Patent Abstract:
the dual microbial preparation contains the microscopic oomycete pythium oligandrum and components of a physiological microbiome . the microscopic oomycete pythium oligandrum and the physiological microbiome component are present in the dual preparation in a form which facilitates their germination , subsequent propagation and colonization of the target tissues . the microscopic oomycete pythium oligandrum is incorporated in a quantity of 10 3 to 10 7 cfu , with 10 4 to 10 5 cfu per one cycle of application preferably . the physiological microbiome component contains 5 × 10 6 to 5 × 10 10 cfu , with 5 × 10 7 to 5 × 10 9 per one cycle of application preferably . the fermented substrate found in the pythium oligandrum oomycete is the source of nutrients for both microbial components . the dual microbial preparation also contains at least one auxiliary substance from a group including a desiccant , components of a buffer system , an anti - caking substance and an agent for the creation of a physiological osmotic environment . the physiological microbiome component is a component of the human microbiome , one of the microbes of the green complex , such as the capnocytophaga sputigena bacterium , or one of the components of the healthy skin microbiome , such as the staphyloccocus epidermidis bacterium , or one of the components of the healthy vaginal microbiome , such as the peroxide producing lactobacillus crispatus . either the microscopic oomycete pythium oligandrum or the physiological microbiome component is present in the dual microbial preparation in the form of inactivated cells , cell extracts or isolated cell fractionation . the microbial activator for the pythium oligandrum oomycete is a yeast autolysate in a quantity of 0 . 1 % to 10 % weight of the total quantity of dual microbial preparation . the auxiliary substances are regulated in a way which allows for application in the form of an ointment , cream , oil or suppository or in the form of a liquid aqueous preparation .

Detailed Description:
the solution which we propose is enabled based on a detailed analysis of the results of laboratory and practical tests of cosmetic products based on patent cz 302 297 b6 [ 34 ] specified above , whose detailed formulation was optimized for products , for example with regard to helping the elimination of symptoms of opportunistic infections in the oral cavity chytrá houba ® pythie ® bioplus , in the area of non - healing wounds biomycosin , on the surface of the membranes of the urogenital tract feelfresh , and with regard to eliminating the agents of fungal infections of the skin chytrá houba ® pythie ® biodeur ® nail and the veterinary product chytrá houba ® ecosin . all these preparations , containing only pythium oligandrum as an active substance underwent toxicological and safety tests according to the principles of european notification of cosmetic products with optimized formulation , or , in the case of the veterinary preparation , were approved by the institute for state control of veterinary biologicals and medicines ( úlstav pro státní kontrolu biopreparát a lé iv brno ), whereby evaluations of the safety of preparations are available from the cpnp european notification portal . individual preparations formulated in this way were then tested in clinical trials under conditions corresponding to stage ii of the clinical trials required for the development of medications . the preparation chytrá houba ® pythie ® bioplus , in the form of effervescent tablets was tested in a multicentric study incorporating three stomatological clinics , and one clinical microbiology department . groups of participants in the study suffering from a periodontal disease ( penodontitis ) were gathered at three dental clinics in prague and kladno in the czech republic , and in ko { hacek over ( s )} ice in the slovak republic . the group in prague consisted of 7 participants , the group in kladno of 15 participants , and the group in ko { hacek over ( s )} ice of 22 participants . the overall number of participants in the study was , therefore , 44 . a control group was treated in parallel using the same method , receiving not a biological , but the classic chemical preparation chlorhexidine gel . the participants in the study were regularly monitored with measurements of pbi ( papilla bleeding index ), which reflects the immediate condition of the inflammation in the gums , and of cpitn ( community periodontal index of treatment needs ), which is a gauge of the long - term condition of the teeth and a predictor of needs from the perspective of their treatment . the study proceeded using the double - blind method , in which envelopes with the preparations were prepared and numbered by an administration worker not actually involved in the course of the study . after carrying out dental hygiene and signing informed consent , the individual patients were instructed how to conduct their evening dental hygiene and apply the preparations . application was undertaken in the form of rinsing every evening after completing oral hygiene for five consecutive days . a dental check - up was conducted at the beginning of the study , after 2 months and after 6 months , whereby the condition of the teeth was checked , and , at the end of the study after six months , the colonization of the microbe of the healthy oral microbiome evaluated based on a genetic test . it was ascertained in a statistical evaluation of the results that the effect of the chemical disinfectant chlorhexidine was only short - term , since there was a statistically significant reduction of the pbi with p ≦ 0 . 05 only after 2 months of monitoring , and not at the end of the study 4 months later . by contrast , the long - term results in the group using the biological preparation were excellent . a statistically significant reduction of the pbi ( p ≦ 0 . 05 ) was achieved in the short - term evaluation , and a statistically highly significant reduction of pbi ( p ≦ 5 0 . 01 ) was achieved in the long - term evaluation . further , stabilization of the condition of the teeth after the period of 6 months , for which the participants in the study were monitored , was observed . however , the results were heterogeneous in the largest group of participants at the dental clinic in ko { hacek over ( s )} ice , and the expected curative effects were not achieved after statistical evaluation , in contrast to the two groups specified above . in order to clarify this situation , details of the clinical records were used , and the complete microbial profile of the oral microflora was monitored in this group of participants using a genetic test before applying the preparation . a mutual correlation of clinical and laboratory results made it possible to state that this heterogeneous group consists of three sub - groups , indicated as low - risk , medium - risk and high - risk groups . the striking feature of the high - risk group is the low content of physiological oral bacteria , the so - called green protective complex bacteria , incorporating the analyzed strains eikenella corrodens and capnocytophaga sputigena . the results are shown in fig1 a , 1b and ic . fig1 a shows the values of the pbi ( papilla bleeding index ) for , on the one hand , patients indicated in the graph as “ unresponsive patients ” under conditions corresponding to application of a preparation with pythium oligandrum , and , on the other , patients indicated in the graph as “ responsive patients ” under conditions corresponding to the application of the dual microbial preparation , all these before application , 2 months after application and 6 months after application . at the beginning of the study , before application ( black column ), there was no significant statistical difference between the groups . the “ unresponsive patients ” had a pbi value of 38 . 8 ± 5 . 8 and the “ responsive patients ” had a pbi value of 44 . 6 ± 4 . 7 . the probability value of the dissimilarity of both groups was p = 0 . 121 ; meaning that both groups of patients were not therefore significantly statistically different . at the end of the study , after 6 months , the “ unresponsive patients ”, under conditions corresponding to the application of pythium oligandrum , showed pbi values that were statistically higher than the “ responsive patients ”, under conditions corresponding to the application of the dual microbial preparation according to the invention . the calculated bleeding index values were after 6 months 39 . 4 ± 10 . 8 for patients under conditions corresponding to the application of pythium oligandrum alone , whereas the values under conditions corresponding to the application of the dual microbial preparation according to the invention were 10 . 6 ± 2 . 4 . the value of the probability of dissimilarity for both groups was p = 0 . 0003 . the results to concern the cpitn ( community periodontal index of treatment needs ) were similar ; however , this is a less immediate indicator given its slower reaction to the condition of the oral cavity , and therefore rather reflects long - term trends . fig1 b shows the dependency of the intensity of hybridization of genetic probes which detect individual groups of oral microorganisms pertaining to the green , orange or red complex . fig1 b shows the results of a genetic analysis of the presence of selected microbes of the green oral complex ( light - gray column ), the orange oral complex ( dark - gray column ) and red oral complex ( black column ) specified in units , corresponding to the number of disintegration per minute of radioactively labeled dna probes . participants in the control group , under conditions corresponding to the application of chytrá houba ® pythie ® bioplus ( on the right of fig1 b ), and in the experimental group , under conditions corresponding to the application of the dual microbial preparation ( on the left of fig1 b ), have a high content of microbes of the orange and red complex , which supports their diagnosis of serious periodontal disease . no statistically significant differences were observed between the two groups . evident in the experimental group of “ responsive patients ”, however , is the statistically significantly higher content of bacteria of the green complex : 14908 ± 1489 ru as opposed to 2750 ± 1171 ru , p & lt ; 0 . 