Pulsed electric field is also another extraction technique that can be applied at room temperature conditions and performed in a matter of seconds requiring low energy to increase cell membrane breakdown in mass transfer which were applied previously in several fruits such as strawberry and grapes.,e analysis of phenolic acids and flavonoids by liquid and/or gas chromatography techniques is the most widely and commonly applied methods for the quantification of phenolics in fruits and vegetables. In addition, spectrophotometric assays are used as nonspecific methods used for evaluating the levels of phenolics in many fruits and vegetables.Although, fruits differ in the quantity and types of phenolic antioxidants, degree of conjugation, and composition of sugar, total phenolic compounds can be estimated in fruits using the reagent proposed by Otto Folin and Vintila Ciocalteu and recently modified by Li et al.. ,is Folin–Ciocalteu method is robust, highly reproducible , convenient, and fast, requiring only a UV spectrophotometer. ,e method is typically standardized with either gallic acid, rutin, or a combination of pinocembrin/galangin. ,e Method is based on a reaction of the chemical reagent with phenolic electron transfer. ,e phenolic compounds are oxidized to phenolates by the reagent at alkaline pH in a saturated solution of sodium carbonate resulting in a blue molybdenum-tungsten complex and can be measured at 765 nm . ,e absorbance of each sample can be compared with those obtained from the standard curve, stacking pots and the obtained data are expressed as µmol gallic acid equivalents per gram of fresh or dry matter.
Because the reaction is quantitative and presumable, the analysis of a mixture of phenols can be recalculated based on any other standard. ,e assay comprises of monophenols and provides predictable reactions based on the phenols and provides measuring of all compounds readily oxidizable under the reaction conditions.Recently, chromatographic techniques such as high performance liquid chromatography , HPLC electrospray ionization mass spectrometry , gaschromatography-mass spectrometry , capillary electrophoresis , and near-infrared spectroscopy techniques are developed for identification, separation, and quantification of phenolics. Phenolic content of plant materials can be measured and identified using HPLC employing different stationary phase-solvent combinations and various detectors. HPLC relies on comparisons of unknown compounds with standard reference compounds to make both qualitative and quantitative analytical measurements. Columns can be selected to impart specific separations based on the stationary phase type and the size and structure of the packing materials to which the stationary phase is bound to. Detector choice can also be manipulated to enhance detection and especially quantifi- cation. Phenolic compounds can easily be measured using UV-Vis, photodiode array detection , fluorometric detection , and electrochemical detection . Each stationary phase-detector combination will provide specific information on the phenolic composition of a sample. For example, UV detection can be used to measure benzoic acid at 246–262 nm, gallic acid at 271 nm, and 275 nm for syringic acid.
Two different wavelengths 225–235 nm and 290– 330 nm can be used to measure cinnamic acids, but the common wavelength of 280 nm is issued for the general analysis of phenolics. However, many factors such as sample purification, column and detector types, solvents used as mobile phase and solvent purity, and their pH affect HPLC analysis of phenolics. It is previously reported that mixtures of water, methanol, acetonitrile, formic and acetic acids, and trifluoroacetic acid are used for mobile phase for phenolic compounds in reversed phase chromatography using octadecyl silica columns. Generally, among the HPLC detectors, UV-Vis and DAD detectors are more common compared to the fluorometric detection . Common stationary phases include C18 RP columns employing an acidified mobile phase and ammonium acetate buffers of organic solvents . Detection efficiency can be improved by using SPE cartridges composed of styrenedivinylbenzene to purify phenolic compounds from crude extracts prior to HPLC analysis, wavelength selected for monitoring phenolics is an important criterion and generally ranges between 190 and 380 nm. Gradient elution is generally preferred rather than isocratic elution. Some of the authors previously reported that phenolics such as flavonones, flavonoids, and flavan-3-ols of plum, blueberry, raspberry, strawberry, orange, apple, and tea are possible to be measured by common HPLC techniques. In general, for identification and quantification of phenolics, individual stock solutions of each standard are prepared in methanol and stored at −20°C until analysis. ,e working standard mixture solutions are made by diluting the appropriate amount of each stock standard solution to obtain at least 5 calibration levels.
