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Sustainable Organic Dyes from Winemaking Lees for Photoelectrochemical Dye-Sensitized Solar Cells

: During the last two decades, Dye Sensitized Solar Cells (DSSCs) have received a great deal of attention as a promising, low-cost alternative to conventional silicon photovoltaic devices. Natural dye molecules can be used as a sensitizer for their low cost, good light absorbance, easy preparation process, and biodegradability. In this study, dyes were obtained from wine lees, the last by-product of winemaking process, supplied by a venetian winery (Italy). Polyphenols, like tannins and anthocyanins, which were extracted from winemaking lees, were adsorbed on a nanostructured ordered mesoporous titanium dioxide, previously treated at di ﬀ erent temperatures (400–600 ◦ C). Both dyes and titania semiconductor samples were studied with di ﬀ erent techniques. The tests were carried out on prototypes to evaluate the cell power and the photocurrent generated under simulated solar light irradiation. The obtained solar energy conversion e ﬃ ciencies are comparable to those that were reported in literature by using organic dyes extracted from vegetables, fruits, and plants. It is signiﬁcant that these dyes are largely available and cost e ﬀ ective, since recovered from a waste otherwise to be disposed of, opening up a perspective of feasibility for inexpensive and environmentally friendly dye solar cells to generate green electricity and transforming agri-food waste into a resource.

Introduction
Global energy consumption is mainly based on non-renewable resources and the increasing energy demand has pushed research on more sustainable and renewable resources [1]. Photovoltaic solar cells are unanimously considered to be very promising for clean energy production, but production costs, material availability, and toxicity sometimes hinder their deployment. An alternative to conventional cells for their much lower environmental impact and production costs are Dye Sensitized Solar Cells (DSSCs), introduced to the world in 1991 by Grätzel and O'Regan [2]. Unlike silicon solar cells, DSSCs charge generation and transport does not happen in a single material, but is split in different layers: a counter-electrode, an electrolyte solution, a sensitizing dye, a nanostructured semiconductor, generally titanium dioxide, and finally, a conductive glass.
One of the most important cells' elements is the dye that is adsorbed on titanium dioxide layer, playing a fundamental role in DSSCs because it is involved in the harvesting and conversion of solar energy to electrical energy. Organometallic dyes, which are based on ruthenium ( Figure 1a) and osmium polypyridyl complexes, have been widely employed as efficient sensitizers [3,4], since they can absorb light in the visible range, have intense charge-transfer and highly efficient metal-ligand charge transfer. Unfortunately, this kind of metal coordination compounds is usually obtained with multistep reactions and expensive chromatographic purification procedures [1]. The use of organic dyes is an alternative to transition metals, leading to a significant reduction of manufacturing costs of the final device [5][6][7]. Many organic dyes are available in nature and they can be used as such. In fact, plants are full of natural pigments that determine their colour, covering the entire solar spectrum from red to purple and that can be employed in DSSCs. Therefore, natural dye molecules can be used like a sensitizer for their low cost, good light absorbance, easily preparation process, and eco-friendly behaviour, as they are completely biodegradable. One of the most important groups of naturally occurring pigments are anthocyanins, which are responsible for red, blue, and purple colours in different plant tissues, like fruits, flower, leaves, and bulbs [8]. These polyphenolic molecules are available in various fruits, such as berries, grapes, pomegranates, raspberries, and many other [9], and, during the impregnation process, can bind the semiconductor titania nanoparticles with their hydroxyl functional group, to obtain an electronic coupling that allows for an efficient electron transfer reaction. Anthocyanins [10,11] and other several natural dyes, such as tannins [12], carotenoids [13,14], flavonoids [5,15], and chlorophylls [16,17] (Figure 1b-c), can be easily extracted from vegetables or fruits and used to build DSSCs [1,5] that are characterized by typical efficiency included between 0 and 2%. This difference is depending, among other things, on the starting material that provides different efficiency, such as 0.64% for eggplants, 0.90% for Radicchio, 1.07% for blackberries, and 0.78% for Red Sicilian orange [9]. Nevertheless, an ethical question arises, since food has to be used and destroyed to obtain dyes from fruits and vegetables. Therefore, developing such a technology on a large scale could be considered incorrect from an ethical point of view and, at the end, also economically disadvantageous. It is necessary to recover and exploit the concept of circular economy, which considers the whole product's life-cycle, from its production to the creation of markets for waste-derived raw materials.
Waste-to-energy is a broad term that encompasses various waste treatment processes generating energy (e.g. in the form of electricity, heat, or produce a waste-derived fuel), each of which has different environmental impacts and circular economy potential.
The innovative idea behind this applied research work, developed in collaboration with a venetian winery, consists of recovering and valorizing the waste of winemaking process, principally lees after fermentation and clarification processes, for the realization of DSSC solar cells.
Winemaking is one of the most important activities of agri-food industries all over the world, with an approximate worldwide production of 250 million hectoliters (OIV, 2017). This kind of Anthocyanins [10,11] and other several natural dyes, such as tannins [12], carotenoids [13,14], flavonoids [5,15], and chlorophylls [16,17] (Figure 1b,c), can be easily extracted from vegetables or fruits and used to build DSSCs [1,5] that are characterized by typical efficiency included between 0 and 2%. This difference is depending, among other things, on the starting material that provides different efficiency, such as 0.64% for eggplants, 0.90% for Radicchio, 1.07% for blackberries, and 0.78% for Red Sicilian orange [9]. Nevertheless, an ethical question arises, since food has to be used and destroyed to obtain dyes from fruits and vegetables. Therefore, developing such a technology on a large scale could be considered incorrect from an ethical point of view and, at the end, also economically disadvantageous. It is necessary to recover and exploit the concept of circular economy, which considers the whole product's life-cycle, from its production to the creation of markets for waste-derived raw materials.
Waste-to-energy is a broad term that encompasses various waste treatment processes generating energy (e.g. in the form of electricity, heat, or produce a waste-derived fuel), each of which has different environmental impacts and circular economy potential.
The innovative idea behind this applied research work, developed in collaboration with a venetian winery, consists of recovering and valorizing the waste of winemaking process, principally lees after fermentation and clarification processes, for the realization of DSSC solar cells.
Winemaking is one of the most important activities of agri-food industries all over the world, with an approximate worldwide production of 250 million hectoliters (OIV, 2017). This kind of activity Appl. Sci. 2020, 10, 2149 3 of 15 produces 2-6% lees on the total volume of wine produced [18,19], therefore it generates a large quantity of waste that must be properly disposed, with considerable costs for wineries.
Wine lees can be defined as 'the residue formed at the bottom of recipients containing wine, after fermentation, during storage or after authorized treatments, as well as the residue obtained following filtration or centrifugation', according to literature and EEC regulation No. 337/79 [20].
Therefore, at the end of the winemaking and clarification wine process, the so-called lees are the precipitate composed of many organic materials like yeast, bacteria, tartaric acid salts and vegetable residues. Furthermore, lees also contain a large amount of bentonite, a natural phyllosilicate clay that is used by wineries to reduce protein instability and precipitate suspended solids responsible for wine turbidity [21]. Usually winemaking lees are re-used for the recovery of tartaric acid [22], but, in recent years, studies showed that the concentration of anthocyanins in these lees are 10 times higher than grape skins [23], and this has increased the interest in these lees. Bentonite slightly bleaches white and red wines absorbing molecules responsible of wines color, such as anthocyanins, tannins, catechins, and polyphenols, which, if properly extracted, can be valorized and used as dyes in DSSCs.
The current work aims to study a method to recover the main waste product of a winery, extracting the organic dyes contained in the lees, reintroducing them into the production cycle, and transforming them into a resource to produce renewable and sustainable electricity from organic photovoltaic solar cells.
To the best of our knowledge, the use of winemaking lees extracts is being reported for the first time as sensitizers for TiO 2 based dye-sensitized solar cells (DSSCs).

Synthesis of Nanostructured Semiconductor
Titanium dioxide powder was synthetized via sol-gel and hydrothermal route, starting from analytical grade reagent without further purifications, following a synthesis that was proposed in the literature [24]. After the synthesis, powder was thermally treated at 400 • C or 600 • C under air flow for one hour (ramp rate 2 • C/min) and named TiO 2 _400 and TiO 2 _600, respectively.

Preparation of Electrodes
Conductive fluorine-doped tin oxide (FTO) glasses, with 7 Ω/sq of surface resistivity, were used as electrodes. The cleaned working electrode was coated with a blocking layer, a thin compact layer that is necessary to reduce the leakage currents and enhance the performance of the device, deposited by spray pyrolysis technique, using an alcoholic solution of titanium diisopropoxide bis(acetylacetonate) on a heated substrate at 400 • C, in order to evaporate the solvent and obtain a compact layer of titanium dioxide. The electrodes coated with TiO 2 blocking layer were heated in air from room temperature (RT) to 500 • C to sinter this compact layer.
The Dyenamo ® screen printer with polyester mesh was used to print on FTO glass a 10 µm thickness [25] active layer of titania semiconductor with an area of 9 × 9 mm 2 . The required viscous titanium dioxide paste was prepared by grinding titanium dioxide powder and then dispersing it with a terpenic dispersing agent and ethyl cellulose in an excess of ethanol (which was evaporated through rotavapor), as described in literature [26].
After the deposition of a porous TiO 2 layer and its sintering at 450 • C for 20 min [24], the plate was immersed in the natural dye solution in a dark container overnight to complete the sensitizer uptake. After soaking the film in this solution, dye molecules were left to be adsorbed on the surface of the titania. A counter-electrode was prepared depositing on FTO platinum by thermal decomposition [27]. An alcoholic solution 1 mM of H 2 PtCl 6 in 2-propanol was sprayed on a cleaned conductive transparent glass that was heated at 250 • C through spray pyrolysis technique that was finally treated at 450 • C for 30 min [24] to obtain metal platinum clusters.

