Uv 1603

The chlorophyll and carotenoid contents of the leaves were determined at the end of each trial. About 0.125 g of fresh leaves were incubated in 10 mL methanol for 24 h in the dark. The concentrations of chlorophylls and carotenoids were measured spectrophotometrically (Shimadzu UV1603) at A₆₆₆ and A₆₅₃. The total chlorophyll and carotenoid content was calculated as described by Wellburn [46 ].
For the determination of photosynthesis, leaves at the end of each Sb treatment were adapted in the dark for 10 min, and then the maximum photochemical efficiency (F_V/F_M) was recorded by a handheld fluorometer (Chlorophyll Fluorometer, OS-30p, Opti-Sciences).

The pH was measured with a pH-meter (Crison Basic 20, Barcelona, Spain) using a penetration probe electrode [27 ]. Water activity (aw) was determined according to the ISO [28 ] by using Aw Sprint equipment (Novasina, Axair Ltd., Pföffikan, Switzerland) and moisture content by oven-drying in accordance with the ISO norm [28 ]. Nitrite content was determined spectrophotometrically (Shimadzu UV-1603, Duisburg, Germany) according to the ISO [29 ]. These parameters were determined in two samples of each batch which were taken on the first to the 21st days of the ripening process.
The total fat content (extractable ether) was determined by using the ISO [30 ] official method. Fat oxidability was measured to determine the thiobarbituric acid reactive substance (TBARS) content of the samples according to the method of Buege and Aust [31 ] and expressed as mg of malonaldehyde per kg of sausage. All the determinations were carried out in duplicate on the 21st day of ripening.

Leaf discs from fresh leaves were taken and incubated in methanol (12.5 mg mL⁻¹) for 24 h in darkness at room temperature. The chlorophyll a, chlorophyll b, and carotenoid contents were determined in a spectrophotometer (Shimadzu UV 1603, Kioto, Japan) by measuring A₆₆₆, A₆₅₃, and A₄₇₀, expressed as µg g⁻¹ FW. The total chlorophyll and carotenoid contents were calculated following Wellburn [33 (link)].
The maximum photosynthetic efficiency (F_V/F_M) was determined on fresh leaves of intact plants, before being collected, using a “ChlorophyllFluorometer OS-30p” device (Opti-Sciences, Hudson, NH, USA). Prior to the excitation, the leaves being sampled were kept in darkness for 10 min, then illuminated so as to measure the fluorescence emitted and calculate the F_V/F_M ratio [34 (link)].

Astaxanthin (Asx) was extracted from the PL, Hx and Ac extracts, as well as from the fresh liposomal dispersions, as previously described [25 (link)], introducing some modifications. Both lipid extracts and liposomal dispersions were mixed with hexane at a ratio of 1:1 (w/v or v/v), shaking vigorously in a vortex for 1 min, followed by centrifugation at 6000× g for 30 min at 4 °C. The supernatants obtained were measured at 470 nm in a spectrophotometer (UV-1603 Shimadzu). Astaxanthin content was determined according to Equation (1): $$\mathrm{Asx}\left(\mathrm{mg}\right)=\frac{\mathrm{A}\ast \mathrm{V}\ast \mathrm{P}}{\mathsf{\epsilon }}$$
where A is the absorbance, V is the dilution volume (mL), P is the molecular weight of astaxanthin (597) and ε is the molar absorption coefficient of astaxanthin (125,100).

The concentration of dye was measured in the supernatant using a spectrophotometer (Shimadzu UV-1603), after the equilibrium was reached. The absorbance was measured before and after the treatment with the polymers at the peak absorbance of the dye (λ_max = 526 nm; ε₅₂₆ = 1065 M⁻¹cm⁻¹) [36 (link)].

Pigments were extracted from 100 to 200 mg ground grapevine leaves using 100% cold acetone and maintained in a cold ultra-sound bath for 2 min. Samples were maintained in the dark overnight at −20 °C to continue extraction [40 (link),41 (link),42 (link)] and centrifuged at 4000 rpm for 15 min at 4 °C. The supernatants were collected and analysed using a dual-beam spectrophotometer (Shimadzu UV-1603). Absorbance spectrums were performed from 350 to 700 nm (0.5 nm steps) and introduced in the Gauss-Peak Spectra (GPS) fitting library, using SigmaPlot Software.
To detect chlorophyll (Chl) a and b and allomer (alloChl) a, pheophytin (Pheo) a and b and allomers (alloPheo) a and b, auroxanthin (Auro), antheraxanthin (Anth), β-carotene (β-Car), trans-lutein (Lut), neoxanthin (Neo), violaxanthin (Vx) and zeaxanthin (Zx) and isomers trans-Zeaxanthin (transZX), Cis-9-Zeaxanthin (cis9Zx), Cis-13-Zeaxanthin (cis13Zx), the algorithm developed by Küpper et al. (2007) [43 (link)] was applied.

The color of the white wine was determined using a ColorFlex 45/0 device (Hunterlab, Virginia, USA). Measurements were carried out at room temperature according to the CIE (Committee International d’Elairage) Lab color notation system. The instrument was calibrated with standard white (L* = 93.17, a* = −0.96, b* = 1.53) and black (L* = 0.29, a* = 0.47, b* = 0.05) tiles, and wine samples were measured in a glass dish (25 mm diameter). Hue angle (h^o) was calculated from tan⁻¹ (b*/a*), and Chroma was calculated as (a*² + b*²)^½. The total colorimetric difference between the color of treated and untreated wine samples is given by the CIELAB color difference [23 ]:
where L₀*, a₀* and b₀* are the color parameters of the untreated samples, and L_F*, a_F* and b_F* are the color parameters of samples treated at fluence F.
The UV spectrum of the wine was determined in a 1:20 dilution of wine in demineralized water measured in a quartz cuvette with a 1 cm optical path length using a spectrophotometer (Shimadzu UV-1603, Japan). Dilution was required because the absorbance spectrum of the undiluted sample could not be measured due to excessive light absorption.