Patent Application: US-201113976291-A

Abstract:
substantially pure biological culture of a strain of the species pseudomonas graminis , deposited with number cbs136973 at the depositary institution “ centraalbureau voor schimmelcultures ” in utrecht , netherlands . use of the cbs136973 culture as an antagonist for the biocontrol of foodborne pathogenic bacteria in fruit intended for human consumption . method for treating the fruit which comprises the step of applying a preparation that comprises a culture of a strain of the species pseudomonas graminis , deposited with number cbs136973 at the depositary institution “ centraalbureau voor schimmelcultures ” in utrecht , netherlands , to the fruit . the application thereof makes it possible to reduce the growth of pathogens during the shelf life of the product , especially when the cold chain is broken .

Description:
it has been observed that strain cbs136973 is very effective against foodborne pathogenic bacteria in fruit , preferably , in fruit cut in pieces and , advantageously , against the microorganisms salmonella spp ., listeria spp . and / or escherichia coli o157 : h7 , which are the main ones in fruits and vegetables . thanks to this , strain cbs136973 may be used as an antagonist against any of said microorganisms , which favours the fulfillment of the microbiological criteria specified especially for fresh - cut fruit or minimally processed fruit , in order to prevent food toxi - infections or illnesses caused by the ingestion of fruit contaminated with bacteria belonging to salmonella spp ., listeria spp . or escherichia coli o157 : h7 genera . in particular , the effectiveness against salmonella spp . has been observed for the species salmonella choleraesuis , whereas the effectiveness against listeria spp . has been observed for the species listeria monocytogenes and listeria innocua . according to a first embodiment of the present invention , the strain of the present invention is used for biocontrol in fruit , preferably fruit cut in pieces , keeping the fruit at a temperature greater than 10 ° c ., preferably a temperature equal to or greater than 20 ° c . at room temperature , it has been observed that strain cbs136973 may slow down , and even reduce , the growth of any of the aforementioned microorganisms , even when these microorganisms are present on the fruit at a concentration equal to or greater than 10 3 cfu / g , which is a very high concentration , difficult to find in real conditions . this use is particularly advantageous since it makes it possible to control the growth of pathogens in those cases where the fruit storage temperature is not the adequate one , or the cold chain of the product is broken during the storage or transport thereof , for example , due to maintenance problems in the fruit refrigeration equipment . it is very important for the strain to be effective at room temperature , since this is the temperature where the pathogenic microorganism can grow the most and , consequently , the risk for the consumer increases . according to a second embodiment , the strain of the present invention is used for biocontrol in fruit , for example fruit cut in pieces , keeping the fruit under refrigeration conditions . refrigeration conditions are understood to mean keeping the fruit at a refrigeration temperature equal to or lower than 10 ° c ., preferably equal to or lower than 5 ° c . surprisingly , the effectiveness of strain cbs136973 against any of the microorganisms salmonella spp ., listeria spp . and escherichia coli o157 : h7 has also been demonstrated at refrigeration temperatures , which are those set by the producer or distributor for storage of the fruit . according to a third embodiment , the strain of the present invention is used for biocontrol in fruit , preferably fresh - cut fruit , keeping the fruit in a modified atmosphere . modified atmosphere is understood to mean an atmosphere with a gas composition different from that of air , in order to improve the fruit storage conditions . the strain of the present invention also shows effectiveness when the fruit is packaged in a modified atmosphere for the storage thereof . thanks to this , the strain may be used under habitual commercialisation conditions ; consequently , it is also possible to guarantee food safety under the conditions of supermarket or displays . advantageously , said fruit is a fruit with a ph ranging between 3 and 7 , for example , fruit such as apple , peach and / or melon . it has been observed that the growth of salmonella spp ., listeria spp . or escherichia coli o 157 : h7 may occur in a wide range of fruits , despite the acidity conditions of certain fruits such as apples . it has also been observed that the growth of the aforementioned bacteria is very rapid in fruits that are less acidic , such as melons . however , thanks to the strain of the present invention , the growth of these pathogens may be controlled in a wide range of fruits . as discussed in the description of the invention , one objective of the present invention is to