DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0040] By “hygiene tissue” is meant any device for wiping skin, for instance, a washcloth, patch, towelette, napkin, wetwipe, and the like.

[0041] By “matrix” is meant any natural or synthetic fiber, such as felt, batting, rayon, cellulose, regenerated cellulose, polyester, polyolefin fibers, textile and the like, or foam, or combinations thereof.

[0042] Preferred “lactic acid producing bacterium” include bacteria from the genera Lactobacillus, Lactococcus and Pediococcus. Preferably the selected bacterium used is from the species Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus curvatus or Lactobacillus plantarum. Even more preferably the lactic acid producing bacterium is Lactobacillus plantarum 931 (deposition No. DSM: 11918).

[0043] By “lipid” is meant a water-insoluble organic molecule with a fatty character. Suitable lipids for the present invention include petroleum-derived lipids, synthetic lipids, and animal- and plant-derived lipids.

[0044] By “additional component” is meant agents commonly added to skin caring products, such as caring agents, water absorbent agents, pH buffering agents (weak organic or inorganic acids, such as lactic acid, ascorbic acid, citric acid or boric acid), perfume, antioxidants, hydrocortisone, other anti-inflammatory steroids, etc. Further details on suitable agents commonly added to skin caring products are given in Harry's Cosmeticology 8th ed., Ed by M M Rieger, Chemical Publishing Co., Inc., New York, 2000.

[0045] By “moisture impervious packing unit” is meant a packing unit having a highest water vapor transmission rate of 6 g/m ² /calendar day in accordance with ASTME 398-83.

[0046] The hygiene tissue can be used for cleaning and simultaneously establishing and maintaining a healthy microbial flora on the skin and in the urogenital area. The hygiene tissue provided can be composed of a matrix comprising any natural or synthetic fiber, such as felt, batting, rayon, cellulose, regenerated cellulose, polyester, polyolefin fibers, textile and the like, or foam, or combinations thereof. The water content of the matrix of the hygiene tissue is preferably 10% or less (by weight), more preferably 5% or less (by weight), and most preferably 1% or less (by weight). The hygiene tissue is impregnated with a suspension of a lactic acid producing bacterium in a lipid. The lipid encapsulates the bacteria and thereby acts to protect the bacteria from moisture, which enhances bacterial survival during manufacturing and storage. Preferably the the lactic acid producing bacteria are present directly in the lipid phase, but the bacteria can also be present in microincapsulated forms. Exposure to moisture during manufacturing and storage of products that comprise lactic acid producing bacteria, causes reactivation of the bacteria, which subsequently leads to their death. Therefore, by protecting the bacteria from moisture, their survival is enhanced and the durability of the product extended. The inventors also found that encapsulating the bacteria in lipid resulted in enhanced transfer rates to and survival of the bacteria on the skin after their delivery to it (see Example 3 below). This can be an effect of the lipid creating a micromilieu, that is beneficial for retaining bacterial viability and that enhances growth of the added bacteria on the skin. Also, the lipid has more adhesive properties than, for example, water, thereby resulting in a higher amount of bacteria actually being transferred to the skin. In addition to its bacterial survival enhancing properties, the lipid also serves as a skin treatment agent that cleans the skin without causing drying out. Once the bacteria have been delivered to the skin, the moisture on the skin reactivates the bacteria, thereby allowing them to perform their intended action, i.e., competitively exclude and minimize colonization of pathogenic microbial species. Optionally, additional substances, including water absorbent agents (such as inorganic salts, e.g., calcium chloride), tensides (non-ionic, amphoteric and anionic surfactants), pH buffering agents (weak organic or inorganic acids, such as lactic acid, ascorbic acid, citric acid or boric acid), perfume, antioxidants, hydrocortisone and other anti-inflammatory steroids, can also be added to the lipid-bacterial suspension, or directly to the hygiene tissue.

[0047] The amount of lipid suspension of lactic acid producing bacteria on the tissue is preferably 0.5-95% by weight.