Extraction of Dyes from Winemaking Lees
Winemaking lees were collected in the first period of the year 2019 from a venetian winery that was situated in the Prosecco DOCG region (Conegliano, Italy). Lees derive from winemaking process of both red and white wines. Several tests were carried out both at RT (room temperature) and with a Soxhlet extraction technique, using different solvent, concentrations, times, and solvent-to-solid ratio. A suitable amount of lees was firstly centrifuged to simulate the process used by the winery to extract residual wine from winemaking lees. Subsequently, the solid waste was recovered and a certain amount (w/w) of solvent (methanol, ethanol, water, and mixtures of them), acidified with 1% v/v with concentrated HCl (37%), was added to maintain pH < 2. The extraction process was monitored by evaluating the maximum of absorbance variation (λ = 525 nm) with an UV-Vis spectrophotometer. The dye solution was stored in the dark at 5 • C after extraction process, and then used for the following impregnation process and analyses.

Dye Sensitized Solar Cell Prototypes Assembling
Dye-titania electrode and counter-electrode were assembled into a sandwich-type cell and then sealed with a gasket that was made by a hot-melt ionomeric resin made by Surlyn ® . Some drops of an iodine-based electrolyte (EL-HPE Sigma Aldrich) solution were introduced into the cell via vacuum backfill through a small circular hole previously done on the counter electrode before the cleaning process. The cell was put in a vacuum chamber and, then, when atmospheric pressure was restored, the electrolyte was pushed inside the internal free space by pressure variation. This method allows for the electrolyte to completely fill the internal space existing between counterelectrode and photoanode, made by 25 µm polymeric Surlyn ® gasket. After this, the dye sensitized solar cells prototype was tested for photocurrent measurements.

Characterization Techniques
Nitrogen physisorption at −196 • C with an ASAP 2010 Micrometrics sorptometer characterized the textural properties of titanium dioxide. The samples (about 0.1 g) were treated first at 130 • C for 12 h at 0.67 Pa and then at RT for 2 h at 1 × 10 −4 Pa. The well-known BET equation was applied and the specific pore volume (V s ) was calculated at P/P 0 = 0.98 to determine the specific surface areas. The pore size distribution was calculated according to the BJH method.
X-Ray Diffraction (XRD) was used to determine the crystalline phases of titania, by using a Philips X'Pert system (Bragg-Brentano parafocusing geometry) with a nickel-filtered Cu Kα 1 radiation at 0.154 nm. The samples (disc shaped pressed powders) were analyzed before and after calcination. The anatase and rutile crystallite average size was determined on XRD peak broadening by the Scherrer equation (line width of the anatase (101) reflection at 2θ = 25.3 • and rutile (110) reflection at 2θ = 27.4 • ): where τ is the crystallite size, K the shape factor, λ is the X-ray wavelength for Cu Kα 1 radiation, β is the line broadening at FWHM, and Θ is the Bragg's angle.
To obtain the anatase/rutile weight fraction, the following equation [28] was used: where I A and I R are the peak intensities of the (101) and (110) reflections for anatase and rutile, respectively. Field-Emission-Gun Scanning Electron Microscopy (FEG-SEM) Zeiss with cutting-edge electro-optical design was used to study the size and morphology of the nanoparticles.
Diffuse Reflectance Spectroscopy (DRS) determined the band gap of titania. The spectra were collected with a Perkin Lambda 35 UV-Vis spectrophotometer. Band gap energy, E g , thoroughly determined the absorption coefficient α value (m −1 ) from a plot of (αhν) 0.5 versus photon energy (hν), where h is the Planck's constant and v is the frequency (s −1 ). The intercept of the tangent to the absorption curves was used to estimate the band gap (E g ) value.
The dye extraction process was monitored by an Agilent 8453 UV-Vis spectrophotometer, following the absorbance at the maximum of UV-Vis spectrum, after water dilution. Dye solutions were characterized by various methods to determine the concentrations of anthocyanins, catechins, tannins, polyphenols, and color intensity. Anthocyanins that were extracted from winemaking lees were determined by the procedure of Ribereau Gayon and Stonestreet (1965) [29] with the bisulphite bleaching as anthocyanins form colorless compound with the bisulphite ion, thus transferring from the red colored flavylium cation to a non-colored form. The catechins were determined according to the method proposed by Zironi et al. [30], while using DAC (4-(dimethylamino)cinnamaldehyde). The assay was performed using a chromogen reagent of 1.0 g DAC dissolved in 250 mL of 37% HCl and 750 mL methanol. To 1 mL of 1:25 (v/v) diluted sample, 5 mL of DAC solution were added and, after 5 minutes, the maximum value of absorption at 640 nm was measured against a blank that was prepared by substituting sample with 1 mL of 10% ethanol. Tannins characterization was carried out following the Weinges and Nader method [31]: the sample was diluted fifty times and 2 mL of solution was added to a tube containing 6 mL of acidized butyl alcohol prepared by dissolving 150 mg Fe(SO 4 ) in 500 mL of 1-butanol and 500 mL of HCl 37%. Half of this solution was put in an oven at 100 • C for 30 minutes. After cooling, the optical density was measured at 550 nm for both solutions against water. The polyphenols content of extracted dyes was evaluated using the Folin-Ciocalteu's method that involves the addition of 0.5 mL of Folin-Ciocalteu's reagent to 0.1 mL dye sample diluted 1:10 in water. After mixing, 2.0 mL of 15% Na 2 CO 3 were added and the volume was brought up to 10 mL with deionized water. The sample was kept in the dark for two hours and then absorbance was measured at 750 nm while using a solution with deionized water as reference sample. The color intensity was determined as a sum of the absorbance at 420 nm, 520 nm, and 620 nm following the Glories method [32], with an optical path of 10 mm.
The measurements of power (P) and photocurrent (J) that were generated by the obtained photovoltaic cell were carried out by irradiating the prototype with a Sunlight Solar Simulator (AM 1.5G filter, 100 W Xenon arc lamp, Abet Technologies) to simulate the solar spectrum on earth surface, and using a digital multimeter (Tecktronix Keithley 2410) that was directly connected to the cell electrodes. The power against voltage P-V curve was obtained by simultaneous measurements of flowing current J and cell voltage V. The efficiency of solar cells η = J max V max /P i were finally calculated, where J max and V max are the current density and voltage at maximum power generation of a solar cell and P i is the incident power of the light source used (1000 W/m 2 ).

