CTAB沉淀培养液中的b型流感嗜血杆菌在不同pH条件下对荚膜多糖得率的影响

doi:10.5246/jcps.2026.02.010

摘要/Abstract

摘要：

b型流感嗜血杆菌(Hib)结合疫苗可预防儿童感染Hib, 该病原体是导致5岁以下儿童严重侵袭性感染的主要原因。在细菌发酵液中, 不同pH值会对阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)沉淀荚膜多糖(CP)的得率具有重要影响。研究在不同pH值(5.25–8.0)条件下, 从3批细菌发酵液中制备12份样本, 采用Gotschlich法纯化得到CP。通过¹H NMR对pH 5.25、7.0和8.0条件下的CP结构进行分析并验证结果。发现3组平行实验的P值均< 0.01(粗品分别为0.001、0.001、0.001, 精品分别为0.002、0.01、0.001), 表明pH值对多糖沉淀得率影响显著。当发酵液pH为6.5时, 多糖回收率较高且结构稳定; 而pH 8.0条件下的¹H NMR谱图显示CP结构已发生变化。因此, Hib细菌发酵液的pH值直接影响CTAB沉淀得到CP的得率和多糖质量。

关键词: pH, 荚膜多糖, b型流感嗜血杆菌, CTAB

Abstract:

Haemophilus influenzae type b (Hib) conjugate vaccines can protect children from Hib infection, which was once the leading cause of severe invasive diseases in children under 5 years of age. The pH of the culture supernatant, in combination with the cationic surfactant cetyltrimethylammonium bromide (CTAB), plays a critical role in determining the yield of capsular polysaccharide (CP). Twelve samples collected from three batches of culture supernatant were adjusted to various pH values (ranging from 5.25 to 8.0). CP purification was carried out according to Gotschlich’s procedure. To confirm the effects of pH, ¹H NMR analyses were performed on CP samples obtained at pH 5.25, 7.0, and 8.0. The P-values for the three parallel experiments were all < 0.01 (0.001, 0.001, and 0.001 for crude samples; 0.002, 0.01, and 0.001 for purified samples), indicating that pH had a significant effect on CP precipitation. When the pH of the supernatant was adjusted to 6.5, CP recovery was maximized, and its structural integrity was maintained. In contrast, ¹H NMR spectra revealed that the structure of CP was altered at pH 8.0. These findings demonstrated that both the yield and quality of CP were strongly influenced by the pH of the culture supernatant.

Key words: pH, Capsular polysaccharide, Haemophilus influenzae type b, CTAB

Supporting:

冯东. CTAB沉淀培养液中的b型流感嗜血杆菌在不同pH条件下对荚膜多糖得率的影响[J]. 中国药学(英文版), 2026, 35(2): 144-150.

Dong Feng. Effect of pH on the yield of capsular polysaccharide precipitated with CTAB from Haemophilus influenzae type b culture supernatant[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(2): 144-150.

图/表 6

Figure 1. The weight of crude pentose precipitated by CTAB in various pH of culture supernatants.

Figure 2. The weight of pentose precipitated by Cetavlon in various pH of culture supernatants.

Table 1. Pentose assay results for final bulk Hib polysaccharide from three consistency runs. Component percentages are expressed relative to the dry weight of the polysaccharide.

Table 2. Results of protein assays performed on final bulk Hib produced for three consistency runs. Percentage of each component was calculated on the dry weight of Hib polysaccharide.

Table 3. Nucleic acid assay results of final bulk Hib polysaccharide from three consistency runs. Percentages are expressed relative to the dry weight of the polysaccharide.

Figure 3. Comparison of the partial one-dimensional 1H NMR spectra of purified CP under pH 5.25 (a), 7.0 (b) and 8.0 (c).

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