Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method

A new spectrophotometric assay for the determination of the polyphenolic content of olive oil is presented. It is a substrate-recycling assay for phenolic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The method offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenolic compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Finally, the enzymatic method demonstrates that there is a linear relationship between the olive oil phenolic content and the antioxidative capacity of oil extracts.     

High-performance liquid chromatography-mass spectrometry profiling of phenolic compounds for evaluation of olive oil bitterness and pungency

Bitterness and pungency are important parameters for olive oil quality. Therefore, two instrumental methods for evaluation of these taste attributes were developed. The first one is based on the photometric measurement of total phenolic compounds content, whereas the second one is based on the semiquantitative evaluation of hydrophilic compounds by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Evaluation of total phenolic compounds content was performed by a modified method for the determination of the K(225) value using a more specific detection based on the pH value dependency of absorbance coefficients of phenols at λ = 274 nm. The latter method was not suitable for correct prediction, because no significant correlation between bitterness/pungency and total phenolic compounds content could be found. For the second method, areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method were correlated with the bitterness and pungency by partial least-squares regression. Six compounds (oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester) show high correlations to bitterness and pungency. The computed model using these six compounds was able to predict bitterness and pungency of olive oil in the error margin of the sensory evaluation (±0.5) for most of the samples.     

Rapid and complete extraction of phenols from olive oil and determination by means of a coulometric electrode array system

Extraction methods to determine olive oil phenols are not exhaustive. A procedure to test their effectiveness, based on the treatment of the extracted oil with 2 N HCl followed by analysis of phenols in the aqueous phase, has been developed. It was concluded, using this test, that 15-40% of phenols remained unextracted when the liquid/liquid extraction method with 80% methanol was applied. Solid phase extraction (C(18) cartridge) succeeded in retaining most of the phenols in the cartridge, but the recovery yield from the sorbent material was low. However, a new extraction method, based on the use of N,N-dimethylformamide (DMF) as an extraction solvent, achieved a complete extraction of phenols from oils. The proposed method requires a lower amount of oil, solvents, energy, and labor than the traditional ones. Because of the low concentration of phenols in the DMF extract, the highly sensitive electrochemical detector (EC) technique was studied. All of the phenols detected by the traditional UV detectors were also detected by EC using a coulometric array system. A rapid and complete analytical methodology of phenols in olive oil has been proposed based on coupling DMF extraction and EC detection.     

Biophenols in table olives

Unprocessed olives are well-known sources of phenolic antioxidants with important biological properties. Processing methods to prepare table olives may cause a reduction of valuable phenols and may deprive the food of precious biological functions. The present work was undertaken to evaluate table olives produced in Greece as sources of biophenols. Commercially available olives were analyzed for their total phenol content by using the Folin-Ciocalteu reagent and for individual phenols by RP-HPLC. Samples were Spanish-style green olives in brine, Greek-style naturally black olives in brine, and Kalamata olives in brine. Most of the types of olives analyzed were found to be good sources of phenols. Hydroxytyrosol, tyrosol, and luteolin were the prevailing phenols in almost all of the samples examined. High levels of hydroxytyrosol were determined mainly in Kalamata olives and Spanish-style green olives, cultivar Chalkidiki (250-760 mg/kg).     

落叶松木材生物油组分分析和表征

为了更加合理、高效的利用落叶松木材快速热解生物油与酚醛树脂制备新型胶黏剂,必须对快速热解生物油的主要组分进行全面、透彻的分析。本文采用气质联用仪（GC-MS）对生物油组分进行定性分析;采用气相色谱（GC）重点对生物油中的酚类物质进行定量分析;采用傅立叶变换红外光谱（FT-IR）对生物油旋转蒸发后得到的物质（重质油）进行结构表征。通过分析得到生物油组分主要包括羧酸类、醚类、酚类、醇类、醛类、烷烃类等有机化合物。结果表明,不同工况条件下生物质油中酚类物质质量分数为4%～15%,最大值为14.15%。     

Chloroguaiacols Change Some Antioxidative Parameters and Affect the Activity of Glutathione S-transferase in the Leaves of Reed Canary Grass (Phalaris arudinacea)

In this work, we evaluated the effect of harmful ecotoxins, 4,5,6-trichloroguaicol (4,5,6-TCG) and tetrachloroguaiacol (TeCG), on the oxidation of the fluorescent probe dihydrorhodamine 123, the content of free phenols and the level of the total, oxidized and reduced glutathione in the leaves of reed canary grass (Phalaris arudinacea). Furthermore, the effect on the activity of guaiacol peroxidase and glutathione S-transferase was investigated. Both 4,5,6-TCG and TeCG increased the activity of guaiacol peroxidase and glutathione S-transferase, they also elevated the content of free phenols and the level of the total glutathione. A stronger effect was exerted by tetrachloroguaiacol, which strongly increased the level of the total glutathione and the content of free phenols on the 3rd and 6th day of the experiment. The activity of glutathione S-transferase was more intensively induced by trichloroguaiacol. Both 4,5,6-TCG and TeCG oxidized dihydrorodamine 123 and the effect was stronger in the presence of magnesium ions.     

Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures

The extraction procedures (solid/liquid SPE and liquid/liquid LLE) and HPLC separation and quantification methods of polyphenolic compounds have been checked in virgin olive oils in order to explain the differences in content reported in the literature. The work has been carried out on oils prepared from one cultivar and produced under the same protocol. The extraction methods are practically equivalent, but the SPE technique is more favorable because it is faster and simpler. It has been proved that the chromatographic features and the method of chemical expression of the concentrations may greatly affect the final values. Thus, under the same analytical method, the total concentration values of polyphenols of the same oil show variations from 18% to 80%, according to the formality of expression as gallic acid, caffeic acid, or tyrosol equivalents. The role of the nature and spectrophotometric features of the phenols and of the internal standard is also discussed, and it was found to be an important source of reported variation. A gradient separation with an eluent mixture acetonitrile-sulfuric acid (0.1 mol/L), detection at 225 nm, and quantitative calculation of polyphenolic compounds in oils (expressed as tyrosol equivalents, THY(eq)) is proposed.     

石榴果实发育期果皮褐变及相关酶活性变化

为探明石榴果实发育期果皮褐变及其相关酶活性、总酚和抗氧化能力的变化规律及相互关系,以泰山红和泰山三白甜石榴为试材,研究各发育期果皮褐变度、多酚氧化酶(PPO)活性、过氧化物酶(POD)活性、苯丙氨酸解氨酶(PAL)活性、总酚含量和DPPH自由基清除率的变化。结果表明,2个品种果皮褐变度、PPO活性、POD活性、总酚含量和DPPH自由基清除率随着发育天数的增加逐渐降低,均在7月15日达到最高。2个品种果实发育期果皮PAL活性呈先降低后升高再降低的变化趋势,分别在7月15日和9月13日出现峰值。泰山红抗褐变能力高于泰山三白甜,二者分别在9月23日和9月13日采收能减轻褐变的发生。相关性分析表明,PAL、PPO、POD、总酚和DPPH自由基清除率均与石榴果实酶促褐变密切相关,石榴果实酚类物质含量高低与其抗氧化能力极显著正相关。本研究结果为深入揭示石榴果实褐变机理提供了理论依据。     

Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.     

Quantitative analysis of red wine tannins using Fourier-transform mid-infrared spectrometry

Tannin content and composition are critical quality components of red wines. No spectroscopic method assessing these phenols in wine has been described so far. We report here a new method using Fourier transform mid-infrared (FT-MIR) spectroscopy and chemometric techniques for the quantitative analysis of red wine tannins. Calibration models were developed using protein precipitation and phloroglucinolysis as analytical reference methods. After spectra preprocessing, six different predictive partial least-squares (PLS) models were evaluated, including the use of interval selection procedures such as iPLS and CSMWPLS. PLS regression with full-range (650-4000 cm(-1)), second derivative of the spectra and phloroglucinolysis as the reference method gave the most accurate determination for tannin concentration (RMSEC = 2.6%, RMSEP = 9.4%, r = 0.995). The prediction of the mean degree of polymerization (mDP) of the tannins also gave a reasonable prediction (RMSEC = 6.7%, RMSEP = 10.3%, r = 0.958). These results represent the first step in the development of a spectroscopic methodology for the quantification of several phenolic compounds that are critical for wine quality.     

