Field of Art

The present invention relates to new strains of lactobacilli producing substances effective against vaginal pathogens, use of these new strains, and sanitary products containing these strains of lactobacilli.

Background Art

Probiotic bacteria are used for treatment and prevention of infections of urogenital tract. Due to changing lifestyle, the number of these infections is growing, especially in young women. The treatment is lengthy, there is a number of means used for treatment, among which the probiotic bacteria are becoming more and more important thanks to minimum side effects. Bacterial infections are mostly caused by Escherichia coli and Staphylococcus aureus, while a growing number of fungal diseases identified as candidiases is caused by massive infection by Candida albicans. An extensive study (over 30 years) published in 2013 has found a significantly higher occurrence of cancer in women suffering from candidiases. Probiotic strains of Lactic Acid Bacteria (LAB) used in the past were efficient mainly against the above-mentioned bacterial strains (for example in CZ 302683, describing probiotic strains of lactobacilli effective against E. coli and S. aureus, however less effective against C. albicans). There are not many probiotic LABs having a comparable effect both against the bacteria and Candida.

Significant is a method of application of these LABs for the prevention of infections of urogenital tract using common ladies sanitary products (tampons, sanitary napkins). Sanitary products containing these probiotic bacteria are known for example from EP 0594628 disclosing sanitary products containing bacteria of the genus Pediococcus, US 2004/0241151 disclosing a polymeric matrix containing the bacteria producing lactic acid, US 6359191, US 7960604, WO 02/28446, and WO 03/038068 disclosing sanitary products containing the bacteria producing lactic acid. Recent patent documents describing the application of probiotic lactobacilli for sanitary sanitary products are EP 1824500 Bl. US 2013/0171253, and WO 2013/093941.

Solutions known in the art use LABs producing lactic acid as the active substance, i.e. acting against bacterial pathogens by lowering the pH, however they are less effective against Candida that are acid-tolerant. Newly isolated LABs that are the object of the present invention produce various ranges of antimicrobial substances, and therefore it is suitable for the application to sanitary products to combine these strains with different effects.

Disclosure of the Invention

The object of the present invention is the strain Lactobacillus fermentwn CCM 8523, producing substances effective against vaginal pathogens. Besides lactic acid, hydrogen peroxide, and protein bacterial toxins, this strain produces a further thermo-Iabile non- proteinous agent excreted to supernatant which is effective against vaginal pathogens (refer below to Example 5, Table VII) - this agent is not deactivated by neutralization, or by the effect of catalase or by proteinase. It is deactivated only by heating to 90 °C. The presence of this agent is unexpected and it allows to inhibit also the pathogens resistant to common active substances of lactobacilli.

The strain Lactobacillus fermentum CCM 8523 can be applied alone or in combination with at least one new strain of lactobacilli producing substances effective against vaginal pathogens, selected from the group comprising:

-Lactobacillus crispatus CCM 7997

-Lactobacillus reuteri CCM 7998

-Lactobacillus crispatus CCM 8522.

All strains specified hereinabove have been deposited in Czech Collection of Microorganisms (CCM) in Brno; the above-presented numbers are the depositary numbers in CCM.

The new strains of lactobacilli identified within the framework of the present invention show combined high antibacterial and antifungal (bactericidal and candidacidal) activity suitable for use for the prevention and treatment of urogenital infections, particularly chronical, for restoration of the natural vaginal microflora, and for elimination of discomfort of women in pre-menstrual and menstrual period. The strains have been isolated from vaginal microflora of healthy women of diversified ethnic origin, thus allowing application in widely diversified population.

Long-term stability of the strains has been proven in lyophilized samples stored separately and bound on a carrier which allows for their formulation into sanitary products. At the same time, the isolates of lactobacilli manifest good auto-aggregation characteristics, allowing for subsequent colonization of urogenital tract.

The present invention thus provides the new strain of Lactobacillus ferment m CCM 8523, optionally in combination with at least one other strain of lactobacillus, for use as a medicament, in particular as a medicament for the prevention and/or treatment of chronic urogenital infections, restoration of natural vaginal microflora, and/or elimination of discomfort of women in pre-menstrual and menstrual period. Furthermore, the present invention provides a medicament containing Lactobacillus fermentum CCM 8523, optionally in combination with at least one other strain of lactobacillus according to the present invention.