001 , in particular the capnocytophaga bacterium , as is shown in fig1 c ( dark column ). these results confirm that the dual microbial preparation according to the invention for use in the oral cavity shows statistically significantly better results in comparison with the standard preparation . based on these results , we considered and looked for a solution for the effective and long - term effect of the pythium oligandrum oomycete in eliminating the symptoms of periodontitis and gingivitis , and tested and developed experiments to prove the cooperation of this microorganism , pythium oligandrum , with the microbial components of the healthy oral microbiome , in particular with the capnocytophaga sputigena bacterium . a total of three versions of the dual microbial preparation were prepared for use in the oral cavity and the stability of such preparations was proven , clinical tests also being carried out in patients with periodontal disease . a detailed description of the results obtained is presented hereunder in more detail in this exemplary embodiment of this invention . altogether , the results of this testing proved that when applying the dual microbial preparation according to the invention in the form of effervescent tablets for oral rinsing , which is the form preferred by most users of the mono - component preparations , there was no negative influence on the stability of any of the components during storage in dry state . after the application of the dual preparation in the form of rinsing of the oral cavity , a simple process for users , there was effective colonization and strengthening of the components of the normal oral microbiome , which led to a significant increase in the success rate of eliminating the symptoms of the person under evaluation according to standard indexes . 1 . the preparation , control and testing of the effectiveness of a dual microbial preparation suitable for eliminating the symptoms of gingivitis and periodontal disease in oral cavity . 1 . 1 protocol for the preparation of a dual microbial preparation for use in the oral cavity . 1 . 1 . 1 preparation of the technical preparation pythium oligandrum m1 atcc 38472 . the inoculum for the cultivation of the microscopic oomycete pythium oligandrum , strain m1 atcc 38472 , is prepared using the “ master cell bank ”-“ working cell bank ” corporate system . the proprietary strain pythium oligandrum m1 atcc 38472 is stored in the master cell bank by way of long - term freeze drying . a limited quantity of aliquots is produced from the master cell bank , according to the protocol , for the working cell bank so that the number of reproductive generations does not exceed a total of 50 . this ensures the genetic stability of the proprietary strain . the cultivation of the inoculum proceeds on a reciprocating shaker with swing of 10 . 5 cm and 96 swings per a minute for a period of 48 hours at a temperature of 28 ° c . in the meantime , we prepare a millet substrate for the solid cultivation of pythium oligandrum m1 : 500 g of hulled panicum miliaceum millet is long - term freezing in the liquid nitrogen during − 150 ° c . in 200 ml distilled water are then dissolved : 50 mg znso 4 , 150 mg kh 2 po 4 , 50 mg mgso 4 and 250 mg cacl 2 . the solution is then heated to boiling point and poured on the washed millet . after boiling , the vessel holding the millet is covered and left to stand for 30 minutes . a thin layer of millet is then spread out on filter paper and left to cool and dry . thirty grams of the millet prepared in this was is then added to a 500 ml erlenmeyer flask , and sterilization proceeds in a steam autoclave at a temperature of 120 ° c . for a period of 40 minutes . sterilization is repeated after 24 hours and the millet is shaken slightly after each cycle of sterilization . we inoculate the sterilized millet with 4 ml of inoculum prepared using the procedure presented above . after inoculation , the millet is shaken thoroughly with the inoculum and finally evened - out into a uniform layer with a light tap . solid cultivation on the millet substrate is conducted in a thermal regulator at a temperature of 28 ° c . and a relative humidity of 70 % for a period of 14 days . the fermented substrate , with pythium oligandrum culture growth , is then dried at 30 ° c . in a drier for a period of 48 hours until reaching a final humidity of 5 %. the fermented substrate obtained in this way is ground using a ball mill into particles of less than 0 . 6 mm in size . the number of oospores per 1 g of preparation and viability are then determined in the preparation of technical quality prepared in this way according to the procedures presented below . around 1 g of the prepared technical preparation of pythium oligandrum is weighed precisely on analytical scales and mixed for 1 minute in a mixer in distilled water so that a concentration of exactly 2 g per liter is maintained . then around 1 ml of the suspension is transferred to a sedgewick - rafter counting chamber and the individual characteristic oospores and the oospores in clusters are counted under a microscope so that a minimum of 100 oospores is counted in total . the entire measurement is repeated three times , whereby the suspension of oospores is carefully mixed before each sample is taken to the chamber . finally , the number of oospores per 1 g of preparation is calculated using the formula n =( a · c )/( b · d · e ), where a is the number of oospores identified ; b is the volume of one square in the chamber ( 1 × 1 × 0 . 1 mm = 1 × 10 − 4 ml ); c is the volume of distilled water used to prepare the sample in ml ( 500 ml ); d is the weight of the sample in g ( 1 g ), and ; e is the number of squares in the chamber in which the identified number of oospores was actually counted . the germination ( viability ) of the technical preparation pythium oligandrum is determined by precisely weighing approximately 10 mg of the technical preparation on analytical scales , and mixing it in water with a vortex mixer at a concentration of 1 mg per ml . we prepare three subsequent tenfold dilutions using the suspension prepared in this way , and then plated 100 μl of each dilution on a mea ( melt extract agar ) medium . we take readings of germination after 8 hours and 16 hours of cultivation in a drier at 28 ° c ., and then take readings of the final number of colonies after 7 days of cultivation . the number of oospores obtained ranges from 0 . 8 to 1 . 4 × 10 6 per gram of preparation according to this invention depending on the batch used . germination ( viability ) usually ranges from 2 to 10 %. the batch used in example of implementation 1 had 1 . 0 × 10 6 oospores per g and germination of 13 . 1 %, meaning that it contained 0 . 131 × 10 6 colony forming units ( cfu ) per 1 g . the original capnocytophaga sputigena ccm3712 culture is stored at a temperature of − 70 ° c . after delivery from the collection of microorganisms . for cultivation , a small amount of the culture is first transferred to a dish with “ chocolate agar ”, containing trypticase soy agar with 0 . 1 % yeast extract and 5 % defibrinated horse blood . the dishes were cultivated in 5 % co 2 at 37 ° c . overnight . the next day , the colonies were transferred to a liquid culture containing trypticase soy substrate comprising 0 . 1 % yeast extract , 0 . 002 % equine hemine iii , 0 . 0001 % menadione and 0 . 1 % sodium bicarbonate . the culture was shaken in an atmosphere of 5 % co 2 in an orbital shaker at 200 revolutions per minute . as soon as the culture reached the middle of the logarithmic phase of 10 ° cells per ml , the bacteria were sedimented with centrifugation 10000 × g av for a period of 20 minutes and then washed three times in 0 . 1 m trisodium citrate buffer solution , ph 6 . 0 . after the final wash , the bacteria sediment was carefully separated from the remainder of the buffer solution and dried using freeze drying ( lyophilization ). one gram of lyophilized bacteria containing 10 9 cfu per one milligram of powder was obtained from one liter of the culture using this procedure . 1 . 1 . 3 preparation of the final formula of the dual preparation for use in the oral cavity . three different version of the preparation were used to prepare the final formula , differing from each other in terms of their relative content of capnocytophaga sputigena ccm3712 bacterium . these three versions were marked with the abbreviated names plaquea , plaqueb and plaquec . the preparations were prepared for pressing into effervescent tablets of a total weight of 3 g for one application and for this reason individual formulae are converted to this weight . the components of individual preparations are shown in table 1 . the pythium oligandrum component is contained in the dual microbial preparation plaquea , plaqueb and plaquec in a quantity of 0 . 6663 × 10 4 cfu per 1 g . the capnocytophaga sputigena ccm3712 component is contained in dual microbial preparation plaquea in a quantity of 0 . 333 × 10 8 cfu per 1 gram ; in the plaqueb preparation in a quantity of 0 . 333 cfu × 10 9 per 1 gram ; and in the plaquec preparation in a quantity of 3 . 333 × 10 10 cfu per 1 gram . the test of the stability of the dual microbial preparation for use in the oral cavity must ascertain whether both microbial components have acted negatively on each other during the period of storage of the prepared preparation , at the very least for the period of conducting an in vitro and in vivo effectiveness test . one tablet with the plaquea , plaqueb or plaquec preparation is dissolved in 100 ml of lukewarm water at a temperature of around 35 ° c . after it has dissolved completely , 1 ml is taken to determine the germination of pythium and 0 . 