低聚甘露糖增强短双歧杆菌抑制具核梭杆菌生长的机制研究

张旭妍; 刘瑞洁; 赵博; 许晓曦

doi:10.13386/j.issn1002-0306.2024020108

低聚甘露糖增强短双歧杆菌抑制具核梭杆菌生长的机制研究

Research on the Mechanism of Mannose Oligosaccharide Enhancing the Inhibitory Effect of Bifidobacterium breve on the Growth of Fusobacterium nucleatum

摘要

摘要: 为探究双歧杆菌与低聚糖协同抑制具核梭杆菌作用及机理，采用6种常见双歧杆菌（动物双歧杆菌、长双歧杆菌、婴儿双歧杆菌、短双歧杆菌、两歧双歧杆菌、青春双歧杆菌）代谢6种不同结构且应用广泛的低聚糖（低聚甘露糖、低聚半乳糖、低聚异麦芽糖、低聚果糖、低聚乳果糖、阿拉伯半乳聚糖），通过硫化氢的生成与生物膜的抑制，确定最佳抑制具核梭杆菌组合，通过扫描电镜、透射电镜与流式细胞仪分析具核梭杆菌菌体细胞完整性，研究抑菌机理，并探讨其酸性代谢产物对具核梭杆菌的抑制作用影响，测定其短链脂肪酸产量，此外还挖掘了双歧杆菌潜在细菌素基因簇。结果显示，低聚甘露糖促进短双歧杆菌抑制具核梭杆菌效果最佳，其组合可显著减少硫化氢的产生与生物膜的形成，与空白对照相比，在添加量达到20%时，对具核梭杆菌的抑制率可达到63.16%±4.48%，其表观结构明显损伤，蛋白和核酸流出，细胞膜的完整性和通透性被破坏。这种抑菌效果会随着上清液pH向中性环境的靠近而下降，表明上清液中酸性代谢产物在发挥抑菌效果中起重要作用，上清液中乙酸、丙酸、丁酸、戊酸分别含量为26.112、10.829、5.106、5.232 mmol/L。此外基因预测到短双歧杆菌包含2个潜在的细菌素合成基因簇。综上，低聚甘露糖配合短双歧杆菌的合生元组合或可作为一种安全、有效的的具核梭杆菌生物防控抑制剂，为预防及辅助治疗结直肠癌等肿瘤疾病提供更为安全有效的新方法。

Abstract: To explore the synergistic inhibitory effect and mechanism of Bifidobacterium and oligosaccharides on Fusobacterium nucleatum, six common Bifidobacterium species (Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium youth) were used to metabolize six widely used oligosaccharides (mannose oligosaccharides, galactose oligosaccharides, isomaltose oligosaccharides, fructooligosaccharides, lactulose oligosaccharides, and arabinogalacia) with different structures. Through the generation of hydrogen sulfide and the inhibition of biofilm, the optimal combination of inhibition of Fusobacterium nucleatum was determined. The cell integrity of Fusobacterium nucleatum was analyzed by scanning electron microscopy, transmission electron microscopy and flow cytometry to study the inhibition mechanism of Fusobacterium nucleatum. The inhibitory effect of acidic metabolites on Fusobacterium nucleatum and the yield of short-chain fatty acids were investigated. In addition, the potential bacteriocin gene clusters of Bifidobacterium were also excavated. The results showed that mannose oligosaccharide had the best effect in promoting Bifidobacterium breve to inhibit Fusobacterium nucleatum, and the combination could significantly reduce the production of H₂S and the formation of biofilm. Compared with the blank control, when the addition amount of CFS reached 20%, the inhibition rate of Fusobacterium nucleatum could reach 63.16%±4.48%. The apparent structure was obviously damaged, protein and nucleic acid flowed out, and the integrity and permeability of the cell membrane were destroyed. This inhibitory effect diminished as the supernatant's pH became closer to neutral, suggesting that the supernatant's acidic metabolites were crucial to the antibacterial action. The supernatant had the following amounts of acid: 26.112 mmol/L for acetic acid, 10.829 mmol/L for propionic acid, 5.106 mmol/L for butyric acid, and 5.232 mmol/L for valeric acid. In addition, Bifidobacterium breve was predicted to contain two potential bacteriocin synthesis gene clusters. In conclusion, the combination of mannose oligosaccharides and Bifidobacterium breve may be used as a safe and effective biological control inhibitor of Fusobacterium nucleatum, which may provide a safer and more effective method for the prevention and adjuvant treatment of colorectal cancer and other tumor diseases.