Abstract：
Lactic Acid Bacteria (LAB) are predominantly probiotic microorganisms and the most are Generally Recognized As Safe (GRAS). LAB inhabit in the human gut ecosystem and are largely found in fermented foods and silage. In the last decades, LAB have also has been found in plant microbiota as a new class of microbes with probiotic activity to plants. For this reason, today the scientific interest in the study and isolation of LAB for agronomic application has increased. However, isolation protocols from complex samples such as plant tissues are scarce and inefficient. In this study, we developed a new protocol (CLI, Complex samples LAB Isolation) which yields purified LAB from plants. The sensitivity of CLI protocol was sufficient to isolate representative microorganisms of LAB genera (i.e. Leuconostoc, Lactococcus and Enterococcus). CLI protocol consists on five steps: i) sample preparation and pre-incubation in 1% sterile peptone at 30 °C for 24-48 h; ii) Sample homogenization in vortex by 10 min; iii) sample serial dilution in quarter-strength Ringer solution, iv) incubation in MRS agar plates with 0.2% of sorbic acid, with 1% of CaCO3, O2 < 15%, at pH 5.8 and 37 °C for 48 h.; v) Selection of single colonies with LAB morphology and CaCO3-solubilization halo. Our scientific contribution is that CLI protocol could be used for several complex samples and represents a useful method for further studies involving native LAB.
摘要：
乳酸菌(LAB)主要是益生菌微生物,并且大多数被公认为安全的(GRAS)。LAB栖息在人类肠道生态系统中，主要存在于发酵食品和青贮饲料中。在过去的几十年里，在植物微生物群中还发现了LAB作为一类对植物具有益生菌活性的新型微生物。出于这个原因,今天，对用于农艺应用的LAB的研究和分离的科学兴趣增加了。然而,从复杂样品如植物组织的分离方案是稀缺和低效的。在这项研究中,我们开发了一个新的协议(CLI，复杂样品LAB分离），从植物中获得纯化的LAB。CLI方案的敏感性足以分离LAB属的代表性微生物(即明串珠菌，乳球菌和肠球菌）。CLI方案包括五个步骤：i）样品制备和在30°C下在1％无菌蛋白胨中预孵育24-48小时；ii）样品在涡旋中均质化10分钟；iii）样品在四分之一强度的Ringer溶液中连续稀释，iv)在含有0.2%山梨酸的MRS琼脂平板中孵育，1%的CaCO3，O2<15%，在pH5.8和37°C下保持48小时。;v)选择具有LAB形态和CaCO3增溶晕的单菌落。我们的科学贡献是CLI协议可用于几个复杂的样品，并代表了涉及天然LAB的进一步研究的有用方法。