Influence of industrial and alternative farming systems on contents of sugars, organic acids, total phenolic content, and the antioxidant activity of red beet (Beta vulgaris L. ssp. vulgaris Rote Kugel)

The contents of sugars, organic acids, total phenolic content, and the antioxidant activity were quantified in the flesh of red beet from conventional (CON), integrated (INT), organic (ORG), biodynamic (BD), and control farming systems using established methods. Significant differences were measured for malic acid, total phenolic content (TPC), and total antioxidant activity, where malic acid content ranged from 2.39 g kg(-1) FW (control) to 1.63 g kg(-1) FW (CON, ORG, and INT). The highest TPC was measured in BD and control samples (0.677 and 0.672 mg GAE g(-1), respectively), and the lowest in CON samples (0.511 mg GAE g(-1)). Antioxidant activity was positively correlated with TPC (r2=0.6187) and ranged from 0.823 μM TE g(-1) FW to 1.270 μM TE g(-1) FW in CON and BD samples, respectively, whereas total sugar content ranged from 21.03 g kg(-1) FW (CON) to 31.58 g kg(-1) FW (BD). The importance of sugars, organic acids, phenols, and antioxidants for human health, as well as for plant resilience and health, gained from this explorative study, is discussed and put into perspective.     

Phenol antioxidant quantity and quality in foods: fruits.

The free and bound phenols have been measured in 20 fruits commonly consumed in the American diet. Phenols were measured colorimetrically using the Folin-Ciocalteu reagent with catechin as the standard after correction for ascorbic acid contribution. On a fresh weight basis, cranberry had the highest total phenols, and was distantly followed by red grape. Free and total phenol quality in the fruits was analyzed by using the inhibition of lower density lipoprotein oxidation promoted by cupric ion. Ascorbate had only a minor contribution to the antioxidants in fruits with the exception of melon, nectarine, orange, white grape, and strawberry. The fruit extracts' antioxidant quality was better than the vitamin antioxidants and most pure phenols, suggesting synergism among the antioxidants in the mixture. Using our assay, fruits had significantly better quantity and quality of phenol antioxidants than vegetables. Fruits, specifically apples and cranberries, have phenol antioxidants that can enrich lower density lipoproteins and protect them from oxidation. The average per capita consumption of fruit phenols in the U.S. is estimated to be 255 mg/day of catechin equivalents.     

快速脱水抑制葡萄干制过程中膜脂过氧化及褐变

为探索脱水速度对无核白葡萄干制过程中膜脂过氧化作用及褐变的影响,以新疆无核白葡萄为试验材料,经过葡萄促干剂处理后,采用快速脱水和缓慢脱水2种处理,质量每减轻10%进行取样,测定脱水过程中果实干基含水率、干燥速率、褐变度、细胞膜透性、丙二醛(malonaldehyde,MDA)含量、脂氧合酶(lipoxygenase,LOX)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、过氧化物酶(peroxidase,POD)、多酚氧化酶(polyphenol oxidase,PPO)活性以及总酚含量的变化。结果表明:与缓慢脱水相比,快速脱水处理显著(P0.05)降低了无核白葡萄褐变度的上升,减少MDA生成量以及膜透性的增加,抑制LOX活性的升高,保持较高的活性氧清除酶SOD、CAT及POD活性及较高的总酚含量,且使PPO活性保持在一个较低的水平。因此认为,快速脱水可以有效地抑制无核白葡萄的膜脂过氧化作用对细胞膜的破坏,保持细胞的完整性,且PPO活性较低,从而减少无核白葡萄脱水褐变的发生。研究结果为快速脱水在无核白葡萄干制中的应用提供参考。     

用毛细吸渗原理快速测量土壤田间持水量的研究

田间持水量是衡量田间土壤保持水分能力的重要指标，视为对作物有效的土壤水分的上限，对农田灌溉和作物水分管理具有十分重要意义。该研究提出了能快速测量土壤田间持水量的新型毛细吸渗法。该方法以田间持水量为无地下水影响下土壤基质所能吸持的最大含水量为物理基础，设计了一套满足田间持水量要求的实验方法、装置和程序，采用5种不同土壤测量了其田间持水量，并与用威尔科克斯法测量得到的田间持水量进行了对比。结果表明：所提出的新型测量方法，无论是基本原理还是室内试验均可行。与威尔科克斯法相比，其测量值略微偏小；两种测量值相关性很好，相差约为9％，产生这种现象的原因主要是土壤的吸湿过程与脱湿过程中土壤含水率的滞后所致。与传统的测量方法相比，该方法可大大缩短测量时间，具有利用前景。     

