Jpn. J. Infect. Dis., 59, 249-253, 2006

Original Article Some Probiotic Properties of Vaginal Lactobacilli Isolated from Healthy Women Belma Aslim* and Emine Kilic Department of Biology, Faculty of Science and Arts, Gazi University, Ankara, Turkey (Received February 7, 2006. Accepted June 15, 2006) SUMMARY: Lactobacilli have long been considered to be protective flora in the vagina. In this study, samples were taken from the lateral vaginal wall of 19 healthy women, and 58 strains of spp. were isolated from 10 of these women. The identification results revealed that Lactobacillus gasseri was the predominant lactobacilli

in healthy women. The amounts of lactic acid and hy drogen peroxide (H2O2) produced by Lactobacillus spp. were determined. The vaginal pH levels induced by Lactobacillus spp. strains ranged from 1.00 to 2.15. The titratable

acidity of the strains ranged from 0.27 to 1.33%. The H2O2 concentration produced in the lactobacilli strains ranged between 1.01 μg/ml and 15.50 μg/ml. The antimicrobial effects of the lactobacilli against the pathogenic Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 2392, Pseudomonas aeruginosa ATCC 29212 and Salmonella enterica serovar Enteritidis RSKK 171 were also determined by the agar diffusion methods.

BV, recurrent urinary tract infections by Escherichia coli and INTRODUCTION acquisition of HIV-1 (12). Hydrogen peroxide-producing Lactobacilli are the dominant bacteria of a healthy human lactobacilli also might exert control over vaginal cancer vagina, and their presence and number are influenced by through specific interaction with reactive oxygen species. estrogen production, which undergoes age- and menstrual In addition to lactic acid, the combination of H2O2 further cycle-dependent changes (1). A healthy vaginal ecosystem is suppresses the endogenous pathogenic bacteria to maintain a dominated by certain species of Lactobacillus which exert a healthy vaginal ecosystem (13). significant influence on the microbiology of the vagina (2,3). The determinative of characteristics of lactobacilli for However, lactobacilli are used in the fermented food indus- probiotic use in the human female urogenital tract have been try as probiotics for human and animal nutrition (4). Lately, suggested (8,14). The selection of Lactobacillus spp. for they have also been suggested as candidate microorganisms to probiotic use in the human vagina as a barrier to infections be included in probiotics for vaginal use (5). There is growing and alternative to long-term use of antibiotics. interest in the use of lactobacilli of human origin as probiotics The aim of this study was to determine the ability of against urogenital tract infections. Lactobacilli have long been Lactobacillus spp. strains isolated from the vaginas of healthy considered to constitute the primary microbiological barrier women to produce acid and hydrogen peroxide. The inhibi- to infection by urogenital pathogens (6). tory effects of these lactobacilli on various pathogenic bacteria

Lactobacilli, particularly those strains that produce H2O2, were also studied. Several of the newly isolated strains that may have a protective effect against vaginal colonization by were determined to have certain desirable properties may be pathogenic species such as those that cause bacterial vaginosis available for use as probiotics. (BV) and possibly human immunodeficiency virus (HIV) and gonorrhea (7). MATERIALS AND METHODS They exert a protective role against pathogen colonization by steric exclusion and production of inhibitory substances. Bacterial strains and identification of lactobacilli: Vagi- Among these substances, lactic acid produced from carbo- nal swabs were taken from the lateral vaginal wall of 19 hydrates helps to maintain a low vaginal pH (8). The healthy healthy women and were inoculated on lactic agar as a selec- vagina is generally found to have a pH of 4 ± 0.5, and this tive medium. Colonies forming a yellow zone were isolated acidity has been shown to be microbicidal for many sexually from the lactic agar (15). To identify the strains, each colony transmitted disease pathogens, including HIV (9). In addition, was subjected to a gram strain test as well as tests for catalase different Lactobacillus spp. produce H2O2 and bacteriocin- activity, cell shape, motility, growth at various temperatures like substances that may affect undesirable or pathogenic (15, 45, and 50°C), and tolerance of different salt levels (MRS strains even on themselves (8). The hydro- broth with 2, 4, and 6.5% NaCl). Production of gas from gen peroxide-producing microorganisms that are present glucose and production of catalase were also tested. Finally, in the vagina of healthy women have been suggested as the carbohydrate fermentation characteristics of all strains the bacterial group that is responsible for the maintenance were determined by using an API 50 CHL system (Bio- of the ecologic balance, mainly in pregnant women (10,11). Merieux, Marcy l’Etoile, France) (16,17). The isolates were Absence of these microorganisms is related to increase in stored at –80°C in MRS broth (de Man, Rogosa and Sharpe Medium; Oxoid, Hampshire, England) containing 30% glyc- *Corresponding author: Mailing address: Department of Biology, erol and regenerated twice before use in the manipulation. Faculty of Science and Arts, Gazi University, Teknikokullar, According to the results of the identification tests, 58 strains Ankara 06500, Turkey. Tel: +90-312-212-6030 ext. 2847, Fax: were identified as Lactobacillus spp. (Figure 1). In prepara- +90-312-212-2279, E-mail: [email protected] tion for the experiments, Lactobacillus spp. strains were