Nanostructured Semiconductor
FEG-SEM microscopy evaluated the morphology of the synthesized TiO 2 . Figure 2 illustrates the presence of aggregated nanoparticles with a size between 5-50 nm. After the screen-printing process of titania paste, a thermal treatment is required to obtain a layer with the most suitable characteristics. As expected for this type of oxide, the aggregation of TiO 2 nanoparticles, as well as their average size, increased by increasing the calcination temperature, passing from about 10 nm for the sample that was treated at 400 • C to about 28 nm for the sample treated at 600 • C. Appl. Sci. 2020, 10 N2 physisorption at -196 °C to evaluate its textural properties as well as the porosity formed during the sol-gel synthesis characterizes the nanostructured titania semiconductor ( Table 1). The isotherms of the samples, both before and after calcination (at 400 °C or 600 °C) are of type IV according to the IUPAC classification, with a hysteresis loop indicating an ordered pore size distribution in the mesoporous region ( Figure 3). Nanostructured titania sample before calcination showed very high specific surface area, SABET, of 206 m 2 g -1 , a pore diameter distribution lower than 20 nm and a pore volume of 0.281 cm 3 g −1 . A correlation between the calcination temperature process and the textural features of these materials can be noticed. By increasing the calcination temperature from 400 °C to 600 °C, the surface area drops from 136 to 12 m 2 g −1 , and the cumulative pore volume significantly decreases, passing from 0.236 to 0.036 cm 3 g −1 , respectively. This phenomenon can be attributed to the reorganization of the crystalline structure towards a thermodynamically more stable structure, with an increase of the nanoparticles average size, as observed by the FEG-SEM micrographs.  N 2 physisorption at −196 • C to evaluate its textural properties as well as the porosity formed during the sol-gel synthesis characterizes the nanostructured titania semiconductor ( Table 1). The isotherms of the samples, both before and after calcination (at 400 • C or 600 • C) are of type IV according to the IUPAC classification, with a hysteresis loop indicating an ordered pore size distribution in the mesoporous region ( Figure 3). Table 1. Textural parameter of titania semiconductor as a function of different thermal treatments. Relative uncertainties are lower than 2%.  N2 physisorption at -196 °C to evaluate its textural properties as well as the porosity formed during the sol-gel synthesis characterizes the nanostructured titania semiconductor ( Table 1). The isotherms of the samples, both before and after calcination (at 400 °C or 600 °C) are of type IV according to the IUPAC classification, with a hysteresis loop indicating an ordered pore size distribution in the mesoporous region ( Figure 3). Nanostructured titania sample before calcination showed very high specific surface area, SABET, of 206 m 2 g -1 , a pore diameter distribution lower than 20 nm and a pore volume of 0.281 cm 3 g −1 . A correlation between the calcination temperature process and the textural features of these materials can be noticed. By increasing the calcination temperature from 400 °C to 600 °C, the surface area drops from 136 to 12 m 2 g −1 , and the cumulative pore volume significantly decreases, passing from 0.236 to 0.036 cm 3 g −1 , respectively. This phenomenon can be attributed to the reorganization of the crystalline structure towards a thermodynamically more stable structure, with an increase of the nanoparticles average size, as observed by the FEG-SEM micrographs.  Nanostructured titania sample before calcination showed very high specific surface area, SA BET , of 206 m 2 g −1 , a pore diameter distribution lower than 20 nm and a pore volume of 0.281 cm 3 g −1 .
A correlation between the calcination temperature process and the textural features of these materials Appl. Sci. 2020, 10, 2149 7 of 15 can be noticed. By increasing the calcination temperature from 400 • C to 600 • C, the surface area drops from 136 to 12 m 2 g −1 , and the cumulative pore volume significantly decreases, passing from 0.236 to 0.036 cm 3 g −1 , respectively. This phenomenon can be attributed to the reorganization of the crystalline structure towards a thermodynamically more stable structure, with an increase of the nanoparticles average size, as observed by the FEG-SEM micrographs. Figure 4 shows the diffractograms of titania samples before and after calcination. The diffraction peaks of the as-prepared and calcined at 400 • C samples can be indexed, which is in agreement with the values of standard cards, as typical anatase phase (JCPDS, PDF card no. 21-1272) of TiO 2 . When the calcination temperature increases to 600 • C, the typical rutile (110) reflection becomes noticeable at 2θ = 27.4 • , showing a partial transformation of anatase phase into rutile polymorph (JCPDS, PDF card no. 21-1276). The peak broadening that was observed for the as-synthetized titania sample suggests the formation of small crystallites during the particle growth in the sol-gel synthesis process. As the calcination temperature increases, anatase and rutile peaks become sharper and more intense, due to particles aggregation, which leads to the growth of larger crystal domains.  Figure 4 shows the diffractograms of titania samples before and after calcination. The diffraction peaks of the as-prepared and calcined at 400 °C samples can be indexed, which is in agreement with the values of standard cards, as typical anatase phase (JCPDS, PDF card no. 21-1272) of TiO2. When the calcination temperature increases to 600 °C, the typical rutile (110) reflection becomes noticeable at 2θ = 27.4°, showing a partial transformation of anatase phase into rutile polymorph (JCPDS, PDF card no. 21-1276). The peak broadening that was observed for the as-synthetized titania sample suggests the formation of small crystallites during the particle growth in the sol-gel synthesis process. As the calcination temperature increases, anatase and rutile peaks become sharper and more intense, due to particles aggregation, which leads to the growth of larger crystal domains. The average size of anatase results to be 6.50 ± 0.07 nm for the as-prepared sample and 28.1 ± 0.4 nm for the titania treated at the highest temperature (600 °C). In Table 1, the weight fractions of the different titania phases are reported, showing the presence of only anatase in both the as-synthesized and the 400 °C treated titania samples. The transformation from anatase to rutile phase is detected at 600 °C, as expected.
In DSSC, an increasing crystallite size, due to high calcination temperature, usually leads to a decrease in traps-level number, thus increasing the final electrical current. On the other hand, thermal treatment at too high temperature reduces the surface area and pore volume of the nanostructured titania, as clearly shown in Table 1, decreasing the available area for dye absorption. For this reason, the titania that was thermally treated at 600 °C was discarded for the realization of a photovoltaic cell.
Diffuse Reflectance Spectroscopy was used to determine the band gap value of the sample calcined at 400 °C. An absorption peak in the UV region, below 400 nm, and due to an intrinsic absorption of titanium dioxide, was recorded ( Figure 5). 20   The average size of anatase results to be 6.50 ± 0.07 nm for the as-prepared sample and 28.1 ± 0.4 nm for the titania treated at the highest temperature (600 • C). In Table 1, the weight fractions of the different titania phases are reported, showing the presence of only anatase in both the as-synthesized and the 400 • C treated titania samples. The transformation from anatase to rutile phase is detected at 600 • C, as expected.
In DSSC, an increasing crystallite size, due to high calcination temperature, usually leads to a decrease in traps-level number, thus increasing the final electrical current. On the other hand, thermal treatment at too high temperature reduces the surface area and pore volume of the nanostructured titania, as clearly shown in Table 1, decreasing the available area for dye absorption. For this reason, the titania that was thermally treated at 600 • C was discarded for the realization of a photovoltaic cell.
Diffuse Reflectance Spectroscopy was used to determine the band gap value of the sample calcined at 400 • C. An absorption peak in the UV region, below 400 nm, and due to an intrinsic absorption of titanium dioxide, was recorded ( Figure 5). Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 15 Figure 5. Diffuse Reflectance Spectroscopy spectra of as-prepared and calcined (400 °C) titanium dioxide samples.
The optical band gap, Eg, of both as-prepared and calcined titania samples was estimated from the linear extrapolation of (h) ½ versus photon energy (h) curves, where h is Planck's constant and  is the frequency (s −1 ). The band gap was approximately 3.2 eV for both samples, as expected for pure anatase polymorphs.

Winemaking Lees Dye Extraction
Dye extraction process was carried out valorizing the lees deriving from red and white wines, characterized by a pH value of 3.7 ± 0.5. After centrifugation of winemaking lees, the colored solid was used to extract the dyes that were used for the realization of a DSSC photovoltaic cell. Firstly UV-Vis spectra of water diluted dyes sample, extracted with ethanol in 1:1 w/w lees-solvent ratio, acidified with concentrated HCl were registered. A peak at 280 nm and a large absorption band centered at 520 nm for red wine lees and at 280 nm for white wine lees can be noticed ( Figure 6). It is evident that the red extracts absorb in the visible region of light spectrum and, hence, fulfil the primary criterion for their use as sensitizers in DSSCs. Considering that the most efficient organic DSSCs are usually based on organic dyes that consist of anthocyanins obtained from red or purple fruits [9], the red extract was further investigated. Using the same conditions reported before, several extractions were conducted with different solvents (methanol, ethanol, water, and mixture thereof). The maximum absorbance of red wine extract was used to monitor the concentration of dye solution during the extraction process, as a function of the extraction time.  The optical band gap, Eg, of both as-prepared and calcined titania samples was estimated from the linear extrapolation of (αhν) 1 2 versus photon energy (hν) curves, where h is Planck's constant and ν is the frequency (s −1 ). The band gap was approximately 3.2 eV for both samples, as expected for pure anatase polymorphs.

Winemaking Lees Dye Extraction
Dye extraction process was carried out valorizing the lees deriving from red and white wines, characterized by a pH value of 3.7 ± 0.5. After centrifugation of winemaking lees, the colored solid was used to extract the dyes that were used for the realization of a DSSC photovoltaic cell. Firstly UV-Vis spectra of water diluted dyes sample, extracted with ethanol in 1:1 w/w lees-solvent ratio, acidified with concentrated HCl were registered. A peak at 280 nm and a large absorption band centered at 520 nm for red wine lees and at 280 nm for white wine lees can be noticed ( Figure 6). It is evident that the red extracts absorb in the visible region of light spectrum and, hence, fulfil the primary criterion for their use as sensitizers in DSSCs.
The optical band gap, Eg, of both as-prepared and calcined titania samples was estimated from the linear extrapolation of (h) ½ versus photon energy (h) curves, where h is Planck's constant and  is the frequency (s −1 ). The band gap was approximately 3.2 eV for both samples, as expected for pure anatase polymorphs.

