Citric acid

Six organic acids (tartaric acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid) were analyzed with an HPLC (1260 series, Agilent, Santa Clara, CA, USA) equipped with a YMC-Triart C18 (4.6 × 250 mm, 5 μm) column. The mobile phase was 25 mM KH₂PO₄ (pH 2.5) and the temperature was set to 30 °C. A 210 nm diode array detector (DAD) and a 1.0 mL/min flow rate were used. The injection volume was 20 μL. Vinegar samples were diluted 10-fold with 0.4% HCl and then filtered with 0.22 μm polyvinylidene fluoride (PVDF) membrane. Standards for six organic acids were also dissolved in 0.4% HCl and used. Concentrations of standards were set to 5, 10, 25, 50, 75, 100, 150, and 200 μg/mL. Correlation values were all over 0.9999. Standards used included L (+)-tartaric acid, 99+% (purity 100%, Thermo Fisher Scientific, Waltham, MA, USA), DL-malic acid (purity 99.2%, TCI, Tokyo, Japan), DL-lactic acid (purity 90.6%, TCI, Tokyo, Japan), acetic acid (purity 99.9%, TCI, Tokyo, Japan), citric acid (purity 99.7%, TCI, Tokyo, Japan), and succinic acid (purity 98.9%, TCI, Tokyo, Japan). The organic acid analysis was performed based on the method in the previous report, with some modifications [26 (link),27 (link)].

The fermenting broth was filtered using Whatman filter paper No. 1 to remove the fungal biomass. The citric acid in the filtrate was recovered by the double precipitation method [44 , 50 (link)] using Ca(OH)₂ and H₂SO₄ in a stepwise manner to separate citric acid from the fermenting broth. To 500 ml of the filtrate, 30 g of Ca(OH)₂ was added and agitated briefly at 60 °C. The calcium citrate precipitate that was formed was recovered, diluted with distilled water (1:10) and further treated with conc. H₂SO₄ (10:1). The sequence of the recovery process of citric acid is depicted in Fig. 3. The quantification of the citric acid was done on HPLC Infinity 1260 (Agilent Technologies, USA) [5 (link), 38 (link)] at a wavelength of 211 nm using a diode array detector (DAD). The citric acid (Sigma-Aldrich, Darmstadt, Germany) was prepared by dissolving 0.001 g in 10 ml of sodium phosphate buffer and used as standard. Acetonitrile buffer at 70:30 (pH 6.5) was used as the mobile phase at a flow rate of 1.0 ml/min. The temperature of the column (Primesep D, 100 Å, 5 μm, 4.6 × 150 mm) was maintained at 35 °C during the analysis.
Fig. 3

Scheme for the recovery and purification of citric acid

Four-μm-thick formalin-fixed, paraffin-embedded serial sections of lung tissues were used for immunohistochemistry analysis of E-cadherin and vimentin expression. The sections were immersed in xylene for 15 minutes before being rehydrated in water by using an ethanol gradient. Then the sections were immersed in citric acid (pH 6.0; DAKO) for 10 minutes, after the samples were cooled to room temperature, the sections were washed with water and PBS buffer for 15 minutes before incubated with 3% H₂O₂ for 10 minutes. Then the sections were blocked with 5% BSA for 30 minutes before incubated with the primary antibody overnight at 4 °C. Color development was performed by using a DAB color development kit (DAKO). Sections were scanned by Pannoramic Scanning Electron Microscope to view the images.