COMPOSITIONS FOR THE VAGINAL AND ORAL ADMINISTRATION OF LACTOBACILLUS AND USES THEREOF |
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| 申请号 | EP11767944.9 | 申请日 | 2011-09-13 | 公开(公告)号 | EP2509610B1 | 公开(公告)日 | 2013-03-27 |
| 申请人 | HSO Health Care GmbH; | 发明人 | KISS, Herbert; UNGER, Frank.M.; DOMIG, Konrad, J.; VIERNSTEIN, Helmut.; KNEIFEL, Wolfgang.; | ||||
| 摘要 | The present invention relates to compositions for the oral and vaginal administration of human Lactobacilli and uses thereof for physiologic restoration of the vaginal flora, physiologic maintenance of Lactobacillus flora in pathologic deficiency to produce Lactobacilli, treatment of asymptomatic bacterial vaginosis and prevention of preterm delivery caused through bacterial vaginosis. | ||||||
| 权利要求 | |||||||
| 说明书全文 | The present invention relates to compositions for the oral and vaginal administration of human Lactobacilli and uses thereof for physiologic restoration of the vaginal flora, physiologic maintenance of Lactobacillus flora in pathologic deficiency to produce Lactobacilli, treatment of asymptomatic bacterial vaginosis and prevention of preterm delivery caused through bacterial vaginosis. The healthy human vagina is dominated by a variety of Lactobacillus species, which play an essential role in protecting women from urogenital infection. Lactobacilli have the ability to adhere to vaginal epithelia, to inhibit the adhesion and growth of pathogens, deplete nutrients otherwise available to pathogens, and modulate the host immune response and microenvironment [1,2]. Most importantly, Lactobacilli metabolize the glycogen contained in the cells of the vaginal vault, forming lactic acid as the final product. Thus, in a healthy vagina, pH-values of 4.0-4.5 are reached, a level at which many pathogens cannot flourish. Because vaginal infection is an important mechanism of disease responsible for preterm birth [3], maintaining the natural, healthy balance of the Lactobacillus flora in the vagina is particularly important during pregnancy. A deficiency in Lactobacilli can upset the microbial balance in the vagina, frequently resulting in the syndrome of bacterial vaginosis [4,5], which may be associated with a quantitative and qualitative shift from normally occurring Lactobacilli to a mixed flora dominated by anaerobic bacteria [6]. According to Nugent et al. [7], bacterial vaginosis is characterized by a complete loss of Lactobacilli and a concomitant increase in Gram-variable and Gram-negative rods, primary among them Gardnerella vaginalis as well as Bacteroides, Prevotella, and Mobiluncus species [4,5]. However, loss of vaginal Lactobacilli also leaves nonpregnant women susceptible to infection which may result in endometritis or even pelvic inflammatory disease [8, 9]. During menopause, involution of the female genital tract occurs, reflecting possibly a built-in biologic life expectancy interrelated with the neurohypophyseal endocrine axis [10]. The major universal change is vaginal atrophy [10]. Vaginal dryness, burning, itching and dyspareunia are frequent complaints along with dysuria, urinary frequency and recurrent infections. The genitourinary atrophy following menopause is associated with a decline in estrogen secretion accompanied by depletion of Lactobacilli and increased colonization by pathogenic microorganisms associated with bacterial vaginosis and urinary tract infections [11]. In post-menopausal women, vaginal estriol therapy reduces E. coli colonization and increases the numbers of Lactobacilli, with the result that the incidence of recurrent urinary and genital tract infections drops significantly [12]. Several species of Lactobacillus have been described to populate the vagina to varying degrees. For some time the flora of healthy women of childbearing age was believed to be dominated by Lactobacillus acidophilus and Lactobacillus fermentum, followed by Lactobacillus brevis, Lactobacillus jensenii and Lactobacillus casei [13]. In their study on the vaginal Lactobacillus flora [14], Vásquez et al. found that the vaginal flora of most participants was dominated by a single Lactobacillus species, with the presence of other species showing wide individual variability. The most frequently occurring species were L. crispatus, L. gasseri, L. iners, and L. jensenii. In another study by Reid et al., the most commonly isolated Lactobacillus strains were L. jensenii, L. acidophilus, L. casei, and L. gasseri [15]. In recent Austrian studies, the predominant Lactobacillus species identified by species-specific PCR, namely L. crispatus, L. gasseri, L. jensenii, and