阴道源抑菌性乳杆菌的筛选及其抑菌能力的评价

Screening and antimicrobial activity evaluation of antimicrobial Lactobacillus strains from vagina

摘要:

【目的】 为开发针对妇科炎症的微生态制剂，本研究对健康妇女阴道的乳酸菌进行分离并对其抑菌特性进行探究。【方法】 采用平板划线、形态学鉴定和16S rRNA基因鉴定相结合的方法，对阴道中的乳杆菌进行分离纯化和鉴定。对分离得到的5株乳杆菌进行生长特性和黏附能力的测定。利用牛津杯法，以大肠杆菌(Escherichia coli) ATCC 25923和金黄色葡萄球菌(Staphylococcus aureus) ATCC 8739为指示菌，测定乳杆菌的抑菌能力；利用微量孔板法测定乳杆菌对白假丝酵母(Candida albicans) CMCC(F) 98001的抑制能力。用有机酸排除和过氧化氢排除法对其抑菌成分进行探究。【结果】 初步分离出5株乳杆菌，其中Q2.1、BHC04和Q8.5为卷曲乳杆菌(Lactobacillus crispatus)，Q6.3和BHG05为格氏乳杆菌(Lactobacillus gasseri)。5株乳杆菌均具有较强的生长和产酸能力，其中Q2.1、BHC04和BHG05的延滞期短，在20 h可达到稳定生长期，BHG05、Q6.3和Q8.5的产酸能力较强，最终pH可达3.80-4.03。卷曲乳杆菌Q2.1、BHC04和Q8.5的黏附能力(疏水能力、自凝集率、与致病菌的共凝集率)显著高于阳性对照菌株德氏乳杆菌(Lactobacillus delbrueckii) DM8909。5株乳杆菌对大肠杆菌、金黄色葡萄球菌和白假丝酵母的抑制能力显著高于阳性对照乳酸链球菌素(nisin)。BHC04、BHG05和Q8.5对大肠杆菌的抑制能力显著高于阳性对照菌株DM8909。BHG05对白假丝酵母的抑制能力显著高于阳性菌株DM8909，可达(73.14±0.14)%。BHC04和Q8.5对白假丝酵母的抑制能力与DM8909无显著性差异，可达(72.80±0.30)%和(72.93±0.10)%。综上，选取BHC04和BHG05作为具有抑菌潜力的优质菌株。5株乳酸菌可以产生有机酸、过氧化氢等发挥抑菌作用。【结论】 筛选出2株具有抑菌能力且益生特性优良的乳杆菌，可作为抑菌微生态制剂的候选菌株来治疗和预防由大肠杆菌、金黄色葡萄球菌和白假丝酵母引起的妇科疾病。

Abstract:

[Objective] To develop probiotics for gynecological inflammation, we isolated and screened out Lactobacillus with antimicrobial and probiotic properties from the vagina of healthy women. [Methods] The plate streaking method was employed to isolate the Lactobacillus strains from vaginal samples, and the isolates were then identified based on morphological and 16S rRNA gene sequencing evidence. The growth and adhesion of the five strains were characterized. The Oxford cup method with Escherichia coli and Staphylococcus aureus as indicator strains was employed to assess the antimicrobial activities of the strains. The microtiter plate method was used to measure the inhibitory effects of the strains on Candida albicans. The antimicrobial components were explored by organic acid elimination and hydrogen peroxide elimination methods. [Results] Five strains of Lactobacillus were isolated, including three strains (Q2.1, BHC04, and Q8.5) of Lactobacillus crispatus and two strains (Q6.3 and BHG05) of Lactobacillus gasseri. All the five strains of Lactobacillus had strong growth and high acid production. Strains Q2.1, BHC04, and BHG05 had a short delay period and reached a plateau growth stage after 20 h, and strains BHG05, Q6.3, and Q8.5 had high acid production, with the culture medium finally reaching pH 3.80-4.03. The adhesion capacity (hydrophobicity, self-agglutination rate, and co-agglutination rate with pathogens) of L. crispatus Q2.1, BHC04, and Q8.5 was significantly higher than that of the positive control strain, L. delbrueckii DM8909. The inhibitory effects of the five strains on E. coli, S. aureus, and C. albicans were stronger than those of the positive control (nisin). Strains BHC04, BHG05, and Q8.5 showcased stronger inhibitory effects on E. coli than DM8909. The inhibitory effect of BHG05 on C. albicans was significantly stronger than that of DM8909, with the inhibition rate reaching up to (73.14±0.14)%. The inhibitory effects of BHC04 and Q8.5 on C. albicans were not significantly different from that of DM8909, with the inhibition rates reaching up to (72.80±0.30)% and (72.93±0.10)%, respectively. According to the results above, we selected BHC04 and BHG05 as high quality strains with antimicrobial potential. The five strains produced organic acids and hydrogen peroxide to exert antimicrobial effects. [Conclusion] Two strains of Lactobacillus with antimicrobial effects and excellent probiotic properties were screened out. They can be used as candidate strains of antimicrobial probiotics for the prevention and treatment of gynecological inflammation caused by E. coli, S. aureus, and C. albicans.