HAMA was synthesized through an esterification reaction in alkaline conditions following the protocol described by Khunmaneeet et al. [28 (link)]. HA (1 g) was dissolved in 100 mL of Milli-Q water in a two necked glass flask for 24 h. MA was added to the HA solution at a MA:HA ratio of 1:1. The mixture was kept at 0 °C using an ice bath and the pH was controlled at 8.5 by adding NaOH (5 M) with the help of an automatic titrator (Metrohm, Switzerland) for 24 h. The final product was purified by precipitation in cold ethanol, subsequently centrifuged (Eppendorf centrifuge 5810 R model, Madrid, Spain), dissolved in double distilled water (ddH2O), and dialyzed for 4 days. After freeze drying, a white powder was finally obtained. HAMA was characterized by proton nuclear magnetic resonance (1H-NMR, Bruker AVANCE IIIHD-400, MA, USA) and attenuated total reflection–Fourier transform infra-red (ATR-FTIR, Perkin-Elmer (Spectrum One), Waltham, MA, USA) spectroscopies. HAMA methacrylation degree was determined by its 1H-NMR spectrum giving a value of 4.5% (Figure S1 ).
Automatic titrator
Manufactured by Metrohm
Sourced in Switzerland
The Automatic titrator is a laboratory instrument used for the automated determination of various analytes in a sample through a titration process. It provides precise and accurate measurements by controlling the addition of titrant, detecting the endpoint, and recording the data.
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3 protocols using automatic titrator
1
Synthesis and Characterization of HAMA
2
Determining Total Soluble Solids and Acidity
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Total soluble solids (°Brix) were measured using an Abbé-type refractometer (Atago RX-5000). Titratable acidity was measured using an automatic titrator (Metrohm, Herisau, Switzerland) with 0.1 N NaOH. Tartaric and malic acids were measured using enzymatic kits from Boehringer 146 Mannheim GmbH (Mannhein, Germany). The methodology used to carry out these analyses is described in OIV (Organisation Internationale de la Vigne et du Vin) (2018) [19 ].
3
Comprehensive Berry Quality Assessment
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Berry fresh weight was determined from a random sample of 200 berries per EU.
Then, 150 berries were crushed and hand-pressed through a metal screen filter to assess must characteristics including total soluble solids (TSS), pH, total acidity (TA), and malic and tartaric acid concentrations. Must TSS were determined by refractometry with a PR-101 refractometer (Series Palette, Atago, Tokyo, Japan), pH and TA were measured in an automatic titrator (Metrohm, Herisau, Switzerland), this latter one using 0.1 N NaOH to an end point of pH 8.2 following the official methods of the Office International de la Vigne et du Vin (OIV 1990) . Berry ripening was assessed using the TSS to TA ratio at harvest as the maturity index (Al-Kaisy et al. 1981) . The concentrations of tartaric and malic acids were measured via infrared spectroscopy with a Bacchus II IR spectrometer (Tecnología Difusión Ibérica, Barcelona, Spain) according to García- Romero et al. (1993) (link).
The remaining 50 berries were homogenized with a blender (Ultraturrax T25, IKA-Werke, Staufen, Germany) for determining phenolic maturity. Anthocyanin and phenolic substances (expressed in malvidin equivalents) were determined in duplicate by UV/VIS spectrophotometry (Iland et al. 2004 ).
Then, 150 berries were crushed and hand-pressed through a metal screen filter to assess must characteristics including total soluble solids (TSS), pH, total acidity (TA), and malic and tartaric acid concentrations. Must TSS were determined by refractometry with a PR-101 refractometer (Series Palette, Atago, Tokyo, Japan), pH and TA were measured in an automatic titrator (Metrohm, Herisau, Switzerland), this latter one using 0.1 N NaOH to an end point of pH 8.2 following the official methods of the Office International de la Vigne et du Vin (OIV 1990) . Berry ripening was assessed using the TSS to TA ratio at harvest as the maturity index (Al-Kaisy et al. 1981) . The concentrations of tartaric and malic acids were measured via infrared spectroscopy with a Bacchus II IR spectrometer (Tecnología Difusión Ibérica, Barcelona, Spain) according to García- Romero et al. (1993) (link).
The remaining 50 berries were homogenized with a blender (Ultraturrax T25, IKA-Werke, Staufen, Germany) for determining phenolic maturity. Anthocyanin and phenolic substances (expressed in malvidin equivalents) were determined in duplicate by UV/VIS spectrophotometry (Iland et al. 2004 ).
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