Li Tianxiao, Fang Yaning, Chai Zhishun, Ji Lingbo, Jiang Zhongrong, Meng Dandan, He Baojiang, Hu Xiaolong, Xi Hui, Jia Xuewei, Li Dongliang
Flavor Research Center, College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, China.
Cigar Fermentation Technology Key Laboratory of Tobacco Industry, China Tobacco Sichuan Industrial Co., Ltd., Chengdu, China.
Front Microbiol. 2025 Apr 1;16:1565502. doi: 10.3389/fmicb.2025.1565502. eCollection 2025.
Cyclic dipeptides represent a class of intriguing molecules with a wide range of biological activities, but their potential application as flavor precursors has not been previously reported. In this study, a flavor-producing bacterium was screened out from 35 isolated endophytic bacteria. Gas chromatography-mass spectrometry (GC-MS) analysis suggested that the fermentation broth of contained flavor compounds and high amount of cyclic dipeptide flavor precursors. Three cyclic dipeptide flavor precursors, namely cyclo (L-prolyl-L-valine) (), cyclo (L-prolyl-L-isoleucine) (), and cyclo (L-prolyl-L-leucine) (), were further isolated from the fermentation broth extraction through Sephadex LH-20 column chromatography and semi-preparative high-performance liquid chromatography (HPLC), and were identified by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Flavor precursors can generate aroma components during pyrolysis, and the pyrolysis of compounds and were performed using pyrolysis GC-MS (Py-GC-MS) to analyze the flavor products. According to the relative odor activity value (ROAV) analysis, the key pyrolysis flavor compounds were revealed as 6-heptyl-5,6-dihydro-2-pyran-2-one, isobutyric acid, 4-methyl-2-oxo-pentanoic acid ester, pyrrole derivatives, and pyrazine derivatives, which could give great contributions to milky, roasting, fruity, sweetness, and nutty aromas. The pyrolysis formation pathway of these flavor compounds was also proposed in detail. Addition of fermentation broth from the flavor-producing bacteria on cigar tobacco leaves significantly enhanced the milky, roasting, fruity, sweetness, and nutty aromas, which further demonstrated the flavor enhancing ability of cyclic dipeptides. This is the first report of flavor enhancing effects of cyclic dipeptides utilized as flavor precursors.
环二肽是一类具有广泛生物活性的有趣分子，但其作为风味前体的潜在应用此前尚未见报道。在本研究中，从35株分离的内生细菌中筛选出一株产香细菌。气相色谱 - 质谱联用（GC - MS）分析表明，该菌的发酵液中含有风味化合物和大量的环二肽风味前体。通过葡聚糖凝胶LH - 20柱色谱和半制备高效液相色谱（HPLC）从发酵液提取物中进一步分离出三种环二肽风味前体，即环（L - 脯氨酰 - L - 缬氨酸）（）、环（L - 脯氨酰 - L - 异亮氨酸）（）和环（L - 脯氨酰 - L - 亮氨酸）（），并通过核磁共振（NMR）光谱和质谱（MS）进行鉴定。风味前体在热解过程中可产生香气成分，利用热解气相色谱 - 质谱联用（Py - GC - MS）对化合物和进行热解以分析风味产物。根据相对气味活性值（ROAV）分析，揭示出关键的热解风味化合物为6 - 庚基 - 5,6 - 二氢 - 2 - 吡喃 - 2 - 酮、异丁酸、4 - 甲基 - 2 - 氧代 - 戊酸酯、吡咯衍生物和吡嗪衍生物，它们对牛奶香、烤香、果香、甜味和坚果香有很大贡献。还详细提出了这些风味化合物的热解形成途径。将产香细菌的发酵液添加到雪茄烟叶上显著增强了牛奶香、烤香、果香、甜味和坚果香，这进一步证明了环二肽的风味增强能力。这是关于环二肽作为风味前体的风味增强作用的首次报道。