[0048] The number of probiotic bacteria on the hygiene tissue is preferably 10 ⁴ -10 ¹¹ colony forming units (CFU) and more preferably 10 ⁶ -10 ¹¹ CFU. The preparation of lactic acid producing bacteria are preferably in a dried form, preferably as a freeze-dried powder. The bacteria can also be provided in microencapsulated forms. Preferably, the water activity of the bacterial preparation is about 0.30 or less, more preferably about 0.25 or less, and most preferably about 0.20 or less.

[0049] In one preferred embodiment the probiotic bacterial strain with antagonistic effect is selected from the genera Pediococcus, Lactobacillus or Lactococcus including combinations thereof. The lactic acid producing bacterial strain is preferably isolated from the skin or urogenital area of a healthy person.

[0050] In another preferred embodiment the probiotic bacterial strain with antagonistic effect is at least a Lactobacillus plantarum strain.

[0051] In an even more preferred embodiment the probiotic bacterial strain with antagonistic effect is at least Lactobacillus plantarum 931 (deposition No. (DSM): 11918).

[0052] Preferable lipids for the present invention support survival of the stored cells so that the maximum decrease in number of culturable cells is 3 log units after 12 months storage. Preferable lipids have a water content of 5% or less (by weight), preferably 3% or less (by weight), most preferably 1% or less (by weight). More preferably, suitable lipids support survival of the stored cells so that the maximum decrease in number of culturable cells is 2 log units after 12 months storage. Most preferably, suitable lipids support survival of the stored cells so that the maximum decrease in number of culturable cells is 1 log unit after 12 months storage.

[0053] Preferable lipids enable transfer of bacteria to the skin of 10 ⁵ or more culturable cells per cm ² . More preferably, suitable lipids enable transfer of bacteria to the skin of 10 ⁶ or more culturable cells per cm ² . Most preferably, suitable lipids enable transfer of bacteria to the skin of 10 ⁷ or more culturable cells per cm ² .

[0054] Preferable lipids are also characterized by supporting survival of the bacterial cells on the skin so that 10 ² or more culturable cells per cm ² can be recovered 12 hours after delivery by use of the hygiene tissue. More preferably, suitable lipids support survival of the bacterial cells on the skin so that 10 ³ or more culturable cells per cm ² can be recovered 12 hours after delivery by use of the hygiene tissue. Most preferably, suitable lipids support survival of the bacterial cells on the skin so that 10 ⁴ or more culturable cells per cm ² can be recovered 12 hours after delivery by use of the hygiene tissue.

[0055] Preferable lipids for the present invention include petroleum-derived lipids, such as paraffinum liquidum (mineral oils, paraffin oils, and vaseline oils), petrolatum (vaseline and petroleum jelly), cera microcrystallina, ozokerite, ceresine and paraffins. Alternatively, synthetic lipids, such as Dimethicone, Cyclomethicone and silicone esters, such as cetearyl methicone can be used. A third alternative is to use animal- or plant-derived lipids, which usually are triglycerides. The naturally animal- or plant-derived lipids are often mixtures of mono-, di- and triglycerides and free fatty acids. The lipids may be purified, hydrogenated, refined, modified and used alone or in different mixtures. Examples of suitable, original animal-derived lipids include bees' waxes, emu oil, lactis lipida, lanolin, shark liver oil and tallow. Examples of suitable plant-derived original lipids include apricot kernel oil, arachis oil, avocado oil/wax, bayberry wax, black currant seed oil, borage seed oil, brazil nut oil, camelia sinensis oil, candelilla wax, canola oil, carnauba wax, castor oil, cocoa butter, coconut oil, corn oil, cotton seed oil, dog rose seed oil, evening primrose seed oil, grape seed oil, illipe butter, jasmine wax, jojoba wax, lavender wax, linseed oil, mango seed oil, olive oil, orange wax, palm oil, palm kernel oil, peanut oil, rice wax, safflower oil, sesame seed oil, shea butter, soybean oil, sunflower seed wax, sweet almond oil and wheat germ oil. The lipids can be used alone or in mixtures comprising at least two lipids.

