A novel integrated bioprocessing strategy for co-production and sequential extraction of menaquinone-7, nattokinase, and γ-polyglutamic acid from Bacillus subtilis natto

Abstract

Bacillus subtilis natto (B. subtilis natto) can produce various beneficial metabolites, including menaquinone-7 (MK-7), nattokinase (NK), and γ-polyglutamic acid (γ-PGA), which were widely applied in healthcare and food industry. This study developed an integrated bioprocessing strategy combining low-cost soybean meal hydrolysates (SMH) with biofilm-based fermentation to promote the synthesis of these metabolites, and established a sequential extraction system to simultaneously extract all three metabolites from a single fermentation matrix. Specifically, using soybean meal hydrolysates prepared with trypsin (SMH-T) as medium achieved maximum yields of 52.4 g/L for MK-7, 2949.3 U/mL for NK, and 11.7 g/L for γ-PGA at a 20 g/L addition under biofilm-based fermentation mode. This was attributed to SMH-T forming more robust biofilms, where the cells had higher membrane potential and appropriate ROS accumulation compared to other nitrogen sources. Moreover, soybean meal hydrolysates prepared with protamex1.6 (SMH-P) were more suitable for submerged fermentation system, achieving fermentation effects comparable to commercial soy peptone. Additionally, based on the distinction in the precipitation ability of ammonium sulfate for various proteins, including NK and MK-7 protein complexes, a sequential extraction system was established, allowing for the sequential extraction of MK-7, NK, and γ-PGA with extraction rates of 75.4 %, 77.1 %, and 71.9 %, respectively. This study provides a promising strategy for enhancing the production and extraction efficiency of valuable metabolites utilizing low-cost substrates.