Measurements of flavanols, hydroxycinnamates, flavonols, and anthocyanins of fruits can be detected at 280, 320, 360, and 520 nm by using HPLC. External standards are used to quantify the phenolic compounds. Stable isotopes can also be used to quantify phenolic compounds when HPLC-ESI/MS is being used as described below.HPLC-ESI/MS is used to increase the range of phenolic compounds detected in a sample and to improve sensitivity as compared with standard chromatographic methods. HPLC-ESI/MS is a robust and selective quantification method that is effective at measuring the complex array of phenolics typically found in fruits and vegetables. Mass spectrometry methods can be performed on a variety of instruments including electrospray ionization ion trap instruments, triple quadrupole instruments , and time-of flight instruments . ,e mass spectrometer is an analytical detector that gives both qualitative and quantitative measurements based on separation of ions by their m/z ratio and 0.01% correction. Mass spectrometry involved three stages: ionization, mass analysis, and detection of ions. Separation of phenolic compounds is best achieved in aqueous-organic extracts of foods with HPLC prior to MS analysis although GC can also be used. ,e most common solvent reduction and ionization technique is electrospray ionization . ,is can be performed using different voltages to create negative or positive pseudomolecular ions that can be accelerated into the mass analyzer, e mass analyzer separates ions based on the flight path as with a magnetic /electric field separation, time-of-flight in a filed free region, or by altering ion trajectories using quadrupole and ion trap mass analyzers. Detection is usually achieved with an ion multiplier tube. Triple quadrupole analyzers and ion trap analyzers are often used when higher sensitivity and specificity, strawberry gutter system or structural information is required for identification. Fidelity of MS measurements can be increased using MS/MS techniques. For example, a common technique is to create product ions through collision-activated dissociation of selected precursor ions in the collision cell of the triple quadrupole mass spectrometer , analyzing the fragment ions in the second analyzer of the instrument . Paper chromatography , thin layer chromatography techniques, high speed counter current chromatography , capillary electrophoresis , and supercritical fluid chromatographic techniques are also used to identify the phenolic compounds.Fruit trees should be planted where they will receive full sun for 6 or more hours per day during the growing season. For maximum production, fruit trees need soil that is deep and well drained. Such soils do not occur everywhere in California, especially in residential areas where the topsoil may have been partially removed by land grading and the remaining soil has been compacted by the weight of construction machinery. With proper irrigation management, however, fruit trees can be grown in as little as 1 to 11 ⁄2 feet of good topsoil. An additional complication in many areas is the presence of an impervious layer of hardpan or claypan beneath the soil surface. Where hard pan is found within 11 ⁄2 to 2 feet of the surface, it should be penetrated to allow for drainage and root growth. It may be possible to break through the hard pan with a pickaxe or by using a powered soil auger or jackhammer . If there is no way to get through it, build a raised bed 1 to 2 feet high and fill it with good quality soil. In any case, use care when irrigating to prevent saturating the soil in the root zone.Although fruit trees may be planted at any time of the year, they are often purchased during the winter months when they are dormant and available as “bare-root” stock. The best trees have a trunk diameter from 1 ⁄2 to 5 ⁄8 inch and usually become established faster than smaller or larger planting stock. If bare-root trees cannot be planted soon after purchase, they should be temporarily “heeled in” by covering the roots with soil, sawdust, or compost, and keeping them moist to prevent drying out.If the soil is compacted, it must be cultivated deeply in a wide area because roots will not grow through densely compacted soil.
It is best not to add soil amendments or fertilizers directly into the planting hole, although it may be beneficial to rototill well-decomposed compost into the soil around the tree before digging the hole. In heavy soils, undecomposed organic amendments may rot or be toxic to new roots. Wait until new growth is several inches long before applying fertilizer. Dig the planting hole at least twice as wide as the spread of the tree’s roots, but no deeper than the depth of the root ball unless the soil is deeply compacted. Leave the bottom of the hole undisturbed to help prevent the tree from settling below its root crown. This reduces the likelihood of crown rot, which often kills trees in poorly drained soil. In soil containing a high percentage of clay, score the sides of the planting hole with a shovel to aid root growth outward from the hole. Container-grown trees may have circling or girdling roots, which should be gently pulled away from the root ball before the tree is planted. Place the tree in the hole with the graft union at least 2 to 4 inches above the soil surface. The protruding notch of the union should be oriented toward the northeast to reduce the likelihood of sunburn . Partially fill the hole with the same soil that was excavated and firm gently, eliminating large air pockets. When properly planted, the soil line on the trunk of the tree should be 1 to 2 inches above the level of the surrounding ground. Placing a shovel handle or other straightedge across the hole can be a helpful way to check this during the filling process. As a general rule, after the soil has settled, the uppermost large root should be just below the soil surface. The soil should slope downward from the tree to prevent water from accumulating near the trunk. Once the tree is properly oriented, finish filling the hole and gently firm the soil into place.All newly planted trees should be watered thoroughly to settle the soil around the root ball. If the soil is heavy loam or clay, care should be taken to avoid keeping the soil overly wet, since too much water eliminates air spaces in heavy soils, creating anaerobic conditions that can kill trees. For this reason, it is not always advisable to form a basin around the tree. However, trees should be basin-irrigated if the soil is sandy to loam, if the clay soil is dry, or if container-grown trees are planted during the growing season. Construct a doughnut-shaped basin for watering the newly planted tree, making sure that water drains away from the trunk. The basin should be slightly wider than the planting hole so that water can be applied to the entire root area and just beyond. Most of the root volume occupies a rather limited area, particularly through the first growing season, so frequent watering may be needed until the roots become established. Fill the basin once or twice a week in hot weather, less often when it is cool or rainy. Water must soak into the root ball of container-grown trees since they cannot obtain water from the surrounding soil until their roots grow into it. Remove the basin in winter so that the tree does not stand in accumulated rainwater. The ground within about 3 feet of the tree trunk should be kept free of grass, weeds, or other vegetation that can compete with the tree for water and nutrients. A layer of mulch 3 to 6 inches thick, such as wood chips, helps control weeds and conserve moisture. Mulch should be kept several inches away from the trunk to minimize the occurrence of crown rot and eliminate hiding places for insect pests.