| Objectives Objective to investigate the distribution of normal flora in female vagina,screen the dominant growth bacteria,and prepare vaginal microecological preparation,so as to provide experimental basis for microecological treatment of bacterial vaginosis.Methods Vaginal secretions of healthy women under regular health checkup in the Department of Gynecological Physical Examination Centers of the Affiliated Hospital of North China University of Science and Technology as well as Tangshan maternal and child health care hospital were collected from October 2018 to March 2019.The vaginal secretions of unhealthy women were removed by routine microscopy and bacterial vaginosis detection kit.Bacterial culture was carried out on the samples meeting the standard,and the colony characteristics were observed and recorded for further pure culture.Preliminary identification of the isolated bacteria was conducted by biochemical identification instrument,and the further molecular biological identification was performed by real-time fluorescent quantitative PCR.The frequency distribution histogram of vagina bacteria in healthy women was drawn,and the dominant bacteria in vagina of healthy women were selected as the candidate seed bacteria for vagina microecological preparation.The best culture conditions and harvest time of seed bacteria were determined by subculture.The mixture of seed bacteria was made into powder vaginal microecological preparation by freeze-drying technology.Uniform design method was used to optimize the protective agents of microecological preparation to obtain the highest viable rate.The acid resistance,water content and preservation condition of microecological preparation were evaluated.Results 1.Investigation of bacterial distribution In 249 healthy women with joint examined by high power microscopy and bacterial vaginosis kit,there were 4 cases with leukocyte > 15 / HP,9 cases with spores,and 1 case with trichomonad.In addition to that,1 sample was positive for sialidase,10 samples with p H value > 4.7.Therefor,25 samples were excluded.The isolated bacteria from 224 vaginal secretions of healthy women were identified by biochemical reaction.The results were as follows: 123 strains of Lactobacillus acidophilus with an isolation rate of 4.9%,98 Lactobacillus fermentum with 43.8%,68 Corynebacterium brevis with 30.4%,37 Streptococcus mitis with 16.5%;32 Staphylococcus epidermidis with 14.3%;28 Staphylococcus equis with12.5%,and 19 Enterococcus faecalis with 8.5%.Gene identification was conducted with corresponding primers according to the results of biochemical reaction,and,the results were as the followings: 122 strains Lactobacillus acidophilus with an isolation rate of 54.5%,98 strains Lactobacillus fermentans with 43.8%,59 strains Corynebacterium parvum with 26.3%,37 strains Streptococcus mitis,with 16.5%,29 strains Staphylococcus epidermidis with 12.9%,28 strains Staphylococcus equi with 12.5%,and 19 Enterococcus faecalis with 8.5%.The distribution frequency histogram of vagina bacteria in healthy women demonstrated that the dominant bacteria in vagina were Lactobacillus fermentans,Lactobacillus acidophilus and Corynebacterium brevis,which would be served as candidate seed bacteria for vagina microecological preparation.2.Preparation of microecological preparation(1)The growth curve of bacteria discovered that both Lactobacillus acidophilus and Lactobacillus fermentans were in the platform stage at 18-22 h,and Corynebacterium brevis was in the platform stage at 20-24 h.(2)Through freezedrying technology,the milky and tasteless vaginal microecological preparation was obtained.(3)The results of single factor analysis of microecological agent unveiled that the maximum viable rate was 70.30±0.28% under the concentration of skimmed milk powder was 14.0%,the maximum viable rate was 60.40±0.41% under the concentration of trehalose was 4.0%,and the maximum viable rate was 64.40±0.37% under the concentration of glycerin was 1.5%.(4)The activity rates of Lactobacillus acidophilus,Lactobacillus fermentans and Corynebacterium brevis were 76.34%,74.32% and 71.3% respectively under the environment p H was 4.0.(5)The water contents of five batches of vaginal microecological preparation were determined.They were 3.86%,4.42%,3.73%,4.06% and 2.62% respectively.(6)During the period of 240 storage days,the number of viable bacteria of microecological preparation kept stable at-20?,about 1×1010cfu / g.While the number of viable bacteria decreased slowly at 4 ?,nevertheless,the number of viable bacteria was far greater than the minimum requirement for the number of probiotic products(106 cfu/g).But the number of viable Lactobacillus in microecological preparation decreased rapidly stored at 25 ? 30?,down to 106cfu/g in 30 days.Conclusions 1.Lactobacillus acidophilus,Lactobacillus fermentans and Corynebacterium brevis were the dominant bacteria in the vagina of healthy women,and they could be used as the candidate seeds for vaginal microecological preparations.2.Microecological preparation(1)Their survival rates were up to the highest after freeze-drying prudoucts revived when Lactobacillus acidophilus and Lactobacillus fermentans were harvested in 20 hours,and Corynebacterium brevis was harvested in 24 hours.(2)When the concentrations of skimmed milk powder,trehalose and glycerin were 14.0%,4.0% and 1.5% respectively,the rate of viable bacteria was the highest(79.68%).(3)At p H 4.0,the survival rate of the three bacteria was the highest.(4)The water content of vaginal microecological preparation meets the national standard(< 5%).(5)The vaginal microecological preparation could be preserved for 8 months at-20 ? and 4 ?.Figure11;Table17;Reference 83... |
PDF Full Text Request