| Summary: | Zearalenone (ZEN, ZEA) contamination in various foods and feeds is a significant global problem. Similar to deoxynivalenol (DON) and other mycotoxins, ZEN in feed mainly enters the body of animals through absorption in the small intestine, resulting in estrogen-like toxicity. In this study, the gene encoding Oxa, a ZEN-degrading enzyme isolated from <i>Acinetobacter</i> SM04, was cloned into <i>Lactobacillus acidophilus</i> ATCC4356, a parthenogenic anaerobic gut probiotic, and the 38 kDa sized Oxa protein was expressed to detoxify ZEN intestinally. The transformed strain <i>L. acidophilus</i> pMG-Oxa acquired the capacity to degrade ZEN, with a degradation rate of 42.95% at 12 h (initial amount: 20 μg/mL). The probiotic properties of <i>L. acidophilus</i> pMG-Oxa (e.g., acid tolerance, bile salt tolerance, and adhesion properties) were not affected by the insertion and intracellular expression of Oxa. Considering the low amount of Oxa expressed by <i>L. acidophilus</i> pMG-Oxa and the damage to enzyme activity by digestive juices, Oxa was immobilized with 3.5% sodium alginate, 3.0% chitosan, and 0.2 M CaCl<sub>2</sub> to improve the ZEN degradation efficiency (from 42.95% to 48.65%) and protect it from digestive juices. The activity of immobilized Oxa was 32–41% higher than that of the free crude enzyme at different temperatures (20–80 °C), pH values (2.0–12.0), storage conditions (4 °C and 25 °C), and gastrointestinal simulated digestion conditions. Accordingly, immobilized Oxa could be resistant to adverse environmental conditions. Owing to the colonization, efficient degradation performance, and probiotic functionality of <i>L. acidophilus</i>, it is an ideal host for detoxifying residual ZEN in vivo, demonstrating great potential for application in the feed industry.
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