249 cultured under anaerobic conditions for 16-18 h (the expo- nential growth phase) at 37°C in MRS broth in isolates. The test bacteria were obtained from the Department of Biology Culture Collection of Gazi University. Determination of lactic acid production ability: The acid production of the lactobacilli strains was determined in 10 ml of sterilized MRS broth. One percent inoculum from an overnight culture was used and the incubation proceeded for 16-18 h at 37°C. After incubation, the pH of samples was measured by an electronic digital type pH meter. In addition, the samples were titrated with 0.1 N NaOH using phenol- phthalein as an indicator. The titratable acidity was expressed as a percentage of that of lactic acid (17).

Determination of hydrogen peroxide (H2O2) production ability: Lactobacilli were added (2% v/v) to 10 ml MRS broth and inoculated at 37°C for 16-18 h. Samples were adjusted to room temperature before adding the desired amount of

H2O2. Ten milliliters of samples were immediately adjusted to pH 4.5 with 0.1 N HCl. Two milliliters of 0.1 M acetate buffer (pH 4.5) were added and the mixture was diluted to 20 Fig. 1. Pie chart of the percentage distribution of 58 strains of ml with distilled water. The samples were then filtered through Lactobacillus spp. isolated from the vagina of Turkish women (n = 10). Whatman no. 42 filter paper to remove the curd. Centrifu- gation of the samples at 3,400×g for 10 min may also be used as a selective medium. Only isolates that formed a used to remove curd. To have the filtrate for each sample yellow colony on lactic agar, indicating a high level of serve as the blank, 5 ml of the filtrate for each sample was acid production, were selected. The 58 Lactobacillus isolates added to a tube containing 1 ml distilled water and 0.1 ml o- were classified as 21% L. gasseri, 16% L. vaginalis, 16% L. dianisidine solution, and then another 5 ml of the filtrate was acidophilus, 14% L. delbrueckii spp. lactis, 14% L. crispatus, added to a tube containing 1 ml peroxidase solution and 0.1 5% L. plantarum, 3% L. cellobiosus, 3% L. jensenii, 3% L. ml o-dianisidine. The assay tubes were then incubated for 10 salivarius, 2% L. curvatus, 2% L. brevis and 2% L. oris (Fig- min at room temperature (23-26°C), 0.2 ml of 4 N HC1 was ure 1). L. gasseri was the predominant Lactobacillus. added to each tube, and the optical density was determined The Lactobacillus spp. were isolated and their hydrogen after an additional 5 min of incubation (18). The H2O2 (30%) peroxide (H2O2) and lactic acid production abilities were was obtained from Merck and was diluted to the desired con- determined (Table 1). The level of pH induced by lactobacilli centration with distilled water just before each assay. H2O2 strains ranged from 1.00 to 2.15. The titratable acidities (%) was quantified by using a H2O2 standard curve, established of the strains were between 0.27 and 1.33%. L. gasseri R4, with concentrations ranging from 1 to 10 μg/ml (19). R5 (1.33, 1.18 %) and L. acidophilus R1, R6 (1.17, 1.12 %) Determination of antimicrobial and bacteriocin activi- strains induced a higher level of acidity than all the other ties: Lactobacillus spp. strains were inoculated in MRS broth lactobacilli strains. at 37°C for 16 to 18 h. Activated cultures were centrifuged at There were striking differences between the amounts of

