Background:To enhance the stress resistance of Lactobacillus plantarum M616, various wall materials including 2-hydroxypropyl-β-cyclodextrin, sorbitol, soybean oil, phospholipids, and enzymatically hydrolyzed soy protein were employed to prepare microcapsule powder. Results:The structural characterization involved Fourier infrared spectroscopy, differential scanning surface calorimetry, cryo-scanning electron microscopy, and fluorescent labeling. The findings revealed the formation of a microcapsule structure between the wall body and Lactobacillus plantarum M616, with the aqueous phase located externally and the oil phase encapsulating both the microcapsule and Lactobacillus plantarum M616.The storage resistance of microcapsule powder was assessed, revealing that the viable bacteria count of microcapsules stored at 4°C for 8 weeks was 1.03×109 CFU/g, indicating favorable storage resistance at 4°C. Subsequently, an in vitro simulated digestion test was conducted on the microcapsules, resulting in a viable bacteria count of 5.1×109 CFU/g after 120 minutes of gastrointestinal fluid digestion. These findings demonstrate that the microcapsule structure effectively shields Lactobacillus plantarum M616 from the detrimental effects of gastric acid's low pH and facilitates successful release in the intestine.The results of in vivo experiments conducted on microcapsules demonstrated that the utilization of microencapsulation could enhance the colonization efficiency of Lactobacillus plantarum M616 within the intestinal tract. Conclusions:Consequently, microcapsules exhibit significant potential as valuable additives for both human and animal food products.