L. rhamnosus were used to generate DNA fingerprints. L. crispatus, L. gasseri, L. jensenii and L. rhamnosus can be regarded as the predominant species in the vagina [16]. To remedy deficiencies in the Lactobacillus flora (and hence, in the protective capability of the vaginal flora), the administration of vaginal suppositories containing Lactobacilli is the most common way of Lactobacilli substitution. Some authors believe that the topical use of Lactobacilli is a safe and promising treatment for the prevention of vaginosis and recurrent urinary tract infections [17]. While vaginal supplementation is a long-standing, widely accepted practice for Lactobacilli substitution, oral administration of a Lactobacillus preparation represents a new concept for the restitution of a normal vaginal flora. Recent results indicate that the probiotic strains L. rhamnosus GR-1 (ATCC 55826) and L. reuteri RC-14 (ATCC 55845) can be taken orally on a daily basis for two months without any side effects [18]. The consumption then resulted in a significant improvement of the vaginal flora in terms of increased Lactobacilli presence and decreased yeast and coliforms. While one group of authors discussed the beneficial effects in terms of an alteration in mucosal immunity or of probiotic bacteria ascending to the vagina from the rectal area [18], another group recently demonstrated, by species-specific PCR-amplification, that L. rhamnosus GR-1 and L. reuteri RC-14 can be delivered to the vaginal environment when administered orally [19]. These preliminary findings were substantiated in a randomized, double-blind, placebo controlled study of oral Lactobacilli to improve the vaginal flora of postmenopausal women. Postmenopausal women with Nugent score between 4 and 6 (indicative of an intermediate floral quality)in initial vaginal swab were randomized in two groups. Women in the intervention group received probiotic capsules containing 2.5 x 109 cfu (colony forming units) each of lyophilized L. rhamnosus GR-1 and L. reuteri RC-14 and women in the control group received an oral placebo once daily, in both groups for 14 days. Final vaginal swabs were taken one day after the last administration of the probiotic. Twenty-one of the 35 subjects (60%) in the intervention group and 6 of the 37 subjects (16%) in the control group showed a reduction in the Nugent score by at least 2 grades (p=0.001). The median difference in Nugent score between baseline and the end of the study was 3 in the intervention and 0 in the control group (p= 0.0001)[20]. Whereas L. rhamnosus GR-1 and L. reuteri RC-14 clearly shifted the quality of the vaginal flora from intermediate to normal in 60% of women of an intervention group, one patient in the intervention group who had a Nugent score of 8 (indicative of bacterial vaginosis) at inclusion, failed to improve despite oral Lactobacillus. This might indicate that administration of Lactobacilli alone lacks therapeutic efficacy in cases of bacterial vaginosis. Indeed, therapeutic studies aimed at curing bacterial vaginosis have used the probiotic combination L. rhamnosus GR-1 and L. reuteri RC-14 as adjunct to Metronidazole or Tinidazole chemotherapy. Thus, in a randomized double-blind, placebo controlled trial, 125 premenopausal women were enrolled who had been diagnosed with bacterial vaginosis. The subjects were treated with oral Metronidazole (500 mg) twice daily from days one to seven and randomized to receive oral L. rhamnosus GR-1 and L. reuteri RC-14 (1x109 cfu each) or a placebo twice daily from day one to 30. Primary outcome was cure of bacterial vaginosis (no symptoms or sign of vaginosis) at day 30. A total of 106 subjects returned for 30 day follow up of which 88% were cured in the antibiotic - probiotic group compared to 40% in the antibiotic placebo group (p<0.001). Of the remaining subjects, 30% in the placebo group and none in the probiotic group had bacterial vaginosis, while 30% in the placebo and 12% in the probiotic group fell into the intermediate category. In summary, the study showed efficacious use of Lactobacilli and antibiotic in the eradication of bacterial vaginosis in African women [21]. In a similar study performed in Brazil, 64 women diagnosed with bacterial vaginosis were randomly assigned to receive a single dose of Tinidazole (2g) supplemented with either 2 placebo capsules or two capsules containing L. rhamnosus GR-1 and L. reuteri RC-14 every morning for the following four weeks. At the end of treatment the probiotic group had a significantly higher cure rate of bacterial vaginosis (87.5%) than the placebo