[0056] Preferably, a liquid lipid is used in the present invention. More preferred lipids include olive oil, canola oil, coconut oil, palm kernel oil, peanut oil, soy bean oil, Dimethicone, paraffin oil, and petrolatum. These lipids are especially preferred because they provide high survival of lactic acid bacteria and high transfer rates of the bacteria to the skin and urogenital area. In addition, these lipids have positive effects on the skin; they have a soothing effect, show skin-protective properties, and are non-toxic and non-allergenic. The preferred lipids are all of non-animal origin.

[0057] In order to protect the probiotic bacteria from moisture during storage and transportation, the hygiene tissue is preferably provided individually packed in a moisture impervious packing unit. The packing unit can be constructed in several different ways. The material used preferably should not have a vapor permeability exceeding 6 g/m ² /calendar day in accordance with ASTME 398-83 at 37.8° C. and 90% relative humidity. Suitable materials to be used to obtain an impervious packing unit include polyethylene, polypropylene, polyester, polyamide, polyvinylalcohol and similar polymers, aluminum foil, aluminum oxide, silicon oxide, and the like. In order to produce packing material that has good wear strength and can readily be sealed, a less expensive material may be used as outer protective wear layers and/or as inner sealing layers. For instance, the packing unit can comprise an inner material that enables a good seal to be obtained, e.g., polyethylene, polypropylene, butyl acrylate, vinyl acetate or wax, an intermediate material that consists of a moisture impervious material, and a stronger outer barrier material, e.g., polyethylene or polypropylene. After placement of the hygiene tissue comprising the lipid suspension containing the lactic acid producing bacteria, openings of the packing unit are tightly sealed. In an exemplary embodiment, the seals do not have a moisture permeability exceeding that of the packing material itself. Further details on the design of suitable impervious packing units are given in WO00/76878, which hereby is incorporated as reference.

EXAMPLES

Example 1

Survival of Lactobacillus plantarum 931 in Olive Oil on Hygiene Tissues

[0058] A preparation of freeze dried L. plantarum 931 cells in skimmilk was ground until a powder of fine grains was formed. 10 g of the L. plantarum 931 powder was added to 120 ml olive oil (Filippo BERIO® extra virgin olive oil) and shaken until a homogenous solution was formed. An additional aliquot of 80 ml of olive oil was added and the resulting 200 ml solution was vortexed for about 2 min. The bacterial suspension was kept at room temperature for 3 hours, with mixing twice an hour. Tissue sheets (Spun Lace Dupont and SCA Absbond) were cut to 6×4 cm squares and placed in sterile stainless steel trays. On each tissue sheet, 2 ml of bacterial suspension was dropped over the tissue to cover it. The tissue sheet was folded in the middle, then from the long side to the middle again and packed in foil bags, which edges were welded. Samples were removed for determination of initial bacterial concentration and the remaining bags were stored at room temperature for viability studies.

[0059] As a comparison to storage of L. plantarum 931 cells in olive oil, L. plantarum 931 cells were stored in suspensions of water and olive oil on hygiene tissues. These were prepared by mixing vigorously 2.5 g of freeze dried L. plantarum 931 cells (prepared as described above) with 45 ml of Milli Q water and 5 ml olive oil (Filippo BERIO, Italy) or 35 ml Milli Q water and 15 ml olive oil to prepare suspensions with approximately 10 or 30% oil, respectively. The bacterial oil-water suspensions were applied to tissue sheets (Spun Lace Dupont) for bacterial survival studies. On each tissue sheet (6×7 cm), 1 ml of bacterial suspension was dropped to cover the tissue. The tissue sheet was folded in the middle, then from the long side to the middle again and packed in foil bags, which edges were welded. Samples were removed for determination of initial bacterial concentration and the remaining bags were stored at room temperature in the dark for viability studies.