Winemaking Lees Dye Extraction
Dye extraction process was carried out valorizing the lees deriving from red and white wines, characterized by a pH value of 3.7 ± 0.5. After centrifugation of winemaking lees, the colored solid was used to extract the dyes that were used for the realization of a DSSC photovoltaic cell. Firstly UV-Vis spectra of water diluted dyes sample, extracted with ethanol in 1:1 w/w lees-solvent ratio, acidified with concentrated HCl were registered. A peak at 280 nm and a large absorption band centered at 520 nm for red wine lees and at 280 nm for white wine lees can be noticed ( Figure 6). It is evident that the red extracts absorb in the visible region of light spectrum and, hence, fulfil the primary criterion for their use as sensitizers in DSSCs. Considering that the most efficient organic DSSCs are usually based on organic dyes that consist of anthocyanins obtained from red or purple fruits [9], the red extract was further investigated. Using the same conditions reported before, several extractions were conducted with different solvents (methanol, ethanol, water, and mixture thereof). The maximum absorbance of red wine extract was used to monitor the concentration of dye solution during the extraction process, as a function of the extraction time.  Considering that the most efficient organic DSSCs are usually based on organic dyes that consist of anthocyanins obtained from red or purple fruits [9], the red extract was further investigated. Using the same conditions reported before, several extractions were conducted with different solvents (methanol, ethanol, water, and mixture thereof). The maximum absorbance of red wine extract was used to monitor the concentration of dye solution during the extraction process, as a function of the extraction time.
The kinetics of the dye extraction process, as shown in Figure 7, prove that the dye extraction from bentonite clay can occur in some hours. For all of the solvents used, absorbance increases very rapidly in the first hour, reaching an asymptotic behavior just after 2 h. These experimental findings suggest that, at the beginning of the extraction process, solvent desorbs dye molecules from the external structure of bentonite layers, and then extracting organic molecules located in the middle of the clay layers. After optimizing the extraction process, the organic molecules were further characterized, and the concentration of the different organic molecules that were involved in color appearance was evaluated. Figure 8 reports anthocyanins, tannins, catechins, and total polyphenols concentrations.
The kinetics of the dye extraction process, as shown in Figure 7, prove that the dye extraction from bentonite clay can occur in some hours. For all of the solvents used, absorbance increases very rapidly in the first hour, reaching an asymptotic behavior just after 2 h. These experimental findings suggest that, at the beginning of the extraction process, solvent desorbs dye molecules from the external structure of bentonite layers, and then extracting organic molecules located in the middle of the clay layers. After optimizing the extraction process, the organic molecules were further characterized, and the concentration of the different organic molecules that were involved in color appearance was evaluated. Figure 8 reports anthocyanins, tannins, catechins, and total polyphenols concentrations. In particular, the anthocyanins responsible for red color resulted in being very concentrated (1 g/L), as compared to the typical values of anthocyanins content in red wines [33]. The presence of water The kinetics of the dye extraction process, as shown in Figure 7, prove that the dye extraction from bentonite clay can occur in some hours. For all of the solvents used, absorbance increases very rapidly in the first hour, reaching an asymptotic behavior just after 2 h. These experimental findings suggest that, at the beginning of the extraction process, solvent desorbs dye molecules from the external structure of bentonite layers, and then extracting organic molecules located in the middle of the clay layers. After optimizing the extraction process, the organic molecules were further characterized, and the concentration of the different organic molecules that were involved in color appearance was evaluated. Figure 8 reports anthocyanins, tannins, catechins, and total polyphenols concentrations. In particular, the anthocyanins responsible for red color resulted in being very concentrated (1 g/L), as compared to the typical values of anthocyanins content in red wines [33]. The presence of water In particular, the anthocyanins responsible for red color resulted in being very concentrated (1 g/L), as compared to the typical values of anthocyanins content in red wines [33]. The presence of water is responsible for the decreasing of dye concentration in the extracted solutions, which was probably due to the different solubility of these molecules in organic and aqueous solutions.
Another important characterization is the color intensity (related to the dye concentration): it was determined as the sum of the absorbance values at 420 nm, 520 nm, and 620 nm. The values of color intensity were 41.3 for methanol extraction, 43.2 by using ethanol, and 32.0 with a mixture ethanol-water. Soxhlet extraction can be another possible way to extract organic dye from solid lees. In this case, the lees are continuously washed with fresh ethanol, allowing for recycling the solvent and perhaps increased the polyphenols content in dye extract. Soxhlet extraction was performed, by using 1:3 w/w lees-solvent ratio. For comparison, solid-liquid extraction was performed with 1:1 w/w or 1:3 w/w lees-solvent ratio.
As reported in Figure 9, the Soxhlet extraction process led to a quite poor absorbance in comparison with both 1:3 w/w lees-solvent ratio and 1:1 w/w extraction. The different absorbance values between the two methods can be due to the different temperature conditions used (RT and reflux temperature, about 80 • C), determining a partial degradation of polyphenols during Soxhlet extraction. Literature reports in fact that these molecules are very sensitive to changes in temperature, following a first-order kinetic model degradation [34]. The different absorbance between 1:3 and 1:1 w/w extracts is due to different volumes used for extraction. Therefore, an alcoholic extraction with 1:1 w/w ratio is the best choice if a concentrated polyphenols solution is needed; conversely, a more diluted solution is preferred if a higher total content of colored polyphenols is required.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 10 of 15 is responsible for the decreasing of dye concentration in the extracted solutions, which was probably due to the different solubility of these molecules in organic and aqueous solutions. Another important characterization is the color intensity (related to the dye concentration): it was determined as the sum of the absorbance values at 420 nm, 520 nm, and 620 nm. The values of color intensity were 41.3 for methanol extraction, 43.2 by using ethanol, and 32.0 with a mixture ethanolwater. Soxhlet extraction can be another possible way to extract organic dye from solid lees. In this case, the lees are continuously washed with fresh ethanol, allowing for recycling the solvent and perhaps increased the polyphenols content in dye extract. Soxhlet extraction was performed, by using 1:3 w/w lees-solvent ratio. For comparison, solid-liquid extraction was performed with 1:1 w/w or 1:3 w/w lees-solvent ratio.
As reported in Figure 9, the Soxhlet extraction process led to a quite poor absorbance in comparison with both 1:3 w/w lees-solvent ratio and 1:1 w/w extraction. The different absorbance values between the two methods can be due to the different temperature conditions used (RT and reflux temperature, about 80 °C), determining a partial degradation of polyphenols during Soxhlet extraction. Literature reports in fact that these molecules are very sensitive to changes in temperature, following a first-order kinetic model degradation [34]. The different absorbance between 1:3 and 1:1 w/w extracts is due to different volumes used for extraction. Therefore, an alcoholic extraction with 1:1 w/w ratio is the best choice if a concentrated polyphenols solution is needed; conversely, a more diluted solution is preferred if a higher total content of colored polyphenols is required. Figure 9. Absorbance of extracts with different extraction method and w/w lees-solvent ratio. A) Soxhlet extraction with 1:3 less-solvent ratio, in ethanol; B) RT extraction with 1:1 wt lees-solvent ratio, in ethanol; and, C) RT extraction with 1:3 wt lees-solvent ratio, in ethanol. Table 2, the total amount of polyphenols evaluated through spectrophotometric analysis is lower for Soxhlet extract than for RT extraction, most probably due to degradation of organic molecules during the extraction process at high temperature. In particular tannins, that absorb visible light around 280 nm, results less concentrated using Soxhlet but, despite different dilution and thermal treatment, anthocyanins content is similar. The method used to determine the concentration of tannins is based on Weinges and Nader method, where procyanidin were depolymerized through the breakdown of their intra-flavonols bonds in an acidic heat medium [35]. This gives rise in carbocation formation partially converted into red cyanidins with a specific absorption at 550 nm. It is possible to assume that also, during Soxhlet extraction, this reaction took place, because of the high temperature and the presence of hydrochloric acid. Tannins content is lower in Soxhlet extract because the sample was already depolymerized and a certain amount of tannins was converted to red cyanidins. Moreover, degradation of anthocyanins is compensated by tannins conversion to anthocyanidin monomers, that absorb close to the wavelength used to determine the anthocyanin contents (520 nm). During Soxhlet extraction of white wine lees (where the anthocyanins content is less than 10 mg/L), a change in color has been noticed from yellow to redorange, confirming the reaction of tannins in acidic heat medium. Finally, RT and Soxhlet extracts of both, red and white lees, were used to impregnate semiconductor porous film of DSSCs. As reported in Table 2, the total amount of polyphenols evaluated through spectrophotometric analysis is lower for Soxhlet extract than for RT extraction, most probably due to degradation of organic molecules during the extraction process at high temperature. In particular tannins, that absorb visible light around 280 nm, results less concentrated using Soxhlet but, despite different dilution and thermal treatment, anthocyanins content is similar. The method used to determine the concentration of tannins is based on Weinges and Nader method, where procyanidin were depolymerized through the breakdown of their intra-flavonols bonds in an acidic heat medium [35]. This gives rise in carbocation formation partially converted into red cyanidins with a specific absorption at 550 nm. It is possible to assume that also, during Soxhlet extraction, this reaction took place, because of the high temperature and the presence of hydrochloric acid. Tannins content is lower in Soxhlet extract because the sample was already depolymerized and a certain amount of tannins was converted to red cyanidins. Moreover, degradation of anthocyanins is compensated by tannins conversion to anthocyanidin monomers, that absorb close to the wavelength used to determine the anthocyanin contents (520 nm). During Soxhlet extraction of white wine lees (where the anthocyanins content is less than 10 mg/L), a change in color has been noticed from yellow to red-orange, confirming the reaction of tannins in acidic heat medium. Finally, RT and Soxhlet extracts of both, red and white lees, were used to impregnate semiconductor porous film of DSSCs. Anthocyanin can show different color and stability depending on the solution pH, from red flavylium form in acidic solution to purple deprotonated quinonodial form at higher pH values. Therefore, the complexation with metal ions can shift to lower energy the absorption band of these molecules. This mechanism can be visible after impregnation process on a porous film, where there is a strongly interaction between hydroxyl or carbonyl group of anthocyanin with titanium(IV) sites of nanostructured semiconductor [10,36] that leads dye color change from red to purple.

DSSCs Efficiencies
The photovoltaic performances of DSSCs using natural winemaking lees dyes as the photosensitizer (TiO 2 -dye/electrolyte containing I, I 3 − /Pt counter electrode) were determined to evaluate the cell power and the photocurrent generated in the presence of the solar simulator. The J-V and P-V curves can be used to determine the open circuit voltage (V OC ), the short circuit current density (J SC ), the fill factor (FF), and the efficiency (η). The same photocurrent tests for the cell using bare TiO 2 _400 electrode determined under identical experimental conditions were also carried out and, as expected, no current was observed. DSSCs was realized with the mesoporous nanostructured titania calcined at 400 • C and using four different organic dyes that were extracted from winemaking lees to impregnate the semiconductor layer. Figure 10 reports the J-V and P-V curves of these solar cells.  Anthocyanin can show different color and stability depending on the solution pH, from red flavylium form in acidic solution to purple deprotonated quinonodial form at higher pH values. Therefore, the complexation with metal ions can shift to lower energy the absorption band of these molecules. This mechanism can be visible after impregnation process on a porous film, where there is a strongly interaction between hydroxyl or carbonyl group of anthocyanin with titanium(IV) sites of nanostructured semiconductor [10,36] that leads dye color change from red to purple.

DSSCs Efficiencies
The photovoltaic performances of DSSCs using natural winemaking lees dyes as the photosensitizer (TiO2-dye/electrolyte containing I, I3 -/Pt counter electrode) were determined to evaluate the cell power and the photocurrent generated in the presence of the solar simulator. The J-V and P-V curves can be used to determine the open circuit voltage (VOC), the short circuit current density (JSC), the fill factor (FF), and the efficiency (η). The same photocurrent tests for the cell using bare TiO2_400 electrode determined under identical experimental conditions were also carried out and, as expected, no current was observed. DSSCs was realized with the mesoporous nanostructured titania calcined at 400 °C and using four different organic dyes that were extracted from winemaking lees to impregnate the semiconductor layer. Figure 10 reports the J-V and P-V curves of these solar cells. Figure 10. J-V curves of the DSSC prototypes realized with red and white winemaking lees extract. R-RT is the extract of red wine lees at RT; R-S extract of red wine lees with Soxhlet; W-RT is the extract of white wine lees at RT; W-S extract of white wine lees with Soxhlet.
As reported in Table 3, the efficiency of cells built with white wine lees has lower efficiency than red wine extract, due to the poor content of anthocyanins that limits the absorption of visible light radiation. Using Soxhlet extraction for both red and white winemaking lees can increase the performance of the cell, most probably due to the tannins reaction, which leads to an increase in redcoloured molecules that can absorb in the visible range and efficiently transfer excited electron to semiconductor layer. The best efficiency η was obtained by red wine extract made with Soxhlet, showing a Voc and Jsc values of 0.492 V and 1.47 mA/cm 2 , respectively. All these data are comparable As reported in Table 3, the efficiency of cells built with white wine lees has lower efficiency than red wine extract, due to the poor content of anthocyanins that limits the absorption of visible light radiation. Using Soxhlet extraction for both red and white winemaking lees can increase the performance of the cell, most probably due to the tannins reaction, which leads to an increase in red-coloured molecules that can absorb in the visible range and efficiently transfer excited electron to semiconductor layer. The best efficiency η was obtained by red wine extract made with Soxhlet, showing a V oc and J sc values of 0.492 V and 1.47 mA/cm 2 , respectively. All these data are comparable with the typical values of an organic dye sensitized solar cell [9] built with dyes that were extracted from vegetables and fruits. Plant kingdom is full of dyes and, depending on fruit, flower or leaf concentration of photosensitive molecules, can significantly vary. Although winemaking lees are widely available, every kind of grape also has different types of polyphenols and different concentrations. Moreover, differences of polyphenols composition depending on the picking times of the grapes are possible, also employing the same type of processed wine.
Two kinds of red wine lees can lead to different efficiency, although lees have been subjected to the same extraction process, as reported in Figure 11.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 12 of 15 with the typical values of an organic dye sensitized solar cell [9] built with dyes that were extracted from vegetables and fruits. Plant kingdom is full of dyes and, depending on fruit, flower or leaf concentration of photosensitive molecules, can significantly vary. Although winemaking lees are widely available, every kind of grape also has different types of polyphenols and different concentrations. Moreover, differences of polyphenols composition depending on the picking times of the grapes are possible, also employing the same type of processed wine. Two kinds of red wine lees can lead to different efficiency, although lees have been subjected to the same extraction process, as reported in Figure 11.  (Table 4) and built while using grapes or wine as dyes. As expected, changing the typology of grapes and wine leads to a widely different efficiency, from 0.025% to 0.57%. For the future it is desirable to further study the variation of winemaking lees composition during the year to understand how the grapes properties can affect the final composition of winemaking lees and eventually purify the extracts to obtain more comparable solutions during the year since vegetables and fruits are natural agricultural products subjected to climatic conditions.  Efficiency, J sc and V oc are comparable with other DSSCs existing in literature (Table 4) and built while using grapes or wine as dyes. As expected, changing the typology of grapes and wine leads to a widely different efficiency, from 0.025% to 0.57%. For the future it is desirable to further study the variation of winemaking lees composition during the year to understand how the grapes properties can affect the final composition of winemaking lees and eventually purify the extracts to obtain more comparable solutions during the year since vegetables and fruits are natural agricultural products subjected to climatic conditions.