provide a method for preparing fruit which comprises the step of applying a preparation that comprises the biological culture of the new strain cbs136973 to the fruit . according to a preferred embodiment of said method , the fruit is cut in pieces prior to applying said preparation . preferably , the concentration of strain cbs136973 in said preparation is equal to or greater than the estimated pathogen concentration that the fruit , preferably the fresh - cut fruit , may contain . according to one embodiment , the concentration of said strain in the preparation is equal to or greater than 10 5 cfu / ml . it has been observed that this concentration is effective against any of the three microorganisms salmonella spp ., listeria spp . and / or escherichia coli o157 : h7 , and at much higher concentrations than those whereat said microorganisms may be found in fresh - cut fruit in real conditions . advantageously , the concentration of said strain in the preparation is equal to or greater than 10 7 cfu / ml . it has been observed that this concentration guarantees a reduction in the pathogenic bacteria of at least two logarithmic units ( two units of the base - 10 logarithmic scale ), regardless of the concentration of pathogenic bacteria in the fruit . according to another embodiment , the method comprises the step of packaging the fruit once said preparation has been applied . advantageously , said method further comprises the step of providing a modified atmosphere to the fruit and / or the step of providing a refrigeration temperature to the fruit , for example , a temperature equal to or lower than 10 ° c ., preferably a temperature equal to or lower than 5 ° c . the modified atmosphere may be provided in a passive manner , for example , by packaging the product using plastic films with different permeabilities to gases , passively creating a favourable modified atmosphere as a result of the permeability of the container wall and factors such as respiration of the product and biochemical changes . packing the product in a modified atmosphere contributes to maintaining the freshness quality of the fresh - cut fruit for a longer period of time , which prolongs the shelf life of the product . as previously discussed , the strain of the present invention is also effective under these conditions of packaging in a modified atmosphere . advantageously , said method for preparing the fruit , preferably fresh - cut fruit , comprises the step of applying an antioxidant to the fruit , prior to applying the suspension that contains the strain . it has been observed that the strain of the present invention is not altered by the use of some antioxidant substances , for which reason said antioxidant substances may be used to delay oxidation of the fruit . in order to better understand what has been presented above , we attach some figures which represent , schematically and strictly for non - limiting purposes , the results of several embodiments . in said drawings , the selected antagonists include those isolated from yeasts and bacteria such as cpa - 1 ( candida sake ), cpa - 2 ( pantoea spp . ), pn5 ( bacillus spp . ), pn6 ( pantoea spp . ), cpa - 5 ( pseudomonas spp . ), m174bal2 ( candida famata ), el8 ( pantoea spp . ), 128 - m ( pantoea spp . ), and c9p21 ( pantoea spp .). fig1 is a graphic representation that shows the population of escherichia coli o157 : h7 in apple cylinders after the inoculation ( initial concentration ), and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include the antagonist cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig2 is a graphic representation that shows the population of salmonella choleraesuis baa - 709 in apple cylinders after the inoculation ( initial concentration ), and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig3 is a graphic representation that shows the population of listeria innocua cect - 910 in apple cylinders after the inoculation ( initial concentration ), after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig4 is a graphic representation that shows the population of escherichia coli o157 : h7 in peach cylinders after the inoculation ( initial concentration ), after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig5 is a graphic representation that shows the population of salmonella choleraesuis baa - 709 in peach cylinders after the inoculation ( initial concentration ), and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig6 is a graphic representation that shows the population of listeria innocua cect - 910 in peach cylinders after the inoculation ( initial concentration ), and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 10 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig7 is a graphic representation that shows the population of salmonella choleraesuis baa - 709 in melon cylinders after the inoculation ( initial concentration ) and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 12 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig8 is a graphic representation that shows the population of listeria