Exopolysaccharides (EPSs) are large-molecular-weight, complex carbohydrate molecules and extracellularly secreted bio-polymers released by many microorganisms, including lactic acid bacteria (LAB). LAB are well known for their ability to produce a wide range of EPSs, which has received major attention. LAB-EPSs have the potential to improve health, and their applications are in the food and pharmaceutical industries. Several methods have been developed and optimized in recent years for producing, extracting, purifying, and characterizing LAB-produced EPSs. The simplest method of evaluating the production of EPSs is to observe morphological features, such as ropy and mucoid appearances of colonies. Ethanol precipitation is widely used to extract the EPSs from the cell-free supernatant and is generally purified using dialysis. The most commonly used method to quantify the carbohydrate content is phenol–sulfuric acid. The structural characteristics of EPSs are identified via Fourier transform infrared, nuclear magnetic resonance, and X-ray diffraction spectroscopy. The molecular weight and composition of monosaccharides are determined through size-exclusion chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography. The surface morphology of EPSs is observed via scanning electron microscopy and atomic force microscopy, whereas thermal characteristics are determined through thermogravimetry analysis, derivative thermogravimetry, and differential scanning calorimetry. In the present review, we discuss the different existing methods used for the detailed study of LAB-produced EPSs, which provide a comprehensive guide on LAB-EPS preparation, critically evaluating methods, addressing knowledge gaps and key challenges, and offering solutions to enhance reproducibility, scalability, and support for both research and industrial applications.
