α amylase

Extrusion trials were performed using commercial chokeberry (Aronia melanocarpa) pomace powder (CPP) (Aronia Original Naturprodukte GmbH, Dresden, Germany) [20 (link)]. Chemicals and reagents of analytical purity grade were obtained from Merck KGaA (Darmstadt, Germany), unless stated otherwise. Amyloglucosidase (E-AMGDFPD, from Aspergillus niger, 36,000 U/g), α-amylase (E-PANAA, from pig pancreas 100,000 U/g), protease (E-BSPRPD from Bacillus licheniformis 9000 U/g), Celite 545 and endo-arabinanase (EC 3.2.1.99, from A. niger, 9 U/mg) were from Megazyme (Bray, Ireland) and Amberlite FPA53 and Ambersep 200 from Rohm and Haas Europe (Frankfurt, Germany). Thermostable α-amylase (Thermamyl 120 L, EC 3.2.1.1, from B. licheniformis, 120 KNU/g), protease (Alcalase 2.5 L, EC 3.4.21.62, from B. licheniformis, 2.5 AU/g), and Amyloglucosidase (AMG 300 L, EC 3.2.1.3, from A. niger, 300 AGU/g) were a gift from Novozymes, (Bagsværd, Denmark). Rotihydroquant C5 and D used for Karl Fischer titration as well as cyanidin-3-O-glucoside (≥96%), cyanidin chloride (≥97%), 5-caffeoylquinic acid (≥97%), quercetin-3-O-glucoside (≥99%), and quercetin dihydrate (≥99%) used as PP standards were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Ultrapure water was used for all experiments.

Thirty milliliters of culture broth were periodically harvested from shaking flask cultures. The sampled broth was centrifuged at 1500×g (4 °C, 1 h) in conical tubes, and the supernatant and precipitate were separated. The collected supernatant was used to assay alizarin concentration and enzymatic activity.
A membrane filter method was used to measure dry cell mass. Briefly, after harvesting and centrifuging 30 ml of culture broth and separating the supernatant and precipitate as described above, the precipitates were re-suspended in 30 ml of 1 mM CaCl₂ and then treated with 10 μl of α-amylase (Novozymes) at 70 °C for 1 h. Thereafter, the suspension was collected on a cellulose acetate membrane filter (pore size, 0.8 μm), which was subsequently dried at 80 °C for 24 h. The dry cell mass was then calculated from the total weight minus the weight of the membrane filter.

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 black wheat seed was provided by the Cotton Research Institute of the Shanxi Academy of Agricultural Sciences (Taiyuan, China). The xylanase (food grade 50,000 U/g), cellulase (food grade 50,000 U/g), high-temperature α-amylase (food grade 40,000 U/g), and acid protease (food grade 700,000 U/g) were obtained from Solarbio (Beijing Solarbio Technology Co., Ltd., Beijing, China). The α-amylase (15 U/mg) and pepsin (474 U/mg) were purchased from Novozymes (Bagsvaerd, Denmark).