group (50%) (p=0.001). In addition, according to the Nugent score, more women were assessed with "normal" vaginal microbiota in the probiotic group (75% vs. 34.4% in the placebo group; p=0.011). This study showed that probiotic Lactobacillus can provide benefits to women being treated with antibiotics for an infectious condition [22]. Together the results of clinical studies indicate that in cases of bacterial vaginosis improvement of Nugent scores with probiotics alone, without administration of an antibiotic or chemotherapeutic can not be achieved with the preparations available in the prior art. The author of the present invention has now found a new probiotic composition, comprising at least four Lactobacillus strains of human origin and capable of improving the vaginal flora. It has now been surprisingly found, that in a controlled trial, oral administration over only two weeks of a combination of Lactobacillus crispatus , Lactobacillus rhamnosus, Lactobacillus jensenii and Lactobacillus gasseri, contrary to the prior art in the absence of antibiotic, the Nugent scores of the participants were lowered from an average of 8 (indicative of bacterial vaginosis) to an average of 6 (indicative of an intermediate floral quality). In addition, the strains of the invention showed significant production of extracellular hydrogen peroxide. As hydrogen peroxide serves to kill noxious microorganisms, this is an important advantage for the composition of Lactobacilli strains of the invention. Therefore, an object of the present invention is a dietetic or pharmaceutical composition based on microbial cultures, preferably lyophilized cultures or liquid cultures such as yogurt and other fermented and/or non fermented drinks, comprising at least four Lactobacillus strains autochthonous with respect to human, said strains being selected from the group consisting of Lactobacillus crispatus LBV 88 (DSM 22566), Lactobacillus rhamnosus LBV 96 (DSM 22560), Lactobacillus jensenii LBV 116 (DSM 22567), Lactobacillus gasseri LBV 150N (DSM 22583), Lactobacillus crispatus LBV 10 (DSM 23744), Lactobacillus crispatus LBV 61 (DSM 23745), Lactobacillus jensenii LBV 8 (DSM 23746) Lactobacillus jensenii LBV 110 (DSM 23747), Lactobacillus rhamnosus LBV 69 (DSM 23748), Lactobacillus rhamnosus LBV 136 (DSM 23749), Lactobacillus gasseri LBV 162 (DSM 23750) and Lactobacillus gasseri LBV 62 (DSM 23751), together with pharmaceutically or nutritionally acceptable carriers, adjuvants and/or excipients. The twelve strains above cited have been submitted to DSMZ for patent deposit (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures). The compositions according to the present invention may comprise additional nutritional components including but not limited to at least one of the following components: vitamins, antioxidants such as preparations from pomegrenade or soybean flavonoids, fibers (inulin and fructooligosaccharides), mineral salts, phytoderivatives, milk (fermented or non-fermented milk, including yogurt). The preparation of the invention comprises Lactobacilli of human origin. These Lactobacilli represent a physiologic composition. Each of the aforementioned Lactobacillus species is present in a concentration ranging from 0.05 x 109 CFU/g to 30 x 109 CFU/g, preferably from 0.5 x 109 CFU/g to 25 x 109 CFU/g. According to a preferred embodiment of the present invention, the the composition comprises a combination of Lactobacillus strains consisting of Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583). In such preferred composition of the invention, Lactobacillus crispatus (DSM 22566) is present in a range of concentration comprised between 3 x 109 and 22 x 109 CFU/g, Lactobacillus rhamnosus (DSM 22560) is present in a range of concentration comprised between 3 x 109 and 22 x 109 CFU/g, Lactobacillus jensenii (DSM 22567) is present in a range of concentration comprised between 0,7 x 109 and 6 x 109 CFU/g, Lactobacillus gasseri (DSM 22583) is present in a range of concentration comprised between 1 x 109 and 8 x 109 CFU/g. In a more preferred embodiment of the invention, the composition of the present invention comprises Lactobacillus crispatus (DSM 22566) in a concentration of 6 x 109 CFU/g, Lactobacillus rhamnosus (DSM 22560) in a concentration of 6 x 109 CFU/g, Lactobacillus jensenii (DSM 22567) in a concentration of 1.2 x 109 CFU/g and Lactobacillus gasseri (DSM 22583) in a concentration of 1.8 x 109 CFU/g. In another more preferred embodiment of the invention, the composition of the present invention comprises Lactobacillus crispatus (DSM 22566) in a concentration