Conclusions
The present work aimed to exploit and valorise the main waste product of a winery, recovering the lees of red and white wines to realize DSSC solar cells and generate a renewable and sustainable photoelectric current. A nanostructured mesoporous titanium dioxide powder was synthetized by the sol-gel process, being thermally treated at different temperatures and characterized by several techniques. Organic dyes (tannins, anthocyanins, polyphenols, catechins), extracted from winemaking lees, were adsorbed on the TiO 2 nanoparticles. DSSCs prototypes were then assembled and tested under simulated solar light irradiation and the generated electric current was measured.
The photocurrent density and photovoltage obtained are comparable with the typical values of an organic dye sensitized solar cell built with dyes that were extracted from vegetables and fruits. More interesting is that these dyes are largely available and cost effective, since being recovered from a waste otherwise to be disposed of, opening up a perspective of feasibility for inexpensive and environmentally friendly dye solar cells. A sustainable and low-cost alternative to the traditional systems could be obtained while using such a peculiar class of organic dyes that were derived from winemaking lees, transforming an agri-food waste in resource to generate green electrical energy.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Encapsulation – A Perspective Improving Functional Properties of Flour-Based Confectionary

Flour-based confectionary is the most popular confectionary and have an important position in food technology in general. Those products present the most often consumed confectionary products and their growth in EU market has been increasing per capita consumption [1] owing to their sweet taste and nice aroma and flavor, easy accessibility–ready to use and well-established habits [2], lower production costs, convenience and long shelf life [3].

Introduction
Flour-based confectionary is the most popular confectionary and have an important position in food technology in general.
Those products present the most often consumed confectionary products and their growth in EU market has been increasing per capita consumption [1] owing to their sweet taste and nice aroma and flavor, easy accessibility-ready to use and well-established habits [2], lower production costs, convenience and long shelf life [3].
Due to consumers demands for the health food the improvements of this type of confectionary are becoming more and more frequent. Biscuits together with chocolate products are on the first place with 8.7% of total European food innovation [1].
By adding different functional components, it is possible to make flour-based confectionary are with functional features offer health benefits that extend beyond their nutritional value by e.g.
incorporating inulin and acacia gum [3]; chickpea and rice bran as a source of dietary fiber and active healthy components to make functional biscuits for lowering blood lipids [4]; addition of the prebiotic ingredient fructo-oligosaccharide found to provide both nutritional and technological advantages in biscuits [5].
The functional foods include foods containing minerals, vitamins, fatty acids, dietary fiber, foods with the addition of biologically active substances such as antioxidants and probiotics [6]. According to Jain [7] functional food ingredients have been brought to the consumers mainly through dairy or confectionery products [7].
Berries such (strawberries, cranberries, blueberries, raspberries and blackberries) in general may offer health promoting benefits and present excellent choice to improve flour-based confectionary from functional aspect, as they are low in calories, and abundant in anthocyanins and polyphenolic components and have a good potential to encapsulate, as well. The importance of cornelian cherry fruits in the class of berry fruit is especially emphasized [8] and traditional wild fruit (rosehip, cornelian cherry, wild strawberry and bilberry) as an important foodstuff in the local population nutrition which has been recognized in folk medicine since ancient times [9][10][11].

Encapsulation
Encapsulation technology is now well developed and accepted within pharmaceutical, chemical, cosmetic, foods and printing industries. In food products, fats and oils, aroma compounds and oleo-resins, vitamins, minerals, colorants, and enzymes could encapsulate while controlling their release and show promising approach to develop functionally active food products.
Microencapsulation describes an even finer degree of encapsulation technology and is one of the earliest examples of applied nanotechnology, which is the science of controlling matter at the atomic and molecular physical level [12]. It defined as a process to entrap active agents within a carrier material (wall material) and presents a useful tool to improve delivery of bioactive molecules and living cells into foods. This technology allows protection of bioactive components by completely enveloping and covering in small capsules that release their contents at controlled rates over prolonged periods and under specific conditions [13,14] and presents a suitable method capable of delivering adequate amounts of viable probiotic bacteria into the gastrointestinal tract [15,16].
Co-encapsulation is especially useful as this concept takes into account the synergistic effect of multiple bioactives in enhancing bioactivity to target specific health benefits [17].

Applying Encapsulation in Flour-Based Confectionary
To produce effective encapsulated products, the choice of coating material and method of microencapsulation process are most important and it also depends on the end use of the product and the processing conditions involved [18]. Lakkis [12] states the spray chilling is the most frequently used processes, due to many benefits such as microcapsule small particle size, good barrier properties, and protection through handling, mixing, and storage of bakery formulations, as well as ability to design the desired mode of release [12]. Nevertheless, other experiences are also presented.
Among the available microencapsulation methods, spray drying offers several advantages, it is a continuous process has an economical potential technology and required short residence time [19,20]. According to Alvim et al. [21] both microencapsulation methods can be used to obtain microparticles as potential protection vehicles in the application of sensitive substances such as ascorbic acid in baked products [21].
As the biscuits or cookies are widely consumed snacks, they are therefore an ideal food for fortification [22]. Incorporation of microencapsulated vitamins into food matrix contributes to improvement food nutritional value, reduce off-flavors, permit timerelease of nutrients, enhance the stability to high temperature and moisture and reduce each nutrient interaction other ingredients [18].
Wilson & Shah [23] have been considering encapsulation efficiency of vitamin C from various studies. Many studies have been done on this vitamin with variables optimized to determine the most stable way of microencapsulating vitamin C to give the highest retention possible and to use these applications in food fortification [23].
According to Shrestha et al. [22] microencapsulation is a technique that provides protection to highly susceptible micronutrients including 5-methyltetrahydrofolic acid which is regarded as one of the most bioactive forms of folate. The study investigated its stability in microencapsulated form and changes the physical properties in biscuits baked at different temperatures and times regimes. They found that microcapsule with pectin and alginate ratio of 80:20, prepared by spray drying, gave the highest retention (68.6%) of the 5-methyltetrahydrofolic acid [22]. also demonstrated that addition of small amount of encapsulated phenolic compounds into cake before baking did not affect its quality parameters adversely [25].

In study by Kulthe et al. [24] laboratory prepared
Encapsulated extract from waste red grape skin in maltodextrin were evaluated on the antioxidant properties and sensory acceptability of whole wheat cocoa biscuits. Extract addition increased the phenolic content and antioxidant capacity of both dough's and biscuits. The results showed that whole wheat cacao biscuits represent an appropriate food matrix to develop pleasant novel products enriched in phenolic compounds from waste grape skins and likeable for regular biscuits consumers [26]. The research of Elsebaie & Essa [27] focused on extraction of biological compounds from red onion peels, and its microencapsulation to evaluate cake quality integrated with microencapsulated polyphenols, and polyphenols stability assessment after baking.
Polyphenols content of encapsulating incorporated cake was enough to claim for the functionality of polyphenols in the cake [27]. Cocoa hulls represent a by-product of cocoa processing and they are a potential source of polyphenols. However, their low stability to oxidation and thermal degradation limits their practical application. The results obtained by Papillo et al. [28] evidenced the usefulness of the microencapsulation process to improve the stability of polyphenolic compounds and antioxidant properties and application in biscuit making [28].
Research of Rosha et al. [29] demonstrated possibility of applying encapsulate oily solutions of lycopene obtained by spray drying technique in cake production. They presented microencapsulation as an alternative to minimize lycopene instability. In addition, it was observed that the microcapsules were able to release pigment and color the studied food system in a homogenous manner [29].  [20]. Encapsulated thyme (Thymus vulgaris) oil presented the potential for use as a natural food preservative that can be applied to cakes to promote their shelf lives and avoid synthetic preservatives [30]. Green tea extract was encapsulated within electro sprayed protein microparticles, and the protective ability of both systems on the green tea catechins was assessed.
Results showed that microencapsulation did not significantly protect during biscuit processing and emphasized the need of assessing the behavior of microencapsulation systems in real food processing conditions [31].
Encapsulation represents a useful method to prevent the formation of potentially harmful compounds in thermally processed foods such as 5-hydroxymethylfurfural (HMF). The potential of sodium chloride encapsulation to mitigate the Maillard reaction on the HMF formation in cookies was tested. The study included 13 cookies samples prepared with recipes containing free or encapsulated NaCl. Increasing NaCl concentration from 0 to 0.65% increases HMF concentration up to 75%, whereas in the presence of encapsulated NaCl the reduction of HMF varied from 18 to 61% [32].
Due to the numerous application possibilities and various methods that enable the most suitable solutions for the incorporation of certain functional ingredients into food matrix, microencapsulation will certainly find its place in the commercial production of flour-based confectionery products.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Impact of fungicides on Rhizoctonia solani Kuhn causing sheath blight disease of rice

Rhizoctonia solani Kuhn, is an important pathogen of rice causing sheath blight disease. The pathogen thrives in soil and is polyphagous in nature. Till date no resistant variety has been registered against the disease which is the main reason of dependence on chemical management of the disease. In the present study six chemicals with different concentrations viz. 50 ppm, 100 ppm and 200 ppm along with control were evaluated in vitro against the pathogen. The chemicals were also tested in vivo for management of sheath blight. The in vitro study was conducted using the ‘poisoned food technique’ and the in vivo study was done in the field condition during kharif season in two subsequent years (2018 and 2019). Spraying of the fungicides was advocated twice after the natural infection i.e. at 45 and 60 days after transplanting. The observations revealed that all the fungicides were capable for significant inhibition of the fungus in ‘in vitro’ and the disease in ‘in vivo’ conditions.