[0060] To test the viability of the L. plantarum 931 cells after storage for different time periods on a tissue sheet, the tissue sheet was transferred to a Stomacher bag and 10 ml of 0.9% NaCl was spread over the tissue sheet. The bag was run for 3 minutes on high effect in the Stomacher bag. The contents of the bag was then transferred to test tubes, diluted in 0.9% NaCl when necessary, and immediately plated onto Rogosa plates. The number of colonies was counted after 2 days of incubation at 37° C. in 5% CO ₂ in air. Two tissue sheets were analyzed at each sampling date.

[0061] The survival of L. plantarum 931 in olive oil on hygiene tissues for a total time period of one year and three months is shown in FIG. 1 . The survival of the L. plantarum 931 cells stored under these conditions was very high for both tissue sheets variants tested. In comparison, storage of oil-water suspensions (10 and 30% olive oil in water) of the bacteria on a tissue sheet ( FIG. 2 ) resulted in a rapid decrease in viability with a drop of more than 10 ³ orders of magnitude over just a three months time period studied.

Example 2

Survival of L. plantarum 931 in Lipids with Different Chemical Compositions

[0062] 497 mg of a powder of L. plantarum 931 in skim milk, prepared as described in Example 1, was mixed with 5 ml of olive oil (Filippo BERIO® extra virgin olive oil, Filippo BERIO, Italy) or rapeseed oil (Felix AB, Sweden). The pH of the oils was about 5. The rapeseed oil also contained, in addition to rapeseed oil, citric acid and vitamin A and D. The suspensions were vortexed for 1 minute and allowed to rest for 1 minute. This was repeated four more times. The bacterial suspensions were kept for 4 hours at room temperature with mixing twice an hour. The suspensions were then divided into 1 ml aliquots and stored in sterile brown glass vials. The initial concentrations of bacteria in the suspensions were determined. The vials were stored in a dark place at room temperature and normal air humidity varying from 30-60%.

[0063] In addition to the experiment described above, to further compare the survival of L. plantarum 931 in lipids with different compositions, 2 g of freeze-dried L. plantarum 931 cells in skimmilk (2×10 ¹⁰ colony forming units/g) were mixed with either 40 ml or 40 g of the different lipid compositions, depending on the lipid consistency. The tested lipids were: white vaseline (petrolatum, Apoteket AB, Umea, Sweden), paraffin (paraffinum liquidum, Apoteket AB, Gothenburg, Sweden), glycerol (“glycerin”, maximum water content 0.5%, Apoteket AB, Gothenburg, Sweden), olive oil (“Olea Europea”, cold pressed, Apoteket AB, Gothenburg, Sweden), dimethiconum (Dimethicone, 350 cSt., Dow Corning 200/350 S fluid, Kebo Lab, Sweden), and Akoline MCM (mono-diglyceride of medium chain fatty acids; primarly caprylic and capric acids, Karlshamns AB, Sweden), Akomed R (caprylic/capric triglyceride from coconut and/or palm kernel oils, deodorized, Karlshamns AB, Sweden), and Akorex L (canola oil, partially hydrogenated, deodorized, Karlshamns AB, Sweden). The samples were stored in sterile, brown, glass vials at room temperature in the dark at normal air humidity (varying from 30-60% relative humidity). To establish the number of viable L. plantarum 931 cells after different storage times, 1 g of the samples was transferred to a stomacher bag and 9 ml of 0.9% NaCl was added. The bag was then run at high effect in Stomacher for 3 minutes. The contents of the bag was transferred to test tubes, diluted when necessary in NaCl and cultured on MRS-plates at 37° C. in 5% CO ₂ in air for 2 days.

[0064] As a comparison to storage of L. plantarum 931 cells in different lipids, L. plantarum 931 cells were also stored in suspensions of water and olive oil. These were prepared by mixing vigorously 2.5 g of freeze dried L. plantarum 931 cells (prepared as described above) with 45 ml of Milli Q water and 5 ml olive oil (Filippo BERIO, Italy) or 35 ml Milli Q water and 15 ml olive oil to prepare suspensions with approximately 10 or 30% oil, respectively. The samples were stored in sterile plastic vials at room temperature at normal air humidity (varying from 30-60% relative humidity). To test survival after different time intervals, samples were removed from the vials, diluted in 0.9% NaCl and incubated at 37° C. in 5% CO ₂ in air for 2 days.