1 ml to determine the cfu of capnocytophaga . in order to determine cfu in capnocytophaga , we diluted the sample using a series of samples with tenfold serial dilution . in order to determine cfu , we use the third dilution for plaquea , for example dilution of 10 3 ×, the fourth dilution of plaqueb , for example dilution of 10 4 ×, and the fifth dilution for plaquec , for example dilution of 10 5 ×. the stability test is conducted immediately after the preparation has been made and again after 1 , 2 , 3 , 4 , 5 and 6 months . the results of a typical stability test are shown in fig2 , which depicts the dependency of the number of colonies on the months of storage . it can be concluded from the result that the emergence of pythium during six months of storage and subsequent use of the preparation firstly declined somewhat and then stabilized at a value corresponding to approximately 75 % of the originally - declared nominal value . by contrast , for capnocytophaga there were no significant changes in viability during the test period , viability remaining at the originally declared value . the result shown in fig2 relates to the preparation termed plaqueb . the results obtained with the plaquea and plaquec preparations were essentially identical and therefore are not shown in the graph ( to ensure that the graph is clearer ). it can be stated , therefore , that neither pressing the original active components into an effervescent tablet nor its activation before use of the dual microbial preparation according to this invention reduced in any significant way the viability of any component of the dual preparation for use in the oral cavity . 1 . 3 effectiveness test of prepared mixtures with the use of an in vitro test . the aim of in vitro effectiveness tests is to prove that the combination of the pythium oligandrum m1 atcc38472 oomycete and the bacterium of the green oral complex capnocytophaga sputigena ccm3712 will act synergistically , meaning that there will be an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . from this perspective , a standard laboratory test was chosen of suppressing the growth of candida albicans pathogenic yeasts of three different hypha - forming strains obtained from clinical isolates at the hospital in pardubice , the czech republic , and transferred for further experiments to the institute of microbiology at the czech academy of sciences ( laboratory of dr . kola { hacek over ( r )} rík ). the actual conducting of the test consisted of the use of a standard application record that is invariably commenced by dissolving a tablet with a content of pythium alone ( the chytrá houba ® pythie ® bioplus product ) or one of the test tablets specified above , termed plaquea , plaqueb and plaquec . the tablets were dissolved in 100 ml of lukewarm water at a temperature of approximately 35 ° c . a quantity of 12 . 5 ml of this suspension was then mixed directly with 12 . 5 ml double - concentrate mixture for the preparation of cda ( czapek dox agar ) agar plates . 10 5 candida albicans pathogenic yeasts were then evenly applied to the plates prepared in this way after cooling . the results of this test are shown below in table 2 . the results show that whereas pythium oligandrum alone had a significant influence on suppressing the growth of yeasts in the experiment dishes , its combination with capnocytophaga produced a further strengthening of the effect in the case of all three pathogenic yeasts obtained from clinical isolates . after a week of cultivation at 28 ° c ., a reading was taken of the number of colonies formed in comparison with the control dish , whereby the reduction in the number of colonies in contrast to the control is a gauge of the effectiveness of pythium . the most effective preparation among the yeast strains under in vitro conditions was preparation plaqueb , with the preparations termed plaquea and plaquec achieving approximately 80 % of this effectiveness . with regard to the demanding nature of clinical trials conducted with a large quantity of dual microbial preparation according to this invention , plaqueb , as the most effective preparation , was chosen as the preparation for preclinical and clinical trials . the cosmetic preparation biomycosin , with active substance pythium oligandrum , was tested on two groups of patients , diabetic and non - diabetic , with microbially inflamed non - healing wounds at the request of the regional hospital in pardubice . the participants in the study were not randomized , but their diabetic status was monitored , which made it possible to divided data into a diabetic and a non - diabetic group at the end of the study . as far as the non - diabetic patients are concerned , their average age was 63 . 7 ± 13 . 1 years , whereby the patients were within an age range of 37 - 79 years . the total number of monitored patients was 19 , 14 women and 5 men , because the incidence of inflamed varicose ulcers is higher in women than in men . the application of biomycosin was conducted at the hospital under the supervision of medical staff . out - patients were hospitalized for the period of application of the preparation . one pack of biomycosin was used for three ( eight hours each ) wet applications , whereby the applications were made over a total of four consecutive days . samples were taken three days after the final application for microbiological examination and out - patients were discharged . regular monthly controls were then conducted in out - patients and hospitalized patients for a period of 6 months following application . the results unambiguously showed a significant reduction in the microbial burden , in particular in g - rods , anaerobic microorganisms and pathogenic yeasts . a negative correlation between a reduction in the microbial burden of g9 + cocci and an improvement in the condition of the clinical infection in non - diabetic patients was clearly proven in a correlative analysis of the results obtained for such patients . this negative correlation was particularly striking for the category of “ other staphylococci ”, of which the staphylococcus epidermidis bacterium in particular is of fundamental importance to the skin . the negative correlation ( y ) for other staphylococci was characterized by the equation y =− 0 . 56x + 85 . 8 , with a correlation coefficient of r 2 = 0 . 87 . with respect to the dependency of the course of treatment in this illness on age and sex , because women are more susceptible , patients first had to be divided into two groups and randomized depending on age and sex . the average age of the patients in the control group corresponding to the conditions of treatment with a preparation containing only p . oligandrum was 77 . 6 ± 9 . 8 years . this parameter was 78 . 0 ± 10 . 3 years in the experimental group corresponding to treatment with a dual preparation . therefore , there was no statistically significant difference between the ages of the patients in both groups . three women and 2 men were included in both groups and the percentage of women in both groups was therefore identical at 67 %: the results obtained after both methods of application are shown in fig3 . the reciprocal relationship between the quantity of removed staphylococci and the reduced improvement of clinical symptoms of infection is absolutely clear from the results obtained . a high content of maintained staphylococci during treatment with the preparation having a content of the p . oligandrum oomycete is absolutely clear for the clinically responsive patients shown in fig3 , on the left - hand side , and a significant improvement in the clinical signs of infection , mostly 20 % to 50 % of the original condition , is also clear . by contrast , the group of clinically unresponsive patients , whose results are shown on the right - hand side of fig3 , show a very distinctive reduction in the number of staphylococci identified by cultivation ( including the staphylococcus epidermidis strain ); however , progress in removing the clinical symptoms of infection was very low in these patients . based on these results , we considered and verified the hypothesis that suppressing certain g + cocci was not desirable since it affected the physiological microflora representative . we tested this working hypothesis by preparing a total of three versions of the dual microbial preparation according to this invention for use in the long - term elimination of the symptoms of clinical infection in non - healing wounds . the stability of such preparations was proven and their effectiveness was monitored using the relevant laboratory test . a detailed description of the results obtained is presented hereunder in more detail in this example . altogether , the results of this testing proved that there was no negative influence on any of the components of the dual preparation when storing the form of a dry preparation . in comparison with the one - component preparation used so far , with a pythium oligandrum base , there was a statistically significant reduction in the microbial yeast load and an improvement in the development of clinical infection when using the microbial dual preparation . 2 . the preparation , control and testing of the effectiveness of a dual microbial preparation suitable for eliminating the symptoms of clinical infection in non - healing wounds in non - diabetics . 2 . 1 protocol for the preparation of a dual microbial preparation for use on non - healing wounds in non - diabetics . 2 . 1 . 1 preparation of the technical preparation pythium oligandrum m1 atcc 38472 . the procedure used for this preparation is identical to the procedure described in section 1 . 1 . 1 . the batch used in exemplary embodiment 2 had 1 . 1 × 10 6 oospores per g and germination of 14 . 3 %, meaning that it contained 0 . 157 × 10 6 colony forming units ( cfu ) per 1 g . the original staphylococcus epidermidis ccm2124 culture is stored at a temperature of − 70 ° c . after delivery from the collection of microorganisms . for cultivation , a small amount of the culture is first transferred to a dish with the tryptone soya agar cm131 oxoid . the dishes were cultivated at a temperature of 37 ° c . overnight . ten well - separated colonies were transferred to a 2 liter erlenmeyer flask containing 500 ml of medium comprising 30 g per liter peptone , 10 g per liter yeast autolysate , 5 g / l nacl , 0 . 1 g cacl 2 . 2h 2 o and 1 ml trybutyrin ; the ph of the medium was regulated at 8 . 0 . the bacteria were cultivated to the middle of the logarithmic phase , sedimented with centrifugation 10000 × g av for a period of 20 minutes and then washed three times in 0 . 1 m trisodium citrate buffer solution , ph 6 . 0 . after the final wash , the bacteria sediment was carefully separated from the remainder of the buffer solution and dried using freeze drying ( lyophilization ). approximately 0 . 6 g of lyophilized bacteria containing 10 8 cfu per one milligram of powder was obtained from one liter of the culture . 2 . 1 . 