Citric pectin

The yeasts under study were inoculated in liquid medium according to Moyo et al. (21 (link)) with modifications and with the following composition in 50 mM citric-citrate buffer, pH=3.8 (in g/L): CaCl₂ 0.05, KH₂PO₄ 0.2, MnSO₄ 0.05, (NH₄)₂SO₄ 1.0, citric pectin 1.0, dextrose 1.0.; Sigma-Aldrich Co. Ltd., Merck KGaA. Cultures were incubated under shaking conditions (130 rpm, i.e. 0.4×g) at 28 °C for 3 days, using a water bath shaker (SHZ-88; Semedix, Buenos Aires, Argentina). Cells were separated by centrifugation (10 000×g for 15 min at 4 °C; Presvac EPF-12R, Buenos Aires, Argentina) and cell-free supernatants were used as enzyme extracts.

SPRs and high sugar tolerant instant dry yeast were provided by Shandong Bio Sunkeen Co., Ltd, Jining, Shandong, China. The cellulase solutions were produced by P. oxalicum JUA10-1, T. reesei T1, T. reesei TX and Aspergillusniger, which are laboratory-maintained filamentous fungi [6 (link)]. T. reesei TX was obtained as follows: the xylanase III gene (Genbank accession number: BAA89465.2) was replaced by β-glucosidase 1 (bgl 1, Genbank accession number: KJ739789.1) derived from A. niger under the control of the T. reesei T1 xylanase III promoter. The methods for cellulase production were as described else [11 (link)]. The commercial pectinase solution was generously provided by Qingdao Vland Biotech, Qingdao, Shandong, China. The α-amylase (Novozymes (China) Biotechnology Co., Ltd) and α-glucosidase (Shandong Longda Bio-products Co., Ltd) were generously provided by Jiang Su Lianhai Biological Technology Co., Ltd. Citric pectin, polygalacturonic acid, soluble starch, and p-nitrophenyl-α-_D-glucopyranoside were purchased from Sigma-Aldrich, St. Louis, MO, USA. Whatman No. 1 filter paper was purchased from Hangzhou Whatman-Xinhua Filter Paper, Hangzhou, Zhejiang, China. All other chemicals were bought from Sinopharm Chemical Reagent, Shanghai, China.

The commercial enzyme preparations used were Pectinex Ultra SPL (Novozymes, Denmark), from Aspergillus aculeatus, and Pectinase P4716 (Sigma-Aldrich, USA), from Aspergillus niger. These preparations were diluted 2500-fold in acetate buffer (0.2 M, pH 4.5) before adding to the substrate mixture. Citric pectin (D-galacturonic acid content>74% and methoxy content>6.7%) and polygalacturonic acid (∼95%, molecular weight 25000–50000), both from Sigma-Aldrich (product numbers P9135 and 81325, respectively), were solubilized in acetate buffer (0.2 M, pH 4.5).