of 20 x 109 CFU/g, Lactobacillus rhamnosus (DSM 22560) in a concentration of 20 x 109 CFU/g, Lactobacillus jensenii (DSM 22567) in a concentration of 4 x 109 CFU/g and Lactobacillus gasseri (DSM 22583) in a concentration of 6 x 109 CFU/g. According to a preferred embodiment, the dietetic or pharmaceutical composition of the present invention has the composition described in Tables 1 or in Table 2. Application forms of the compositions according to the inventions suitable for oral intake or topical vaginal administration are preferred. As an example, probiotic bacteria for female use can be administered orally in the form of capsules, or (filled in sachets) suspended in a drink, or in the form of fermented milk (yogurt). When administered orally, they are expected to survive passage through the stomach and duodenum (displaying a certain stability towards acid and bile) and temporarily colonize the gut. From there, small numbers of bacteria will ascend to the vagina and (again temporarily) colonize the vaginal mucosa. Probiotic bacteria can also be used conventionally as vaginal capsules or suppositories and directly applied to the vagina. The protective effect of Lactobacilli against potential pathogens in the vagina is generated through the metabolic activity of the Lactobacilli. The bacteria consume glycogen and other sources of glucose and produce lactic acid. The low pH generated in this manner is harmful to the less desirable bacteria and fungi and thus protects the vaginal mucosa against infections. Therefore, pharmaceutical compositions according to the invention may be administered in the form of suppositories, vaginal capsules for vaginal administration or as coated capsules, tablets, sachets, pills, pearls, vials for oral intake as well as yogurt, yogurt drinks, fermented milk, juices, and other fermented drinks and foods. The present invention further relates to dietetic or pharmaceutical compositions based on microbial cultures of Lactobacilli, preferably lyophilized cultures or liquid cultures such as yogurt and other fermented and/or non fermented drinks, comprising at least four Lactobacillus strains, autochthonous with respect to human, selected from the group consisting of Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567), Lactobacillus gasseri (DSM 22583), Lactobacillus crispatus (DSM 23744), Lactobacillus crispatus (DSM 23745), Lactobacillus jensenii (DSM 23746) Lactobacillus jensenii (DSM 23747), Lactobacillus rhamnosus (DSM 23748), Lactobacillus rhamnosus (DSM 23749), Lactobacillus gasseri (DSM 23750) and Lactobacillus gasseri (DSM 23751), for use in the treatment of vaginal and female urogenital infections caused by Lactobacillus deficiency, preferably selected between vaginosis or vaginitis, chronic bacterial vaginosis and chronic yeast infection, chronic urinary tract infection in menopause, atrophic vaginitis or vaginosis and similar infections as abacterial vaginosis. In fact, these Lactobacilli are for oral or vaginal administration for physiologic restoration of the vaginal flora and for a physiologic maintenance of a Lactobacillus flora in pathologic deficiency to produce Lactobacilli. In addition, the compositions according to the invention are particularly suitable for use in the treatment or prevention of asymptomatic and recurrent bacterial vaginosis in pregnancy or preterm delivery caused by bacterial vaginosis. Further embodiments of the present invention are represented by integrators, dairy products, drinks and/or food products for human nutrition characterized in that they contain a dietetic composition as above defined. Dairy products of the present invention can consist of milk, yogurt, cheese, homogenized products (based on milk, cheese, fruit), fermented or non-fermented milk (including powdered milk, non-lactose containing milk, milk shakes) containing probiotics. Therapeutic cheese can be obtained by the addition of suitable probiotic microorganisms in a concentrated dried form in a certain processing phase of the cheese in order to guarantee the supply of the dose of the microorganisms necessary for the organism. The drinks can be instantaneous drinks or water containing the compositions according to the present invention. Said integrators, dairy and food products are particularly suitable for use in the treatment or prevention of asymptomatic or recurrent bacterial vaginosis, vulvo-vaginal candidosis, preterm delivery caused by bacterial vaginosis or recurrent urinary tract infections. Finally, the invention is directed to a combination of four probiotic strains autochthonous with respect to human consisting of Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583) for medical use in the treatment of affections of the urogenital tract of women. The present invention is now described for illustrative but non-limiting purposes, according to its preferred embodiments in the following examples and enclosed Figure wherein:
