This study aimed to investigate the effects of various growth conditions and nutritional stress on the rate of cell development, auto and co-aggregation abilities, cell surface hydrophobicity, adherence to HT-29 cells, and changes in cellular fatty acid composition of Limosilactobacillus fermentum JNU532 (JNU532). The strain JNU532 was exposed to different stressors, including temperature variations (37°C and 42°C), salt (3% NaCl), acid (pH 4), and alkali (pH 8). Following exposure to temperature stress at 42°C, the strain demonstrated the highest levels of auto- and co-aggregation, but the lowest levels of hydrophobicity and adhesion abilities. Under acidic conditions, the strain's development remained stable, but its auto and co-aggregation abilities were enhanced compared to those grown at a temperature of 37°C and pH 6.5. Moreover, JNU532 grown at pH 4 showed relatively high cell surface hydrophobicity and adhesion abilities. However, the strain exhibited the greatest adhesion capacity when subjected to alkaline stress compared to the control and other stress groups. Gas chromatography was used to isolate and quantitatively assess the levels of intracellular oleic acid and its post-synthetic derivatives, including cis-9,10-methyleneoctadecanoic acid (C19:0 cyclo ɷ8c) and C19:0 cyclo ɷ10c/19ɷ6. The JNU532 exhibited the highest amount of oleic acid during temperature stress at 42°C. Furthermore, JNU532 demonstrated the highest yield of the oleic acid cyclopropanation product C19:0 cyclo ɷ10c/19ɷ6 under acidic conditions (pH 4). Therefore, this study the application of JNU 532 as a probiotic in the food and milk industry has laid a theoretical foundation.