CLI: A new protocol for the isolation of Lactic Acid Bacteria from complex plant samples

Area of Science:

• Microbiology
• Plant Science
• Agricultural Science

Background:

• Lactic Acid Bacteria (LAB) are recognized probiotics, commonly found in the human gut and fermented foods.
• Recent discoveries show LAB also exist in plant microbiota, exhibiting probiotic potential for plants.
• Increased scientific interest focuses on isolating LAB for agronomic applications, but efficient protocols for plant samples are lacking.

Purpose of the Study:

• To develop and validate a novel, efficient protocol for isolating Lactic Acid Bacteria (LAB) from complex plant samples.
• To establish a reliable method for obtaining purified LAB for further agronomic research.

Main Methods:

• The developed protocol, Complex samples LAB Isolation (CLI), involves five key steps: pre-incubation, homogenization, serial dilution, selective culturing on MRS agar with specific additives (sorbic acid, CaCO3, controlled oxygen, pH 5.8), and colony selection.
• Incubation conditions included 37°C for 48 hours under microaerophilic conditions (O2 < 15%).
• Selection criteria focused on typical LAB morphology and the presence of a CaCO3-solubilization halo.

Main Results:

• The CLI protocol demonstrated sufficient sensitivity to isolate representative LAB genera, including Leuconostoc, Lactococcus, and Enterococcus, from plant tissues.
• The method successfully yielded purified LAB colonies with characteristic morphology and calcium carbonate solubilization halos.

Conclusions:

• The CLI protocol offers an efficient and sensitive method for isolating Lactic Acid Bacteria from complex plant matrices.
• This new protocol is a valuable tool for future research on native LAB and their potential applications in agriculture.