The object of the present invention are also sanitary products, such as sanitary napkins or tampons, containing the strain Lactobacillus fermentum CCM 8523, optionally in combination with at least one other strain of the lactobacillus according to the invention; and also use of the strain Lactobacillus fermentum CCM 8523, optionally in combination with at least one other strain of lactobacillus, for the preparation of sanitary products for the prevention or treatment of chronic urogenital infections, restoration of natural vaginal microflora and/or for the elimination of discomfort of women in pre-menstrual and menstrual period. Methods for incorporation of microorganisms into sanitary products are known in the art, for example from the documents cited in the section describing the background art.

In a preferred embodiment, the lactobacilli, when used according to the present invention or in the sanitary products according to the present invention, are in lyophilized form. However, they may be also in another suitable form, for example prepared using spray drying.

Brief description of Drawings

Fig. 1 shows auto-aggregation of fresh strains of Lactobacillus sp., at the temperature of 37 °C ₅ for the period of 24 h, under aerobic conditions (Example 4). Lbc. crispatus CCM 8522, Lbcfermentum CCM 8523, Lbc. crispatus CCM 7997, Lbc. reuteri CCM 7998. Fig. 2 shows auto-aggregation of lyophilized strains of Lactobacillus sp., at the temperature of 37 °C, for the period of 24 h, under aerobic conditions (Example 4). Lbc. crispatus CCM 8522, Lbc fermentum CCM 8523, Lbc. crispatus CCM 7997, Lbc. reuteri CCM 7998.

Fig. 3 shows viability of selected lyophilized lactobacilli at the temperature of 5 °C (Example 6). 8522 - Lbc. crispatus CCM 8522, 7998 - Lbc. reuteri CCM 7998, 8523 - Lbc. fermentum CCM 8523 , 7997 - Lbc. crispatus CCM 7997.

Examples of carrying out the Invention

Example 1: Isolation and characterization of strains

New strains have been isolated from vaginal swab of healthy women of diversified ethnic origin.

Newly isolated strains have been standardized and identified using three methods:

1. Phenotyping of isolates based on their biochemical activities

2. Genotyping of isolates based on rep-PCR and detection of the gene coding 16S rRNA using PCR with the use of species specific primers for Lactobacillus sp

3. Exact genotyping has been performed using the amplification of the gene coding 16S rRNA with the use of species specific primers and subsequent sequencing

Table I:

Phenotyping of lactobacilli isolates determined using the test API 50 CH

Isolate CCM 8523 CCM 7997 CCM 8522 CCM 7998

+ pos tive reaction; - negat ve react on; Rib - ribose; Glu - glucose; Ng - N-acetyl glucosamine; cellobiose; Fru - fructose, Gal - galactose; Lac - lactose; Mai - maltose; Sac - saccharose; Mel - melibiose; Raf - rafinose; Gd - potassium gluconate; Ara - arabinose; Esc - esculin; Xyl - xylose; Tre - trehalose; Tur - turanose; Sal - salicin; Amd - amidone; Mdx - methyl-p,D-xylopyranoside; Mdg - methyl-a,D- glukopyranoside (all saccharides are in the form of D-isomer, exception: Lara - L-arabinose)

Table II Sequence of 16S rRNA of the tested strains. Isolates of the genus Lactobacillus have been identified on the the level of species using sequencing of the 16S rRNA gene (generic primers W001 and W002).

Sequence (5 '- 3') Identification

(BLAST- NCBI), depositary number

GTTACCCC¾CC6ACrrTGGGTGTTACAAACT rCATGGTGTGACGGGCGeTGTGTACAAGGCCCGGGAACGTATTCA

CCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCA GCCTGCAGTCCGAACTGAGA Lbc. fermentum ACGG I 1 1 lAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTGTACCGTCCATTGTAGCACGTGT GTAGCCCAGGT CCM 8523

CATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCA GTCrCACTAGAGTGCCCAACT

TAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT CACGACACGAGCTGACGACG ACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCA AGATGTCAAGACCTGGT AAGGTTCTTCGCGTAGCTTCGMTTAAACCACATGCTCCACCGCTTGTG

CCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCrCCGGCACTGAAGG GCGGAAACCCTCCMCACCT

AGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCn TCGAGTCTCAGCGTCAG

TTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTC ATATATCTACGCATTCCACCGCTACACATGGAGTTCCAC

TACCCTCrrCTGCACTCAAGTTATCCAGTTTCCGATGCACrr TCCGGTTAAGCCGAAGGCTTTCACATCAGAC^

AAACCGCaGCACTCTCTTTACGCCCAATAAATCCGGATMCGCFTGCCACCTACGTATTAC CGCGGCTGCTGGCACG

TAGTTAGCCGTGACTTTCTGGTTAMTACCGTCAACGTATGMCAGTTACTCT

GCTTrACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCWTCAGGCrrGCGCC CATTGTGG GATTCCCTACT

GCTGCCTCCC6TAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCA ACTCGGCTATGCATCATC

GCCTTGGTAGGCCGTTACCCCACCAAC GCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCGAGAAGCCATCrr

TTMGCGnGTTCATGCGAACAACGTrGmTGCGGTATOGCATCTGmCCAMTGnGTCCCCCGC rrCTGGGCAG

GTTACCTACGTGTTACTCACCCGTCCGCCACTCGTTGGCGACCAAAATCAATCAGGTGCA AGCACCATCAATCAATTG

GGCCAACGCGTCGACTGC

GGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTA CMGGCCCGGGAACGTATT

CACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTC AGA rGCAGTCCGAACTGAG Lbc. crispatus

MCAGCTnCAGAGATrCG TTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAG CCM 7997

GTCAT GGGGCATGATGACTTGACGTCATCCCCACC rCCTCCGGTTTGTCACCGGCAGTCT ATTAGAGTGCCCAA

CTT TGCTGGCAACTAATMCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAA ATCrCACGACACGAGCrGACGA

CAGCCATGCACC^CCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCA CrAGATGTCAAGACCTGG

TMGGTTmCGCGTTGmCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT TCCmGAGTTTCA

ΑθατβοΰατοστΑα^εοΑσααοσΑατΰατΑ� �ταοΰπΑβαοοΑσοΑ σΑσΑΰοαασΑΑΑο αοΑΑθΑ

TAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTMTCCTGTrCGCTACCCATGCT rrCGAGC CAGCGTCA

GTTGCAGACCAGAGAGCCGCCTTCGCCA TGGTGTT TTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCC ACT c mCTGCACTCAAGAAAAACAGmCCGATGCAGTTCCrCGGTTMGCCGAGGGCmCACATCAGA CTTA

TTCTTCCGCCTGCGCTCGCTITACGCCCAATAAATCCGGACAACGCTTGCCACCTAC GTATTACCGCGGCTGCTGGCAC GTAGTTAGCCGTGACrrTCTGGTTGATTACCGTCAAATAMGGCCAGTTACTACCT rTTCL 1 1 CH CACCAACAACA6 AGCTTTACGATCCGAAMCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCA TTGTGGAAGATTCCCTAC

TGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCT CTCAACTCGGCTATGCATCAT

CGCCTTGGTAAGCCTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATA GCGACAGCTTACGCCGCCTTT

ΤΑΑΑΑ6 6ΑΤεΑΤ6α6ΑΤ 607ΤΤ ΤΑΤ€066ΤΑ Α60Ααα6ΤΓΓ€{_ΑΑατε5ΤΑΤθαεΑ6ΑΟΤΑΤ66 660Α6

GTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACC6 GTAAATCTGTTAGTCCGCTCGCT

CG

GGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTA CAAGGCCCGGGAACGTATT

CACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTG CAGACTGCAGTCCGAACTGA Lbc. crispatus

GMCAGCTTTCAGAGATCCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCAT TGTAGCACGTGTGTAGCCCA CCM 8522

GGTCATMGGGGCATGATGACnGACGTCATCCCCACOTCCTCCGGTTTGTCACCGGCA GTCTCATTAGAGTGCCCA

ACTTMTGCTGGCAACTAATMCAAGGGTTGCGC CGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACG

ACAGCCATGCACCACCTGTCTTAGCGTCCCCG GGG CTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTG

GTAAG 1 1 CTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGG GTGCGGGCCCCCGTCAATTCCTTTGAGTTTC

AACCTrGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGG CGGAAACCTCCCAACAC

TTAGCACTCATCGTTTACGGCATGGA rACCAGGGTATC TCCTGTTCACTACCCATGCrrTCGAGC rCAGCGTCA

GTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCAC CGCTACACATGGAGTTCC

A ac C FCTGCACTCMGAAA CAGmcCGATGCAGnc CGGn GCCGAGGGCmCACATCAGACnA

TTCTTCCGCCRGCGCTCGCTTTACGCCCMTAAATCCGGACAACGCTTGCCACCTACGTAT TACCGCGGCTGCTGGCAC

GTAGTTAGCCGTGAC1TTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTC TATC I I ^' CI T CACCAACAACAG

AGCTTTACGATCCGAAMCCTTCT CACTCACGCGGCGTTGCTCCATCAGACRRGCGTCCATTGTGGAAGATRCCCTA^

TGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTC AACTCGGCTATGCATCAT

CGCCTTGGTMGCCRRRACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAG CGACAGCTTACGCCGCCTTT

TAAAAGCTGATCATGCGATCTGCVUCTOTCCGGTATOGCACCTGTTTCCAAGTGGTA TCCCAGACTATGGGGCAG

GTTCCCCACGTGTTACTCACC ATCCGCCGCTCGCTTTCCTMCGTCATTACCGAAGTA TCTGTTAGTTCCGCTCGC

TCGACTGCA

TAAAGGTTAGGCCCCGACTTTGGGCGTTACAAACTCCCATGGTGTGACGGGCGGTGT GTACAAGGCCCGGGAACGT

ATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAG TTGCAGCCTACAGTCCGAACT Lbc. reuteri

GAGAACGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTTGCGACRCGTTGTACCGTC CATTGTAGCACGTGTGTAGCC CCM 7998

CAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCAC CGGCAGT TCACTAGAGTGCC

CAACRT TGCTGGCAACTAGT CAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGA

CGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTATCT AAGGTTAGCGCAAGATGTCAAGAC

CTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGG GCCCCCGTC AATTCCTTTGAG

TTrCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCAC TGAAGGGCGGAAACCCTCCA

ACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACC CATGCTTrCGAGCCTCAGC

GTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATT CCACCGCTACACATGGAG

TTCCACTG7CCTCTTCTGCACTCAAGTCGCCCGGTTTCCGATGCACTTGTCGGTTAA GCCGAAGGCTTTCACATCAGA

CCTAAGOWCCGCCTGCGCTCGCTTTACGCCCAATAMTCCGGATAACGCTTGCCACCTACG TATTACCGCGGCTGCT

GGCACGTAGTTAGCCGTGACTTTCTGGTTGGATACCGTCACTGCGTGAACAGTTACT CTCACGCACGTTCTTCTCCAA

C CAGAGCTTTACGAGCCGAMCCCTTCTTCACTCACGCGGTGTTGGt!CATCAGGCrrGCGC CCATTGTGGAAGAT

TCCCTA TGCTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTC AACTCGGGrAT

GCATCATCGCCrrGGTAAGCCGTTACCrrACCAACTAG TAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGC

CATCTTTCAAACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCAT TGTTTCCAAATGTTATCCCCCGCT GGGCA6GTTACCTACGTGTTACTCACCC6TCCGCCACTCACTGGTGATCCATCGTCAATC AGGTGCAAGCACCATCAA

TCAGTTGGGCCAGTGCGTACGACTGC

Identification of the isolated strains with anti-microbial activity has been performed using the method 16S rRNA and it is shown in Table III.

Table III. Identification and standardization of isolated strains

Based on Gram staining, it has been found that all strains are gram-positive. The strains have been deposited in the Czech Collection of Microorganisms (CCM) in Brno.

