Casein

Methanol, acetonitrile, and acetic acid, chromatographically pure, were purchased from Thermo Fisher Technology China Co., LTD. (Shanghai, China). Sodium hydroxide was provided by the Tianjin Tianshun Lye Co., LTD. (Tianjin, China), edible salt was purchased from the Jiangxi Jiangyan Huakang Industrial Co., LTD. (Jiangxi China), and copper sulfate (food grade) was provided by the Xilong Technology Co., LTD. (Guangdong, China). Casein, trichloroacetic acid, and folinol were purchased from the Beijing Solarbio Technology Co., LTD. (Beijing, China). Sulfosalicylic acid was purchased from the Tianjin Damao Chemical Reagent Factory (Tianjin, China). Standard products: 5′-cytidine monophosphate (CMP), 5′-uridine monophosphate (UMP), succinic acid, 5′-adenosine monophosphate (AMP), 5′-guanosine monophosphate (GMP), 5′-inosine monophosphate (IMP), and 5′-xanthosidic monophosphate (XMP) were purchased from the Beijing Solarbio Technology Co., LTD. (Beijing, China).

The A. oryzae strain 3.042 was purchased from China General Microbial Culture Collection Center (CGMCC No. 3.00951, Beijing, China) and used as the original strain for ARTP mutagenesis. Strain B-2 (CGMCC No. 8199) was a mutant strain with the supposedly highest acid protease activity by ARTP treatment of A. oryzae strain 3.042 in this study.
The bran and bean pulp were kindly supplied by Tianjin Tianli Mature Vinegar Co., Ltd. (Tianjin, China). Casein and Folin-Ciocalteu’s phenol reagent were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). All other reagents were of analytical grade.
Potato dextrose agar (PDA) medium was used for the conventional culture and subculture of A. oryzae strain 3.042 and its mutants (Zhao et al., 2012 (link)). The screening medium was composed of 1% non-fat dry milk and 1.5% agar, which was sterilized at 115°C for 20 min before mixing, and the medium was adjusted to pH 3.0 using HCl under sterile conditions. The solid-state fermentation medium consisted of bean pulp, bran, and tap water at a ratio of 6:4:8 and was sterilized at 121°C for 30 min.