3 preparation of the final formula of a dual microbial preparation for use on non - healing wounds in non - diabetics . three different versions of the preparation were used in order to prepare the final formula , differing in terms of their relative content of the staphylococcus epidermidis ccm2124 bacterium ; these three versions were designated using the abbreviated working titles of nonheala , nonhealb and nonhealc . the preparations were prepared for use in one package containing 10 g of loose preparation for one application . the components of the individual preparations are presented below in table 3 on the next page . the pythium oligandrum component is contained in the dual microbial preparation nonheala and nonhealb in a quantity of 7 . 85 × 10 3 cfu per 1 g and in nonhealc in a quantity of 7 . 85 × 10 3 cfu per 1 g . the staphylococcus epidermidis ccm2124 component is contained in the dual microbial preparation nonheala in a quantity of 1 × 10 6 cfu per 1 g ; in the nonhealb preparation in a quantity of 1 × 10 t cfu per 1 g ; and in the nonhealc preparation in a quantity of 1 × 10 8 cfu per 1 g . 2 . 2 stability test of dual microbial preparation for use on non - healing wounds in non - diabetics . the test of the stability of the dual microbial preparation for use on non - healing wounds must ascertain whether both microbial components have acted negatively on each other during the period of storage of the prepared preparation , at the very least for the period of conducting in vitro and in vivo effectiveness tests . one package , 10 g of the nonheala , nonhealb or nonhealc preparation , is re - suspended in 250 ml of lukewarm physiological solution 9 g / l nacl at a temperature of around 35 ° c . after complete resuspension , 1 ml is taken to determine the germination of pythium and 0 . 1 ml to determine cfu of staphylococcus . in order to determine cfu for staphylococcus , we further dilute the sample using tenfold serial dilutions , and to determine cfu we use 1 × dilution for nonheala , for example dilution of 10 ×, 2 × dilution for nonhealb , for example dilution of 10 2 ×, and 3 × dilution for nonhealc , for example dilution of 10 3 ×. the stability test is conducted immediately after the preparation has been made and again after 1 , 2 , 3 , 4 , 5 and 6 months . the results of a typical stability test are shown in fig4 . it can be concluded from the result that the viability of pythium during six months of storage and subsequent use of the preparation firstly declined somewhat and then stabilized at a value corresponding to approximately 75 % of the originally - declared nominal value . by contrast , for staphylococcus there were no significant changes in viability during the test period , viability remaining et almost the originally declared value . the result shown in fig4 relates to the preparation termed nonhealb . the results obtained with the nonheala and nonhealc preparations were very similar and therefore are not shown in the graph ( to ensure that the graph is clearer ). it can therefore be stated that the viability of any of the components of the dual microbial preparation for use on non - healing wounds was not reduced by drying the original active components and storing them in the presence of silica gel without activation before the use of the preparation according to this invention . 2 . 3 effectiveness test of prepared mixtures of dual microbial preparation using an in vitro test . the aim of in vitro effectiveness tests is to prove that the combination of the pythium oligandrum m1 atcc38472 oomycete and the bacterium of the normal skin microbiome staphylococcus epidermidis ccm2124 will act synergically , meaning that there will be an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . from this perspective , a standard laboratory test was chosen of suppressing the growth of candida albicans pathogenic yeasts of three different hypha - forming strains obtained from clinical isolates at the hospital in pardubice , the czech republic , and transferred for further experiments to the institute of microbiology at the czech academy of sciences . the actual conducting of the test consisted of the use of a standard application protocol , which was invariably commenced by re - suspending a dry preparation containing only pythium ( the biomycosin preparation ) or one of the above - mentioned test preparations designated as nonheal a , b or c . preparations were re - suspended in 250 ml of lukewarm physiological solution at a temperature of around 35 ° c . a quantity of 12 . 5 ml of this suspension was then mixed directly with 12 . 5 ml double - concentrate mixture for the preparation of cda ( czapek dox agar ) agar plates . 10 5 candida albicans pathogenic yeasts obtained from clinical isolates was then evenly applied to the plates prepared in this way after cooling . after a week of cultivation at 28 ° c ., a reading was taken of the number of colonies in comparison with the control dish , whereby the reduction in the number of colonies in contrast to the control is a gauge of the effectiveness of pythium . the results of this test are shown below in table 4 . the results show that whereas pythium alone had a significant influence on suppressing the growth of yeasts in the experiment dishes , its combination with staphylococcus produced a further strengthening of the effect . the most effective preparation among the three pathogenic yeast strains under in vitro conditions was preparation nonhealb , with the preparations termed nonheala and nonhealc achieving approximately 50 % of this effectiveness . with regard to the demanding nature of clinical trials conducted with a large quantity of dual microbial preparation according to this invention , the most effective preparation , nonhealb , was proposed as the starting preparation for preclinical and clinical trials . the example shown is not limited to the use of the dual microbial preparation specified in this exemplary embodiment for non - healing wounds on skin . there are naturally other possible uses not specified here ; for example use in different cases of opportunistic microbial infections accompanied by dysbiosis on the skin , such as atopic eczema and psoriasis . convincing evidence was taken from clinical observations in the area of care for oral cavity health , non - healing wounds and suppressing yeasts in vaginal candidiasis that pythium oligandrum suppresses and kills pathogenic yeasts of the candida and malassezia strains . this conclusion was then independently confirmed by a laboratory cultivation test at the institute of microbiology of the czech academy of sciences in prague . a total of 4 clinical strains of the pathogenic yeast candida albicans , isolated at pardubice hospital , were tested in a laboratory competition test , in that this ascertained their ability to pass to hyphal growth , which acts as evidence of the clinical aggression of these strains . the strains involved were candida albicans 2508 , 2548 , 2558 and 2944 . pythium oligandrum fully outgrew all four tested strains in the competition test in the case of using mea ( malt extract agar ) as the medium and pda ( potato dextrose agar ) as the medium . after 10 days of the experiment , meaning once the experiment had come to an end , the remains of the yeast strains overgrown with pythium were transferred to a medium of cda ( czapek - dox agar ). yeasts grow in this medium , but not pythium oligandrum . nonetheless , there was no growth of yeasts in this test medium , which shows that pythium oligandrum not only overgrew the pathogenic yeast cells , but killed them . therefore , following on from the results of these laboratory test results , systematic studies were conducted in cooperation with doctors at gynecological surgeries in prague , rychnov nad kn { hacek over ( e )}{ hacek over ( z )} nou , vysoké mýto , klá { hacek over ( s )} terec nad oh { hacek over ( r )} í , pilsen and uhlísk6 janovice comparing the effects of the chemical antimycotic clotrimazol and the feelfresh preparation among women with recurring incidence of vaginal candidiasis . a clinical evaluation of the women was conducted by a doctor at the beginning of testing on a six - point scale that considered discomfort , the presence of vaginal discharge , irritation , pain , burning and reddening . an initial microbiological examination was conducted to identify the presence of pathogenic yeasts of the candida strain and of lactobacilli , which are the dominant microorganisms in the normal vaginal microbiome . application was made for a period of 5 consecutive days , either clotrimazol being applied or a hip bath with the feelfresh preparation being used . a microbiological control was then conducted after 10 days and 1 month . a final clinical study and a microbiological control were then carried out three months following application . the intensity of clinical symptoms in the control group with the clotrimazol application and in the experimental groups with the feelfresh application was comparable at the beginning of the study . after 3 months , however , the results of assessing clinical symptoms ( the number of clinical symptoms identified ) were statistically better for the group applying feelfresh in comparison with the chemical antimycotic clotrimazol : 2 . 57 ± 0 . 53 and 1 . 00 ± 1 . 52 , p = 0 . 035 . it was possible to monitor the intensity of yeast infection by way of cultivation after 10 days , 1 month and 3 months , meaning more frequently than the clinical picture . the results were also very interesting in this regard since they showed a rapid and statistically significant reduction in the incidence of pathogenic yeasts for clotrimazol after 10 days , when the initial values in arbitrary units were 3 . 71 ± 0 . 75 and 3 . 71 ± 0 . 48 for the application with clotrimazol and feelfresh . after 10 days , however , the corresponding values in arbitrary units were 1 . 28 ± 0 . 48 and 1 . 85 ± 0 . 38 , p = 0 . 04 . nonetheless , after 1 month , or even 3 months , the situation had reversed completely and the reduced level of pathogenic yeasts was stabilized only among patients using feelfresh , when the values after 1 month for control and application in arbitrary units of feelfresh were 2 . 42 ± 0 . 53 and 1 . 28 ± 0 . 48 , p = 0 . 008 . the values in arbitrary units after 3 months were 3 . 14 ± 0 . 69 and 1 . 42 ± 0 . 78 , p = 0 . 