This work aimed to isolate, and identify Lactic Acid Bacteria LAB from Egyptian immature citrus honey, and characterize their secondary metabolites, as well as determine the antibacterial activities and transcription of virulence genes (stx1, stx2, and eae) influenced by these bacterial secondary metabolites. From twenty hives, twenty immature citrus bee honey samples were taken. Traditional cultural and biochemical testing were used, followed by molecular confirmation. Further, LAB isolates' antibacterial and cytotoxic properties were investigated. 16S rRNA gene sequencing were assessed and, two lactic acid bacterial isolates were identified as Lactobacillus acidophilus Ch₂ and Levilactobacillus brevis Ch₁. Both isolates have good antagonistic action against clinical pathogens, with Levilactobacillus brevis Ch₁ exhibiting the best antibacterial activity against all indicator pathogens examined. When compared to untreated cancer cells, the isolates demonstrated significant cytotoxic activity. Ch₁ and Ch₂ cell viability percentages were 39.5% and 18.76%, respectively. Furthermore, when exposed to Levilactobacillus brevis Ch₁ metabolites, Shiga-producing Escherichia coli (STEC) virulence gene expression was suppressed. To identify bacterial secondary metabolites, a high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF) approach was developed. Twenty-seven metabolites from diverse chemical classes were discovered in the crude extracts with antibacterial and anticancer characteristics. This is the first thorough investigation on the metabolic profile of LAB isolated from immature Egyptian honey and the findings suggested that isolates or their secondary metabolites could be used in the food sector as medicinal alternatives or as a biocontrol agent.

本工作旨在从埃及未成熟柑橘蜂蜜中分离和鉴定乳酸菌 LAB，并表征其次级代谢产物，并确定受这些细菌次级代谢产物影响的抗菌活性和毒力基因（stx1、stx2 和 eae）的转录。从二十个蜂箱中采集了二十个未成熟的柑橘蜂蜂蜜样品。使用传统的培养和生化测试，然后进行分子确认。此外，还研究了 LAB 分离物的抗菌和细胞毒性特性。对 16S rRNA 基因测序进行了评估，两种乳酸菌分离株被鉴定为嗜酸乳杆菌Ch ₂和短乳杆菌Ch ₁。两种分离株均对临床病原体具有良好的拮抗作用，其中短乳杆菌Ch ₁对所有检测的指示病原体均表现出最佳的抗菌活性。与未经处理的癌细胞相比，分离物表现出显着的细胞毒活性。Ch ₁和 Ch ₂细胞活力百分比分别为 39.5% 和 18.76%。此外，当暴露于短乳杆菌Ch ₁代谢物时，产志贺大肠杆菌(STEC) 毒力基因的表达受到抑制。为了鉴定细菌次级代谢物，开发了高效液相色谱-四极杆飞行时间质谱（HPLC-QTOF）方法。在粗提物中发现了来自不同化学类别的二十七种具有抗菌和抗癌特性的代谢物。这是对从未成熟埃及蜂蜜中分离出的乳酸菌的代谢特征进行的首次彻底调查，研究结果表明分离物或其次生代谢物可在食品领域用作药物替代品或生物防治剂。