Liquid hot water pretreatment of olive tree pruning residues

Olive tree pruning generates an abundant, renewable lignocellulose residue, which is usually burnt on fields to prevent propagation of vegetal diseases, causing economic costs and environmental concerns. As a first step in an alternative use to produce fuel ethanol, this work is aimed to study the pretreatment of olive tree pruning residues by liquid hot water. Pretreatment was carried out at seven temperature levels in the range 170-230 degrees C for 10 or 60 min. Sugar recoveries in both solid and liquid fractions resulting from pretreatment as well as enzymatic hydrolysis yield of the solid were used to evaluate pretreatment performance. Results show that the enzyme accessibility of cellulose in the pretreated solid fraction increased with pretreatment time and temperature, although sugar degradation in the liquid fraction was concomitantly higher.     

Quick assessment of the economic value of olive mill waste water

Background

Olive biophenols are emerging as a valued class of natural products finding practical application in the food, pharmaceutical, beverage, cosmetic and nutraceutical industries due to their powerful biological activity which includes antioxidant and antimicrobial properties. Olive mill waste water (OMWW), a by-product in olive oil manufacturing, is rich in biophenols such as hydroxytyrosol and tyrosol. The amount of biophenols depends on the cultivar, the geographical area of cultivation, and the seasonal conditions. The goal of this study was to develop a straightforward method to assess the economic value of OMWW via quantification of hydroxytyrosol and tyrosol.

Results

The amount of hydroxytyrosol and tyrosol phenolic compounds in the OMWW from four different cultivars grown in four different regions of Sicily was analyzed using liquid–liquid and solid–liquid analytical protocols developed ad hoc. Results showed significant differences amongst the different cultivars and their geographical origin. In all samples, the concentration of hydroxytyrosol was generally from 2 to 10 times higher than that of tyrosol. In general, the liquid–liquid extraction protocol gave higher amounts of extracted biophenols. The cultivar Cerasuola had the highest amount of both hydroxytyrosol and tyrosol. The cultivar Nocellara Etnea had the lowest content of both biophenols.

Conclusions

A quick method to assess the economic value of olive mill waste water via quantification of hydroxytyrosol and tyrosol in olive phenolic enriched extracts is now available.

     

Supercritical fluid extraction and HPLC determination of relevant polyphenolic compounds in grape skin

The polyphenols determined are: (+)-catechin, (-)-epicatechin, rutin, quercetin and trans-resveratrol. Suitable conditions of supercritical fluid extraction were established using ethanol as a modifier of the polarity solvent (supercritical carbon dioxide). Final extraction conditions were: 20% v/v ethanol, 60degreesC, 250 bars and flow rate 2 mL/min. Static step time and dynamic step time were established using a selected grape skin sample. The extract was collected in water; the more polar polyphenols ((+)-catechin and (-)-epicatechin) remain in solution but rutin, quercetin and trans-resveratrol precipitate in this medium, thereby the solution of the extracted polyphenols was filtered. (+)-Catechin and (-)-epicatechin were determined in the liquid fraction, while the solid fraction, containing rutin, quercetin and trans-resveratrol, was solved with ethanol/H20 (40:60). HPLC determination was carried out at C18 stationary phase, with ethanol/water/acetic acid as mobile phases and UV-visible diode array detection. Due to the significant differences between the polarity of the polyphenols, two different mobile phases were used. An ethanol/water/acetic acid (5:93:2) mobile phase was used to determine (+)-catechin (280 nm) and (-)-epicatechin (280 nm). On the other hand, rutin (254 nm), quercetin (254 nm) and trans-resveratrol (306 nm) were resolved using ethanol/water/acetic acid (40:58:2) as mobile phase. Instrumental parameters were optimised and analytical parameters obtained. The analytical method was validated and applied to five different varieties of Vitis vinifera from the geographical area of Valencia.     

Optimum extraction Process of polyphenols from the bark of Phyllanthus emblica L. based on the response surface methodology

Phyllanthus emblica L. is an economic plant used in Chinese medicine for the treatment of various diseases. The bark of P. emblica is rich in polyphenols and its extractions have shown strong antioxidative and radical scavenging activity. Response surface methodology (RSM) was used to assess the optimal extraction of polyphenols from P. emblica bark. Various extraction parameters including ethanol concentration, extraction time, temperature, solid–liquid ratio, and extraction times were chosen to identify their effects on polyphenols extraction. Among these parameters, extraction times and solvent concentration were found to have significant effect on polyphenols extraction. RSM was applied to obtain the optimal combination of solvent concentration, extraction time, temperature, and extraction time for maximum rate of extraction. The most suitable condition for the extraction of polyphenols was at ethanol concentration 75%, extraction time 25 min, extraction temperature 45°C, and extraction times 3. At these optimal extraction parameters, the maximum extraction of polyphenols obtained experimentally was found to be very close to its predicted value. The extraction rate of polyphenols was 19.78% at the optimum conditions. The mathematical model developed was found to fit with the experimental data of polyphenols extraction.     