Introduction
Sheath blight of rice is considered as the second most economically important disease of rice after blast (Manibhusanrao, 1995) [3] . It is mainly aggravated in the field when the crop is in active tillering stage. In Odisha, the crop is mostly affected by the disease during late September to October as the weather is more favourable for spread of the disease. Sheath blight disease is favoured by the high humidity condition, water logging situation near the sheath, close spacing and indiscriminate use of nitrogenous fertilizer (Kagale, 2004) [7] . Monocropping with high yielding varieties for several years is also one of the causes of prevalence of the disease due to genetic homogeneity. No resistant variety till now is registered against the disease. By observing the degree of incidence of the disease in favourable weather conditions, chemical management as the last tool under Integrated Disease Management (IDM) is very essential sometimes as the rate of spread of the disease is very high crossing Economic Threshold Level within a short span of time. Keeping in view the disease severity, a study was undertaken with some commercially available new generation chemicals along with standard chemicals, which were previously reported efficient against the disease, both in vitro and in vivo to evaluate their efficacy against the disease.

Materials and Method
In vitro Study of Chemicals against Rhizoctonia solani An in vitro study was conducted by taking six chemicals along with control (untreated) viz. (Tebuconazole 50% + Trifloxystrobin 25%) 75 WG, Propiconazole 25% EC, Azoxystrobin 25% SC, Validamycin 3L, Carbendazim 50% WP, Hexaconazole 5% SC in different concentrations of 50 ppm, 100 ppm and 200 ppm using the 'poisoned food technique' (Nene & Thapliyal, 1982) [6] . The media used for the purpose was potato dextrose agar. After calculation, the desired amount of chemicals were weighed and mixed with the calculated amount of the molten media to get the desired concentration. First 1% tween 20 was added to the emulsified chemicals for their thorough mixing and then poured into the molten media. The poisoned media with different concentrations were poured in 90 mm petri dishes. 5 mm mycelial discs were cut from the edges of 5 days old fresh culture plates of Rhizoctonia solani and inoculated at the centre of the plate.
Each treatment was replicated thrice and the design of the experiment was Completely Randomized Design (CRD). The whole process was carried out in aseptic condition by taking utmost care. The plates were incubated at 27 ± 1 °C for 24 hours and observations were taken at regular interval. The untreated plate with the fungus was maintained along with treated plates as control. The final observations were taken when the control plate attained its maximum mycelial growth and the treated plates were compared with the control plate to evaluate the inhibition of radial growth by the following formula (Vincent, 1927) [17] . Azoxystrobin 25% SC 1ml Triazole

Bio-Efficacy Study of Chemicals against Rhizoctonia solani under Field Condition
After observing efficacy of the chemicals against the pathogen in vitro, a field experiment was conducted in the Regional Research and Technology Transfer Station (OUAT), Ranital, Bhadrak for two consecutive years i.e. 2018 kharif and 2019 kharif to study the efficacy of the chemicals in field condition. The design of the experiment was Randomized Block Design (RBD) with seven treatments along with the control having three replications for each treatment. The rice variety 'Swarna (MTU 7029)' was taken as it is susceptible to sheath blight disease. The plot size was (6X3) m 2 . Standard agronomic practices with judicious fertilizer application were followed in the experiment. The crop was allowed to have natural incidence of the sheath blight infection. The chemicals were sprayed twice in respective plots at 15 days interval at active tillering stage (45 DAP) and at 60 DAP. The control plot was sprayed with only water. The data on disease incidence were recorded

Statistical Analysis
The observations recorded in the various experiments of the study were statistically analysed by OPSTAT agriculture data analysis tool, CCSHAU, Hissar.

Results and Discussion
The present in vitro study on the chemicals showed that all the chemicals significantly reduced the mycelial growth at all the concentrations. The inhibitory effect of all test chemicals increased with the increase of concentrations.  [14] reported the combination of strobilurin and azole compounds in (Tebuconazole 50% + Trifloxystrobin 25%) 75 WG could affect the quinines and sterol biosynthesis in the fungal pathogen. The effect of Hexaconazole 5% SC and Propiconazole 25% EC was also found significant with least PDI 28.99% and 26.81% with an increase in yield by 27.29% and 22.19% over control, respectively. Kumar et al. (2014) [10] and Chandra et al. (2016) [2] reported Propiconazole 25% EC at 1ml/l as most efficient with disease severity of 7.77% and 7.50% with gain yield of 3486.04 kg/ha, 3985 kg/ha, respectively. Likewise Swamy et al. (2009), Bhuvaneswari and Raju (2012) reported Hexaconazole at 2ml/l to be limiting the disease severity within 25% and 31.06% with gain yield of 5087 kg/ha and 6661 kg/ha, respectively. The promising effect of Hexaconazole 5% SC and Propconazole 25% EC also corroborates with the finding of Nagaraju (2013). This was followed by Carbendazim 50% WP with PDI 31.84% and Validamycin 3L with PDI 32.24% with grain yield of 47.77 q/ha and 44.97 q/ha, respectively and were found to be at par with each other. The finding was supported with the work of Nagaraju et al. (2017) [5] who found the effect of Carbendazim 50% WP and Validamycin 3%L to be at par in their performance with PDI of 24.80% & 21.60% with higher grain yield of 69.21% and 73.83%, respectively in field trial but contrast to the finding of Prakash (2015) i.e. Carbendazim 50% WP is most effective against the disease with low PDI of 24.81%. The effect of Azoxystrobin 25% SC on the fungus was not that promising (with PDI 24.98 and grain yield 45.75 q/ha) in the finding of Bag et al. (2016). The report revealed that Azoxystrobin 25% SC @1 ml/l effectively controlled the disease (with the lowest disease severity 16.4%) and improved grain yield 5225 kg/ha.


text:
COMPARISON OF ADAPTATION ASSESSMENT OF FOUR LOCAL SPRING WHEAT VARIETIES IN KABUL CLIMATIC CONDITIONS

A field experiment was conducted for two consecutive spring seasons of 2017 and 2018 at experimental farm of the Kabul University, to study effects of varieties on growth, yield components and yield of wheat under Agro-climate conditions of Kabul. The experimental was laid out in randomized complete block design with four treatment and three replications. Four local spring varieties i.e., V1-Zardana Baghlan,V2Sorkha-Andarab, V3-Safida-Kunduz and V4Safida-Andarab. Among the varieties Safida-Andarab (V4), produced shorter plant height but significantly higher number of tillers (416.67 and 425.33 m2), spikes (397.33 and 419.6), number of grains per spike (50.67 and 53.33), grains per spike length (3.00 and 3.00), test weight (40.33 and 41.33 g) and grain yield of (4.17 and 4.43 t/ha) of wheat, being at par with variety of Safida-Kunduz during both the consecutive years, respectively.

INTRODUCTION
Afghanistan is a semi-arid country where where drought occurs frequently due to fluctuation in a rain-fed region. Wheat cultivation in Afghanistan depends either directly on precipitation or on irrigation (FAO/WFP, 2003). A large part of the wheat crop is grown in the Northern provinces with the majority of the crop being dependent on seasonal precipitation. The total wheat production in Afghanistan is 5.00 t/ha which is achieved from an area of 2.30 million hectare with the annual productivity of 2.17 ton/ha (USDA and MAIL-Afghanistan, 2019).
Adaptation to drought stress is considered an important progress in the selection of wheat genotypes. However drought stress is found to be major constraint for crop production globally. It affect the growth and productivity of wheat by including many biochemical and molecular response of the crop under variable environmental condition Allahverdiyev et al. (2015). The ability of a cultivar to produce high and satisfactory yield over a wide range of stress and nonstress environments is very important. Finlay (1968) believed that stability over environments and yield potential are more or less independent from each other. The most widely used criteria for selecting high yield performance are mean yield, mean productivity (average yield performance under stress and non stress conditions) and relative yield performance in droughtstressed and more favorable environments Rashid et al. (2003). Bari (1990) found that the among eleven tested wheat genotypes, the wheat variety of Kanchann produced significantly higher grain yield. Chatterjee et al. (1980) carried out an experiment with three wheat cultivars (Janak, UP262 and Sonalika) and observed that significantly higher grain yield of wheat was obtained with Janak and lowest with Sonalika. Keeping all the above fact selection of resistant varieties with high adoptability to specific environmental condition is necessary to for higher grain yield.

OBJECTIVE
To assess the comparative performance of four local spring wheat varieties under Kabul climatic condition.