monocytogenes lm230 / 3 in melon cylinders after the inoculation ( initial concentration ), and after 2 days of incubation at 20 ° c . without an antagonist ( control ) and with 12 of the selected antagonists , which include strain cbs136973 . the values represent the mean of 6 values ( 2 assays with 3 repetitions each ) and the bars represent the standard error . the numbers in brackets indicate the mean reduction obtained . fig9 is a graphic representation that shows of the population of escherichia coli o157 : h7 in apple cylinders coinoculated or not with a suspension of strain cbs136973 ( 10 8 cfu / ml ) and stored at 5 ° c . fig1 is a graphic representation that shows the population of escherichia coli o157 : h7 in peach cylinders coinoculated or not with a suspension of strain cbs136973 ( 10 8 cfu / ml ), and stored at 5 ° c . and at 10 ° c . fig1 is a graphic representation that shows the population of salmonella choleraesuis baa - 709 in peach cylinders coinoculated or not with a suspension of strain cbs136973 ( 10 8 cfu / ml ) and stored at 5 ° c . fig1 is a graphic representation that shows the population of salmonella choleraesuis baa - 709 in melon cylinders coinoculated or not with a suspension of strain cbs136973 ( 10 8 cfu / ml ), and stored at 5 ° c . and at 10 ° c . fig1 is a graphic representation that shows of the population of a cocktail of strains of listeria monocytogenes ( cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ) in melon cylinders coinoculated or not with strain cbs136973 at different concentrations and stored at 10 ° c . fig1 is a graphic representation that shows the population of a cocktail of strains of salmonella choleraesuis ( baa - 707 , baa - 709 , baa - 710 and baa - 711 ) in melon cylinders coinoculated or not with strain cbs136973 at different concentrations and stored at 10 ° c . fig1 is a graphic representation that shows of the population of a cocktail of strains of salmonella choleraesuis ( baa - 707 , baa - 709 , baa - 710 and baa - 711 ) in cut apple treated with antioxidant and inoculated or not with strain cbs136973 at 10 7 cfu / ml , by means of immersion for 2 min , and stored in modified atmosphere packaging ( map ) at 5 ° c . and 10 ° c . fig1 is a graphic representation that shows of the population of a cocktail of strains of listeria monocytogenes ( cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ) in cut apple treated with antioxidant and inoculated or not with strain cbs136973 at 10 7 cfu / ml , by means of immersion for 2 min , and stored in modified atmosphere packaging ( map ) at 5 ° c . and 10 ° c . below we present different assays , which must be interpreted to be an auxiliary tool for a better understanding of the invention and not as limitations to the object thereof . the antagonistic effect was assayed in different strains of the genera salmonella and listeria , and in a strain of escherichia coli o157 : h7 . these pathogens are the major ones in fruits and vegetables . table 3 , attached , shows the strains of foodborne pathogenic microorganisms used in the assays . firstly , we describe the examples performed in order to demonstrate the effectiveness under laboratory conditions . secondly , we describe the examples performed to demonstrate the effectiveness under conditions that simulate commercial production . assays designed to demonstrate the effectiveness of the strain cbs136973 against the major foodborne pathogens in fresh - cut fruit under laboratory conditions below we describe a number of examples of assays performed under laboratory conditions which demonstrate the effectiveness of strain cbs136973 applied at different doses , in different fruits , at room temperature and under refrigeration conditions . the strains of pathogenic microorganisms used in these assays were : listeria innocua ( cect - 910 ), escherichia coli o157 : h7 ( nctc - 12900 ) and salmonella choleraesuis ( baa - 709 , baa - 707 , baa - 709 , baa - 710 and baa - 711 ), and , in some cases , listeria monocytogenes ( cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ) ( see table 3 ). the fruits were previously disinfected by means of spraying with 70 % ethanol . subsequently , cylindrical pieces of the fruit to be assayed were prepared , by means of a punch , with the dimensions of 1 . 2 cm in diameter and 1 cm in height , which is approximately equivalent to 1 g of fruit . these pieces were introduced into sterile test tubes and inoculated with 15 μl of a suspension that contained the two microorganisms ( pathogen and antagonist ), a process called coinoculation . in the control treatment , the pathogen was added to a tube with 10 ml of sterile water ( without an antagonistic microorganism ). following the coinoculation , the fruit was allowed to dry at room temperature . subsequently , 3 tubes were collected , and the initial pathogen concentration was measured by means of seeding in specific culture media . the other tubes were stored at 20 ° c ., 10 ° c . or 5 ° c ., depending on the assay . after 2 days ( assays at 20 ° c . ), or 2 - 3 days , 5 - 7 days and 10 days ( assays at 5 ° c . or 10 ° c . ), the pathogen concentration per piece of fruit was once again determined in the samples with an antagonist ( treatment with an antagonist ) and in those that did not have an antagonist ( control treatment ). the concentration data were transformed to log 10 cfc . in order to calculate the pathogen growth reduction value , the following formula was used : reduction log 10 cfc = log 10 c t − control − log 10 c t + antagonist , where : c t − control is the pathogen concentration in the control treatment after “ t ” days of storage , and c t + antagonist is the pathogen concentration in the treatment with an antagonist after “ t ” days of storage . positive reduction values indicate that the growth of the pathogen on the fruit assayed in the presence of the antagonist is lower than the same without an antagonist . the higher the value , the greater the effectiveness against the pathogen studied . in the assays at room temperature ( 20 ° c . ), in order to obtain the suspension of strain cbs136973 and the other strains assayed , production in nyda medium incubated at 25 ° c . for 48 h was used . isolated colonies were taken , suspended in sterile de - ionised water and , from said suspension , another suspension was prepared , which was adjusted , by means of a spectrophotometer , to different transmittances ( λ = 420 nm ) that correspond to the different concentrations of antagonists assayed ( 10 5 cfu / ml , 10 6 cfu / ml , 10 7 cfu / ml and 10 8 cfu / ml ). in the assays under refrigeration conditions ( 5 ° c . or 10 ° c . ), in order to obtain the suspension of strain cbs136973 , production in a liquid medium was used . to this end , an erlenmeyer flask was inoculated containing 50 ml of tsb and incubated at 30 ° c . for 20 - 24 h . it was centrifuged for 10 min at 10000 rpm and cells resuspended with 25 ml of sterile de - ionised water . the pathogens were inoculated in tubes containing 10 ml of tsb medium ( salmonella choleraesuis baa - 707 , baa - 709 , baa - 710 and baa - 711 , and escherichia coli o157 : h7 ) or tyseb medium ( listeria innocua cect - 910 and listeria monocytogenes cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ), which were incubated at 37 ° c . for 20 - 24 h . subsequently , they were centrifuged at 8000 rpm for 10 min and cells resuspended with 5 ml of saline solution ( 0 . 85 g / l of nacl ). by measuring the transmittance at 420 nm and a curve previously obtained in the laboratory for each of the pathogens , the estimated pathogen concentration was determined . the pathogen concentrations assayed ranged between 10 5 cfu / ml and 10 7 cfu / ml . however , under real conditions , it is estimated that the 10 5 cfu / ml pathogen concentration , which in the assays performed corresponds to 10 3 cfu / g of product , is even a very high pathogen concentration ( salleh et al ., 2003 6 ; nguz et al ., 2005 7 ); for this reason , the results obtained are presented under unfavourable / adverse conditions for strain cbs136973 . effectiveness of strain cbs136973 against different foodborne pathogens on “ golden delicious ” apples at 20 ° c . fig1 to 3 show an example of the results obtained against strains of escherichia coli o157 : h7 , salmonella choleraesuis baa - 709 and listeria innocua cect - 910 , in “ golden delicious ” apples , comparing the effectiveness of strain cbs136973 with other strains isolated in the laboratory and which were assayed under the same conditions . the suspension of strain cbs136973 was inoculated at approximately 10 8 cfu / ml and the pathogens at 10 7 cfu / ml . the ph of the apples was 3 . 8 ± 0 . 2 and the acidity ranged between 1 . 6 and 2 . 9 g malic acid / l . as shown in fig1 , the initial concentration of e . coli o157 : h7 was 5 . 1 log 10 cfu / g and it increased to 6 . 8 log 10 cfu / g in the control treatment ( without an antagonist ). on the contrary , in the pieces coinoculated with different antagonists , there was lower growth , with reductions between 1 . 0 and 1 . 6 log units . in the case of strain cbs136973 , the pathogen population after two days of storage at 20 ° c . was even lower than the initial one ( 2 . 4 log 10 cfu / g ), which indicates an effective reduction of 4 . 5 logarithmic units . similar results were obtained when the effectiveness of different strains was tested against salmonella baa - 709 ( fig2 ), with reductions ranging between 0 . 3 and 1 . 0 log units in the case of the other antagonists and of 4 . 7 log units in the case of the strain cbs136973 . the increase of listeria innocua population on apples not treated with an antagonist after storage at 20 ° c . for 2 days was 2 . 5 log 10 cfu / g . in the case of some of the antagonists isolated in the laboratory , there was a significant reduction , between 1 . 1 and 2 . 2 log units , but strain cbs136973 showed much better results , with a reduction of 5 . 