Assessment of phenolics-enriched extract and fractions of olive leaves and their antioxidant activities

Recent studies suggest that olive leaf is a significant source of bioactive phenolic compounds comparable to olive oil and fruits. Identifying appropriate extraction methods is thus an important step to increase the yield of such bioactive components from olive leaf, which is otherwise agricultural waste. The present study evaluates phenolic contents and compositions of olive leaf extracted by several solvent methods and to further establish their antioxidant activities using various radical scavenging systems. Total flavonoid and phenolic contents were significantly higher in the 80% ethanol extract, butanol, and ethylacetate fractions than hexane, chloroform and water fractions (p < 0.05). Oleuropein was identified as a major phenolic compound with considerable contents in these major three fractions and the extract that correlated with their higher antioxidant and radical scavenging. These results indicate that olive leaf contains significant amounts of oleuropein and phenolics, important factors for antioxidant capacity, which can be substantially modified by different extraction methods.     

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.     

Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata

Proteins have been extracted from the edible seaweeds Ulva rigida Agardh and Ulva rotundata Bliding using classical or enzymatic procedures. The protocols using NaOH under reductive conditions or a two-phase system (PEG/K₂CO₃) produced the best protein yields. The cleavage or the limitation of the linkages between proteins and polysaccharides caused by these experimental conditions probably explains the efficiency of these protocols. In SDS PAGE, the protein fraction obtained after NaOH extraction from U. rotundata is characterised by the presence of three major bands with apparent molecular weights of 45 600, 31 800 and 18 600. The protein fraction from U. rigida presents two specific bands with apparent molecular weights of about 27 000 and 12 000. These fractions are mainly rich in aspartic and glutamic acids, alanine, glycine and contain few hydroxyproline residues (0.91–2.44% total amino acid content). The use of cellulase does not significantly improve the extraction of algal proteins in comparison with the blank procedure (without enzymes). The weak accessibility of the substrates in the intact cell wall could explain these experimental data. The improvement of protein yield after the use of the polysaccharidase mixture (-glucanase, hemicellulase, cellulase) partially confirms this hypothesis.     

Optimization of the Cellulase−Ultrasonic Synergistic Extraction Conditions of Polysaccharides from Lenzites betulina

Lenzites betulina has been recognized as a rich source of chemical components, including polysaccharides, sterides and sugar alcohols. In this study, cellulase?ultrasonic synergistic extraction method was applied to extract polysaccharides from L. betulina, and the response surface methodology was used to optimize the extraction conditions. The eight basic factors affecting extraction yield were evaluated by Plackett?Burman design (PBD). Then, the four important factors significantly affecting the yield of polysaccharides from L. betulina, including enzymolysis temperature, enzymolysis time, ultrasound time and ultrasound temperature, were optimized by Box?Behnken design (BBD). Maximum extraction yield of L. betulina polysaccharides was 13.64±0.09 % at a cellulase dosage of 0.8 %, enzymolysis temperature of 60 °C, enzymolysis time of 180 min, pH of 4.5, liquid‐solid ratio of 45 ml/g, ultrasound power of 300 W, ultrasound time of 20 min and ultrasound temperature of 45 °C. Subsequently, the characteristic structure of crude polysaccharides was determined by FT‐IR. Results indicated that cellulase?ultrasonic synergistic treatment is suitable for L. betulina polysaccharides extraction, and it has good prospect for development and utilization.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

Effect of cultural factors on cellulase activity and protein production by Aspergillus terreus

Protein production by Aspergillus terreus GN1 grown on 1.0% alkali-treated bagasse was studied under various cultural conditions. The maximum biomass protein content of 20.1% and protein recovery of 11.2% was obtained with an initial pH of 4.0, with 1/5 (v/v) inoculum in continuously shaken cultures grown for seven days. Protein content of the alkali-treated bagasse was 3.0%. Highest crude protein percent also corresponded with highest carboxymethyl cellulase and filter paper enzyme activities.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Meetings & Symposia

Optimization of the extraction process of phenolics from Bryophyllum pinnatum was carried out using response-surface methodology (RSM). The effect of different variables such as ratio of solvents, plant material/solvent ratio, extraction time, and temperature were investigated. An optimal phenolics yield of 7.952 mg/g gallic acid equivalence (GAE) was achieved at reduced levels of methanol/water ratio (1:1, v/v). During optimization, the product yield was enhanced by ～2-fold at reduced extraction solvent (methanol/water) up to 37%. Validation of the RSM model for extraction of total phenolic content (TPC) was confirmed by high-performance liquid chromatography (HPLC) analysis. The obtained experimental values were in good agreement with the predicted values, thereby indicating the appropriateness of the model generated. Phenolic extracts from B. pinnatum were further examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods for determining the radical scavenging activities. EC₅₀ values of B. pinnatum extracts (BPEs) obtained by these methods were in accordance with the amount of phenolics present in the extract. Significant correlation was found between total phenolic content and antioxidant activities (p < 0.05).     