Example 2: Determination of anti-candida and anti-staphylococcus activity

The isolated strains were tested for anti-candida activity, which was determined as follows: The tested strain Lactobacillus spp. was first cultivated in the MRS growth broth (Merck Milipore) for the period of 24 h in anaerobic conditions at the temperature of 37 °C. Inoculum with the concentration corresponding to 2 McF (McFarland) was prepared from the freshly grown culture using saline solution. 1 ml of this inoculum was poured over with 15 ml of MRS Agar and cultivated at 37 °C in anaerobic conditions for the period of 24 h. Subsequently, Petri dishes were overlaid with 10 ml of ME Agar (Merck Milipore), on the surface of which 100 μΐ of the suspension of C. albicans 2186 with the concentration of 0.5 McF was spreaded. Anaerobic cultivation followed, first at 37 °C for the period of 24 h, and further at the room temperature (25 °C) for the period of 24 h. For each sample, a blind sample was prepared, which was prepared from 15 ml of MRS agar overlaid with 10 ml of ME agar with the spread of 100 μΐ of the suspension of C. albicans 2186 with the concentration of 0,5 McF. The blind sample was cultivated under the same conditions as the other samples. Evaluation of the inhibition of C. albicans 2186 by the individual strains of lactobacilh was evaluated according to the ratio of growth of the yeast on the surface of the dish as compared to the blind sample.

Further, the isolated strains were tested to anti-staphylococcus activity, which was determined using the puncture method as follows: melted agar soft BHI was inoculated with 1 % (vol.) of inoculum of freshly grown indicator strain of S. aureus CCM 4516 with the density of approximately 10 ⁵ KTJ ml ^"1 , then distributed in 10 mi portions to Petri dishes. After solidifying and pre-drying, 10 μΐ of living cells of the tested strain Lactobacillus spp. or its adjusted supernatant was dosed by injection into the agar. The dishes were subsequently covered with petrifilm and cultivated at the temperature 37 °C for 18 h. Anti-staphylococcus activity was evaluated according to the diameter of growth inhibition zones formed around the point of injection of the tested strain to agar with the inoculated indicator strain.

Anti-candida and anti-staphylococcus activity was evaluated as follows:

+ ... inhibition zone < 5 mm (low activity)

++ ... inhibition zone 5 to 10 mm (medium activity)

+++ ... inhibition zone > 10 mm (high activity)

The results of the anti-staphylococcus activity of lactobacilli are shown in Table IV and they are expressed as average values of three measurements.

Table IV. Anti-staphylococcus and anti-candida activity of strains

Example 3: Effect of lyophilisation on anti-staphylococcus activity of lactobacilli Anti-staphylococcus activity of lactobacilli was tested using the agar injection method (see Example 2). Selected vaginal isolates of lactobacilli were used as tested microorganisms, in the form of fresh and lyophilized cultures. In case of lyophilized cultures, the anti-staphylococcus activity of lactobacilli was tested after re-hydration of the lyophilisate (0.1 g) in saline solution (1 ml) and after re-cultivation of the lyophilized culture (0.1 g) in the MRS broth (pH 5.6 before sterilization, 5 ml) at the temperature of 37 °C, for the period of 24 h, under aerobic conditions. Testing of lyophilized cultures was performed immediately after lyophilization and subsequently after storage of thus prepared cultures at the temperature of 25 °C for the period of 1 month. Staphylococcus aureus CCM 4516 was selected as indicator strain for testing the anti-staphylococcus activity of lactobacilli. Anti-staphylococcus activity was evaluated according to the diameter of inhibition zones formed around the point of injection of the tested strain to agar with the inoculated indicator strain as follows:

+ ... inhibition zone < 5 mm (low activity)

++ ... inhibition zone 5 to 10 mm (medium activity)

+++ ... inhibition zone > 10 mm (high activity)

The results of the anti-staphylococcus activity of lactobacilli are shown in Tables V and VI and they are expressed as average values of three measurements.

Table V. Anti-staphylococcus activity of vaginal isolates of Lactobacillus sp. (measured immediately after lyophilization)

* after re-hydration of lyophilisates in saline solution (1 ml), ** after re-cultivation of lyophilisates in broth MRS (pH 5.6 before sterilization) the temperature of 37 °C for the period of 24 h, under aerobic conditions. + ... inhibition zone < 5 mm (low activity), ++ ... inhibition zone 5 thru 10 mm (medium activity), +++ ... inhibition zone > 10 mm (high activity). Table VI. Anti-staphylococcus activity of vaginal isolates of Lactobacillus (measured after storage of lyophilisate at 25 °C for the period of 1 month)