The twelve preferred strains chosen have been submitted to DSMZ for patent deposit (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures). They have been selected from an initial sample of 168 colonies isolated from 84 women. An initial selection process led to 41 strains considered candidates for inclusion in a commercial product (9 strains of L. rhamnosus, 6 strains of L. jensenii, 8 strains of L. crispatus and 18 strains of L. gasseri). In a second selection step, 12 strains were selected, as follows: Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583), Lactobacillus crispatus (DSM 23744), Lactobacillus crispatus (DSM 23745), Lactobacillus jensenii (DSM 23746) Lactobacillus jensenii (DSM 23747), Lactobacillus rhamnosus (DSM 23748), Lactobacillus rhamnosus (DSM 23749), Lactobacillus gasseri (DSM 23750), Lactobacillus gasseri (DSM 23751), together with pharmaceutically or nutritionally acceptable adjuvants and/or excipients. The sample cultures transmitted by DSMZ for back-checking have been re-typed and found in agreement with the initially transmitted cultures. The compositions according to the invention will be administered in capsule form. The first step consists in the preparation of the lyophilized cultures of the probiotic strains intended to be used (in a preferred embodiment of the invention, the four strains Lactobacillus rhamnosus LBV 96 (DSM 22560), Lactobacillus crispatus (DSM 22566), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583)). The common steps followed in the manufacture of the lyophilized culture are: activation of the strain (laboratory sub-cultivations), industrial fermentation, separation of the bacterial biomass, generally by centrifugation or nanofiltration, washing of the biomass, cryoprotection of the biomass, freeze-drying, grinding of the "cake" and standardization of the number of viable cells per gram. After the manufacture of the strains, a pilot or industrial mixing of probiotics with the other excipients to be used follows (if necessary, proper pre-mixes of minority components could be performed). After the manufacture of this final blend, the capsules are filled with a proper laboratory or industrial capsule filler. The machine could be manual, semiautomatic or completely automatic. Such process is well-known to a person skilled in the art. The active formulation used in the clinical study, i.e. the composition 1, is depicted in the following Table 3: The corresponding product formulation (considering a 5 times overdosage), i.e. composition 2, is detailed in the following Table 4: A stability assessment of the four strains Lactobacillus jensenii LbV 116 (DSM 22567), Lactobacillus rhamnosus LbV 96 (DSM 22560), Lactobacillus gasseri LbV 150N (DSM 22583) and Lactobacillus crispatus LbV 88 (DSM 22566) in standard, pasteurized fresh milk has been made. The daily dose of milk has been considered as 167 ml (approximately the content of a glass). The aim is to guarantee 2.5 billion of viable cells at the end of shelf-life. The formulation proposal has been defined according to the product which is currently being clinically tested at University Hospital of Vienna. The overage considered for our first evaluation was 5 times. In the following Table 5 the detailed formulation is shown: In the following Table 6 preliminary stability data of the four strains in milk stored at 4°C are reported: The mean shelf-life of an industrial, pasteurized fresh milk is 6 days. In any case, we decided to assess stability up to 15 days in order to gain a more complete overview of the behavior of bacterial cells in milk. As can be inferred from the above data, the probiotics of the invention are very stable in fresh industrial milk. The data suggest that the project is feasible with only a little overage at manufacturing time. Another aspect to be considered is that pH remained substantially unchanged even after 15 days storage at 4°C. This means that probiotics are not metabolically active in these conditions, which is a certainly desirable aspect. Molecular typing of the selected strains in comparison with a range of competitor strains was performed according to several methods (RAPD-PCR, GTG5 REP-PCR, PFGE). The following competitor strains available on the market were examined.