Simultaneous extraction of proteins and DNA by an enzymatic treatment of the cell wall of <Emphasis Type="Italic">Palmaria palmata</Emphasis> (Rhodophyta)

The extraction of proteins and DNA from seaweed tissue is difficult due to the presence of cell wall anionic polysaccharides that, after cell disruption, remain in the extraction medium as hydrocolloidal compounds. These compounds increase medium viscosity, thus limiting access to and, consequently, quantification of the soluble macromolecules such as proteins or DNA. This study describes a protocol that enables the simultaneous isolation of proteins and DNA from the red seaweed Palmaria palmata. It is based upon a specific form of hydrolysis using a mixture of cellulase and xylanase at different concentrations. This approach was carried out on samples collected during April and July, seasons in which differences in protein content have previously been reported. Our results confirm this report and also show that protein yield depends on the enzyme concentration used. As for DNA, this enzymatic digestion results in a much higher yield compared with the control. However, no seasonal differences are found for DNA and there is no clear link between the increase in DNA yield and the enzyme mixture concentration.     

Solid-substrate fermentation of alfalfa for enhanced protein recovery

Solid-substrate fermentations for extraction of protein from pressed alfalfa residues with Aspergillus sp. QM 9994 aspergillus niger QM 877, and Rhizopus nigricans QM 387 were conducted in shake flasks. Upon reimbibing and second pressing, total protein recovery from alfalfa was increased from 47.2% for control samples and up to 64.5% for fermented samples. Analysis of juice from fermented samples indicated the presence of cellulase as well as pectinase activities. Dialysis cultures of cellulase-producing fungi showed that total biomass production and solids consumption were much higher than those of a mutant strain lacking the ability to produce cellulase, indicating significant utilization of cellulosic materials in alfalfa. The biomass yields in the former case ranged from 39–47% based on total solids consumption. Since some of the cellulosic and other carbohydrate constituents in alfalfa may be converted into fungal protein, final alfalfa residues following protein extraction in a commercial process would be less bulky for storage and handing and would be more digestible as a nonruminant animal feed.     

Properties of Rice Stem Extracts Obtained by Using Subcritical Fluids

Rice stems were subjected to a subcritical fluid treatment at 230 °C, using ethanol or acetone at a dilution of 0–100% in water. The obtained extracts were determined for their yield, carbohydrate content, phenolic content, DPPH radical scavenging ability, and color. The highest yield and carbohydrate content were achieved with the subcritical 20% (v/v) organic solvent, while the highest phenolic content was obtained with subcritical 80% (v/v) acetone. The highest radical scavenging ability was achieved with subcritical 60% (v/v) ethanol and 80% (v/v) acetone. The lightness of the extracts obtained with subcritical ethanol and acetone was negatively correlation with their radical scavenging ability (R=?0.85). The relationship between the lightness and phenolic content of the extracts was not significant, suggesting that other substances in the extract could also possess radical scavenging ability.     

Olive mill wastes: Biochemical characterizations and valorization strategies

The olive mill waste generated from olive oil extraction is a major environmental issue, particularly in Mediterranean areas. The extraction of olive oil is achieved through discontinuous or continuous processes. The two processes yield three fractions: a solid residue and two liquid phases (oil and olive mill wastewater). The characterization of these two by-products showed that they are mainly composed of phenolic compounds, carbohydrates, organic acids and mineral nutrients variably distributed depending on the process employed and the agronomic practices. Untreated olive by-products discharged between November and March into the environment are a major ecological issue for olive oil-producing countries due to their high toxic organic loads, low pH, and high chemical and biological demands. In this context, recent research studies highlight on the treatment approaches and valorization options for dealing with olive mill waste residues, predominantly those allowing for the recovery of valuable natural components such as phenolic compounds, dietary fibers, animal feed, biofuel, biogaz, enzymes, polymers and other. The impact of the chemical heterogeneity and water content of olive mill by-products about these processes of valorization and bioconversion is discussed.     

拟松材线虫纤维素酶活性与其致病性关系

【目的】研究拟松材线虫是否分泌纤维素酶以及纤维素酶与其致病力的关系。【方法】对拟松材线虫的不同致病性种群以及松材线虫的虫体蛋白提取液、分泌液的纤维素酶活性进行定量测定,比较群体间纤维素同工酶谱型差异。【结果】拟松材线虫也含有纤维素酶,并向体外分泌;且不同致病性种群的纤维素酶活性与其致病性有一定相关性,致病性越强纤维素酶活性越强。【结论】纤维素酶活性大小是导致拟松材线虫不同种群间致病性差异的重要原因,这对于全面认识松树线虫萎蔫病的致病机理有重要意义。     

