Beef extract

Fresh soybean residue was provided by Kangle Tofu Workshop (Changchun, China). Soybean residue should be dried for 24 h at 55 °C before reuse. Saccharomyces cerevisiae strain BNCC337309 was obtained from the BeNa Culture Collection (Beijing, China). The KC205 LAB strain was isolated and screened from homemade pickled cabbage juice by the food microbiology team of the Agro-product Process Institute of the Jilin Academy of Agricultural Sciences, and has the characteristics of strong acid production and acid resistance.
High temperature-resistant α -amylase (2 × 10⁴ U/mL), papain (800 U/mg), and amyloglucosidase (1 × 10⁵ U/mL), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Peptone, yeast extract fermentation, glucose, NaCl, beef extract, anhydrous sodium acetate, sodium citrate, K₂HPO₄, MgSO₄, MnSO₄ and Tween 80 were all purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals were of analytical grade or better.

The strains and plasmids used in this study are shown in Table 2. E. coli strains were grown in Luria-Bertani medium (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl [pH 7.0]) at 37°C. L. enzymogenes strains were grown at 28°C in Luria-Bertani medium and 10% TSB. M813 modified medium (4 g glucose, 3 g K₂HPO₄, 1.2 g NaH₂PO₄, 1 g NH₄Cl, 0.3 g MgSO₄, 0.15 g KCl, 10 mg CaCl₂, and 2.8 mg FeSO₄·7H₂O, per liter) was used for the growth of L. enzymogenes OH11 (32 (link)). For the preparation of culture medium, tryptone, peptone, beef extract, and yeast extract were purchased from Sangon Biotech (Shanghai, China). When required, antibiotics were added (100 μg/ml sodium ampicillin, 30 μg/ml kanamycin sulfate, and 50 μg/ml gentamicin) to the E. coli or L. enzymogenes cultures. The bacterial growth in liquid medium was determined by measuring the optical density at 600 nm (OD₆₀₀) using a Bioscreen-C automated growth curves analysis system (Oy Growth Curves, Helsinki, Finland).