add titratable acidity assay and protocol

assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md

+## Titratable acidity
+
+Approximately 30 mg of ground leaf material was incubated in 500 µl 50% (v/v) methanol at 90 °C while shaking. After centrifugation at 13,000 g for 5 min at room temperature, 450 µl of the supernatant was collected and the remaining pellet was extracted once more. Both fractions were pooled and stored at –20 °C until use. Titratable acidity was measured using bromothymol blue (Carl Roth) as a pH indicator: 60 µl of extract was mixed with 40 µl distilled H2O and 4 µl bromothymol blue (1 mg/ml stock). Using the microinjector of a Synergy H1 (BioTek) plate reader, samples were titrated with 1 mM KOH in 5 µl steps. After each step, absorbance at 445 nm and 615 nm was recorded, and the ratio of the absorbances at 615/445 nm was calculated. Based on the 615/445 ratio of buffer standards (pH 4.6–7.5), the pH of analysed extracts was determined. From the volume of KOH required to titrate extracts to pH 7.0, the titratable acidity was calculated. Two extractions were performed from each sample, and each extract was titrated in three replicates.
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