007 . the positive correlation between improvement in clinical condition and the concentration of lactobacilli on the one hand and between the elimination of yeast infection and the concentration of lactobacilli on the other was also very interesting . these results clearly show , as with the previous examples presented , that the curative effect of the pythium oligandrum microorganism contained in the feelfresh preparation is significantly potentiated by the presence of healthy microflora , in this case lactobacilli . a working hypothesis could be drawn from these results that the presence of lactobacilli significantly increases the effectiveness of suppressing yeasts through the pythium oligandrum oomycete . a study to prove the role of lactobacilli proceeded using the double - blind method , in which envelopes with the preparations were prepared and numbered by an administration worker not actually involved in the course of the study . after confirming the clinical diagnosis in the first microbiology sample , the patients signed informed consent and were instructed how to apply the preparations . the application was made in the form of a rinse over five consecutive days using a provided vaginal applicator . a gynecologist undertook a professional examination at the beginning of the study and again after 3 months , in that the level of yeast infection and the level of colonization of the vaginal membrane with lactobacilli were invariably checked . the results of the clinical study are clearly presented in graphic format in fig5 a , 5b and 5c . from the perspective of clinical evaluation at the beginning and at the end of the study , the control group , applying the classic preparation containing only pythium oligandrum , is variable : there was effective elimination of symptoms among certain women , but not in other patients , as is clear from fig5 a . for this reason patients were divided into two groups of clinically responsive patients on the left - hand side of fig5 and clinically unresponsive patients on the right - hand side of the figure . there was a significant reduction in the incidence of the candida albicans yeast among clinically responsive patients after three months in all cases , whereas there was no such reduction among unresponsive patients ( one patient actually experienced an increase — fig5 b ). nonetheless , it is still interesting that the incidence of lactobacilli was far higher among responsive patients in all cases , increasing to physiologically high values during the study ( fig5 c ), whereas the incidence of yeast was considerably lower among unresponsive patients , accompanied in two cases by further reduction . these results confirm that the dual microbial preparation for use in suppressing and eliminating pathogenic yeasts shows statistically significantly better results in comparison with a standard preparation in all parameters . we therefore verified and tested this hypothesis : a total of three versions of the dual microbial preparation according to this invention were prepared for use for vaginal candidiasis , cavity and the stability of such preparations was proven , clinical tests also being carried out in patients with such a diagnosis . a detailed description of the results obtained is presented in example 3 for this invention below . the aggregate results of this testing showed that the effective microbiological components had no negative influence on each other during drying and storage under dry conditions . the results of laboratory tests show the greatest effectiveness in the dual preparation vaginalb . 3 . preparation , control and testing of the effectiveness of the dual microbial preparation according to this invention for application on skin and membrane which is susceptible to the incidence of pathogenic yeasts . 3 . 1 protocol for the preparation of the dual microbial preparation for application on skin and membrane which is susceptible to the incidence of yeasts . 3 . 1 . 1 preparation of the technical preparation pythium oligandrum m1 atcc 38472 . the procedure used for this preparation is identical to the procedure described in section 1 . 1 . 1 . the batch used in exemplary embodiment 3 had 0 . 8 × 10 6 oospores per g and germination of 12 . 8 %, meaning that it contained 0 . 102 × 10 6 colony forming units ( cfu ) per 1 g of preparation . the original lactobacillus crispatus ccm7010 culture is stored at a temperature of − 70 ° c . after delivery from the collection of microorganisms . for cultivation , a small amount of the culture is first transferred to an agar dish with medium for the cultivation of lactobacilli , containing 5 g per liter yeast autolysate , 10 g per liter bovine extract , 10 g per liter peptone , 20 g per liter glucose , 5 ml per liter tween 80 , 2 g per liter k 2 hpo 4 , 5 g per liter sodium acetate , 2 g per liter diamonium citrate , 0 . 2 g per liter mgso4 . 7h 2 o and 0 . 05 g per liter mnso 4 . 7h 2 o . we regulate the ph after adding all components at a value of ph 6 . 2 - 6 . 6 . the dishes were cultivated at a temperature of 37 ° c . overnight . ten well - separated colonies were transferred to a 2 - liter erlenmeyer flask containing 500 ml of medium 6 , having the components specified above . the bacteria were cultivated to the middle of the logarithmic phase , sedimented with centrifugation 10000 × g , for a period of 20 minutes and then washed three times in 0 . 1 m trisodium citrate buffer solution , ph 6 . 0 . after the final wash , the bacteria sediment was carefully separated from the remainder of the buffer solution and dried using freeze drying ( lyophilization ). approximately 0 . 8 g of lyophilized bacteria containing 10 9 cfu per one mg of powder was obtained from one liter of the culture . 3 . 1 . 3 preparation of the final formula of the dual microbial preparation for application on skin and membrane which is susceptible to the incidence of yeasts . three different versions of the preparation were used in order to prepare the final formulation , differing in terms of their relative content of the lactobacillus crispatus ccm7010 bacterium ; these three versions were designated using the abbreviated titles of vaginala , vaginalb and vaginalc . the preparations were prepared for use in one package containing 2 g of loose preparation for one application . the components of the individual preparations are presented below in table 5 on the next page . the active components in the preparation are pythium and lactobacillus . silica gel is added as a drying agent in order to preserve the original properties and the emergence of both active components . a small amount of added sodium chloride aids better activation of the biological agent , chamomile aroma acts as perfume . the pythium oligandrum component is contained in the dual microbial preparation vaginala and vaginal b in a quantity of 19 . 2 × 10 3 cfu per 1 g and in vaginalc in a quantity of 19 . 2 × 10 3 cfu per 1 g . the lactobacillus crispatus ccm7010 component is contained in the dual microbial preparation vaginala in a quantity of 0 . 5 × 10 8 cfu , in the vaginalb preparation in a quantity of 0 . 5 × 10 9 cfu per 1 g and in the vaginalc preparation in a quantity of 0 . 5 × 10 10 cfu per 1 g . 3 . 2 stability test of dual preparation for use on skin and membrane which is susceptible to the incidence of yeasts . the test of the stability of the dual microbial preparation for use on skin and membrane which is susceptible to the incidence of yeasts must ascertain whether both microbial components have acted negatively on each other during the period of storage of the prepared preparation , at the very least for the period of conducting in vitro and in vivo effectiveness tests . one pack of 2 g of the vaginala , vaginalb or vaginalc preparation is re - suspended in 500 ml of lukewarm water at a temperature of around 35 ° c . after complete resuspension , 1 ml is taken to determine the germination of pythium oligandrum and 0 . 1 ml to determine the cfu of lactobacillus units . in order to determine cfu for lactobacillus , we further dilute the sample using tenfold serial dilutions , and to determine cfu we use 1 × dilution for vaginala — dilution of 100 ×- 2 × dilution for vaginalb — dilution of 10 ×- and 3 × dilution for vaginalc — dilution of 10 4 ×. the stability test is conducted immediately after the preparation has been mixed and again after 1 , 2 , 3 , 4 , 5 and 6 months . the results of a typical stability test are shown in fig6 . it can be concluded from the result that the viability of pythium during six months of storage and subsequent use of the preparation firstly declined somewhat and then stabilized at a value corresponding to approximately 80 % of the originally - declared nominal value . by contrast , there was only a slight reduction in viability of around 10 % for lactobacillus during the test period . the result shown in fig6 relates to the preparation termed vaginalb . the results obtained with the vaginala and vaginalc preparations were very similar and therefore are not shown in the graph ( to ensure that the graph is clearer ). it can therefore be stated that the emergence of any of the components of the dual microbial preparation for use in the oral cavity was not significantly reduced by drying the original active components and storing them in the presence of silica gel or its activation before the use of the preparation according to this invention . 3 . 3 . effectiveness test of prepared mixtures with the use of an in vitro test . the aim of in vitro effectiveness tests is to prove that the combination of the pythium oligandrum m1 atcc38472 and healthy bacterium of the normal vaginal microbiome lactobacillus crispatus ccm7010 will act synergically , meaning that there will be an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . from this perspective , a standard laboratory test was chosen of suppressing the growth of candida albicans pathogenic yeasts of three different hypha - forming strains obtained from clinical isolates at the hospital in pardubice , the czech republic , and transferred for further experiments to the institute of microbiology at the czech academy of sciences ( laboratory of dr . kola { hacek over ( r )} ik ). the actual conducting of the test consisted of the use of a standard application protocol , which was invariably commenced by re - suspending a dry preparation containing only pythium oligandrum ( the feelfresh preparation ) or one of the above - mentioned test preparations designated as vaginala , vaginalb or vaginalc . preparations were re - suspended in 500 ml of lukewarm water of a temperature of around 35 ° c . and 12 . 