocysteine (SeCys) was detectable in leaves at 2.2 mg kg⁻¹ Se treatment; SeCys, selenite [Se(IV)]and selenate [Se(VI)] were detected in flue-cured tobacco leaves at Se treatment (≥4.4 mg kg⁻¹), which accounted for 4.6–10%, 9–18.7% and 71–86% respectively; SeCys, selenomethionine (SeMet) and Se(IV) were detected in roots, and organic selenium(66–84%) was the main Se species at Se ≤ 11.1 mg kg⁻¹ treatment; four Se species [SeCys, SeMet, Se(IV) and Se(VI)] were detected in flue-cured tobacco roots, and the main Se species was inorganic Se (60%) at 22.2 mg kg⁻¹ Se treatment. That was to say, the percentage of organic Se species (SeCys and SeMet in flue-cured tobacco leaves and root) declined, whereas the ratio of inorganic Se species [Se(IV) and Se(VI)] increased with the growth of Se levels. The correlation analysis showed that the superoxide dismutase (SOD) activity as well as the glutathione (GSH) and MDA contents were positively correlated with the Se(IV) and Se(VI) contents at P < 0.01 and excessive inorganic Se might destruct the reactive oxygen species (ROS) balance and enhance the MDA content, thus causing damage to the plant growth. In a word, the present study suggested that the ratio of inorganic Se [Se(IV) and Se(VI)] was closely related with the growth and the antioxidant capacity of flue-cured tobacco and the excessive application of Se led to the higher proportion of inorganic Se and poorer antioxidant capacity, which ultimately inhibited the growth of flue-cured tobacco.     

Treatment of dye-rich wastewater by an immobilized thermophilic cyanobacterial strain: Phormidium sp.

The removal of Remazol Blue and Reactive Black B by the immobilized thermophilic cyanobacterial strain Phormidium sp. was investigated under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye removal. In the experiments, performed at pH 8.5, with different initial dye concentrations between 9.1 mg l⁻¹ and 82.1 mg l⁻¹ and at 45 °C, calcium alginate immobilized Phormidium sp. showed high dye decolorization, with maximum uptake yields ranging from 50% to 88% at all dye concentrations tested. When the effects of high dye concentrations on dye removal were investigated, the highest uptake yield in the beads was 50.3% for 82.1 mg l⁻¹ Remazol Blue and 60.0% for 79.5 mg l⁻¹ Reactive Black B. The highest color removal was detected at 45 °C and 50 °C incubation temperatures for all dye concentrations. As the temperature decreased, the removal yield of immobilized Phormidium sp. also decreased. At about 75 mg l⁻¹ initial dye concentrations, the highest specific dye uptake measured was 41.29–41.17 mg g⁻¹ for Remazol Blue and 47.69–43.82 mg g⁻¹ for Reactive Black B at 45 °C and 50 °C incubation temperatures, respectively, after 8 days incubation.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Simple GC-FID method development and validation for determination of α-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min^− 1. Calibration curves were linear over the concentration range 1–30 μg ml^− 1 (for standard solutions and solutions without endogenous α-tocopherol in plasma) and 5–34 μg ml^− 1 (for solutions with endogenous α-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml^− 1 and 0.30 μg.ml^− 1, respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Evaluation of phosphorus distribution and bioavailability in sediments of a subtropical wetland reserve in southeast China

The phosphorus (P) fractions and bioavailable P in the sediments from the Quanzhou Bay Estuarine Wetland Nature Reserve were investigated using chemical extraction methods for the first time to study the distribution and bioavailability of P in the reserve sediments. A hypothesis was presented suggesting that the bioavailable P in the sediments could be evaluated using the P fractions. The total phosphorus (TP), inorganic phosphorus (IP), organic phosphorus (OP), non-apatite phosphorus (NAIP), and apatite phosphorus (AP) contents in the sediments were in the ranges of 303.87–761.59 mg kg⁻¹, 201.22–577.66 mg kg⁻¹, 75.83–179.16 mg kg⁻¹, 28.86–277.90 mg kg⁻¹, and 127.36–289.94 mg kg⁻¹, respectively. The water soluble phosphorus (WSP), readily desorbable phosphorus (RDP), algal available phosphorus (AAP), and NaHCO₃ extractable phosphorus (Olsen-P) contents in the sediments were in the ranges of 0.58–357.17 mg kg⁻¹, 80.77–586.75 mg kg⁻¹, 1.09–24.12 mg kg⁻¹, and 54.96–676.82 mg kg⁻¹, respectively. The correlation analysis results showed that the NAIP was the major component of the bioavailable P and that the impact of the AP on the bioavailable phosphorus may be minimal. Due to the low TP content in the sediments of the Quanzhou Bay Estuarine Wetland Nature Reserve, the potential pollution risks of P in the sediments may not be very high. The results also show that the bioavailable P concentrations in the sediments of the Quanzhou Bay Estuarine Wetland Nature Reserve could not be evaluated by measuring the P fractions and that the hypothesis was untenable.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Chromate reduction by resting cells of Achromobacter sp. Ch-1 under aerobic conditions