4,000 rpm for 15 min and the clear supernatants were steri- H2O2 production (range, 1.01 to 15.50 μg/ml H2O2), and 23 lized by filtration (0.45 μl) to obtain cell-free filtrates. The strains of lactobacilli did not produce any detectable amount test bacteria on the petri dishes with 20 ml of nutrient agar of H2O2. The H2O2 production of L. vaginalis H5 was the (Oxoid) solidified; the dishes were stored in a refrigerator highest. for 2 h. Four wells (Ø 6 mm) were then made and filled using The antimicrobial effect of Lactobacillus spp. was studied 100 μl of cell-free filtrate. The inoculated plates were incu- by using the agar diffusion technique on the test bacteria (P. bated at 37°C for 24 h, and the diameter of the zone was aeruginosa ATTC 29212, S. aureus ATTC 2392, S. enterica measured with calipers. The measurements recorded were serovar Enteritidis RSKK 171 and E. coli ATTC 11230) which the radius from the edge of the zone to the edge of the well. caused infections in the genital and urogenital system. The Previously inoculated with 0.1 ml of a 24 h nutrient broth strains of Lactobacillus spp. had an 85% inhibitory effect on culture of test bacteria (E. coli ATCC 11230, S. aureus ATCC P. aeruginosa ATTC 29212, a 48% inhibitory effect on S. 2392, P. aeruginosa ATCC 29212, S. enterica serovar Enter- aureus ATTC 2392, a 71% inhibitory effect on S. enterica itidis RSKK 171) (20). serovar Enteritidis RSKK 171, and a 43% inhibitory effect Determination for bacteriocin production: Lactobacillus on E. coli ATTC 11230. Although most of the strains were spp. were to have bacteriocin effect on the test bacteria. Some found to produce inhibition zones against some pathogenic supernatants were neutralized with 1 N NaOH to pH 6.5, and bacteria, the strains L. gasseri O2, L. vaginalis S1, L. salivarius inhibitory from the hydrogen peroxide was eliminated by the S2, and L. plantarum T1 did not show antimicrobial activity addition of catalase (5 mg/ml) for determination of bacterio- against any of the test bacteria. P. aeruginosa ATTC 29212 cin or bacteriocin-like substances (21). was the most sensitive, while E. coli ATTC 11230 was the most resistant to the supernatants of Lactobacillus strains (Table 2). RESULTS In this study, the samples were taken from the lateral vaginal DISCUSSION wall of 19 healthy women. Fifty-eight strains of Lactobacillus spp. were isolated from 10 of these women. Lactic agar was Lactobacilli are used in the fermented food industry and as

250 Table 1. The lactic acid and hydrogen peroxide levels produced In this study, vaginal swabs were collected from the lateral by Lactobacillus spp. strains vaginal wall of 19 healthy Turkish women, and lactobacilli