5 ml of this suspension was then mixed directly with 12 . 5 ml of double - concentrate mixture , cooled to a temperature of 45 ° c ., for the preparation of cda ( czapek dox agar ) agar plates . 10 5 candida albicans pathogenic yeasts obtained from clinical isolates were then evenly applied to the plates prepared in this way after cooling . after a week of cultivation at 28 ° c ., a reading was taken of the number of grown colonies in comparison with the control dish , whereby the reduction in the number of colonies in contrast to the control is a gauge of the effectiveness of pythium oligandrum . the results of this test are shown below in table 6 . the results show that whereas pythium oligandrum alone had a significant influence on suppressing the growth of yeasts in the experiment dishes , its combination with lactobacillus produced a further strengthening of the effect . the most effective preparation among the three pathogenic yeast strains under in vitro conditions was the vaginalb preparation , with the preparations termed vaginala and vaginalc achieving approximately 50 % of this effectiveness . with regard to the demanding nature of clinical trials conducted with a large quantity of preparation , vaginalb , as the most effective dual microbial preparation , was chosen for preclinical and clinical trials . the significant positive correlation between the presence of yeasts and improvement in the clinical condition of infection in non - healing wounds was seen in the same way for diabetic and non - diabetic patients . the relevant correlation coefficients were 0 . 4 and 1 . 0 . this correlation led to consideration of whether the presence of yeasts or their components might cause activation of the pythium oligandrum microorganism . with respect to the fact that biological tests concurrently containing three different microorganisms are very complicated from the perspective of their execution and evaluation , alternatives were chosen including dead saccharomyces cerevisiae baker &# 39 ; s yeasts in the form of so - called yeast autolysate , which is commonly commercially available . a standard plate test on an agar mea was used . its components were as follows : 20 g per liter malt extract , 20 g per liter glucose , 1 g per liter peptone and 20 g per liter agar . interaction was monitored of the pythium oligandrum oomycete and the dermatophytes trichophyton interdigitale dmf2477 and microsporum fulvum p245 , for which previously tests were conducted to show that they suppress the growth of these dermatophytes to around 50 % uninhibited growth . however , pythium was activated after adding yeast extract in a concentrate of 1 g per liter to the agar carrier and there was an increase in growth inhibition to 75 % for trichophyton and 80 % for microsporum . therefore , this result also showed how the result of the biological abilities of pythium to suppress certain dermatophytes is dependent on its physiological state , in that its biological ability could be significantly enhanced by the presence of even inactivated target organisms or another form of their processed components and extracts , in this case commercial yeast autolysate . a working hypothesis could be drawn from these results that even inactivated yeasts have an influence on increasing the effectiveness of the pythium oligandrum oomycete . the optimum dual preparation to eliminate the agents of mycosis ( dermaphytosis ) was tested in patients with mycotic diseases of the feet incorporating dermatophytic infection on the soles , between the toes and under the nails — onychomycosis . the curative effect was clinically evaluated by dermatologists and microbiological tests were also carried out at the beginning and the end of the period under consideration . patients were randomized into two groups . in the first group , treatment was provided with a one - component preparation with pythium oligandrum oomycete content and in the second group a combined preparation with yeast autolysate content was administered . the results showed good clinical effectiveness for the preparation containing only pythium oligandrum , but the clinical effectiveness for the dual microbial preparation was even better ( control 3 . 0 ± 0 , experiment 3 . 6 t 0 . 5 , t - test , p = 0 . 01 ). the improvement in microbiology according to fig9 c was similar ( control 2 . 0 ± 0 , experiment 2 . 6 ± 0 . 5 , t - test , p = 0 . 01 ). these results confirm that the dual microbial preparation according to the invention for use on mycosis of the feet shows statistically significantly better results in comparison with the standard one - component preparation . a total of 25 cavia porcellus guinea pigs underwent a study to monitor the effects of a one - component preparation containing the pythium oligandrum oomycete . the effect of the preparation was evaluated by veterinarians and confirmed by taking microbiological samples at the beginning and at the end of the period under consideration , whereby all specimens were negative for the presence of dermatophytic funguses and yeasts at the end of the experiment . the overall score of the clinical evaluation of the effects of the preparation was 1 . 72 ± 0 . 87 , p & lt ; 0 . 05 , in that 1 = excellent effect and 4 = no effect . the zoonotic characteristic of dermatophytic infections which scientists frequently describe guarantees us that the mechanism of these infections in animals is identical to mycotic infections in humans . with respect to the demanding nature of clinical tests and the fact that the results obtained in evaluating the effectiveness of the one - component preparation corresponded to them in a comparison of tested human and animal groups , section 4 . 3 may be referred to for further verification of the effectiveness of the dual preparation . both series of the experiments carried out above showed the potentiating influence of yeasts or products of their metabolism on the effectiveness of preparations containing pythium oligandrum in eliminating the agents of dermatophytosis . this potentiating effect could be caused by the activation by live yeasts or the activation of their metabolites without the immediate requirement of the incidence of live yeasts . the final option showed itself to be more likely in systematic research and the hypothesis was therefore put forward that pythium oligandrum is stimulated in its abilities to eliminate dermatophytes by certain components relating to the yeast metabolism . this working hypothesis was therefore tested : a total of three versions of the dual microbial preparation according to this invention were prepared for use on mycosis of the feet and the stability of such preparations was proven , clinical tests also being carried out on patients having this disease . a detailed description of the results obtained is presented in example 4 for this invention . altogether , the results of this testing showed that , in comparison with the live version of dual microbial preparations , a relatively large quantity of the inactivated , dead partner microorganism or its components must be added , the addition of up to around 10 weight percent proving effective in the case of the yeast autolysate tested . there really was a statistically significant increase in effectiveness in the preparation prepared in this way against the classic one - component preparation . this increase was equivalent to the increase observed in the case that the yeast infection appeared endogenously as a result of naturally - occurring co - infection , which occurs in approximately 20 % of cases . 4 . the preparation , control and testing of the effectiveness of the dual microbial preparation according to this invention as suitable for application in the case of mycosis of the feet , including onychomycosis in people and dermatophytes in animals . 4 . 1 protocol for the preparation of a dual preparation for use on mycosis in humans and animals . 4 . 1 . 1 preparation of the technical preparation pythium oligandrum m1 atcc 38472 . the procedure used for this preparation is identical to the procedure described in section 1 . 1 . 1 . the batch used in exemplary embodiment 4 had 1 . 3 × 10 6 oospores per g and germination of 14 . 5 %, meaning that it contained 0 . 189 × 10 6 colony forming units ( cfu ) per 1 g of preparation . 4 . 1 . 2 yeast autolysate , the second component in the preparation , was bought from oxoid . 4 . 1 . 3 preparation of the final formula of the dual preparation for application on mycosis . three different version of the preparation were used to prepare the final formula , differing from each other in terms of their relative content of yeast autolysate . these three versions were given the abbreviated names of mycosina , mycosinb and mycosinc . the preparations were prepared for pressing into effervescent tablets of a total weight of 3 g and for this reason individual formulae are converted to this weight . the components of individual preparations are shown below in table 7 . the pythium oligandrum component is contained in the dual microbial preparation mycosina , mycosinb and mycosinc in a quantity of 12 . 6 × 10 3 cfu per 1 g . the content of yeast autotysate is contained in the mycosina dual microbial preparation in a quantity of 50 mg per 3 g tablet , meaning a quantity of 1 . 66 % weight for one tablet ; in the mycosinb preparation in a quantity of 100 mg per 3 g tablet , meaning a quantity of 3 . 33 % for one tablet ; and in the mycosinc preparation in a quantity of 150 mg per 3 g tablet , meaning a 5 % weight for one 3 g tablet . a stability test was not conducted for the preparation in question since it is well - known from literary sources that the presence of yeast autolysate in a dried preparation does not influence the properties of the pythium oligandrum oomycete . 4 . 