The selected strains were distinguishable from all competitor strains examined by RAPD-PCR and Primer 4. PFGE following restriction with ApaI or SmaI also showed differences between the strains examined (data not shown). Tests to explain the antimicrobial mechanism of action were carried out using two strains per target species (Lactobacillus crispatus: LBV 10 (DSM 23744), LBV 88 (DSM 22566); Lactobacillus gasseri: LBV 162 (DSM 23750), LBV 150N (DSM 22583); Lactobacillus jensenii: LBV 110 (DSM23747), LBV 8 (DSM 23746); Lactobacillus rhamnosus: LBV 69 (DSM 23748), LBV 96 (DSM 22560)) on three pathogenic vaginal strains (Gardnerella vaginalis Ga 3, Candida albicans Cd 30 and Candida glabrata Cd 34- see Table 5) using a smear-spotting method. Here the antimicrobial effect of the live active Lactobacilli strains was tested against live active pathogenic strains. Parallel smears of the Lactobacilli strains are first placed on an agar plate at a separation of about 2 cm. When antimicrobial substances are formed and released by the Lactobacilli strains, these diffuse into the growth medium and form an antimicrobial inhibitory strip into which the live pathogenic strains are spotted. At the same time spotting of the same pathogenic strains is carried out outside the parallel smear as a growth control. No growth or poor growth of the pathogenic strains within the smear compared to the growth control outside the smear indicates the formation of antimicrobial substances by the Lactobacilli being tested [23]. A requirement for the smear-spotting method is a method of cultivation in which both the Lactobacilli and the pathogenic strains demonstrate good growth, which proved to be a problem mainly in the cultivation of Gardnerella vaginalis. In the course of the tests several methods of cultivating Gardnerella vaginalis were tested where neutralised MRS starch agar (pH 6.7, 1% starch) proved to be the best growth medium.
The detailed results are depicted in
Mutual negative interactions on growth using a spotting-coating method for most combinations of the 12 selected strains have been investigated.
The strength of the mutual inhibition was assessed visually on a scale between - (no inhibition) and +++ (very strong inhibition) as shown in the following Table 9. Large differences were established in the inhibitory effects of the 12 strains against each other. The three Lactobacillus crispatus strains demonstrated either no inhibitory effect or only a weak inhibitory effect on the strains of the other species. The strains themselves were however considerably inhibited by all of the other strains. The Lactobacillus jensenii and Lactobacillus gasseri strains mainly inhibited the Lactobacillus crispatus strains while the inhibitory effect towards the other strains was rather weak. The strongest inhibitory effect was established for the Lactobacillus rhamnosus strains. These very strongly inhibited the three Lactobacillus crispatus strains and strongly inhibited the representatives of Lactobacillus jensenii and Lb gasseri. Each strain also showed a strong inhibitory effect towards both of the other Lactobacillus rhamnosus strains. Mutual interactions are minimal for the preferred combination of Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583). Under the latter conditions, only the strain Lactobacillus crispatus DSM 22566 is strongly inhibited by Lactobacillus rhamnosus DSM 22560. In a unique pilot study the human Lactobacilli composition (cultivation out of human Lactobacilli) was tested in vivo. These human Lactobacilli were isolated out of a study with pregnant women. Lactobacilli in pregnancies play an important role to protect women from vaginal infections in order to protect the fetus maintaining a healthy flora in pregnancy. Vaginal infection in pregnancy is associated with preterm delivery, preterm labour and poor pregnancy outcome. To date data of a study with 9 women (neovagina, no communication with the abdominal cavity, no uterus) are available; Lactobacilli (Composition 1, Table 1) were orally administered, vaginal samples were taken before oral administration, then one week after oral daily intake and third vaginal samples after one week without probiotic treatment were taken to see a long term effect. The ability of the orally administered preparation of four probiotic lactobacillus strains according to the invention [Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583)] to improve the quality of the flora of neovagina was investigated in a placebo-controlled, double-blind trial (n=70). To date 31 women completed trial. The protocol treatment was the following:
Lactobacilli strains of the prior art :
in the following named "Ellen" strains. Lactobacilli strains of the invention:
The following analysis were carried out on "Ellen" strains in comparison with the strains of the invention.