An improved method for the extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex

An improved technique is described for the extraction and analysis of corticosterone (11β,21-dihydroxy-4-pregnene-3,20-dione) from homogenates and subcellular fractions of the rat adrenal cortex. Factors influencing complete extraction of corticosterone were the nature of the organic solvent system and the concentration of the tissue being extracted. The continued activity of steroidogenic enzymes during subcellular fractionation was prevented by 0.1 mM 1-benzylimidazole. For optimum extraction, homogenates were diluted 1:12 (v/v) in 0.25 M sucrose, containing 0.1 M potassium hydroxide. Dilute homogenate was mixed with absolute ethanol (1:10, v/v) and extracted three times with diethyl ether (1:5, v/v). Following extraction, corticosterone in each sample was isolated by thin-layer chromatography (TLC), quantitated by radioimmunoassay (RIA), and corrected by measuring the recovery of added ³H corticosterone. With these procedures, 90–100% of corticosterone found in extracts of adrenal homogenates was recovered in extracts of subcellular fractions of the homogenates.     

Haltbarkeit von Silagen und die sie beeinflussenden Faktoren

Content of p‐coumaric (PCA) and ferulic (FA) acid was determined by the HPLC method in fourteen forbs with a potential utilization as forages (range of nutrient content per kg DM: 100 to 244gCP, 339 to 528 g NDF and 180–369 g ADF. PCA and FA were determined after methanol extraction in four fractions: free phenolic acids extracted into ether, ester‐bound phenolic acids after alkaline hydrolysis, glycoside‐bound phenolic acids after acid hydrolysis, and cell wall‐bound phenolic acids after alkaline hydrolysis of the solid residue after the extraction with methanol.

Cell wall‐bound phenols were quantitatively the most important fraction (50% of total PCA and 47% of total FA, respectively). The differences among plant species in total PCA plus FA content were significant (F‐value 775, P < 0.01). The range of total phenol content was 31.3 to 416.3 mg/100g DM, the overall mean was 84 mg/100g DM.

Content of phenolic acids was correlated neither with ADF, NDF or ADL content (R ² = 1–3%, P > 0.05) nor with CP degradability (R ² = 3% and R ² = 1% for PCA and FA, respectively, P > 0.05).

95.4% and 30.9% of total PCA, and 98.3% and 72.5% of total FA disappeared in the rumen from the sample of Glechoma hederacea (species with the highest phenol content) and from the sample of Galium aparine (species with low phenol content), respectively, within the four hour incubation interval.

It is presumed that in comparison with grasses, PCA and FA concentration in tested forbs represents a much lower risk in potential ruminant nutrition.     

Proteomic study of a model causative agent of harmful red tide,Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis

A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Significant correlation of Cd,Cu, Pb and Zn in sediments and oysters (Saccostrea cucullata) collected from Qeshm Island,Persian Gulf,Iran

Abstract

The relationships are evaluated between the heavy metal species in sediments and the accumulation by the soft tissues of Saccostrea cucullata collected from the northern and southern coasts of Qeshm Island, Persian Gulf. The sequential extraction technique was employed to fractionate the sediment into non resistant and resistant fractions. Data from sequential extractions indicated that the metals were mainly associated with the residual fraction at the north coast and with non residual fractions at the south coast. The results showed that the best correlations were observed between heavy metals concentrations in soft tissues and the fractions in the southern sediment. Note that the bioavailability of trace metals is influenced by the chemical characteristics and properties of the sediment. Therefore, the present results generally supported the use of soft tissues of S. cucullata as a more accurate biomonitoring organism for Cu, Pb and Cd in sediments from the southern coast of Qeshm Island, Persian Gulf.     

Degradabflity and phenolic components of cell walls of wheat in relation to susceptibility to Puccinia striiforntis

Cell walls of tips and bases of laminae of flag leaves from 11 cultivars of winter wheat of varying susceptibility to Puccinia striiforntis yellow rust) were compared by examining their phenolic components released by treatment with a commercial cellulase. Isolated cell walls of both susceptible and resistant cultivars released water-soluble carbohydrate esters of phenolic acids, the major acid detected being trans-ferulic. There was no relationship between the amount of phenolic esters released by cellulase and susceptibility to yellow rust. The leaf lamina tips, which were much more susceptible to yellow rust than the corresponding bases, had lower cell wall contents than the bases and their walls were more degradable by cellulase. Examination of transverse sections of leaf laminae of cv. Little Joss showed that cells whose walls contained phenolic groups that gave a red colour reaction with p-nitrobenzene diazonium tetrafluoroborate, occupied a larger area of leaf lamina bases than of the tips. A greater percentage of the abaxial epidermal cells of the bases, compared with the tips, had walls that gave this red colour reaction. We suggest that the presence of phenolic groups in these walls may be related to the greater resistance of the bases to P. striiforntis.