3 effectiveness test of prepared mixtures with the use of an in vitro test . the aim of in vitro effectiveness tests is to prove that the combination of pythium oligandrum m1 atcc38472 oomycete and inactivated yeast components contained in yeast autolysate acts synergically , meaning that there is an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . from this perspective , a standard laboratory test was chosen of suppressing the growth of four types of dermatophytes , for which it is proven that they are known agents of mycotic diseases of a zoonotic character . specifically , the tests were conducted with the use of four common dermatophytes , two different strains of each being used : trichophyton interdigitale ( ti ), trichophyton erinacei ( te ), microsporum fulvum ( mf ) and microsporum canis ( mc ). the results of determining the activities of individual preparations are presented in fig7 , in that the number of formed colonies of dermatophytes is related to the control without an active substance . the results shown in fig7 concern the mycosinb preparation , the results obtained with the mycosina and mycosinc preparations being qualitatively similar , but with resultant ability to suppress dermatophytes of only around 30 % of the increase on the control . the result in fig7 clearly shows that a simple preparation containing only pythium oligandrum as an active substance has the ability to suppress dermatophytes , suppression at a level of 50 - 60 % of the control growth being achieved . yeast autolysate itself had no ability to suppress the growth of dermatophytes in the test used , the results being at roughly the same level as the control experiment . a statistically significant increase in effectiveness was shown for the dual preparation at a level of approximately 10 % of infections in control samples . with regard to the demanding nature of clinical trials conducted with a large quantity of preparations according to this invention , the most effective preparation , mycosinb , was proposed as the starting preparation for preclinical and clinical trials . the activation of the other unique abilities of the microscopic oomycete pythium oligandrum by the components of healthy microflora is also worth noting . the unique ability of our technical substance , pythium oligandrum , to disrupt the biofilms formed by pathogenic bacteria populating non - healing wounds was proven in our previous studies . the results of the study were presented at the 12 th international “ interdisciplinary collaboration in the treatment of wounds and skin defects ” congress held on 23 and 24 january at the faculty of health studies at the university of pardubice . the measurements conducted showed that 129 of the 160 tested bacterial strains clinically isolated from non - healing wounds created biofilm . a 70 % reduction in the creation of biofilm was also proven in biofilm - positive strains after the addition of germs of the pythium oligandrum oomycete using the biomycosin preparation , in that the reduction of biofilm was very significant in 43 % of the tested strains , there occurring a reduction in the activity of biofilm creation of more than 50 %. no change in the intensity of creation of biofilm was recorded in 17 % of the strains examined in vitro , while no reproducible results were obtained in the remaining 13 % of the monitored strains . it was possible to influence the production of biofilms at least in vitro in the case of the observed stenotrophomonas maltophilia and pseudomonas aeruginosa strains . if , however , bacteria of the normal skin microbiome staphylococcus epidermidis were added to the incubation mixture in addition to germs of the oomycete , there was a reduction in the creation of biofilms of more than 50 % even in the two awkward strains already mentioned . this interesting phenomenon , together with the knowledge already published [ 32 ], shows that in addition to their metabolic regulatory functions , the components of the normal physiological microbiome might also play a part in reducing the intensity of biofilm created with the participation of pathogenic microorganisms . the aim of in vitro effectiveness tests is to prove that the combination of the pythium oligandrum m1 atcc38472 oomycete and the healthy bacterium of the normal skin microbiome will act synergically , meaning that there will be an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . another extremely important factor which must be verified is the specificity of such action . components of the physiological microbiome were often applied in the previous experiments in probiotic preparations , without of course maintaining topological specificity . the effectiveness of preparations provided in this way was mostly very low , which correlates well with the differing microbial composition of physiological microbiomes in topologically different locations of the human body with different levels of exposure to the outside environment and different metabolic niches . a simple laboratory test of the creation of biofilms was therefore used to verify this hypothesis , as is described in this exemplary embodiment . the creation of biofilm was measured based on the absorption of blue microbial dye , depicting the intensity of the creation of biofilm , whereby each experiment was conducted three times independently in triplicate [ 38 ]. this viability was measured using a test of vital staining according to literature [ 39 ] in order to prove the influence of the viability of the microorganisms in biofilms . the results of these determinations , conducted with preparations containing only the microscopic oomycete p . oligandrum according to invention cz 302 297 b on the one hand and with the optimized dual microbial preparations described in exemplary embodiments 1 , 2 and 3 according to the invention submitted on the other , unambiguously showed that the optimum disruption of biofilms always occurred only in the case of dual preparations comprising the physiological microbial component contained in the microbiome of the relevant topology . biofilms of the oral cavity , therefore , were most effectively disrupted by a dual microbial preparation containing the dominant physiological microbe for this location , whereas the effect of other physiological components was minor . similar evidence of specificity was also shown in the case of the skin and vaginal microbiome . the results obtained in this way are very important because they show that there is no universal probiotic or dual microbial preparation which is suitable for long - term suppression of the symptoms of opportunistic microbial infections — only a preparation having a content of topologically relevant microbial components is invariably effective . in this test , simple laboratory tests were conducted on the creation and viability of biofilms using two oral cavity bacteria , streptococcus gordonii and fusobacterium nucleatum , as the monitored components . both these bacteria have a key role in the creation of microbial biofilms of the oral cavity : streptococcus is the only bacterium capable of directly catching on tooth enamel in the aggressive environment of the oral cavity , whereas the large ( in terms of dimensions ) bacterium of the fusobacterium genus creates important retaining centers for the colonization of bacteria of the orange and red complex in invasive pathogens of type aggregatibacter actinomycetemcommitans . the results of the laboratory test are shown in table 8 ( above ). the results clearly show that whereas the influence on biofilm was lesser for a simple preparation containing only the p . oligandrum oomycete , only the use of the plaqueb dual preparation led to very marked disruption and restriction of the viability of the oral biofilm . the somewhat more significant disruption of biofilm caused by the feelfresh preparation and vaginalb can be ascribed to the higher percentage content of pythium in this preparation . 5 . 2 the influence of dual microbial preparations on the disruption of biofilms containing bacteria in non - healing wounds in this test , simple laboratory tests were conducted on the creation and viability of biofilms using two bacteria creating biofilms in non - healing wounds , stenotrophomonas maltophilia and pseudomonas aeruginosa , as the monitored components . these bacteria were used for the reason that in previous tests conducted by dr . karel mencl at the department of clinical microbiology at pardubice hospital , these two strains were resistant to the disruption of biofilm using simple preparations having only the pythium oligandrum oomycete . the results shown in table 9 obviously clearly show that a dual microbial preparation intended for improving clinical infection in non - healing wounds and containing the normal skin microbiome component staphylococcus epidermidis was able to effectively overcome this shortcoming and ensure effective disruption of biofilm in these resistant types . a certain lesser influence was also recorded for the vaginalb preparation containing lactobacilli , for which such activity has been described [ 33 ]. in this test , simple laboratory tests were conducted on the creation and viability of biofilms using the candida albicans yeast as the monitored component . more effective disruption of this biofilm occurred with the use of the dual microbial preparation vaginalb , as is evident from the results shown in table 10 above . this knowledge could be of general significance , because the pathogenic yeast candida albicans frequently creates combined . in this experiment were used the simple laboratory tests and viability of the biofilm using as monitored component candida albicans . the most effective ruption of the biofilm occurred by using the dual microbial preparation vaginalb , as is evident from the results in table 10 the above . these knowledges may have a general significance , because the pathogenic yeast candida albicans often creates the combined microbial biofilms in combination with certain types of pathogenic bacteria . a major asset of the dual microbial preparations according to the submitted invention is the opportunity to use them for prevention , for which values of active components which are up to ten times lower can be used based on the possibility of effective colonization and long - term propagation at the point of application . the effective mycoparasitism of the p . oligandrum oomycete and the anti - fungal action of certain other microbial strains may obviously also be used to protect the living and working environment from molds . it is well described in medical literature that the molds occurring in the living and working environment can shoot harmful spores into their surroundings . if their