All strains were successfully grown in MRS broth from cryocultures stored at -80°C. All cultures used for investigation were not more than 24 h old. Assessment of the intensity of growth in aerobic or anaerobic culture is an important criterion for the selection of strains. Assessment of growth was on the basis of fractionated smear cultures of the strains on MRS-agar plates, incubated 48h at 37°C under aerobic or anaerobic conditions. Colony diameters were optically evaluated (+++, ++, +). Agar plates were documented with the aid of digital photographs. Results on the basis of two analyses are shown in the following Table 10: Both Lactobacillus rhamnosus strains (ELLEN LN 113-2 and L. rhamnosus DSM22560) grow well. Under aerobic conditions, there is slightly better growth of the L. gasseri strain DSM 22583 versus the ELLEN L. gasseri strain LN 40. The pH lowering capabilities of the seven strains were determined following incubation under anaerobic conditions for 48h at 37°C in 10 ml MRS-broth (starting pH = 5.8). The pH-values resulting in the broths were determined in triplicate using a standard pH-meter. Resulting capability to lower pH (arithmetic mean of three measurements) are shown in the following Table 11. While the ELLEN strain L. gasseri LN 40 is capable of lowering the pH below the corresponding level achieved by L. gasseri DSM 22583 (pH = 3.73 vs. pH 4.15), the L. rhamnosus strains showed an inverse result (LN 113-2: pH = 3.82 vs. DSM 22560 pH = 3.66). The most pronounced lowering of pH value is achieved by the L. crispatus strain DSM22566 (pH = 3.64). Stability under acid stress of 3 hours duration of the Lactobacillus strains was determined in duplicate in MRS broth adjusted to pH values of 2, 2.5, 3, 3.5 and 4 with 1 M hydrochloric acid. From a starter culture in MRS broth, 9.9 ml portions of the acidified broths were inoculated with 100 µl and incubated at 37° C for 3 h under anaerobic conditions. The determination of the starting bacterial count (present in each inoculum) was performed in parallel using the plate count method (in duplicate, 37°C, 48 h, anaerobic conditions). Following 3 hours incubation, the plate counts of the cultures incubated at different pH values were determined using a similar procedure. Stability was calculated in relation to the respective starting plate count. Results are shown in the following Table 12 as arithmetic mean of two independent investigations. L. gasseri DSM 22583 showed the highest stability under acid stress. The plate count of this strain did not decrease but even increased at a pH value as low as 2.0. In contrast with ELLEN strain L. rhamnosus LN 113-2, L. rhamnosus DSM 22560 showed increasing plate counts at pH values of 2.5, 3.0, 3.5 and 4.0, showing decreased counts only at pH = 2. Lowering of pH is an important mechanism of action by which lactobacilli kill undesired microorganisms. Concerning stability in gastric juice and growth under acidic conditions the Lactobacilli strains of the invention show clear advantages. Stability toward bile salts was investigated using fractional smears of the 7 strains on MRS agar plates with different concentrations of bile salts (0%; 0.1%; 0.2%; 0.3%; 0.4% und 0.5% (B8756-100G, Bile Salts, Sigma). Following anaerobic incubation at 37°C for 48h, the amount of growth was visually assessed: very good (+++), good (++), slight (+), no growth (-). The following Table 13 shows the results of stability toward bile salts (mean of duplicate determinations). All the strains of the invention showed better tolerance of bile salts (0.2 %). Higher concentration of bile salts in the duodenum result in the death of many bacteria. The strains of the invention show clear advantages here, important especially for oral application. Two methods for determination of H2O2 were selected and applied.
While none of the ELLEN strains produced measurable hydrogen peroxide, the strain L. rhamnosus LBV 96 (DSM22560) showed slight production and the other strains of the invention showed significant production of extracellular hydrogen peroxide (see Tables 14 and 15). As hydrogen peroxide serves to kill noxious microorganisms, this is an important advantage for the composition of Lactobacilli strains of the invention.
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