H2O2 Titratable were isolated from only 10 of these women. Lactobacilli are Strains production pH acidity dominant in this habitat, at 107 to 108 cfu/g of vaginal fluid of (μg/ml) (%) healthy premenopausal women (23). As a result of the identifi- L. gasseri H14 12.30 ± 0.00 1.45 ± 0.15 0.50 ± 0.04 cation tests, 58 Lactobacillus spp. isolates were identified as L. gasseri H15 11.72 ± 0.12 1.89 ± 0.06 0.66 ± 0.04 12 L. gasseri, 9 L. vaginalis, 9 L. acidophilus, 8 L. delbrueckii L. gasseri H16 11.00 ± 0.05 1.89 ± 0.01 0.73 ± 0.07 L. gasseri I2 4.52 ± 0.03 2.15 ± 0.00 0.94 ± 0.00 spp. lactis, 8 L. crispatus, 3 L. plantarum, 2 L. cellobiosus, 2 L. gasseri F1 1.39 ± 0.06 1.83 ± 0.08 0.85 ± 0.01 L. jensenii, 2 L. salivarius, 1 L. curvatus, 1 L. brevis, and 1 L. L. gasseri O2 1.06 ± 0.01 1.59 ± 0.01 0.83 ± 0.02 oris (Figure 1). L. gasseri was predominant among the other L. gasseri R3 3.91 ± 0.04 1.77 ± 0.00 1.06 ± 0.01 lactobacilli; these bacteria represented 21% of the isolates, L. gasseri R4 – 1.66 ± 0.01 1.33 ± 0.00 L. gasseri R5 – 1.23 ± 0.14 1.18 ± 0.00 followed by L. acidophilus (16%) and L. vaginalis (16%) in L. gasseri R7 – 1.78 ± 0.00 1.04 ± 0.00 Turkish women. Similar results have been reported by other L. gasseri R10 3.98 ± 0.09 1.00 ± 0.07 0.49 ± 0.00 authors. Benno et al. (24) reported that L. gasseri was the L. gasseri L1 – 1.71 ± 0.00 0.95 ± 0.01 dominant species among lactobacilli isolated from the in- L. acidophilus A1 – 1.65 ± 0.00 0.75 ± 0.06 testinal tracts of Japanese people. Also, Song et al. (25) re- L. acidophilus G6 – 1.85 ± 0.02 0.48 ± 0.09 L. acidophilus G7 1.86 ± 0.12 1.89 ± 0.00 0.58 ± 0.01 ported that L. crispatus and L. gasseri were the predominant L. acidophilus G8 5.00 ± 0.01 1.99 ± 0.02 0.86 ± 0.01 lactobacilli in the vaginas of Japanese women. However, Reid L. acidophilus R1 – 1.34 ± 0.05 1.17 ± 0.11 and Bruce emphasized that L. iners were the most common L. acidophilus R6 1.03 ± 0.01 1.59 ± 0.00 1.12 ± 0.06 organisms, at least in one mainly white population (26). L. acidophilus I4 5.14 ± 0.02 2.10 ± 0.02 1.02 ± 0.01 L. acidophilus R9 – 1.00 ± 0.10 0.78 ± 0.04 Among them, those belonging to the L. acidophilus group L. acidophilus G11 – 1.85 ± 0.00 0.45 ± 0.05 and L. fermentum are most frequently isolated, although L. vaginalis H1 3.51 ± 0.01 1.74 ± 0.00 0.39 ± 0.04 others, such as L. plantarum, L. brevis, L. jensenii, L. casei, L. vaginalis H2 – 1.79 ± 0.00 0.41 ± 0.03 L. delbrueckii, and L. salivarius are isolated as well (27). A L. vaginalis H3 4.35 ± 0.02 2.02 ± 0.03 0.80 ± 0.01 new species, L. vaginalis, was recently isolated; this species L. vaginalis H4 – 1.66 ± 0.04 0.60 ± 0.02 L. vaginalis H5 15.50 ± 0.00 1.64 ± 0.02 0.52 ± 0.05 resembles L. fermentum and L. reuteri, but is phylogenetically L. vaginalis H6 1.22 ± 0.00 1.65 ± 0.11 0.93 ± 0.01 most closely related to L. oris and L. reuteri (14). The inhibitory L. vaginalis H7 2.33 ± 0.01 1.56 ± 0.05 0.54 ± 0.09 effect on growth of the pathogen thus involved a combina- L. vaginalis H8 5.36 ± 0.02 1.93 ± 0.18 0.61 ± 0.08 tion of both hydrogen peroxide and acidity. That was pro- L. vaginalis S1 4.79 ± 0.01 1.07 ± 0.07 0.29 ± 0.01 L. crispatus G1 – 2.01 ± 0.02 0.77 ± 0.00 duced during the lactobacilli growth. One of the mechanisms L. crispatus G2 3.84 ± 0.04 1.96 ± 0.00 0.85 ± 0.05 suggested to control the vaginal ecosystem is the production of L. crispatus G3 – 1.97 ± 0.00 0.72 ± 0.07 these antagonistic substances. Tomas et al. (28) reported that L. crispatus G5 – 1.90 ± 0.00 0.67 ± 0.00 when the vaginal pH is <4.5, colonization of the introitus by L. crispatus G9 2.33 ± 0.03 1.83 ± 0.04 