In SituAnalysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

Abstract

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q 2 ] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Spontaneous and solid-state fermentation has been used for thousands of years for the preparation of traditional foods such as bread, tempeh, yogurt, and cheese (1-3). Spontaneously fermented foods are generally characterized by a wide variety of tastes and textures, and sensory properties of the foods are generally determined by the involved microflora, besides the raw material (4, 5). With this in mind, it is therefore important to identify the functional microorganisms contributing to the formation of food flavors.The metabolic characteristics of lactic acid bacteria (LAB) and yeasts have been studied extensively (6-8), and their effects on food flavor have been highlighted through the detection of the corresponding metabolites in pure cultures. Although these studies have provided valuable information regarding the individual species, they take no account of the fact that the level of volatiles produced by a single species may be influenced by other species in a mixed-culture system. The different flavors of foods would be determined to a large extent by the in situ metabolic characteristics of different microorganisms. Several studies have already described the metabolic characteristics of mixed cultures with the aim of identifying the effects of individual strains on the generation of volatile compounds (9-12). For instance, the metabolic footprints of each strain of Saccharomyces in monoculture and mixed cultures were derived during wine fermentation (9). This showed that the volatile compounds produced in the mixed culture could not be predicted based on the volatiles observed in the monocultures of the same yeast species. Recent work i...

Flavor Profile of Chinese Liquor Is Altered by Interactions of Intrinsic and Extrinsic Microbes

“…uvarum and S. servazzii were unique strains in another type of Chinese light-style liquor, and they produced special flavors in liquor fermentation (15). To improve flavor, they were added to this intrinsic Fen liquor coculture.…”

“…The following strains were isolated from the Chinese light-style liquor-making process (15) Mixed-culture fermentations. The fermentation medium was prepared with sorghum as previously reported (16).…”

“…Six compounds (ethyl acetate, ethyl hexanoate, isobutanol, 3-methylbutyric acid, 2-phenylethanol, and geranyl acetone) were significant by the combination of the two qualitative measures. These compounds are also the main flavor compounds in Chinese light-style liquor (15). The intrinsic species culture produced more geranyl acetone than any of the single cultures.…”

“…Each sample (10 g) was mixed with 90 ml sterile saline (0.85% NaCl) and soaked at 4°C for 30 min. Yeast enumeration was carried out on Wallerstein Laboratory nutrient (WLN) medium (17), in which the five strains showed different macroscopic features, which were detailed in a previous report (15). According to the macroscopic features (texture, surface, margin, elevation, and color), colonies of different types on the WLN medium were counted separately (15,16).…”

“…After ultrasonic treatment at 0°C for 30 min in a capped bottle, the mixture was centrifuged at 8,000 ϫ g at 4°C for 10 min. The supernatant was filtered through a 0.22-m filter and stored at Ϫ20°C until analysis (15). Volatile compounds in fermented sorghums were assayed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (6890N gas chromatograph and 5975 mass selective detector; Agilent) and on a DB-Wax column ( Standard deviations were calculated from triplicate repetitions of the fermentation.…”