Chromate reduction was carried out by resting cells of Achromobacter sp. Ch-1 with lactate as electron donor under aerobic conditions. The reduction activity of the samples supplemented with lactate was two times as those without lactate. The reduction rate was influenced by initial pH and lactate concentration. Under the optimal conditions, pH 9.0 and 4000 mg l⁻¹ lactate supplement, reduction rate was 5.45 mg l⁻¹ min⁻¹. The reduction rate decreased with increasing of Cr(VI) concentrations and increased with cell densities proportionally. The maximum reduction limit of Ch-1 cells was obtained at 2107 mg l⁻¹ of Cr(VI).     

Multiple regeneration pathways in Spathoglottis plicata Blume — A study in vitro

Asymbiotic germination of immature seeds (embryos), and mature seeds and micropropagation of Spathoglottis plicata were described. Effects of three nutrition media namely, Murashige & Skoog (MS); Phytamax (PM); and Phyto-Technology orchid seed sowing medium (P₇₂₃), two carbon sources such as glucose and sucrose at 2–3% (w/v), two plant growth regulators such as 6-benzylaminopurine (BAP; 0.5–3.0 mg l^− 1) and α-naphthalene acetic acid (NAA; 0.5–2.0 mg l^− 1) and peptone (2.0 g l^− 1) were examined on seed germination, early protocorm development and micropropagation. The maximum germination of mature seeds (95%) was recorded in PM medium supplemented with 2% (w/v) sucrose + 2.0 g l^− 1 peptone. For germination of embryos P₇₂₃ medium supplemented with 1.0 mg l^− 1 BAP proved best. Multiple shoot buds or protocorm-like bodies (PLBs) were produced from stem segments of in vitro raised seedlings. Both direct organogenesis and embryogenesis were observed and the morphogenetic response was initiated by different concentrations and combinations of PGRs. The optimum PGR combination for maximal PLB regeneration was 1.0 mg l^− 1 NAA + 2.5 mg l^− 1 BAP, while 1.0 mg l^− 1 NAA + 1.0 mg l^− 1 BAP for shoot bud development. Strong and stout root system was induced in half strength PM medium supplemented with 0.5 mg l^− 1 IAA. The well-rooted plantlets were transferred to pots containing a potting mixture composed of saw dust, coconut coir, humus, and coal pieces at 1:1:1:2 (w/w) with 80% survival in outside environment and flowered after two years of transfer.     

Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Effects of salt stress on volatile compounds,total phenolic content and antioxidant activities of Salvia mirzayanii

Salvia mirzayanii is a medicinal and aromatic plant belonging to the Lamiaceae family, which is an endemic plant in Iran. In this study, the effects of different salt concentrations on total phenolic content, antioxidant activities and volatile components of the aerial parts of S. mirzayanii were studied. The results showed that total phenolic content increased with the increase in salt concentration. The increase was more pronounced under moderate salinity (3.8 mg GAE g^− 1 DW at 6.8 dS m^− 1 NaCl). Plants grown at 6.8 dS m^− 1 NaCl displayed the highest DPPH˚ scavenging activity with the lowest IC₅₀ value (2.13 mg ml^− 1) compared to the control. The volatile components were identified and analyzed by HS (headspace)-GC–MS using the Combi PAL System technique. The main components of control plants were α-terpinyl acetate, 1,8-cineole and bicyclogermacrene. The proportions of these main compounds were differently affected by salinity stress. The results showed that the synthesis of both total phenolic and some important volatile components was induced by moderate salinity.     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.