Novel human milk-derived probiotics delays senescence and improves antioxidant capacity in <em>Caenorhabditis elegans</em> by affecting the insulin signaling pathway

Probiotics have received considerable attention owing to their potential benefits in maintaining intestinal health, modulating immune function, and antioxidant activity. The antioxidant capacity of recently isolated and screened probiotics have not been characterized. Therefore, the present study used <em>Caenorhabditis elegans</em> (<em>C. elegans)</em> as a model to investigate the effects of three novel probiotics (<em>Limosilactobacillus reuteri</em> B1-10, <em>Lactobacillus paragasseri</em> B1-26, and <em>Lacticaseibacillus rhamnosus</em> B2-1) and their probiotic complexes, isolated from breast milk, on the ability to delay aging and antioxidant capacity. Probiotics significantly prolonged the lifespan of nematodes by reducing lipofuscin accumulation, and their average lifespan was prolonged by 19.40%, 7.72%, 19.72% and 20.30%, respectively. The probiotics reduce nematode fecundity and retard their growth and development, possibly due to dietary restriction. Additionally, the probiotics reduced the accumulation of reactive oxygen species (ROS) and malondialdehyde content (MDA), the reduction was 9.7% - 44.5% and 18.69% - 46.99%, respectively, and increased the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), the increases were 26.22 to 41.72%, 30.33 to 83.20% and 77.13% to 171.85%, respectively. Which improved the <em>C. elegans</em> resistance to hydrogen peroxide stressors, and the complex probiotic was the most effective. Furthermore, the probiotics acts on the insulin/insulin-like pathway to regulate the expression of <em>daf-16</em>, which in turn upregulates the expression of <em>sod-3</em>, <em>ctl-1</em>,<em> sod-1</em>, down-regulated the expression of<em> age-1</em>, and promoted the nuclear localization of DAF-16::GFP nematodes to prolong the lifespan and antioxidant capacity of<em> C. elegans</em>. These results provide a new basis and research direction for further analyses of the role of probiotics in maintaining the health, improving stress resistance, and prolonging the lifespan of the host. They also provide potential insights into the development of functional food to facilitate anti-aging management in humans.