0.48 ± 0.06 E. coli is not frequent. It was found that the titratable acidity L. crispatus G10 – 1.87 ± 0.00 0.50 ± 0.04 L. crispatus G14 1.24 ± 0.02 1.95 ± 0.00 0.76 ± 0.00 of L. gasseri R4 (1.33%) was highest. New strains isolated L. crispatus O3 – 1.36 ± 0.07 0.65 ± 0.06 from healthy women, which are able to high acid production, L. delbrueckii spp. lactis G4 – 1.94 ± 0.01 0.68 ± 0.00 may be used as probiotics. L. delbrueckii spp. lactis G12 – 1.90 ± 0.00 0.47 ± 0.07 In this study, only 10 strains from the 58 human vaginal L. delbrueckii spp. lactis G13 – 1.74 ± 0.05 0.55 ± 0.05 L. delbrueckii spp. lactis H9 – 1.76 ± 0.00 0.65 ± 0.02 isolates inhibited all the test bacteria. In their study, Tomas L. delbrueckii spp. lactis H11 5.34 ± 0.02 1.48 ± 0.03 0.32 ± 0.04 et al. (28) reported that only 6 of 134 isolated strains of L. delbrueckii spp. lactis H12 11.99 ± 0.01 2.02 ± 0.03 0.90 ± 0.09 vaginal lactobacilli were able to inhibit the growth: all the L. delbrueckii spp. lactis H13 7.92 ± 0.00 1.99 ± 0.02 0.80 ± 0.03 pathogens such as E. coli, S. aureus, Enterecoccus faecalis, L. delbrueckii spp. lactis R8 3.00 ± 0.00 1.74 ± 0.03 0.96 ± 0.02 and Klebsiella sp. L. plantarum H17 11.26 ± 0.02 1.85 ± 0.05 0.74 ± 0.08 L. plantarum T1 – 1.00 ± 0.05 0.27 ± 0.00 Lactic acid bacteria have been shown to inhibit the in vitro L. plantarum H10 1.02 ± 0.01 1.98 ± 0.16 0.80 ± 0.06 growth of many enteric pathogens and have been used in both L. jensenii R11 4.55 ± 0.05 1.00 ± 0.04 0.78 ± 0.02 humans and animals to treat a broad range of gastrointestinal L. jensenii R12 2.57 ± 0.01 1.00 ± 0.02 0.87 ± 0.02 disorders (29). L. gasseri R4 and L. cellobiosus L2 have been L. salivarius I1 – 1.84 ± 0.01 0.75 ± 0.01 L. salivarius S2 1.59 ± 0.01 2.06 ± 0.04 0.81 ± 0.00 shown to strongly inhibit the enteropathogenic bacteria E. L. cellobiosus L2 1.69 ± 0.00 1.08 ± 0.07 0.72 ± 0.06 coli ATCC 11230 and S. enterica serovar Enteritidis RSKK L. cellobiosus I3 7.65 ± 0.00 2.06 ± 0.00 0.91 ± 0.00 171 in vitro. Similar results have been reported (30,31) for L. brevis R2 3.00 ± 0.00 1.75 ± 0.01 1.04 ± 0.01 other strains of Lactobacillus isolated from chickens that L. curvatus L3 1.01 ± 0.01 1.62 ± 0.03 0.89 ± 0.00 inhibited Salmonella and E. coli growth. In the present L. oris A2 – 1.80 ± 0.00 0.78 ± 0.60 study, L. salivarius isolated from the cloaca and vagina pro- Values are the means ± standard deviations of triplicate measurements. duced significant inhibitory activity against the S. enteritidis. P. aeruginosa ATCC 29212 was more sensitive against probiotics for human and animal nutrition; lately, however, lactobacilli strains than other test bacteria. Collins and Aramaki they have also been suggested as candidate microorganisms (32) have shown that some L. acidophilus strains were able to be included in probiotics for vaginal use (22). There is to inhibit the growth of certain Pseudomonas spp. by produc- growing interest in the use of lactobacilli of human origin as ing levels of 40-55 μg/ml H2O2. L. gasseri O2, L. vaginalis probiotics against urogenital tract infections (6). Recently, S1, L. plantarum T1, and L. salivarius S2 exhibited no anti- however, the use of lactobacilli isolated from dairy products bacterial activity against any of the test bacteria. Also, these as probiotics have been investigated. The potential use of bacteria showed weak H2O2 and acid production. However, vaginal lactobacilli isolated from healthy women as probiotics Aroutcheva et al. (13) and Annuk et al. (33) revealed that has not been examined in detail. there was no correlations among bacteriocin activity, lactic

