酱香型白酒堆积酒醅中象牙色克罗彭斯特德菌的分离筛选及其代谢特性解析

Isolation, screening, and metabolic characterization of Kroppenstedtia eburnea from the fermented grains of Jiang-flavor Baijiu

摘要:

【目的】高温堆积发酵是酱香型白酒生产中的关键工艺环节，克罗彭斯特德菌属(Kroppenstedtia)是堆积酒醅中的优势细菌属，研究其生长和代谢特征对于理解堆积发酵的关键作用至关重要。【方法】采用胰酪大豆蛋白胨(tryptic soy broth, TSB)培养基从酒醅中筛选克罗彭斯特德菌，通过形态学和16S rRNA基因测序确定其分类学地位，结合菌株纯培养及不同温度(45 ℃和50 ℃)下的高粱固态发酵实验，研究其生长和挥发性化合物代谢特征。【结果】从酱香型白酒堆积酒醅中分离筛选到3株克罗彭斯特德菌，经鉴定为象牙色克罗彭斯特德菌(Kroppenstedtiaeburnea)。液态培养时菌株K. eburnea 1613促进吡嗪类物质的产生，为对照的2.66倍。在固态发酵高粱中检出的挥发性化合物主要为醇类和酸类，总含量随发酵时间的推移而逐渐增加。50 ℃发酵高粱有利于醇类、酸类和吡嗪类物质的积累，而45 ℃下酯类物质含量较高。3株菌以高粱为基质进行固态发酵时主要代谢产物是苯乙醇和异戊酸，其中K. eburnea 1615在50 ℃下发酵15 d时苯乙醇和异戊酸含量最高，为(31.17±0.14) µg/g和(16.75±0.76)µg/g。菌株K. eburnea 6E22在50℃下发酵15 d时2,5-二甲基吡嗪含量最高，为(1.67±0.14) µg/g。菌株K. eburnea 1613在发酵15 d时己酸含量最高，为(3.74±0.19) µg/g。50 ℃下发酵高粱自身中醛酮类物质积累明显。偏最小二乘判别分析(partial least squares discrimination analysis, PLS-DA)显示，温度和时间对3株菌发酵高粱中挥发性化合物的组成有显著影响。【结论】象牙色克罗彭斯特德菌有助于堆积发酵酒醅风味化合物的产生，特别是醇类、酸类和吡嗪类等酱香型白酒特征风味物质。

Abstract:

[Objective] High-temperature stacking fermentation is a key process in the production of Jiang-flavor Baijiu. Kroppenstedtia is a predominant bacterial genus in the stacked fermented grains, and investigating its growth and metabolic characteristics contributes to comprehensively understanding the crucial role of stacking fermentation. [Methods] The tryptic soy broth was used to screen Kroppenstedtia strains from the stacked fermented grains, and the taxonomic status of each strain was determined by morphological observation and 16S rRNA gene sequencing. Furthermore, the growth characteristics and volatile compound metabolism of each strain were investigated by solid-state fermentation experiments on sorghum at different temperatures (45 ℃ and 50 ℃) in combination with pure culture. [Results] Three strains of Kroppenstedtia were isolated from the fermented grains of Jiang-flavor Baijiu and identified as K. eburnea. The liquid culture of strain K. eburnea 1613 significantly enhanced the production of pyrazines, which was 2.66 folds of that in the control group. The volatile compounds of sorghum in solid-state fermentation predominantly consisted of alcohols and acids, the total content of which increased over the fermentation time. The fermentation at 50 ℃ promoted the accumulation of alcohols, acids, and pyrazines, and that at 45 ℃ facilitated the accumulation of esters. The primary metabolites produced by the three strains during solid-state fermentation with sorghum as the substrate were phenethyl alcohol and isovaleric acid. The fermentation with K. eburnea 1615 at 50 ℃ for 15 days produced the highest levels of phenethyl alcohol and isovaleric acid, which reached (31.17±0.14) µg/g and (16.75±0.76) µg/g, respectively. The fermentation with K. eburnea 6E22 and K. eburnea 1613 at 50 ℃ yielded the highest levels of 2,5-dimethyl pyrazine [(1.67±0.14) µg/g] and hexanoic acid [(3.74±0.19) µg/g], respectively, after 15 days. The accumulation of aldehydes and ketones was significant in sorghum fermented at 50 ℃. The partial least squares-discriminant analysis (PLS-DA) revealed significant influences of temperature and time on the volatile compound composition of sorghum fermented by the three strains. [Conclusion] K. eburnea contributes to the production of flavor compounds in fermented grains, particularly the characteristic flavor compounds such as alcohols, acids, and pyrazines of Jiang-flavor Baijiu.