METHODOLOGY
A field experiment was conducted for two consecutive spring seasons of 2017 and 2018 at experimental farm of the Kabul University. The experiment was laid out in a randomized complete block design consisting of three replications and four treatments. The underground water level of site of the experiment was expected to be 10 to 11 m below the soil surface. The fertilizers were applied through Urea 200kg/ha and DAP 175 kg/ha as basal. The crop was sown with the seed rate of 120kg/ha on March 10 under rainfed condition, only one supplemental irrigation was given on May 28 was during both the years. All the growth and yield parameters were measured as per standards at different observations taken during both the experimental years. The crop was harvested with the help of sickles, bundled separately, tagged properly and sundried. Later the crop was threshed, and grains were cleaned and weighted as per plot, separately during both the experimental years.

RESULTS AND DISCUSSION
The plant height of wheat was significantly influenced by different varieties during both the years. The significantly taller plant height of 88.4 and 86.8 cm were recorded with V1. Sorkha-Andarab during both the consecutive years, respectively, while during both the years the lowest plant height of wheat was observed with Safida-Andarab (table 1)  The significantly higher grain yield of 4.17 and 4.43 t/ha of wheat was produced by Safida-Andarab followed by Safida-Kuduz during both the consecutive years, respectively. The significantly higher grain yield of wheat with mentioned treatments are due to higher yield attributing character of wheat which theses characters contributed to the final yield of wheat. Similar result was also observed by Kabir (2009)

CONCLUSION
Among the varieties Safida-Andarab (V4), produced produced shorter plant height but significantly higher number of tillers (416.67 and 425.33 m2), spikes (397.33 and 419.6), number of grains per spike (50.67 and 53.33), grains per spike length (3.00 and 3.00), test weight (40.33 and 41.33 g) and grain yield of (4.17 and 4.43 t/ha) of wheat, being at par with variety of Safida-Kunduz during both the consecutive years, respectively.


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Seed vigor of Piptadenia moniliformis Benth. subjected to thermal stress

The objective of this work is to evaluate the influence of accelerated aging test at 41oC on the germination and seed vigor of Piptadenia moniliformis Benth. Two experiments were conducted. Experiment 1: seeds were submitted to dormancy overcoming treatments. They were immersed into sulfuric acid for 0 (intact seeds), 5, 10, 15, 20 and 25 minutes. Experiment 2: two methods were evaluated for the accelerated aging test at 41oC, that is, the traditional method and the saturated NaCl solution method with exposure periods of 0, 24, 48, 72 and 96 hours. After each treatment, the seeds were subjected to electrical conductivity and germination tests. Seeds overcame their dormancy when immersed into concentrated sulfuric acid for 5, 10, 15, 20 or 25 minutes, but the germination was faster when immersed for 20 minutes. The different methods for the accelerated aging test, decreased the germination potential and seed vigor of P. moniliformis after 24 hours of exposure. Regarding the method used, the saturated solution provided, in general, the best results since the seed water content was lower, thus reducing the proliferation of fungi. The electrical conductivity test showed that, by increasing the aging period regardless of the method, there is an increase in seed deterioration, corroborating with germination results, which showed that the correlation was moderate and negative and indicated that the higher the conductivity, the lower the percentage and the germination speed index. The immersion of seeds of P. moniliformis into concentrated sulfuric acid for 20 minutes provides a fast and increased germination. The methods for accelerated aging at 41oC, traditional and saturated solution, can be used as vigor tests to evaluate the physiological quality of seeds.

Introduction
The duration and intensity of the seed deterioration process during storage are determined mainly by the interaction between genetic inheritance, water content/relative humidity and ambient temperature, causing physiological, biochemical, physical and cytological changes. Thus, there is a reduction of vigor, culminating in the death of the seed (Marcos Filho, 2015). In order to simulate what occurs during storage, the seeds are submitted to the accelerated aging test, considered a stress vigor test.
Accelerated aging is one of the most widespread vigor tests due to its accuracy and sensitivity in detecting quality differences among seed lots with a similar germination (Pereira, Torres, & Linhares, 2015). This test aims to evaluate seed resistance after a period of exposure to high temperatures and relative humidity (Marcos Filho, 2015). Temperatures above 40°C are known to cause a decreased capacity for RNA and protein biosynthesis, mitochondrial degeneration, increased chromosome fragmentation, membrane lipid peroxidation, and enzymatic changes such as reduced activity of lipases, amylases, proteases, peroxidases, among others (Dotto & Silva, 2017;Bewley, Bradford, Hilhorst, & Nonogaki, 2013;Mahjabin & Abidi, 2015). This results in a decreased germination speed and percentage, as well as in an increased formation of abnormal seedlings (Marcos Filho, 2015). Thus, seed lots that maintain a high germination rate after accelerated aging are considered of high vigor when compared to lots with a reduced viability (Pereira, Martins Filho, & Laviola, 2012).
The species Piptadenia moniliformis Benth., belonging to the Fabaceae family, is a native species to the Northeastern Brazil. It has a height of four to nine meters, and its wood is heavy, of a medium texture, a medium mechanical resistance and a good natural durability (Lorenzi, 2002). This species presents dormancy due to impermeability of the integument to water, characteristic of this family and others such as Malvaceae, Geraniaceae, Chenopodiaceae, Convolvulaceae, Solanaceae, and Liliaceae (Carvalho & Nakagawa, 2012). Due to its small size, Lorenzi (2002) emphasized that this species can be used in small works of civil construction, in light joinery and in coal production. It is also indicated for heterogeneous reforestation for preservation purposes due to its rusticity and fast growth.
Considering the increased demand for seeds of high-quality native forest species, mainly to support mixed reforestation works, it is essential that seed quality assessment methods be efficient. The objective of this work is to evaluate the influence of accelerated aging test on the physiological quality of P. moniliformis seeds.

Experiment site
The experiments were carried out at the Universidade Federal Rural de Pernambuco -Serra Talhada Academic Unit, Brazil. The seeds of P. moniliformis were granted by the Ecology and Environmental Monitoring Center (NEMA) of the city of Petrolina, PE, Brazil.

Overcoming the integument dormancy of P. moniliformis seeds
To overcome dormancy, the seeds were submitted to different periods of immersion into concentrated sulfuric acid (95-97%), i.e., 0 (intact seeds), 5, 10, 15, 20 and 25 minutes, totaling six treatments and five replications of 20 seeds per treatment. After immersion, the seeds remained for ten minutes under running water to eliminate any acid residues.
Subsequent to dormancy overcoming treatments, the seeds were sown on two sheets of blotting paper arranged in Gerbox plastic boxes previously moistened at 2.5 times their dry weight. The germination test was conducted in a climate-controlled room at an average temperature of 24ºC and an average RH of 35%, constant light, for ten days. Daily counts of the number of germinated seeds were performed until germination stabilization, adopting as a criterion seeds with a primary root protrusion of 2 mm. During the conduction of the germination test, the germination speed index (GSI) (Maguire, 1962) and the mean germination time (MGT) were obtained (Labouriau, 1983). At the end of the test, the germinated seeds were counted to determine germination percentage (GP). The immersion period in the concentrated sulfuric acid that provided the best germination in the shortest time (20 minutes) was used for overcoming the dormancy of seeds submitted to the accelerated aging test.

Evaluation of the methodology of accelerated aging test of P. moniliformis seeds
After immersing seeds of P. moniliformis into sulfuric acid for 20 minutes, the seeds were subjected to two methods for the accelerated aging test at 41ºC and five aging periods (0, 24, 48, 72 and 96 hours) in a 2x5 factorial design.
Accelerated aging test (traditional method): it was conducted using Gerbox plastic boxes containing 40 mL of water inside and a suspended stainless-steel mesh tray, where the seeds were distributed forming a uniform layer. The boxes were kept in an incubator for four aging periods: 24, 48, 72 and 96 hours at 41ºC and 100% relative humidity, besides the control.
Accelerated aging test using saline solution: instead of using only H 2 O inside the plastic boxes, 40 mL saline solution prepared with sodium chloride (40 g of NaCl in 100 mL of distilled water) were used to adjust the relative humidity to 76%, according to Jianhua and McDonald (1996).
Before and after each aging period, regardless of the method, the seed water content was determined by the greenhouse method at 105 ± 3ºC for 24 hours, according to the Seed Analysis Rules (RAS) (Brasil. Ministério da Agricultura, Pecuária e Abastecimento, 2009). After each treatment, the seeds were subjected to electrical conductivity and germination tests.
The electrical conductivity test was performed with four replicates of 25 seeds per treatment. Seeds were initially weighed and placed in plastic cups containing 75 mL of distilled water for 24 hours at 25°C. After this period, the electric conductivity of the imbibition solution was determined by a benchtop conductivity meter, and the results were expressed as µS.cm -1 .g -1 .
Germination test: four replications with 25 seeds from each treatment were sown in Gerbox plastic boxes on two sheets of blotting paper previously moistened with distilled water at 2.5 times their dry weight. The germination test was conducted in a climate-controlled room at an average temperature of 24ºC and an average RH of 35%, constant light, for ten days. Daily counts were performed of the number of germinated seeds until germination stabilization. The seed that emitted a primary root of at least 2 mm was considered germinated. The germination speed index (GSI) and the mean germination time (MGT) were evaluated according to Maguire (1962) and Labouriau (1983), respectively. On the tenth day, all germinated seeds were counted to determine germination percentage (GP).
The Pearson's simple correlation coefficients (r) were also calculated among electrical conductivity (EC) and germination percentage (GP), and germination speed index (GSI) and mean germination time (MGT) for the different methods for the accelerated aging test using the Excel software.

Experimental design and statistical analysis
The experiment was completely randomized. Data were submitted to analysis of variance (ANOVA), and the means of quantitative factors were submitted to regression analysis using the software SISVAR (Ferreira, 2011).

