| In the traditional Chinese liquor industry, Bacillus licheniformis is an important strain,which has an important contribution to the quality of Chinese liquor. Scientific understandingof the metabolites of Bacillus licheniformis and its mechanism in Chinese liquor production,will help us to realize the brewing mechanism and provide theoretical guidance for liquorproduction. However, research about the unique characteristics and features of the metabolitesof Bacillus licheniformis in the solid-state fermentation conditions is still rare. Thus, this thesisis on the metabolism of Bacillus licheniformis in the solid state fermentation, detection andanalysis of the important metabolites by Bacillus licheniformis, and the influence mechanismof the metabolites on aroma compounds in the liquor. This work is not only important forapplications of liquor quality and upgrading traditional industries, but also important forimplications of Chinese traditional brewing mechanism and its flavoromics.In this study, the application of microbial metabolism analysis, helps to analyze thedifferences in the physiological characteristics and metabolic characteristics between Bacilluslicheniformis MT-6and the type strain in solid state fermentation conditions. Meanwhile, it isthe first time we used highly sensitive mass spectrometry combined with NMR analysis thatthe non-volatile components of the liquor is characterized as lichenysin. It is the first time thatwe studied about the interactionof flavor compounds inthe liquor inthe molecular level. Basedon microbial metabolism, we enhanced the quality of liquor using Bacillus licheniformis asstarters. The main contents are as follows:(1) In Chinese Moutai liquor, Bacillus licheniformis is considered as a most importantstrain. The research about its metabolism in solid state fermentation has been a hot spot. Thewater activity in fermentation process affected the metabolites produced by Bacilluslicheniformis, whose cell morphology varied according to the condition. We compared themetabolites of MT-6from Moutai Daqu with ATCC14580, using swarm expansion assay, cellmorphology, cell counting and HS-SPME method. MT-6has a better swarming ability thanATCC14580, and produced more C4compounds, pyrazines, volatile acids and phenoliccompounds in solid state fermentation. In solid state fermentation (low water activity aw=0.960), MT-6produced more metabolites, which has a better swarming ability.(2) Little information has been reported about the nonvolatile compounds in Chinesedistilled spirits. MT-6produced lichenysin in the solid state fermentation as its characters. Forthe first time, we isolated and identified a cyclic lipopeptide “lichenysin” as a novel nonvolatilecompound in Chinese liquors, using preparative high performance liquid chromatography(HPLC) coupled with quadrupole-time-of-flight (Q-TOF) mass spectrometer and nuclearmagnetic resonance (NMR). The concentrations of lichenysin were quantified in14main typesof bottled Chinese liquors by using ultra performance liquid chromatography (UPLC) coupledwith triple quadrupole (TQD) mass spectrometer. The concentrations of lichenysin in Chineseliquor ranged from0.01to111.74μg·L-1, where the concentration was the highest in Dongjiuliquor. Lichenysin could be a novel nonvolatile compound that selectively affected aromavolatility in Chinese liquors. (3) Nonvolatile compounds play important roles in quality of alcoholic beverages. In ourprevious work, a type of cyclooctapeptide “lichenysin” was newly identified in Chinese liquor.In this work, it was found that lichenysin could selectively affect aroma volatility in three typesof liquor. Interaction of lichenysin and volatile phenolic compounds (off-odors in strong-aromatype liquor) was characterized using headspace solid phase microextraction coupled with gaschromatography-mass spectrometry (HS-SPME-GC-MS). HS-SPME results indicated thatlichenysin very efficiently suppressed the volatility of phenolic compounds by36-48%(P <0.05). Thermodynamic analysis showed that the binding process was mainly mediated byhydrogen bonding. Furthermore, the mixture of lichenysin and4-ethylguaiacol revealedintermolecular cross-peaks between the aH (Val) of lichenysin and the1H of4-ethylguaiacol,by using nuclear overhauser effect spectroscopy (NOESY). This study will help to furtherunderstand the interaction mechanisms between flavor and nonvolatile matrix components inChinese liquors.(4) The potential of Bacillus licheniformis as a starter culture for aroma concentrationimprovement in the fermentation of Chinese Moutai-flavor liquor was elucidated. The volatilecompounds produced by B. licheniformis were identified by GC-MS, in which C4compounds,pyrazines, volatile acids, aromatic and phenolic compounds were the main ingredients. Thestrains B. licheniformis (MT-6and MT-15) produced more volatile compound concentrations,mainly C4compounds, than the type strain of B. licheniformis (ATCC14580) at thefermentation temperature of55°C. Meanwhile, more volatile compound concentration wereproduced by B. licheniformis in solid state fermentation than in submerged state fermentation.Thus, the strains MT-6and MT-15were used as the Bacillus starter culture for investigatingMoutai-flavor liquor production. The distilled liquor inoculated with Bacillus starter culturewas significantly different from the liquor without inoculum. This was particularly evident inthe fore-run part of the distilled sample which was inoculated with Bacillus starter culture,where volatile compounds greatly increased compared to the control. Furthermore, the distilledliquor with Bacillus starter culture showed improved result in sensory appraisals. These resultsindicatedthatB. licheniformiswasoneofthe main species influencingthearomacharacteristicsof Chinese liquor. |
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