* After re-hydration of lyophilisates in saline solution (1 ml), ** after re-cultivation of lyophilisates in broth MRS (pH 5.6 before sterilization) at the temperature of 37 °C for the period of 24 h, under aerobic conditions, + ... inhibition zone < 5 mm (low activity), inhibition zone 5 thru 10 mm (medium activity), +++ ... inhibition zone > 10 mm (high activity)

Example 4: Auto-aggregation of lactobacilli

Auto-aggregation of lactobacilli was tested in 4 selected vaginal isolates of lactobacilli. For testing auto-aggregation of lactobacilli, both fresh cultures of these bacteria and cultures after lyophilization were used. Auto-aggregation of fresh cells of lactobacilli was tested in the MRS broth (pH 5.6 before sterilization, 10 ml), at the temperature of 37 °C, for the period of 24 h. For testing auto-aggregation, a sample (1 ml) was taken at time points 0, 2, 4, 7, and 24 h from the surface of the suspension, and the absorbance was measured in said sample at the wavelength of 600 nm. Percentage of auto-aggregation of lactobacilli was calculated accordin the following equation:

Autoaggregation = loo [%]

. absorbance at the time 0 h [-]

absorbance at the time 2, 4, 7 and 24 h [-]

Auto-aggregation of lyophilized cells of lactobacilli was measured according to the same methodology as for the fresh cells. However, the lyophilized strains had to be re-cultivated prior to measurement in the MRS broth (pH 5.6 before sterilization, 5 ml) at the temperature of 37 °C, for the period of 24 h, under aerobic conditions and subsequently once more re-inoculated (1 % vol. of the culture) into the MRS broth (pH 56 before sterilization, 5 ml) and cultivated under the same conditions.

The results of auto-aggregation are summarized in Figure 1 and 2 and they are expressed as arithmetic averages of three parallel experiments.

From the results shown in Figure 1, it is evident that the strains Lbc. crispatus CCM 7997 and Lbc. reuteri CCM 7998 reached 100% auto-aggregation after 24 h of measurement. For the strain Lbc. crispatus CCM 8522, the auto-aggregation found after 24 h of measurement was at least 50 % and for the strain Lbc. fermentum CCM 8523 34 %.

Fig. 2 shows the effect of lyophiiization process on the ability of auto-aggregation of lactobacilli. For most tested strains, the same or even increased ability of auto-aggregation was found. The strains Lbc. crispatus CCM 7997 and Lbc. reuteri CCM 7998 reached, as in the previous case, 100% auto- aggregation after 24 h of measurement. In vaginal isolates Lbc. crispatus CCM 8522 and Lbc. fermentum CCM 8523 then, the ability of auto- aggregation was increased by up to 40 % in 24 h of measurement.

Example 5: Determination of characteristics of the active antimicrobial substance

To characterize the antimicrobial active substances produced by the lactobacilli, the supematants obtained by centrifugation (9000 rpm, 5 min, 4 °C) of each tested strain were used. Supematants were further neutralized using 10% (wt.) NaOH solution, or 10% (vol.) HC1 solution to pH 7, to neutralize the acid activity. To determine thermal stability of the antimicrobial substance, the neutralized supematants (pH 7) were kept in water bath at the temperature of 90 °C for the period of 10 min. To eliminate the activity of hydrogen peroxide, the neutralized supematants (pH 7) were treated with catalase. To eliminate the activity of proteinous antimicrobial substance (bacteriocine), the neutralized supematants (pH 7) were treated by proteinase K. The enzymes were always prepared by dissolving in sterile demineralized water and subsequently added to supernatant to the final concentration of 1 mg ml ^"1 . Supematants with the enzyme applied were incubated at the temperature of 37 °C for the period of 2 h. The treated supematants were tested using the agar injection method. The results of the analysis are summarized in Table VII. To determine the production of hydrogen peroxide, MRS was inoculated with 1 % (vol.) of inoculum of the tested strain and cultivated in aerobic conditions at the temperature of 37 °C for 24 h. After this period of time, the suspension of cells was centrifuged (5000 rpm, 15 min, 4 °C). The supernatant obtained was decanted and the cells were twice washed with phosphate buffer and centrifuged again. After the last centrifugation, chilled phosphate buffer (20 ml) was added to the cells. The resulting cell suspension was cultivated at the temperature of 5 °C for 48 h. After cultivation, the cell suspension was centrifuged (5000 rpm, 1 min, 4 °C), and the resulting supernatant was used to determine H ₂ 0 ₂ .