Results and discussion
Overcoming the integument dormancy of P. moniliformis seeds The analysis of variance of the germination percentage and germination speed index (Table 1) of P. moniliformis seeds showed significant effects (p<0.05) when seeds were submitted to different periods of immersion into sulfuric acid to overcome dormancy since the mean germination time was not influenced by the treatments. Significant effect at 1% (**), at 5% (*) and not significant effect ( ns ); coefficient of variation (CV); degree of freedom (DF) For seed germination percentage of P. moniliformis seeds submitted to different periods of immersion into sulfuric acid, a quadratic behavior was verified for the different times. The values obtained for 5, 10, 15, 20 and 25 minutes of acid immersion were 78, 86, 83, 88 and 86%, respectively, while intact seeds showed a germination of 47% ( Figure 1A).
By using different pre-germination treatments for P. moniliformis seeds, Azeredo, Paula, Valeri, and Moro (2010) found that periods of immersion into sulfuric acid (20, 25 and 30 minutes) provided a higher germination (values higher than 80%), while the immersion for 15 minutes resulted in less than 50% germination, whereas for the control, seeds that were not submitted to any germination treatment presented a germination of 15%. In turn, Benedito, Torres, Ribeiro, and Nunes (2008), using sulfuric acid, observed a germination of 81 and 82% when seeds were immersed for 10 and 15 minutes, respectively, while the immersion for 20 minutes resulted in a germination of 58%. The variation in germination found in the present study and by Azeredo et al. (2010) and Benedito et al. (2008), using seeds immersed into sulfuric acid for the same period, may be related to the degree of dormancy in which the seed was, since the intensity of dormancy varies depending on seed age from plant to plant, or even among seeds of a same plant, besides the influence of environmental conditions in which the seeds formed.
The proportion of hard seeds is higher when the plant is exposed to relative water deficiency during the seed maturation process, more specifically during the dry mass accumulation period (Marcos Filho, 2015). In this context, in the Northeast region of Brazil, characterized by an irregular distribution of rainfalls and high temperatures, there are many native forest species whose seeds present integumentary dormancy as a survival strategy. Still, the degree of dormancy may vary depending on seed size, in which smaller seeds have a higher proportion and degree of dormancy.
Regarding vigor characterized by GSI, the control presented a lower germination speed for the other treatments, as the seed immersion period increased the speed index. It can be observed that seeds immersed for 20 minutes provided a higher germination speed. The use of sulfuric acid is one of the most common methods to overcome dormancy of species with an impermeable integument. It modifies the permeability of membranes, allowing the water to be absorbed, which is essential to germination. It thus starts the germination process (Dousseau, Alvarenga, Castro, Arantes, & Nery, 2007). In addition, ruptures in the integument allow gas exchanges and suppresses mechanical resistance to protrusion of the primary root. It also facilitates embryo expansion.
By overcoming dormancy, the immersion process into water accelerates. The seeds tend to germinate faster, as can be observed for seeds that were immersed into sulfuric acid for 20 minutes. Therefore, the consumption of energy reserves during the germination process will be lower. Such reserves thus contribute to the development of seedlings (Souza, Araújo, Pinto, & Brito, 2016).

Evaluation of the method for accelerated aging test of P. moniliformis seeds
Seeds after dormancy overcoming and subsequent submission to the accelerated aging test at 41ºC for different periods were evaluated as for water content, as it may interfere with the results. According to Bewley and Black (1994), drier seeds, because they have a low matric potential, absorb water quickly when placed in a humid atmosphere. The seed water content values obtained before and after the accelerated aging test (Table 2) show that, according to the traditional accelerated aging test method, there is a marked increase in seed moisture considering that water content was initially 9.06% and that after overcoming dormancy (immersion into sulfuric acid for 20 minutes), the observed content was 22.17%. After 24 hours of aging, a value of 44.49% was reached, reaching 54.77% after 96 hours of exposure to accelerated aging. Initial water content *before and **after overcoming seed dormancy.
When the seeds aged using the saturated solution method, the initial water content was similar as the previous method (22.19% after overcoming dormancy). However, a decrease in water content was observed over the aging periods, reaching 20.39% after 24 hours and 18.71% after 96 hours. According to Marcos Filho (2015), the water content corresponds to the hygroscopic equilibrium point, which increases or decreases due to relative humidity. The salt present in the solution contributes to a lower evaporation of water molecules. Therefore, the seeds absorb less water, which reflects in a lower water content. Thus, seeds in contact with air, whose relative humidity is 100%, will present a higher water content than seeds in contact with air at 76% relative humidity. By subjecting seeds of Tabernaemontana fuchsiaefolia to accelerated aging using both traditional and saline methods, Moraes, Lopes, Farias, and Maciel (2016) observed a higher incidence of fungal attacks in traditional aging treatments, corroborating the observations of the present study. Carvalho and Nakagawa (2012) emphasized that the increase in water content favors the increase in seed temperature due to the activation of the respiratory process and the greater activity of microorganisms.
Due to the high humidity found in seeds exposed for 72 and 96 hours during the accelerated aging test using the traditional method (exclusive use of water inside the plastic box), there was proliferation of microorganisms. It is therefore a disadvantage to use the traditional method in comparison to the saturated solution method.
The results of analysis of variance of the variables germination percentage, germination speed index and electrical conductivity had significant effects (p<0.05) when the seeds were subjected to different methods and periods of exposure to accelerated aging (Table 3). Both factors evaluated interfered simultaneously with the seed vigor of P. moniliformis seeds. Mean germination time was not influenced by the treatments.  Significant effect at 1% (**), at 5% (*) and not significant ( ns ); coefficient of variation (CV); degree of freedom (DF) By using the traditional accelerated aging test method, there was a marked decrease in germination between the 0-and 24-hour periods (Figure 2A). The germination was 84 and 44%, respectively. The aging periods 48, 72 and 96 hours provided a very low germination. The period of 96 hours provided 1% of germination, possibly because of the higher degree of deterioration. With the reduction in vigor, there is also a decrease in respiratory rates, indicating a decrease in the number and/or efficiency of mitochondria, which are organelles responsible for producing a great amount of ATP, thus culminating in deleterious interferences with vital processes of seeds (Marcos Filho, 2015).
A similar behavior was observed using the NaCl saturated solution method (Figure 2A). The seeds that did not age presented a germination of 74%. For the 24-hour aging period, the germination was 52%. Therefore, there was a reduction of 22% in relation to the control (absence of aging). For the periods 48, 72 and 96 hours, the germination was 26, 20 and 13%, respectively. Salt, by limiting the evaporation of water molecules, contributed to a lower water content, thus reflecting a lower degree of deterioration, i.e., a greater vigor, as can be observed by germination values and germination speed index. Temperatures above 40°C are known to cause protein denaturation, both structural and enzymatic, causing changes in the degradation and mobilization of stored seed reserves, thus reducing the energy available for germination (Dotto & Silva, 2017).
Data regarding the germination speed index (GSI) of P. moniliformis seeds in function of the different treatments presented a decreasing linear behavior. Using the traditional methodology, the periods 0, 24, 48, 72 and 96 hours provided values of 4.68, 2.14, 0.88, 0.96 and 0.05, respectively, in relation to the means obtained using the saturated solution method: 4.18, 2.77, 1.45, 0.98 and 0.71, respectively ( Figure 2B). Comparing both methodologies in the periods of 24, 48 and 96 hours, the saturated solution method provided a higher germination rate. As the seeds aged, there was a decrease in vigor determined by the electrical conductivity test (Figure 3). It detected a significant increase in the amount of leachate from seeds subjected to aging using the traditional method. The seeds that aged for 96 hours presented a higher amount of leachate compared to seeds that aged during the other periods. Regarding the methodology using saturated solution, the values did not oscillate much among aging periods. The membrane system, which delimits the organelles and other cellular components, gradually organizes itself during seed maturation, reaching its maximum organization near physiological maturity. The deterioration process causes the loss of integrity of this system, thus releasing essential constituents to germination (Marcos Filho, 2015). By using the electrical conductivity test on Erythrina velutina Willd seeds previously submitted to accelerated aging, Guedes et al. (2009) found that seed vigor was greatly affected during accelerated aging, as evidenced by the increase in the amount of leachate released by seeds.
When the accelerated aging test methodologies were compared, the saturated solution caused a lower leachate release compared to the traditional method for the 72-and 96-hour periods. This resulted in a lower intensity of deterioration due to a lower evaporation rate of water molecules obtained by adding NaCl to water until saturation.
When correlating electrical conductivity with the percentage and germination speed index (Table 4), it is possible to observe a negative correlation of using both accelerated aging test methodologies, i.e., as there was an increase in the values of electrical conductivity, germination and speed index decreased. For the mean germination time, there was a positive correlation when seeds aged by the traditional method because, as the release of exudates increased, there was an increase in the time required for seeds to germinate.  The disruption of the cell organelle membrane system, evidenced by the progressive increase in the amount of leachate, i.e., higher EC values, makes seeds more susceptible to the deleterious effects of O 2 . In view of a stress situation, there are increases in the concentration of reactive oxygen species (ROS) in cells, culminating in membrane damage from protein degradation and from DNA and RNA molecules, impaired enzymatic activity, carbohydrate oxidation, and lipid peroxidation (Mittler, 2002). Reductions in the activity of lipase, amylase, protease, peroxidase, among others, decrease the rate of digestion of reserve substances, consequently reducing both the rate and the percentage of germination. Therefore, there is a process called oxidative stress, which corresponds to the oxidative destruction of the cell (Choudhury, Rivero, Blumwald, & Mittler, 2017). According to Bewley and Black (1994), oxidation causes reserves to be consumed faster, reflecting in the reduction of seed germination potential, which can be verified in the present work. Furthermore, the researchers point out that unsaturated fatty acids are the main targets of free radicals. Because mitochondrial membranes are very rich in unsaturated fatty acids, there is an increased likelihood of lipid peroxidation. In addition to affecting membrane permeability, peroxidation acts on the respiratory activity, directly affecting seed performance. It is noteworthy that the visible mycelial growth in seeds aged for 72 and 96 hours using the traditional method, a growth caused by a higher water content, contributed to intensify the disorganization of the cell membrane system, consequently favoring a greater loss of leachate.

Conclusion
The immersion of P. moniliformis seeds into concentrated sulfuric acid for 20 minutes provides a fast and increased germination.
The methods for accelerated aging test at 41ºC, i.e., traditional and saturated solution, affect the physiological quality of P. moniliformis seeds.