School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China.
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China.
J Dairy Sci. 2019 May;102(5):3933-3938. doi: 10.3168/jds.2018-15972. Epub 2019 Mar 7.
Lactic acid bacteria are often preserved as starter cultures by freezing to extend shelf stability as well as maintain cell viability and acidification activity. Previous studies showed that the endocyte extracted from gradient-freezing pretreated cells could act as lyoprotectant in the lyophilization process of Lactococcus lactis ssp. lactis. In this study, the molecular mechanisms of L. lactis in response to gradient freezing exposure are described using high-throughput sequencing. Nineteen of 56 genes were upregulated after gradient freezing, whereas 37 genes were downregulated. Further validation results of quantitative real-time PCR experiments were consistent with the RNA sequencing. Gene Ontology (http://www.geneontology.org/) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathway were used to analyze the differentially expressed genes. Several pathways, such as glutathione metabolism, ATP-binding cassette transport, metabolism of cell wall and cell membrane components, and stress response-related pathways, were affected by gradient freezing. Six genes relevant to freezing stress response were selected for quantitative real-time PCR, including 3 upregulated genes (hisK, eutD, dukA) and 3 downregulated genes (als, yedF, pepN). The Gene Ontology enrichment and KEGG pathway analyses showed these genes may influence stress response-related pathways, improving the survival of the L. lactis under freezing stress. The identification of these genes deepened an understanding about their response under freezing stress, helping us find potential genes or pathways related to gradient freezing for further research on lyoprotectants.
乳酸菌通常通过冷冻保存为起始培养物，以延长货架稳定性并保持细胞活力和酸化活性。先前的研究表明，从无梯度冷冻预处理细胞中提取的内体可以在干酪乳杆菌乳亚种的冷冻干燥过程中充当保护剂。在这项研究中，使用高通量测序描述了乳杆菌对梯度冷冻暴露的分子机制。梯度冷冻后有 56 个基因中的 19 个上调，而 37 个基因下调。定量实时 PCR 实验的进一步验证结果与 RNA 测序一致。GO（http://www.geneontology.org/）富集和京都基因与基因组百科全书（KEGG；https://www.genome.jp/kegg/）途径用于分析差异表达基因。几个途径，如谷胱甘肽代谢、ABC 转运、细胞壁和细胞膜成分代谢以及应激反应相关途径，受到梯度冷冻的影响。选择了 6 个与冷冻应激反应相关的基因进行定量实时 PCR，包括 3 个上调基因（hisK、eutD、dukA）和 3 个下调基因（als、yedF、pepN）。GO 富集和 KEGG 途径分析表明，这些基因可能影响与应激反应相关的途径，从而提高了乳杆菌在冷冻应激下的存活率。这些基因的鉴定加深了对其在冷冻应激下反应的理解，有助于我们发现与梯度冷冻相关的潜在基因或途径，以进一步研究保护剂。