The samples were prepared by adding 0,001% (wt.) of aqueous solution of HRP (1 ml) and 1% (wt.) of methanol solution of o-dianisidine (0,1 ml) to 5 ml supernatant/H^C^ solution (calibration curve). The thus modified samples were cultivated at the temperature of 37 °C for the period of 10 min. Subsequently, the reaction was stopped by adding 4 mol Γ ¹ of HCI (0,2 mi). The absorbance of samples was measured at 400 nm (A400). The results of the analysis are summarized in Table VII.

For the interpretation of the values measured using this method, it was necessary to make the calibration lines for Η ₂ 0 ₂ . First calibration solutions of H ₂ 0 ₂ in phosphate buffer were prepared with the following H ₂ 0 ₂ concentrations (0,5; 0,8; 1,0; 2,0; 3,0 a 4,0 mg I ^"1 of phosphate buffer). The calibration line was expressed as the relation of absorbance vs. concentration of H ₂ 0 ₂ .

To determine the production of acids, the tested strains were cultivated for 24 h at the temperature of 37 °C in MRS broth pH 5.4 after sterilization (inoculated 1% inoculum). Then the pH of the bacterial suspension was determined. The results of the analysis are summarized in Table VII.

Table VTI. Characterization of anti-microbial substances at the identified isolates of lacto bacilli

Isolate Lbc. Lbc. Lbc. Lbc.

crispatus reuteri fermentum crispatus

CCM 8522 CCM 7998 CCM 8523 CCM 7997

LC 8.3 ± 0.5 11.7 ± 0.9 15.0 ± 0.8 10.0 ± 0.8

S 0 5.3 ± 0.5 12.0 ± 0.0 7.0 ± 0.0

SN7 0 3.7 ± 0.9 10.5 ± 0.5 0

SNI7 0 0 0 0 SNP7 0 0 10.0 ± 1.0 0

SNK7 0 4.3 ± 0.9 11.0 ± 1.0 0

C _H 202 [mg. 'j 5.87 3.60 0.08 3.35

CH20I [mg. ¹ ] produced by one cell * 10 ¹² 1190 690 3.17 86.6

Production of acids [pH after 12 h of 4.27 4.10 4.11 4.16

cultivation]

Key: LC - living cells; S - supernatant, not modified; SN7 - supernatant, neutralized to pH 7; SNI7 - supernatant, neutralized to pH 7 and heat treated by heating at the temperature of 90 °C for the period of 10 min; SNP7 - supernatant, neutralized to pH 7 and treated with proteinase K; SNK7 - supernatant, neutralized to pH 7 and treated with catalase.

Example 6: Storage tests of selected strains (32 weeks)

The results of storage tests of the new strains of Lactobacillus sp. lyophilized in cryo- protective medium at the temperature of 5 °C, during 48 weeks, under aerobic conditions, are shown in Figure 3.

It is apparent from the results of the storage tests of the lyophilized isolated of the lactobacilli shown in Figure 3, that all the strains tested preserved high viability during the storage at 5 °C for the period of 48 weeks. The quantity of viable lactobacilli ranged at this temperature between 10 ⁸ to 10 ¹⁰ KTJ.g ^"1 of the lyophilisate.

Example 7: Preparation of a functional sample of ladies sanitary product with probiotic effect

Functional samples were prepared according to the procedure described in WO 02/28446 Al as follows:

Lyophilisate of the each probiotic strain of lactobacilli (Lbc. renter i 291, Lbc. fermentum Gl) was mixed into a sterile carrier. The resulting mixture was applied to sanitary products under sterile conditions. Commercially available sanitary products by Johnson & Johnson, G.m.b.H, Germany have been used for the preparation of functional samples. The samples prepared were aseptically inserted individually into sterile closable sample closures and stored at the temperature of 25 °C in normal atmosphere.

Industrial applicability New strains of lactobaciili are usable as active ingredients of medicaments and sanitary products for the prevention and treatment of urogenital infections, for the restoration of natural vaginal microflora, and/or for elimination of discomfort of women in pre-menstrual and menstrual period.