[0065] FIG. 3 shows the level of survival of the L. plantarum 931 cells in olive and rapeseed oil over the more than one year time period studied. Storage in vaseline, paraffin, Dimeticonum, Akoline MCM, Akomed R, and Akorex L also resulted in survival over extended time periods ( FIG. 4 ). However, storage in the more hydrophilic glycerol resulted in a decrease in survival over time ( FIG. 4 ). Also, the Akoline MCM, known to have bacteriostatic properties, could not support survival of the cells ( FIG. 4 ). In addition, it can be seen that storage in the more hydrophilic environment provided in the oil-water suspensions ( FIG. 5 ) results in a very rapid initial decrease in viability by more than 10 ² orders of magnitude over the first month of storage. During the next two months studied, the decrease in survival rates flattened out, but still the decrease in survival is much higher than what is observed when the bacterial cells were stored in a lipid.

Example 3

Study on Transfer Efficacy to and Survival Rates on Skin of L. plantarum 931 Suspended in Olive Oil, Paraffin Oil or Milli Q Water

[0066] 2.00 g of freeze-dried L. plantarum 931 were added to a sterile glass vial and 40 ml of olive oil, paraffin oil or Milli Q water were added. The suspensions were shaken until homogenous solutions formed and were left at room temperature for four hours. Tissue sheets with L. plantarum 931 were prepared by cutting tissue sheets to 7×8 cm pieces and dropping 2 ml of the different bacterial suspensions, prepared as described above, to cover the tissue. The tissue sheets were folded in the middle, then from the long side to the middle again and packed in foil bags which edges were welded. Samples were removed for determination of initial bacterial concentrations and the remaining bags were stored at room temperature for viability studies. Two of the prepared tissue sheets with L. plantarum 931 for each preparation were used in the bend of the arm on five subjects (i.e., one tissue was used for three subjects and the other one for two). Before the test, samples were taken to assure that no L. plantarum 931 cells were initially present on the skin. On each subject, in one bend of the arm a tissue sheet with L. plantarum 931 in Milli Q water was streaked and a tissue sheet with L. plantarum 931 in olive oil was similarly used in the other. The skin was then sampled for presence of L. plantarum 931 cells at 0, 4, 6 and 24 hours after streaking. The sampling procedure was as follows: a sterile stick provided with a cotton wool top was dipped in 0.9% NaCl and rolled 4 times over an area of 1 cm ² at the site of application of the bacteria. The stick was then dipped in 1 ml of 0.9% NaCl and mixed. The samples were diluted in 0.9% NaCl and immediately plated onto Rogosa plates. The plates were incubated at 37° C. in 5% CO ₂ in air for 2 days. After 24 hours the bend of the arm, where L. plantarum 931 cells in Milli Q water were applied, was rinsed with Sumabac (Diversey Lever, Huddinge, Sweden) and a tissue sheet with L. plantarum 931 cells in paraffin oil was streaked in that bend of the arm, which, as previously, was sampled at 0, 4, 6 and 24 hours after streaking to assess the amount of L. plantarum 931 cells on the skin.

[0067] As shown in FIGS. 6 - 8 , the amount of L. plantarum 931 cells transferred to the skin was usually more than 10 times higher when the bacteria were suspended in olive ( FIG. 6 ) or paraffin oil ( FIG. 7 ) compared to when they were suspended in Milli Q water ( FIG. 8 ). Suspending the bacteria in lipid instead of water therefore was demostrated to enhance the transfer rate of the bacteria to the skin. Also, once the L. plantarum 931 cells were on the skin, the lipid also enhanced bacterial survival, since the initial drop in bacterial counts was slower and the final level of bacteria on the skin after 24 hours was higher after use of a tissue where the bacteria were suspended in olive or paraffin oil ( FIGS. 6 and 7 ), compared to what was found when water was used as suspending agent ( FIG. 8 ).