Ultrasound-assisted extraction, HPLC analysis, and antioxidant activity of polyphenols from unripe apple

The polyphenols were extracted from the unripe apple assisted by a highly efficient and simple method of the ultrasound. Response surface methodology was used to investigate the effects of processing parameters, including ultrasound power, extraction time, temperature, and ethanol concentration on total polyphenols yield and polyphenols composition was analyzed by HPLC. Antioxidant activity of the polyphenols was evaluated as 2, 2-diphenyl-1-picrylhydracyl scavenging activity and inhibition activity of lipid peroxidation. The results showed that 10-100 times higher total polyphenols yield was obtained from the unripe apple than those from the reported apple pomace. The optimum extraction conditions were ultrasonic power of 519.39 W, extraction time of 30 min, extraction temperature 50°C, ethanol concentration of 50% gave the total polyphenols yield of 13.26 ± 0.56 mg GAE/g. HPLC analysis indicated that (-)-epicatechin, procyanidin B2, chlorogenic acid, and procyanidin B1 were the predominant polyphenols in unripe apple, which contributed to the higher antioxidant activity to 2, 2-diphenyl-1-picrylhydracyl of unripe apple polyphenols than other apple polyphenols. The extracted polyphenols had higher ability to inhibit lipid peroxidation than butylated hydroxy toluene, which demonstrated that the unripe apple polyphenols have the potential to be used as a substitute of some synthetic antioxidants.     

Ultrasound-assisted extraction of pesticides from olive branches: a multifactorial approach to method development

A rapid and simple method has been developed for the analysis in olive branches of two insecticides currently used in olive pest control, dimethoate and α-cypermethrin. The effects of analytical conditions on pesticide recovery and the optimal extraction conditions were evaluated by means of a factorial design. The use of this chemometric tool in analytical method development allows the identification of the principal and interaction effects of the extraction conditions on the recovery of pesticides. It also gives information about the location of pesticide maximum recovery with minimal experimental investment. Extraction was carried out with an ultrasonic bath and the experimental conditions studied included the volume of extractant, the time of extraction, the number of extraction steps and the sample weight. The sample was further cleaned up using a Florisil solid-phase extraction cartridge. For the overall extraction procedure, recoveries of 99 % for α-cypermethrin and 90 % for dimethoate from the spiked samples were found for 1 g of sample extracted three times with 35 mL hexane, sonicating for 2 min in each step. The complete process including ultrasonic extraction and filtration will not require more than 15–20 min, in contrast with several hours for conventional liquid–solid extraction techniques. The proposed method allows a high sample throughput, as commonly required in monitoring studies.     

Phenolic compounds and antioxidant activity of olive leaf extracts

The total phenolic content and antioxidant activities of olive leaf extracts were determined. Plant material was extracted with methanol and fractionated with solvents of increasing polarity, giving certain extracts. The qualitative changes in the composition of the extracts were determined after the storage of leaves for 22?h at 37°C, before the extraction. Total polyphenol contents in extracts were determined by the Folin-Ciocalteu procedure. They were also analysed by liquid chromatography-mass spectrometry. Their antioxidant activities were evaluated using the diphenyl picrylhydrazyl method and the β-carotene linoleate model assay. Moreover, the effects of different crude olive leaf extracts on the oxidative stability of sunflower oil at 40°C and sunflower oil-in-water emulsions (10% o/w) at 37°C, at a final concentration of crude extract 200?mg?kg(-1) oil, were tested and compared with butylated hydroxyl toluene.     

Ethanol production from olive oil extraction residue pretreated with hot water

The olive pulp fraction contained in the residue generated in olive oil extraction by a two-step centrifugation process can be upgraded by using the cellulose fraction to produce ethanol and recovering high value phenols (tyrosol and hydroxytyrosol). Olive pulp was pretreated in a laboratory scale stirred autoclave at different temperatures (150–250°C). Pretreatment was evaluated regarding cellulose recovery, enzymatic hydrolysis effectiveness ethanol production by a simultaneous saccharification and fermentation process (SSF), and phenols recovery in the filtrate. The pretreatment of olive pulp using water at temperatures between 200°C and 250°C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp at 210°C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated at 230°C.     

Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.     