弱光条件下光质和光周期对水培生菜生长与品质的影响

在弱光条件的人工光植物生长室中,设置两种不同红蓝光比例光质和两种不同光周期,水培盆栽红叶生菜（R）、绿叶生菜（G）和紫叶生菜（P）,采收后测定3种生菜的生物量指标以及抗氧化物、可溶性糖等营养品质指标,以研究弱光条件下光质和光周期对3种叶色水培生菜产量和品质的影响。结果表明：（1）生菜产量主要受光周期影响,营养品质指标受光质和光周期的综合影响。光照时间延长显著增加了3种生菜叶面积。绿叶生菜和红叶生菜单位叶面积鲜重在不同光质及光周期下无显著差异。紫叶生菜单位叶面积鲜重对光周期变化的反应则因光质环境而异。延长光周期使3种叶色生菜地上部鲜重提高一倍多,但3种生菜地上部鲜重受光质影响不显著。（2）延长光照时长对3种生菜营养品质的影响因光质而异。在红蓝1:2光质下,延长光照时长降低了绿叶生菜总酚和类黄酮相对含量以及抗坏血酸和可溶性蛋白含量,提高了红叶生菜花青素和可溶性糖含量,但降低了抗坏血酸和可溶性蛋白含量,紫叶生菜总酚相对含量得到提高,但抗坏血酸含量显著降低。在红蓝2:1光质下,延长光照时长显著提高了绿叶生菜总酚和类黄酮相对含量,抗坏血酸含量有所降低,红叶生菜总酚和类黄酮相对含量、抗坏血酸、可溶性糖和可溶性蛋白含量均显著降低,紫叶生菜总酚含量显著提高。说明在弱光条件下延长光照时长可以显著提高3种不同叶色生菜产量。在红蓝1:2光质下,延长光照时长使红叶生菜的花青素含量和可溶性糖含量得到显著提高。在红蓝2:1光质下,延长光照时长可以提高绿叶生菜和紫叶生菜营养品质。     

Phenolic compounds profile of cornicabra virgin olive oil

This study presents the phenolic compounds profile of commercial Cornicabra virgin olive oils from five successive crop seasons (1995/1996 to 1999/2000; n = 97), determined by solid phase extraction reversed phase high-performance liquid chromatography (SPE RP-HPLC), and its relationship with oxidative stability, processing conditions, and a preliminary study on variety classification. The median of total phenols content was 38 ppm (as syringic acid), although a wide range was observed, from 11 to 76 ppm. The main phenols found were the dialdehydic form of elenolic acid linked to tyrosol (p-HPEA-EDA; 9 +/- 7 ppm, as median and interquartile range), oleuropein aglycon (8 +/- 6 ppm), and the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA; 5 +/- 8 ppm). In many cases the correlation with oxidative stability was higher when the sum of the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) and oleuropein aglycon (r (2) = 0.91-0.96) or the sum of these two and hydroxytyrosol (r (2) = 0.90-0.97) was considered than was observed with HPLC total phenols (r (2)= 0.91-0.95) and especially with colorimetric determination of total polyphenols and o-diphenols (r (2) = 0.77-0.95 and 0.78-0.92, respectively). 3,4-DHPEA-EDA, p-HPEA-EDA, the aglycons of oleuropein and ligstroside, and HPLC total phenols content presented highly significant differences (p = 0.001-0.010) with respect to the dual- and triple-phase extraction systems used, whereas colorimetric total polyphenols content did not (p = 0.348) and o-diphenols showed a much lower significant difference (p = 0.031). The five variables that most satisfactorily classified the principal commercial Spanish virgin olive oil varieties were 1-acetoxypinoresinol, 4-(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC), ligstroside aglycon, p-HPEA-EDA, and RT 43.3 contents.     

主成分分析法优选枸杞乳酸菌发酵饮品发酵剂

乳酸菌发酵作为果蔬汁的一种绿色加工技术,不仅可以赋予产品独特的风味,还可以转化其中的活性物质,提高产品的营养价值和保健功效。该研究以湖北杂交枸杞为原料,使用6种乳酸菌(植物乳杆菌、嗜热链球菌、嗜酸乳杆菌、鼠李糖乳杆菌、干酪乳杆菌及发酵乳杆菌)进行发酵,研究发酵前后枸杞果汁理化特性、主要活性成分及体外抗氧化变化,并利用主成分分析进行综合评价优选出理想的发酵菌株。结果表明,6种乳酸菌在枸杞果汁中生长良好,活菌数均能达到10.0 lg CFU/mL以上。发酵后的枸杞果汁中总糖和还原糖含量显著降低(P0.05),且植物乳杆菌和嗜热链球菌产酸能力更强,发酵后总酸含量达6.74、6.07g/kg。与未发酵枸杞果汁相比,经植物乳杆菌、嗜热链球菌、鼠李糖乳杆菌和发酵乳杆菌发酵的枸杞果汁中总酚含量增加了13.76%～28.07%,而嗜酸乳杆菌和干酪乳杆菌发酵后无显著性差异(P0.05)。6种乳酸菌发酵后枸杞果汁中总黄酮含量增加了55.80%～161.97%。发酵枸杞果汁的抗氧化活性与发酵前相比均有显著提高(P0.05)。基于主成分分析的综合评价函数显示经植物乳杆菌、发酵乳杆菌发酵的枸杞果汁品质更优,适宜作为开发枸杞高值化绿色加工饮品的发酵剂。     

