Phenolphthalein

Refined food grade canola oil was purchased from a local store in Hong Kong while crude Jatropha oil was produced from cold-pressed oil extractor using Jatropha seed which was supplied by a local store in China. Manganese (II) acetate tetrahydrate ((CH₃COO)₂Mn·4H₂O, 97%) was obtained from Fraco Chemical Supplies. Glycerol (C₃H₈O₃, >99%), cobalt (II) acetate tetrahydrate ((CH₃COO)₂Co·4H₂O, >98%) and iron (II) acetate ((CH₃COO)₂Fe, 95%) were purchased from Acros. Zinc acetate dihydrate ((CH₃COO)₂Zn·2H₂O, 98.5%) was provided by BDH Chemical Ltd. Ethanol (C₂H₅OH, 99.0%) of reagent grade was supplied by ACS. Oleic acid (C₁₈H₃₄O₂, 99.9%) was purchased as laboratory reagent grade from Fisher Chemical. Rice wine (24.4 wt.% ethanol) and glutinous rice wine (14.1 wt.% ethanol) were purchased from local stores in Hong Kong. Methyl yellow, methyl red, neutral red, bromothymol blue and phenolphthalein were supplied from Sigma Aldrich. Sodium hydroxide (NaOH, 96.0%) of analytical reagent was purchased from UNI-CHEM.

The growth of bacterial and yeast cells was measured as the absorbance at a wavelength of 600 nm using a spectrophotometer (BioSpec-mini; SHIMADZU, Kyoto, Japan). Samples for analysis were prepared by periodic removal from the fermentation broth and centrifugation at 13,000× g for 1 min, followed by collection of the supernatant. To determine the titratable acidity, 9 mL of sample was added to 10 mL of distilled water and titrated with 0.1 N sodium hydroxide (Samchun Chemical, Seoul, Korea) using phenolphthalein (Sigma-Aldrich, St. Louis, MO, USA) as an indicator. The volume of NaOH used in the titration was expressed as the titratable acidity (%) for neutralizing lactic acid as in Equation (1): $\mathrm{Titratable}\mathrm{acidity}(\%)=\frac{0.1\mathrm{N}\mathrm{NaOH}\left(\mathrm{mL}\right)\times 0.009\times 100}{sample weight \left(\mathrm{g}\right)}$
where, 0.009 was lactic acid equivalent.
The pH and sugar content (°Brix) were determined using a pH meter (FE20; Mettler Toledo, Columbus, OH, USA) and refractometer (MT-325; Atago Co., Ltd., Tokyo, Japan), respectively. The ethanol concentration was determined using the Roche Ethanol Assay Kit (Cat. No. 10 176 290 035; Roche, Basel, Switzerland) according to the manufacturer’s protocol.

The N-hexane, ethyl acetate, methanol, ethanol, Folin–Ciocalteu reagent, gallic acid, Sodium carbonate, potassium hydroxide, diethyl ether, phenolphthalein, starch indicator were purchased from Sigma-Aldrich (Steinheim, Germany). Wijs solution (0.1 mol∙L⁻¹ iodine monochloride, acetic acid) was purchased from (Fluka (Steinheim, Germany)), chloroform, acetic acid by (lachner), potassium iodide, sodium thiosulfate by (CHEMPUR (Poland)). The methylene chloride, sodium chloride, sodium sulfate were provided by (Acros Organics (Great Britain)). All of the chemicals used for the analyses were of analytical grade, and all of the test solutions were freshly prepared prior to use.