251 Table 2. Antimicrobial activity of Lactobacillus spp. strains on test bacteria Inhibition zone (mm) Strains S. enterica P. aeruginosa S. aureus E. coli serovar Enteritidis L. gasseri H14 7.50 ± 0.35 3.40 ± 0.40 6.80 ± 0.00 – L. gasseri H15 6.60 ± 0.00 10.20 ± 0.00 5.90 ± 0.05 – L. gasseri H16 – 4.20 ± 0.00 – 1.90 ± 0.80 L. gasseri I2 4.20 ± 0.00 6.90 ± 0.30 2.10 ± 0.00 – L. gasseri F1 3.10 ± 0.55 – 4.00 ± 0.00 – L. gasseri O2 – – – – L. gasseri R3 9.40 ± 0.70 4.90 ± 0.40 4.50 ± 0.20 1.60 ± 0.40 L. gasseri R4 4.80 ± 0.00 5.80 ± 0.40 6.70 ± 0.15 8.70 ± 0.50 L. gasseri R5 4.00 ± 0.00 6.00 ± 0.00 6.90 ± 0.05 6.60 ± 0.30 L. gasseri R7 4.70 ± 0.05 6.60 ± 0.50 3.50 ± 0.15 5.30 ± 0.10 L. gasseri R10 7.70 ± 0.25 – 5.10 ± 0.85 1.30 ± 0.00 L. gasseri L1 10.00 ± 0.00 – 6.50 ± 0.05 – L. acidophilus A1 6.20 ± 0.05 3.90 ± 0.70 6.40 ± 0.00 – L. acidophilus G6 8.80 ± 0.10 – 4.50 ± 0.45 1.60 ± 0.10 L. acidophilus G7 5.80 ± 0.30 – 3.20 ± 0.00 – L. acidophilus G8 5.70 ± 0.35 – 4.40 ± 0.00 1.80 ± 0.20 L. acidophilus R1 10.90 ± 0.25 7.50 ± 0.10 4.70 ± 0.45 5.40 ± 0.30 L. acidophilus R6 4.30 ± 0.35 4.10 ± 0.10 2.60 ± 0.00 – L. acidophilus I4 4.70 ± 0.55 10.00 ± 0.00 3.90 ± 0.35 4.40 ± 0.00 L. acidophilus R9 7.50 ± 0.45 – 5.20 ± 0.10 – L. acidophilus G11 6.10 ± 0.35 – 5.20 ± 0.00 – L. vaginalis H1 4.10 ± 0.45 6.10 ± 0.50 4.20 ± 0.10 – L. vaginalis H2 3.10 ± 0.25 6.70 ± 0.30 – 2.00 ± 0.00 L. vaginalis H3 3.40 ± 0.00 4.90 ± 0.50 7.20 ± 0.40 – L. vaginalis H4 4.00 ± 0.50 7.70 ± 0.70 5.70 ± 0.35 – L. vaginalis H5 5.40 ± 0.00 5.30 ± 0.90 5.00 ± 0.00 2.50 ± 0.90 L. vaginalis H6 – – 3.00 ± 0.00 – L. vaginalis H7 4.20 ± 0.00 – 3.70 ± 0.15 1.60 ± 0.00 L. vaginalis H8 6.10 ± 0.25 4.10 ± 0.50 4.30 ± 0.15 – L. vaginalis S1 – – – – L. crispatus G1 4.60 ± 0.20 6.90 ± 0.50 – 1.90 ± 0.40 L. crispatus G2 6.20 ± 0.10 5.00 ± 0.00 – 1.40 ± 0.20 L. crispatus G3 6.00 ± 0.20 – – 1.50 ± 0.20 L. crispatus G5 8.30 ± 0.45 – – – L. crispatus G9 4.50 ± 0.25 – 4.30 ± 0.05 2.90 ± 0.85 L. crispatus G10 4.30 ± 0.05 – – 1.50 ± 0.10 L. crispatus G14 6.40 ± 0.00 7.20 ± 0.10 6.00 ± 0.00 2.30 ± 0.30 L. crispatus O3 4.20 ± 0.00 – – – L. delbrueckii spp. lactis G4 7.10 ± 0.45 – 4.20 ± 0.00 2.30 ± 0.10 L. delbrueckii spp. lactis G12 5.40 ± 0.00 – 4.70 ± 0.25 – L. delbrueckii spp. lactis G13 5.60 ± 0.30 5.30 ± 0.90 – – L. delbrueckii spp. lactis H9 – 5.40 ± 0.20 3.20 ± 0.00 1.70 ± 0.00 L. delbrueckii spp. lactis H11 7.10 ± 0.35 – 4.10 ± 0.15 – L. delbrueckii spp. lactis H12 2.40 ± 0.00 – – – L. delbrueckii spp. lactis H13 – 3.50 ± 0.30 – – L. delbrueckii spp. lactis R8 7.80 ± 0.30 – 6.10 ± 0.45 1.60 ± 0.10 L. plantarum H17 – – – 2.10 ± 0.10 L. plantarum T1 – – – – L. plantarum H10 4.10 ± 0.05 3.70 ± 0.10 3.80 ± 0.30 1.50 ± 0.00 L. jensenii R11 7.90 ± 0.35 – 4.80 ± 0.00 – L. jensenii R12 10.20 ± 0.40 – 7.20 ± 0.00 – L. salivarius I1 4.60 ± 0.00 4.20 ± 0.00 – – L. salivarius S2 – – – – L. cellobiosus L2 6.00 ± 0.00 – 9.50 ± 0.55 – L. cellobiosus I3 4.60 ± 0.00 8.90 ± 0.30 2.60 ± 0.00 – L. brevis R2 11.30 ± 0.45 5.80 ± 0.00 3.50 ± 0.15 4.10 ± 0.10 L. curvatus L3 5.00 ± 0.00 – 8.80 ± 0.00 – L. oris A2 4.60 ± 0.30 – 6.40 ± 0.00 – Values are the means ± standard deviations of triplicate measurements. acid and hydrogen peroxide production. They also found promising candidates for use in the preparation of probiotic that some Lactobacillus strains produced H2O2, but did not products and for use as health-promoting bacteria. demonstrate any inhibitory effect. We obtained similar results in the present study: L. gasseri R4, R5, R7 and L. acidophilus ACKNOWLEDGMENTS R1 strains produced a high level of lactic acid, but did not produce H2O2. Also, no correlation was found between anti- This study was supported by Gazi University BAB project microbial activity with lactic acid and H2O2 production of no. FEF 05/2002-54 and TR Prime Ministry State Planning strains. Our results indicated that new strains of Lactobacillus Organization project no. 2003 K 12470-06. spp. isolated from the vaginas of healthy women could be

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