Application of analytical standards in method development: selected chlorinated phenols and herbicides

Pure analytical standards are key components in the successful development of accurate analytical methods. When such compounds are unavailable commercially, a laboratory synthesis activity can provide such materials for use in method development. An example of such an approach is the synthesis of ethyl ethers of chlorinated phenols and ethyl esters of several phenoxy herbicides. Individual synthesis steps and the characterization of such materials are described. The use of such analytical standards in method development is vital for defining method characteristics. In addition, such materials are helpful in diagnosing chromatographic and mass spectral problems in the course of this work. Key mass spectral information including parent and daughter scans obtained from these analytical standards by using tandem mass spectrometry provide the basis for a new analytical method with increased sensitivity and specificity.     

Feasible application of a portable NIR-AOTF tool for on-field prediction of phenolic compounds during the ripening of olives for oil production

Olive fruits of three different cultivars (Moraiolo, Dolce di Andria, and Nocellara Etnea) were monitored during ripening up to harvest, and specific and total phenols were measured by HPLC (High Pressure Liquid Chromatography). On the same olive samples (n = 450), spectral detections were performed using a portable NIR (Near Infrared)-AOTF (Acousto Optically Tunable Filter) device in diffuse reflectance mode (1100-2300 nm). Prediction models were developed for the main phenolic compounds (e.g., oleuropein, verbascoside, and 3,4-DHPEA-EDA) and total phenols using Partial Least Squares (PLS). Internal cross-validation (leave-one-out method) was applied for calibration and prediction models developed on the data sets relative to each single cultivar. Validation of the models obtained as the sum of the three sample sets (total phenols, n = 162; verbascoside, n = 162; oleuropein, n = 148; 3,4-DHPEA-EDA, n = 162) were performed by external sets of data. Obtained results in term of R(2) (in calibration, prediction and cross-validation) ranged between 0.930 and 0.998, 0.874-0.942, and 0.837-0.992, respectively. Standard errors in calibration (RMSEC), cross-validation (RMSECV), and prediction (RMSEP) were calculated obtaining minimum error in prediction of 0.68 and maximum of 6.33 mg/g. RPD ratios (SD/SECV) were also calculated as references of the model effectiveness. This work shows how NIR-AOTF can be considered a feasible tool for the on-field and nondestructive measurement of specific and total phenols in olives for oil production.     

Antioxidant compounds and antioxidant activity in "early potatoes"

The antioxidant content and the antioxidant capacity of both hydrophilic and lipophilic antioxidant extracts from four "early potato" cultivars, grown in two different locations (Racale and Monteroni), were examined. There was a considerable variation in carotenoid content and weak differences in the ascorbic acid concentration of the examined cultivars of "early potato" and between the harvested locations. An increase in both methanol/water (8:2 v/v) and phosphate buffer soluble (PBS) free phenols (70%) and bound phenols (28%) in the extracts from the cultivars grown at Racale site was found and discussed. Examination of individual phenols revealed that chlorogenic acid and catechin were the major phenols present in potato tuber extracts; a moderate amount of caffeic acid and ferulic acid was also detected. The total equivalent antioxidant capacity (TEAC) was higher in the Racale extracts and a highly positive linear relationship ( R (2) = 0.8193) between TEAC values and total phenolic content was observed. The oxyradical scavenging capacity (TOSC) of methanol/water and PBS extracts of peel and whole potatoes against the reactive oxygen species (ROS) peroxyl radicals, peroxynitrite, and hydroxyl radicals was also analyzed. A highly significant linear correlation ( R (2) = 0.9613) between total antioxidant capacity (as a sum of peroxyl radicals + peroxynitrite) and total phenol content of methanol/water extracts was established. Moreover, proliferation of human mammalian cancer (MCF-7) cells was significantly inhibited in a dose-dependent manner after exposure to potato extracts. These data can be useful for "early potato" tuber characterization and suggest that the "early potato" has a potential as a dietary source of antioxidants.