含羞草中总黄酮的提取与分离纯化

确定含羞草中总黄酮的最佳提取部位,并对其初步分离纯化。通过正交试验,分别筛选含羞草根部、茎叶及种子中总黄酮的最优提取条件,比较三者黄酮总含量,最终确定提取部位为茎叶,最佳提取条件:以70%乙醇为溶剂,按照1∶8配料比,在55~58℃下超声50 min。提取液依次用石油醚、乙酸乙酯液液萃取,蒸干上样,用乙醇水溶液以4 m L/min梯度洗脱Diaion HP–20大孔树脂,收集洗脱液并用液相色谱监测每个梯度洗脱液的总黄酮含量,得到分离纯化过的黄酮类物质。当40%乙醇洗脱部位总黄酮含量最高,达57.7%。该工艺确定了含羞草中茎叶部位总黄酮含量最高,大孔树脂初步纯化黄酮类物质有效,为含羞草中黄酮类物质的应用提供了依据。     

蔗汁酒精废液色素的提取及其抗氧化活性研究

针对蔗汁酒精废液中含有的大量色素物,采用大孔树脂吸附提取色素,对蔗汁酒精废液中色素的提取工艺及其抗氧化活性进行了研究。通过静态吸附实验筛选出吸附树脂D101及对应的洗脱溶剂乙醇。最佳流速为3BV/h,最佳洗脱条件为40%的乙醇在pH6～7范围内进行洗脱。经测定,色素的黄酮含量为茶多酚的27.2%,但对自由基的抑制率却为茶多酚的88.06%,表明蔗汁酒精废液中提取的色素具有较高的抗氧化活性。     

Optimization of microwave-assisted extraction of polyphenols from apple pomace using response surface methodology and HPLC analysis

A simple and efficient microwave-assisted extraction of polyphenols from industrial apple pomace was developed and optimized by the maximization of the yield using response surface methodology. A Box-Behnken design was used to monitor the effect of microwave power, extraction time, ethanol concentration and ratio of solvent to raw material (g/mL) on the polyphenols yield. The results showed that the optimal conditions were as follows: microwave power 650.4?W, extraction time 53.7?s, ethanol concentration 62.1% and ratio of solvent to raw material 22.9:1. Validation tests indicated that the actual yield of polyphenols was 62.68±0.35?mg gallic acid equivalents per 100?g dry apple pomace with RSD=0.86% (n=5) under the optimal conditions, which was in good agreement with the predicted yield and higher than those of reflux and ultrasonic-assisted extraction methods. HPLC analysis indicated that the major polyphenols of apple pomace consisted of chlorogenic acid, caffeic acid, syrigin, procyanidin B2, (-)-epicatechin, cinnamic acid, coumaric acid, phlorizin and quercetin, of which procyanidin B2 had the highest content of 219.4?mg/kg.     

Analytical determination of polyphenols in olive oils

The increasing popularity of olive oil is mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of human disease. An overview of analytical methods for the measurement of polyphenols in olive oil is presented. In principle, the analytical procedure for the determination of individual phenolic compounds in virgin olive oil involves three basic steps: extraction from the oil sample, analytical separation, and quantification. A great number of procedures for the isolation of the polar phenolic fraction of virgin olive oil, utilizing two basic extraction techniques, LLE or SPE, have been included. The reviewed techniques are those based on spectrophotometric methods, as well as analytical separation (gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE)). Many reports in the literature determine the total amount of phenolic compounds in olive oils by spectrophometric analysis and characterize their phenolic patterns by capillary gas chromatography (CGC) and, mainly, by reverse phase high-performance liquid chromatography (RP-HPLC); however, CE has recently been applied to the analysis of phenolic compound of olive oil and has opened up great expectations, especially because of the higher resolution, reduced sample volume, and analysis duration. CE might represent a good compromise between analysis time and satisfactory characterization for some classes of phenolic compounds of virgin olive oils.     

木槿叶中混合氨基酸的提取工艺研究

以浓盐酸和乙醇溶液为提取剂,分别通过加热回流和超声波组织破碎法提取了木槿叶中的混合氨基酸,确定了氨基酸提取的最佳工艺条件。结果表明,用盐酸回流提取,盐酸与木槿干粉的液料比为25∶1,盐酸浓度6 mol/L,100℃下回流提取5 h,提取3次,氨基酸的提取效果最佳,提取率可达16.76%,产品收率可达8.6%。用80%乙醇溶液,室温下超声波提取30 min,提取3次,提取效果最好,提取率可达17.42%,产品收率可达8.93%。     

Supercritical fluid extraction of phenol compounds from olive leaves

A clean, highly selective supercritical fluid extraction (SFE) method for the isolation of phenols from olive leaf samples was examined. Total phenol extracts were determined using the Folin-Ciocalteu reagent. Dried, ground, sieved olive leaf samples (30 mg) are subjected to SFE, using carbon dioxide modified with 10% methanol at 334 bar, 100 degrees C (CO(2) density 0.70 g ml(-1)) at a liquid flow-rate of 2 ml min(-1) for 140 min. Diatomaceous earth is used to reduce the void volume of the extraction vessel. The influence of extraction variables such as modifier content, pressure, temperature, flow-rate, extraction time, and collection/elution variables, were studied. Supercritical fluid extracts were screened for acid compounds such as carboxylic acids and phenols using Electrospray-MS (in the negative ionization mode). SFE was found to produce higher phenol recoveries than sonication in liquid solvents such as n-hexane, diethyl ether and ethyl acetate. However, the extraction yield obtained was only 45%, using liquid methanol.     

