接种地衣芽孢杆菌发酵的普洱茶品质与微生物群落分析

Analysis of Quality and Microbial Communities of Pu-Erh Tea Through Inoculation Fermentation with Bacillus licheniformis

为通过接菌发酵提高普洱茶的品质,将从普洱茶中分离鉴定到的地衣芽孢杆菌(Bacillus licheniformis)L1接种于灭菌和未灭菌的晒青茶中,分别进行纯菌发酵与强化发酵。结果表明:纯菌发酵后茶叶中茶多酚质量分数和5种儿茶素[儿茶素(C)、表儿茶素没食子酸酯(ECG)、表没食子儿茶素没食子酸酯(EGCG)、儿茶素没食子酸酯(CG)、没食子儿茶素没食子酸酯(GCG)]质量比较自然发酵显著降低(P<0.05),茶褐素质量分数显著增加(P<0.05)。与自然发酵相比,强化发酵中茶褐素[(6.75±0.13)%]、茶红素[(1.04±0.09)%]和可溶性糖[(5.76±0.10)%]质量分数显著升高(P<0.05);而CG[(0.39±0.03)mg/g]、表儿茶素[(0.86±0.05)mg/g]、GCG[(0.20±0.03)mg/g]、C[(2.28±0.14)mg/g]、表没食子儿茶素[(1.69±0.11)mg/g]、EGCG[(0.33±0.02)mg/g]6种儿茶素质量比显著降低(P<0.05)。强化发酵茶汤甜味(3.33±0.78)和厚重感(6.11±0.71)感官评分增加,苦味(1.33±0.50)、涩味(0.67±0.50)和酸味(0.33±0.44)分数降低。色差仪检测发现,强化发酵茶汤的a*值(25.70±0.68)、b*值(77.06±1.07)增加,L*值(71.58±0.83)降低。细菌16S rRNA和真菌ITS序列的扩增子测序发现,地衣芽孢杆菌L1在整个过程中并未一直是优势细菌(发酵中后期仅为0.2%~0.5%),但改变了微生物群落结构,表现在短杆菌(Brevibacterium)、食腺嘌呤芽生葡萄孢酵母(Blastobotrys adeninivorans)和Rogersella griseliniae的相对丰度显著升高(P<0.05);而无色杆菌(Achromobacter)、伯克氏菌(Burkholderia)、柠檬酸杆菌(Citrobacter)、欧文氏菌足杆菌(Erwinia)、埃希氏菌(Escherichia)、克雷白氏菌(Klebsiella)和微小根毛霉(Rhizomucor pusillus)7种微生物的相对丰度显著降低(P<0.05)。因此,接种地衣芽孢杆菌L1发酵普洱茶,可能通过与其他微生物的协同作用改变茶叶中的特征成分,从而提高普洱茶的感官品质。收稿日期:20210809基金项目:国家自然科学基金资助项目(31760225;32160728);云南省高层次人才培养计划青年拔尖人才专项(YNWR-QNBJ-2018-366);云南省科技厅科技计划项目(202104BI090008)。第一作者:刘琨毅,男,副教授,博士,主要从事传统发酵食品方面的研究。*通信作者:赵明,男,教授,博士,主要从事茶叶生物化学方面的研究。

In order to improve the quality of Pu-erh tea through inoculation fermentation,Bacillus licheniformis L1 isolated and identified from Pu-erh tea was inoculated in sterilized or unsterilized sun-dried green tea leaves to develop pure culture fermentation and enhanced fermentation(EF)of Pu-erh tea.The results showed that the mass fraction of tea polyphenols and the mass ratio of five catechins[(+)-catechin(C),(-)-epicatechin-3-gallate(ECG),(-)-epigallocatechin-3-gallate(EGCG),(-)-catechin gallate(CG)and(-)-gallocatechin gallate(GCG)]in tea leaves decreased significantly compared with control group(P<0.05),while the mass fraction of theabrownins increased significantly(P<0.05)after pure culture fermentation.Compared with normal fermentation(NF),the mass fraction of theabrownins[(6.75±0.13)%],thearubigins[(1.04±0.09)%]and soluble sugar[(5.76±0.10)%]in EF were significantly higher(P<0.05),whereas the mass ratio of six catechins were significantly lower(P<0.05),such as CG[(0.39±0.03)mg/g],(-)-epicatechin[(0.86±0.05)mg/g],GCG[(0.20±0.03)mg/g],C[(2.28±0.14)mg/g],(-)-epigallocatechin[(1.69±0.11)mg/g],EGCG[(0.33±0.02)mg/g].In sensory evaluation for tea infusion,the scores of sweetness(3.33±0.78)and thickness(6.11±0.71)of EF were higher,while the scores of bitterness(1.33±0.50),astringency(0.67±0.50)and acidity(0.33±0.44)were lower.Colorimeter detection showed that the value of a*(25.70±0.68)and b*(77.06±1.07)of tea infusion in EF were higher and the value of L*(71.58±0.83)was lower.Amplicon sequencing of bacterial 16S rRNA and fungal ITS sequences found that B.licheniformis L1 was not always the dominant bacteria in the whole EF process(only 0.2%~0.3%in the middle and late stage of fermentation),the microbial communities in EF were different from those in NF.The relative abundances of Brevibacterium,Blastobotrys adeninivorans and Rogersella griseliniae in EF were significantly higher(P<0.05),and the relative abundances of seven microorganisms(Achromobacter,Burkholderia,Citrobacter,Erwinia,Escherichia,Klebsiella and Rhizomucor pusillus)in EF were significantly lower(P<0.05).Therefore,characteristic components were changed and sensory quality of Pu-erh tea were improved by the synergistic effect of B.licheniformis L1 with other microorganisms in enhanced fermentation.