高温大曲中大曲慢生芽孢杆菌的分离鉴定与特性分析

Isolation and characterization of Lentibacillus daqui from high-temperature Daqu

摘要:

【背景】随着细菌新物种的陆续报道，原先被注释为枝芽孢杆菌(Virgibacillus sp.)的高温大曲优势16S rRNA基因序列被重新归类为大曲慢生芽孢杆菌(Lentibacillusdaqui)，但对该物种在制曲过程中的发酵特性和代谢功能尚不了解。【目的】研究大曲慢生芽孢杆菌在高温大曲中的分布规律、生长和代谢特性。【方法】采用高通量测序技术分析大曲慢生芽孢杆菌在三类高温大曲中的分布规律；采用纯培养技术分离大曲慢生芽孢杆菌，研究其生长的温度、pH、NaCl浓度等范围；采用小麦固态发酵试验，研究大曲慢生芽孢杆菌在不同温度(30、45、60 ℃)的产酶和产香特征。【结果】从48个高温大曲样品中测序获得3条属于大曲慢生芽孢杆菌的16S rRNA基因扩增子序列变体(amplicon sequence variant, ASV)序列，平均相对丰度之和为21.4%，均存在于大曲慢生芽孢杆菌ZS110521^T的基因组上，共有6个基因拷贝。菌株ZS110521^T生长的温度范围10-55 ℃，pH 5.5-10.0，NaCl浓度范围0-20%。菌株ZS110521^T具有产生糖化酶、纤维素酶与蛋白酶的能力，在45 ℃实验组酶活最高。相较于30 ℃实验组，45 ℃和60 ℃发酵试验组的吡嗪类、酚类、酸类、酮类、酯类化合物含量均显著升高，而醇类化合物含量显著降低(P<0.05)。菌株ZS110521^T发酵小麦中含量超过180 ng/g的优势挥发性化合物有10种，包括川芎嗪、2,3,5-三甲基吡嗪、2,5-二甲基吡嗪、2-乙基-3,5,6-三甲基吡嗪、4-乙基愈创木酚、愈创木酚、苯乙醇、苯甲醛、异戊酸和乙偶姻，并且其含量均随着培养温度而显著增加。【结论】大曲慢生芽孢杆菌是高温大曲中的优势细菌，在45-60 ℃温度下具有良好的产吡嗪类和愈创木酚类物质的能力。研究结果为了解大曲慢生芽孢杆菌的发酵功能与应用潜力提供了依据。

Abstract:

[Background] With the update of the database, the dominant 16S rRNA gene sequence in high-temperature Daqu that was originally annotated as Virgibacillus sp. has been reclassified as Lentibacillus daqui. However, the fermentation characteristics and metabolic functions of this species in the process of Daqu production remain unknown. [Objective] To study the distribution, growth, and metabolic characteristics of L. daqui in high-temperature Daqu. [Methods] High-throughput sequencing was used to analyze the distribution of L. daqui in three types of high-temperature Daqu. L. daqui was isolated by pure culture and its growth conditions including temperature, pH, and NaCl concentration were studied. A wheat solid-state fermentation experiment was then carried out to study the enzyme production and aroma production characteristics of L. daqui at different temperatures (30 ℃, 45 ℃, and 60 ℃). [Results] Three distinct amplicon sequence variants (ASVs) from 16S rRNA gene sequences, obtained from 48 high-temperature Daqu samples, were annotated as L. daqui and constituted 21.4% of the total relative abundance. All the three ASVs were presented in the genome of L. daqui ZS110521^T, comprising a total of six gene copies. The strain ZS110521^T could grow at 10-55 ℃, pH 5.5-10.0, and the NaCl concentrations of 0-20%. ZS110521^T could produce glucoamylase, cellulase, and protease, with the highest enzyme activity at 45 ℃. Compared with the fermentation at 30 ℃, the fermentation at 45 ℃ and 60 ℃ showed increased content of pyrazines, phenols, acids, ketones, and esters and decreased content of alcohols (P<0.05). Ten dominant volatile compounds with the content exceeding 180 ng/g were detected in ZS110521^T-fermented wheat, including tetramethylpyrazine, 2,3,5-trimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 4-ethyl-2-methoxyphenol, guaiacol, phenethyl alcohol, benzaldehyde, isovaleric acid, and acetoin. Moreover, their content significantly increased with the rise in temperature. [Conclusion] L. daqui is the dominant bacterium in high-temperature Daqu, and it demonstrates high production of pyrazines and guaiacols at 45-60 ℃. The results provide a basis for understanding the function and application potential of L. daqui.