Ethanol production from lignocellulosic byproducts of olive oil extraction

The recent implementation of a new two-step centrifugation process for extracting olive oil in Spain has substantially reduced water consumption, thereby eliminating oil mill wastewater. However, a new high sugar content residue is still generated. In this work the two fractions present in the residue (olive pulp and fragm ented stones) were assayed as substrate for ethanol production by the simultaneous saccharification and fermentation (SSF) process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam explosion was the most effective treatment for increasing enzymatic digestibility; however, a pretreatment step was not necessary to bioconvert the olive pulp into ethanol. Theolive pulp and fragmented olive stones were tested by the SSF process using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive stones provided SSF yields significantly lower than those obtained at standard SSF procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g of substrate.     

Bioguided extraction of polyphenols from grape marc by using an alternative supercritical-fluid extraction method based on a liquid solvent trap

The biological activity of polyphenols extracted from grape marc was studied with a view to finding a new use for this winery waste. Polyphenols were extracted by using an alternative supercritical-fluid extraction method based on the use of a liquid trap that allows extracted polyphenols to be retained in a saline buffer, thus avoiding the need for the organic solvent required to elute polyphenols from a solid trap. The major extraction variables influencing the performance of the liquid trap (viz. CO₂ modifier content, flow-rate, extraction time and trap volume) were optimized. The proposed method was applied to the supercritical-fluid extraction extraction of 0.3 g grape marc with CO₂ modified with 3% methanol at 350 bar at 50 °C (CO₂ density 0.9 g mL^-1) for 20 min, using a liquid flow-rate of 0.9 mL min^-1. The polyphenol extracts thus obtained exhibited cytotoxic effects that induced apoptosis in tumour cells.     

Ethanol production from pretreated olive tree wood and sunflower stalks by an SSF process

Olive tree wood and sunflower stalks are agricultural residues largely available at low cost in Mediterranean countries. As renewable lignocellulosic materials, their bioconversion may allow both obtaining a value-added product, for fuel ethanol, and facilitating their elimination. In this work, the ethanol production from olive tree wood and sunflower stalks by a simultaneous saccharification and fermentation (SSF) process is studied. As a pretreatment, steam explosion at different temperatures was applied. The water insoluble fractions of steam-pretreated sunflower stalks and steamed, delignified olive tree wood were used as substrates at 10% w/v concentration for an SSF process by a cellulolytic commercial complex and Saccharomyces cerevisiae. After 72-h fermentation, ethanol concentrations up to 30 g/L were obtained in delignified steam-pretreated olive tree wood at 230°C and 5 min. Sunflower stalks pretretated at 220°C and 5 min gave maximum ethanol concentrations of 21 g/L in SSF experiments.     

New possibilities for the valorization of olive oil by-products

In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200 °C (EC(50) 18.6 μg/mL) and liquid ethanol at 150 °C (EC(50) 27.4 μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542 mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819 mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves.     

Reflux Extraction Optimization and Antioxidant Activity of Phenolic Compounds from Pleioblastus amarus (Keng) Shell

Phenols were extracted from the Pleioblastus amarus (Keng) shell (PAS) using ethanol. A Plackett–Burman assessment indicated that the factors affecting polyphenol extraction included the ethanol concentration, extraction temperature, liquid to solid ratio, extraction time, and reflux extraction times; the best extraction parameters were the ethanol concentration of 75%, a 20:1 liquid to solid ratio, and an extraction time of 2.1 h. The number of polyphenols was 7.216 mg/g. Furthermore, the phenol composition analysis showed the presence of p-Coumaric acid (196.88 mg /mL) and rutin (312.9 mg /mL), which were used for the in vitro extraction and determination of the antioxidant activity. According to the A, B, C, and D antioxidant activity assays, the ethyl acetate phase was the strongest with low IC₅₀ values of 0.169 ± 0.01 mg/mL, 0.289 ± 0.01 mg/mL, 0.372 ± 0.01 mg/mL, and 1.029 ± 0.03 mg/mL, respectively, confirming high antioxidant activity. For the n-butanol and petroleum ether phases, antioxidant activity was lower. This study showed that the polyphenol extract from Pleioblastus amarus (Keng) shell displayed excellent antioxidant activity, enhancing its practical application.