Sub-high temperature Daqu, a traditional solid fermenting agent used in Chinese strong-aroma Baijiu production, is abundant in diverse microorganisms, including bacteria, yeasts, molds, and actinomycetes. Among these, yeasts are pivotal for ethanol production and flavor formation. However, counting yeasts in Daqu is challenging due to interference from molds and bacteria. Antibiotics are employed to inhibit bacterial growth, but there is no practical way to suppress molds without affecting the growth of yeasts. In this study, short-chain carboxylates (C1-C6) were added to the culture medium at various pH conditions to investigate their effects on the growth of molds and yeasts. The results demonstrated distinct inhibitory effects of the short-chain carboxylates, depending on both pH and concentration. Several tested short-chain carboxylates effectively suppressed mold growth on agar plates while leaving yeast growth unaffected. This suggests a simple and feasible method for enhancing the efficiency of yeast isolation and counting in Daqu. Such an approach is valuable for studying yeasts in diverse and complex habitats.

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 Rhizopus being the dominant fungal genus. Six Rhizopus 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 Rhizopus oryzae based on morphological characteristics and molecular biological analyses. This strain was subsequently designated as Rhizopus oryzae M1 (R. oryzae 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 Rhizopus 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 Rhizopus 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 Rhizopus in driving the esterifying enzyme activity. This study successfully screened a high esterifying enzyme-producing strain, R. oryzae 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.

Lactic acid (LA) is a significant byproduct in Baijiu fermentation, with both D(-)-lactic acid (D(-)-LA) and L-lactic acid (L(+)-LA) being present due to the diverse microbial metabolism involved. L(+)-LA, a key industrial raw material, is metabolizable by humans, unlike D(-)-LA. This study aims to isolate microorganisms from fermented grains that preferentially produce L(+)-LA and apply these strains to Baijiu fermentation processes. We analyzed 106 commercially available Baijiu samples, along with fermented grains, yellow water, and Baijiu samples from 10 Baijiu factories, confirming the coexistence of D(-)-LA and L(+)-LA in fermentation products. Notably, a strong correlation was found between L(+)-LA in fermented grains and L-ethyl lactate (L(+)-EL) in Baijiu. Microbial community analysis revealed that Lactobacillus and Bacillus were the dominant bacterial groups, with a succession of microbial communities throughout fermentation. Two strains, A6 and B12, identified as Bacillus licheniformis and Bacillus cereus, respectively, were isolated for their high selectivity in L(+)-LA production. Both strains demonstrated a high yield of L(+)-LA in fermentation tests. Incorporating these strains into both liquid- and solid-state fermentation systems markedly increased the proportion of L(+)-EL relative to total ethyl lactate in Baijiu. This study successfully isolated and applied L(+)-LA-producing strains to fermentation models, offering a practical strategy for increasing L(+)-LA content in Baijiu.