Example 4

Transfer of L. plantarum 931 Suspended in Olive Oil Applied to a Tissue Sheet Used in the Urogenital Area

[0068] A bacterial suspension was prepared by adding 5.051 g of a powder of L. plantarum 931 (prepared as described in Example 1) to 100 ml olive oil (Filippo BERIO® extra virgin olive oil). The powder was first added to 75 ml of olive oil and shaken to form a homogenous solution before addition of another 25 ml, followed by vortexing for 2 min. The bacterial suspension was kept at room temperature for 2 hours, with mixing twice an hour. A tissue sheet was prepared and inoculated with the bacteria as described in Example 1. Four girls were treated with the tissue sheet in the urogenital area. Samples were collected from the urethra and perineum, using sterile cotton sticks, before using the tissue sheet, to ensure that no lactobacilli were initially present. Thereafter samples were collected immediately after treatment, and after 2, 4, 6, and 17 hours, to monitor transfer and survival of the lactobacilli by dipping a sterile stick provided with a cotton wool top in MRS-broth and rolling it three times over an area of 1 cm ² at the site of application of the bacteria. The stick was then put in one ml MRS-broth. In the same way a stick was rolled over the uretra over an area of ¼ cm ² and was the put in another tube of MRS-broth. The samples were plated onto Rogosa plates containing 128 μg/ml vancomycin. The plates were incubated at 37° C. in 5% CO ₂ in air for two days.

[0069] The girls were not allowed to take a bath or shower during the study, but were not given instructions in any other respect. The results shown in FIGS. 9 and 10 demonstrate that there was a high level of transfer of L. plantarum 931 to the urethra ( FIG. 9 ) and perineum ( FIG. 10 ) and that a surprisingly high amount of bacteria remained in these areas during the time period studied.

Example 5

Viability of L. plantarum 931 After Storage in Olive Oil on a Hygiene Tissue

[0070] Growth of L. plantarum 931 suspended in olive oil on tissue sheets that had been stored for 6 days was compared to growth of L. plantarum 931 that were not suspended and stored in olive oil. The tissue sheets were wetted with 10 ml 0.9% NaCl and run in Stomacher for 3 minutes on high effect. The solution was then transferred to test tubes and 10 ml were inoculated into 10 ml MRS broth. The bacterial concentration was followed during growth at 37° C. in 5% CO ₂ in air by quantifying the number of colony forming units (CFU) and the optical density (OD) at 0, 2, 4, 6, 8, 10, 12 and 24 hours. Duplicate samples were taken for the analysis. The results shown in FIG. 11 demonstrate that the capability of growth of the L. plantarum 931 cells had been suspended and stored in olive oil on a tissue sheet is just as good as growth of cells from an overnight culture of L. plantarum 931. Accordingly, the L. plantarum 931 cells were not negatively affected by the storage in olive oil.

Example 6

Difference in Transfer Rates and Maintenance Levels of LB931 and ATCC 8014 Provided via Panty Liners, in Urethra and Perineum Areas

[0071] Lactobacillus plantarum strain LB931 (DSM11918) was obtained from Deutche Sammlung von Microoganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweg, Germany. Lactobacillus plantarum strain ATCC 8014, as described in U.S. Pat. No. 5,705,160, was obtained from America Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA.

[0072] LB931 and ATCC were grown on MRS agar plates for two days at 37° C. in CO ₂ (5%). One colony of each strain was inoculated into 5 ml of MRS broth (start tube) and incubated in the same way as above, but overnight. 50 μl from the start tube was placed into 5 ml of MRS broth, preheated to 37° C., and incubated at 37° C. for eight hours. 500 μl was put in 50 ml of preheated MRS broth and is incubated at 37° C. for 16 hours.

