A High Esterifying Enzyme-Producing <i>Rhizopus</i> Strain for Fortified Daqu: Screening, Preparation, and Microbial Community Characterization.

Abstract

To explore microbial resources for ester production in sub-high-temperature Daqu, this study first established that the esterifying enzyme activity in Daqu predominantly originated from fungi, with <i>Rhizopus</i> being the dominant fungal genus. Six <i>Rhizopus</i> strains capable of decomposing esters were isolated and purified from Daqu. Following secondary screening, strain M1 exhibited the highest esterification activity (40.26 U/mL) and was identified as <i>Rhizopus oryzae</i> based on morphological characteristics and molecular biological analyses. This strain was subsequently designated as <i>Rhizopus oryzae</i> M1 (<i>R. oryzae</i> M1). Using mycelial powder of strain M1 as the inoculum and sterilized wheat bran as the substrate, a pure-culture Fuqu was prepared. Orthogonal array design experiments were conducted to optimize the preparation process of this Fuqu, using esterifying enzyme activity as the evaluation index. Under the optimal conditions, the spore count and esterification activity of the pure-culture Fuqu reached 1.73 × 10⁹ CFU/g and 80.13 U/g, respectively. This pure-culture Fuqu was subsequently used as an inoculum to produce fortified Daqu. Following orthogonal optimization of the Daqu preparation process, the esterification activity of the fortified Daqu reached 103.22 U/g, and its key physicochemical indices met the requirements for high-quality sub-high-temperature Daqu. Analysis of the microbial community structure revealed that <i>Rhizopus</i> was the dominant fungal genus in the fortified Daqu, with its relative abundance increased by 35% compared to the non-fortified Daqu. Consistent with this, the esterifying enzyme activity of the fortified Daqu was 51.79% higher, suggesting that <i>Rhizopus</i> may have been largely responsible for the increase in esterification capacity. In laboratory-scale Baijiu brewing trials, this fortified Daqu produced a base Baijiu with a total ester content of 2.74 g/L, representing a 40.5% increase over the non-fortified Daqu and further confirming the pivotal role of <i>Rhizopus</i> in driving the esterifying enzyme activity. This study successfully screened a high esterifying enzyme-producing strain, <i>R. oryzae</i> M1, systematically optimized its enzyme production and Qu-making processes, and provides an excellent microbial strain and process reference for the preparation of fortified Daqu and the enhancement of Baijiu flavor.