average concentration in the air exceeds a value of 500 viable spores per 1 m 3 , harmful effects leading to allergies , respiratory illnesses and even depression could be manifested in full according to the standards set by the world health organization ( who ). the anti - mold product biorepel , containing p . oligandrum and used mainly to eliminate mold from walls , ceilings , floors and other areas of contaminated rooms , was developed and is successfully used based on invention cz 302 297 . other microbial preparations that are capable of eliminating mold based on the principle of antibiosis and that primarily use for such purposes the abundantly widespread bacillus amyloliquefaciens bacillus is also used for this purpose . the effectiveness of a dual preparation in eliminating molds and yeasts from the living environment based on a combination of the two above - mentioned microorganisms is tested in field experiments under the conditions of their actual use . the experimental object is , for example , a wall in a damp room with even incidence of the black mold aspergillus niger , in that we measure the concentration of spores in the rooms before application and 6 months after application and quantitatively evaluate the presence of mold directly in smears of material taken from walls in a standard way . for application , 3 g of the preparation is divided into two bags , bag a containing 1 g of the preparation and bag b containing 2 g of the preparation . bag a is re - suspended in 10 liters of lukewarm water and the whole of the affected area is rubbed with a sponge soaked in this preparation . after a gentle drying , the same area is rubbed with a sponge soaked in solution b , which is prepared by re - suspending bag b in 1 . 5 liters of lukewarm water . samples to ascertain effectiveness are taken after 1 month , 3 months and 6 months , at which time the field experiment is ended . a preparation is considered effective in the case in which the number of spores in the fall is reduced to less than 500 ( who standard ) and the presence of mold ascertained using a cultivation test is reduced to level 1 ( present sporadically only after cultivation ). the specific result of field experiments at two different locations is depicted in fig8 . it is clear that the required reduction in the concentration of spores in the atmosphere was reduced to a value of less than 500 in both locations observed only when using the dual preparation , even though both its components showed certain reduction against the control ( fig8 a ). similarly , only the dual preparation reduced the actual incidence of mold on the wall , proven by a smear test , to the target value of around 1 . 0 . in this case too , the effect of individual preparations was only partial and the target values were not achieved ( fig8 b ). these results confirm the hypothesis that the dual microbial preparation for the elimination of mold and yeasts from the living environment shows better results than a standard preparation . we further tested and verified this hypothesis . as part of this verification , we prepared , according to the procedures described in this exemplary embodiment , three different versions of the combined preparation and verified their effectiveness on the target mold under in vitro conditions . 6 . the preparation , control and testing of the effectiveness of a dual microbial preparation suitable for the elimination of mold and yeasts from the living environment . 6 . 1 protocol for the preparation of a dual microbial preparation for use in the elimination of mold and yeasts from the living environment 6 . 1 . 1 . preparation of the technical preparation pythium oligandrum m1 atcc 38472 . the procedure used for this preparation is identical to the procedure described in section 1 . 1 . 1 . the batch used in exemplary embodiment 6 had 1 . 0 × 10 6 oospores per g and germination of 12 . 6 %, meaning that it contained 0 . 126 × 10 6 colony forming units ( cfu ) per 1 g of preparation . the original bacillus amyloliquefaciens ccm1084 culture is stored at a temperature of − 70 ° c . after delivery from the collection of microorganisms . for cultivation , a small amount of the culture is first transferred to a dish with agar and medium 10 for the cultivation of bacilli comprising peptone , 5 g per liter , bovine extract , 3 g per liter , and mnso 4 . h 2 o , 0 . 01 g per liter ; the ph of the medium was regulated at 7 . 0 . the dishes were cultivated at a temperature of 37 ° c . overnight . ten well - separated colonies were transferred to a 2 - liter erlenmeyer flask containing 500 ml of medium 10 , having the components specified above . the bacteria were cultivated to the middle of the logarithmic phase , sedimented with centrifugation 10000 × g , for a period of 20 minutes and then washed three times in 0 . 1 m trisodium citrate buffer solution , ph 6 . 0 . after the final wash , the bacteria sediment was carefully separated from the remainder of the buffer solution and dried using freeze drying ( lyophilization ). approximately 0 . 6 g of lyophilized bacteria containing 10 8 cfu per one gram of powder was obtained from one liter of the culture . 6 . 1 . 3 . preparation of the final formula for a dual preparation for use in eliminating molds and yeasts from the living environment three different versions of the preparation were used in order to prepare the final formula , differing in terms of their relative content of the bacillus amyloliquefaciens ccm1084 bacterium ; these three versions were designated using the abbreviated titles of molda , moldb , and moldc . the preparations were prepared for use in one pack containing 3 g of loose preparation ( bag a containing 1 g and bag b containing 2 g of loose preparation ). the components of individual preparations are shown in table 11 below . the test of the stability of the dual preparation for use in eliminating mold and yeasts from the living environment must ascertain whether both microbial components have acted negatively on each other during the period of storage of the prepared preparation , at the very least for the period of conducting in vitro and in vivo effectiveness tests . one pack ( 3 g ) of the preparation molda , moldb or moldc is re - suspended in 10000 ml of lukewarm water ( temperature of around 35 ° c .). after complete resuspension , 10 ml is taken to determine the germination of pythium and 1 ml to determine the cfu of the bacillus . in order to determine the cfu in the bacillus , we further dilute the sample using tenfold serial dilutions , and to determine the cfu we use undiluted preparation for molda ( dilution of 1 ×), 1 × dilution for moldb ( diluted of 10 ×) and 2 × dilution for moldc ( dilution of 10 2 ×). the stability test is conducted immediately after the preparation has been made and again after 1 , 2 , 3 , 4 , 5 and 6 months . it can be concluded from the result that the viability of pythium during six months of storage and subsequent use of the preparation first declined somewhat and then stabilized at a value corresponding to approximately 75 % of the originally - declared nominal value . by contrast , for bacillus there were no significant changes in the viability during the test period , viability remaining et almost the originally declared value . it can therefore be concluded that the emergence of any of the components of the dual preparation is not significantly affected by drying the original active components and storing them in the presence of silica gel . 6 . 3 effectiveness test of prepared mixtures with the use of an in vitro test . the aim of in vitro effectiveness tests is to prove that the combination of the pythium oligandrum m1 atcc38472 oomycete and the bacterium of the environment bacillus amyloliquefaciens will act synergically , meaning that there will be an increase in certain measurable in vitro activity of the pythium oligandrum oomycete . from this perspective , a standard laboratory test was chosen of suppressing the growth of contaminating aspergillus niger mold on walls . the actual conducting of the test consisted of the use of a standard application protocol , which is invariably commenced by re - suspending a dry preparation containing only pythium ( the biorepel preparation ) or one of the above - mentioned test preparations designated as molda , b or c . preparations were re - suspended in 250 ml of physiological saline solution of a temperature of around 35 ° c . and 12 . 5 ml of this suspension was then mixed directly with 12 . 5 ml of double - concentrate mixture for the preparation of mea agar plates . 10 5 fungal microconidia aspergillus niger was then applied evenly to the plates prepared in this way after cooling . after a week of cultivation at 28 ° c ., a reading was taken of the number of colonies formed in comparison with the control dish , whereby the reduction in the number of colonies in contrast to the control is a gauge of the effectiveness of pythium . the results of this test are shown below in table 12 . the results show that whereas pythium alone had a significant influence on suppressing the growth of yeasts in the experiment dishes , its combination with staphylococcus produced a further strengthening of the effect . the most effective preparation among the three pathogenic yeast strains under in vitro conditions was the moldb preparation , with the preparations termed molda and moldc achieving approximately 50 % of this effectiveness . with respect to the demanding nature of field tests , the most effective preparation , moldb , is proposed for further field tests . the example shown , for the use of the dual microbial preparation specified in this exemplary embodiment , is not limited to the elimination of mold or yeasts from the surrounding environment . this type of dual microbial preparation can be positively used as prevention of the incidence of mold and yeasts in the living and working environment , which could be important to people suffering from opportunistic microbial infections . the solution is intended for application to ensure a healthy oral cavity , on non - healing wounds such as varicose ulcers , on the skin and to suppress yeasts occurring on the above - mentioned places and on the mucous membrane of the urogenital tract and for other places on the human body , in particular on skin 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