[0073] The suspension was centrifuged for 20 minutes at 3000×g at 4° C., and the pellet was washed three times in 10-20 ml NaCl (0.9%). After washing, the pellet was resuspended in 2.5 ml NaCl (0.9%)+2.5 ml skim milk, thereby concentrating the sample 10×. This suspension was transferred to an actuator vial. Each 10+1 (test) panty liners were sprayed with about 150 mg of bacterial suspension. The panty liners were dried for 6 hours at room temperature before being packaged one by one into mini-grip bags.

[0074] Control of colony forming units (CFU) of the test panty liner: 50 ml of NaCl (0.9%) was poured over the panty liner in a stomacher bag and run at high speed for 3 minutes. The fluid is diluted and 100 μl was spread on MRS agar plates. The number of CFU/panty liner for ATCC 8014 was 3×10 ⁹ and for LB931 was 1×10 ⁹ .

[0075] Women took a time zero sample (perineum and urethra) before wearing a panty liner supplied with either of the L plantarum strains for 5 hours. The panty liner was removed and sample 2 was taken. After 24 hours (from time zero sample), sample 3 was taken. Transfer is defined as % of total women that after 24 hours still carry bacteria.

[0076] As shown in FIG. 12 , more of the women who had used panty liners with LB931 still harbored the LB931 bacteria in the perinuem 24 hours after the time zero sample was taken (92% still harbored the bacteria). In comparison, of the women who had used panty liners with ATCC 8014 only 50% harbored the tested bacterial strain at the same time point in the perineum ( FIG. 13 ).

[0077] A similar difference was observed when the number of bacteria that still were present in the urethra was determined 24 hours after the time zero sample was taken. In this case, 62% of the women who had used panty liners with LB931 harbored the bacteria ( FIG. 14 ), compared to 25% of women who had used panty liners with ATCC 8014 ( FIG. 15 ).

Example 7

Comparison of Ability of Growth Inhibition and Killing of Pathogenic Bacteria Between LB931 and ATCC 8014

[0078] LB931 and ATCC 9014 were grown as described above, but to a final volume of 100 ml each. The suspension was centrifuged and resuspended in 10 ml of PBS, in a Falcon tube (size 50 ml). The tube was incubated at 37° C. for 5 hours. Thereafter the suspension was centrifuged and the supernatant (PBS sup) collected, containing inhibiting substances secreted by the lactic acid bacteria. The PBS sup was stored at −20° C. until use. For controls, PBS buffer, pH adjusted (pH 5.1) and unadjusted (pH 7.3) were used.

[0079] The ability of the two tested Lactobacillus plantarum strains, LB931 and ATCC 8014, to inhibit growth of common urogential pathogens was tested. The tested pathogens were Escherichia coli, Enterococcus, Staphylococcus aureus, and Group B Streptococcus (GBS). These bacteria were grown in TH broth and diluted 1:100 before 10 μl was inoculated into 1 ml of PBS sup. The number of pathogens was determined for the tested pathogenic strains at 0, 4, 6, and 24 hours.

[0080] As can be seen in FIGS. 16 - 19 , LB931 is superior over ATCC 8014 in inhibiting growth and killing pathogenic bacteria. In the case of E. coli, Enterococcus, and S. aureus, the growth of these pathogenic strains was not or was only marginally inhibited by the ATCC 8014 strain, while the pathogenic bacteria are completely abolished when placed in the supernatant of LB931. In the case of GBS, LB931 supernatant killed all the bacteria within 4 hours, while ATCC 8014 required 24 hours to do the same.

[0081] It is apparent from Examples 6 and 7 that the ability of LB931 to be transferred and maintained in the urogenital area is higher than it is for another L. plantarum strain, ATCC 8014. There is a very pronounced difference between the two L. plantarum strains in their ability to inhibit growth of and killing pathogenic bacteria, with LB931 being superior over ATCC 8014. Thus, a clear advantage can be gained in using LB931 as a probiotic bacterium in hygiene products such as embodiments of the present invention over other L. plantarum strains.

[0082] Although the present invention has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without department from the spirit and scope of the invention as defined in the appended claims.