嗜琼胶卵链菌β-琼胶酶Y3R1酶解工艺优化及产物对肠道菌群体外发酵的影响

Optimization of the enzymatic hydrolysis process of β-agarase Y3R1 from Catenovulum agarivorans and effects of the products on intestinal microbiota during in vitro fermentation

摘要:

为了优化β-琼胶酶Y3R1酶解琼脂糖的工艺条件，并探究其产物新琼寡糖对肠道菌群的益生作用，本研究以还原糖生成量为评价指标，对嗜琼胶卵链菌β-琼胶酶Y3R1酶解工艺进行优化。通过3,5-二硝基水杨酸法和苯酚-硫酸法分别测定反应生成的还原糖及总糖含量，计算酶解产物的平均聚合度(average degree of polymerization, DP)，通过响应面法优化，确定β-琼胶酶水解琼脂糖的最佳工艺条件为：50 mL反应体系中，琼脂糖底物浓度41.9 mg/mL，加酶量7.5 U，在温度65 ℃、pH 6.5下反应120 min。经质谱与高效液相色谱分析鉴定，β-琼胶酶Y3R1酶解琼脂糖的产物为新琼二糖、新琼四糖和新琼六糖，三者峰面积比例为4%:77%:17%。进一步基于体外发酵模型，通过α、β多样性分析，门和属水平菌落结构差异LEfSe分析以及短链脂肪酸含量测定，探究新琼寡糖(neoagaro-oligosaccharides, NAOS)对肠道菌群的调节作用，结果表明，NAOS主要通过促进拟杆菌属和乳杆菌属等有益菌群的生长从而调节菌群结构，同时通过短链脂肪酸(尤其是乙酸、丙酸和丁酸)的变化发挥益生作用。上述结果证实NAOS具有良好的益生元潜力，本研究为其在功能性食品中的应用提供了理论依据。

Abstract:

This study aimed to optimize the hydrolysis conditions of agarose by β-agarase Y3R1 and to investigate the prebiotic effects of its product, neoagarooligosaccharides, on gut microbiota. Using reducing sugar yield as the evaluation index, the enzymatic hydrolysis process of β-agarase Y3R1 from Catenovellum agarivorans was optimized. The content of reducing sugar and total sugar generated during the reaction were determined via the 3,5-dinitrosalicylic acid (DNS) method and the phenol-sulfuric acid method, respectively, and the average degree of polymerization (DP) of the enzymatic hydrolysis products was calculated. Through response surface methodology, the conditions for β-agarase hydrolysis of agarose were optimized as follows: a 50 mL reaction system, an agarose (substrate) concentration of 41.9 mg/mL, an enzyme dosage of 7.5 U, and reaction conducted at 65 ℃ and pH 6.5 for 120 min. The products of agarose hydrolysis by β-agarase Y3R1 were identified by MS and HPLC as neoagarobiose, neoagarotetraose, and neoagarohexaose, with the peak area ratio of 4%:77%:17%. Furthermore, an in vitro fermentation model was employed to investigate the regulatory effects of neoagaro-oligosaccharides (NAOS) on intestinal microbiota through alpha and beta diversity analyses, comparison of intestinal microbiota structure at the phylum and genus levels, LEfSe, and short-chain fatty acid (SCFA) content determination. The results indicated that NAOS primarily modulated the intestinal microbiota structure by promoting the growth of beneficial bacteria such as Bacteroides and Limosilactobacillus, while also exerting prebiotic effects through changes in levels of SCFAs, especially acetic acid, propionic acid, and butyric acid. The above findings confirm the promising prebiotic potential of NAOS and provide a theoretical basis for its application in functional foods.