9 log units following the storage , which indicates that the population of listeria innocua on apples that had been treated with strain cbs136973 was lower than 2 log 10 cfu / g ( fig3 ). effectiveness of strain cbs136973 against different foodborne pathogens on different peach varieties at 20 ° c . fig4 to 6 show an example of the results obtained against strains of escherichia coli o157 : h7 , salmonella choleraesuis baa - 709 and listeria innocua cect - 910 , in peach , comparing the effectiveness of strain cbs136973 with other strains isolated in the laboratory and which were assayed under the same conditions . peaches of the “ merry o &# 39 ; henry ”, “ tardibelle ”, “ roig d &# 39 ; albesa ”, “ placido ”, “ royal glory ” and “ elegant lady ” varieties were used . the suspension of strain cbs136973 that was inoculated had a concentration of approximately 10 8 cfu / ml and the pathogens were inoculated at 10 7 cfu / ml . the ph of the peaches used was 3 . 6 - 5 . 3 , and the acidity ranged between 2 . 8 and 7 . 8 g malic acid / l . as shown in fig4 , the initial concentration of e . coli o157 : h7 on peach was 4 . 9 log 10 cfu / g , and after 2 days of storage at 20 ° c . it increased by approximately 3 log units in the pieces not treated with an antagonist . in the pieces inoculated with some of the isolated antagonists , the concentration of e . coli o157 : h7 was reduced by between 1 . 8 and 3 . 0 log units , whereas the reduction with strain cbs136973 was 4 . 3 logarithmic units , the concentration at 2 days being even lower than the initial one , which demonstrates its great effectiveness . fig5 shows the results of the same assay , but performed with the strain of salmonella baa - 709 . in this case , the increase in the population after two days of storage at 20 ° c . was lower than that of e . coli o157 : h7 , an approximately 2 . 5 log increase . in general , the reductions with the other antagonists were lower , between 0 . 4 and 1 . 8 logarithmic units , but the reduction of salmonella baa - 709 was greatest in those pieces of apple treated with strain cbs136973 , 2 . 8 log units . fig6 shows the data relative to listeria innocua on peach . in this fig6 , we may observe that the population of listeria innocua in the control treatment ( without an antagonist ) also increased by approximately 3 log units in each piece of peach , whereas in those pieces inoculated with different antagonists isolated in the laboratory the population was lower , with reductions between 0 . 7 and 2 . 1 log units being observed . again , the reduction obtained with strain cbs136973 was greater , 4 logarithmic units , and , once again , the final population was even lower than the initial one . effectiveness of strain cpa - 7 against different foodborne pathogens on melon at 20 ° c . fig7 and 8 show an example of the results obtained against strains of salmonella choleraesuis baa - 709 and listeria monocytogenes lm230 / 3 , on melon , comparing the effectiveness of strain cbs136973 with other strains isolated in the laboratory and which were assayed under the same conditions . melon is a fruit that has a more neutral ph and less acidity than apple and peach ( ph 5 . 7 - 6 . 5 , acidity 0 . 7 - 1 . 9 g citric acid / l , generally ). in this case , the problem of pathogens is more significant , because the ph does not act as a barrier to the growth of foodborne pathogens . the suspension of the strain cbs136973 was inoculated at approximately 10 8 cfu / ml and the pathogen was inoculated at 10 7 cfu / ml . fig7 shows the results for different strains of antagonistic microorganisms , including cbs136973 , against salmonella on pieces of “ toad skin ” melon . in this case , the reduction values ranged between 1 . 5 and 3 . 2 log units , the greatest reduction being obtained with strain cbs136973 , a total of 3 . 5 log units . it may be observed that the growth of salmonella on melon after 2 days of storage at 20 ° c . was very high in the control treatment ( without an antagonist ), 4 . 2 logarithmic units , with final population being greater than 10 8 cfu / g of product . fig8 shows the effectiveness of strain cbs136973 against the strain of listeria monocytogenes lm230 / 3 , in melon stored for 2 days at 20 ° c . as may be observed , in this case there is also a reduction in the pathogen with respect to the untreated control . effectiveness of strain cbs136973 against escherichia coli o157 : h7 on “ golden delicious ” apple under refrigeration conditions fig9 shows the result of the effectiveness of strain cbs136973 against e . coli o157 : h7 on “ golden delicious ” apple stored at 5 ° c . the assay was performed by the coinoculation of 15 μl of a suspension that contained both strains , e . coli o157 : h7 at 10 7 cfu / ml and cbs136973 ( 30 % transmittance , approximately 10 8 cfu / ml ). as may be seen in the figure , at 5 ° c . no growth of e . coli o157 : h7 was observed on cut apple in the control treatment ; on the contrary , in the samples coinoculated with the antagonist , there was a reduction from the second day of storage and , after 7 days , the population decreased to less than 10 cfu / g . effectiveness of strain cbs136973 against different foodborne pathogens on peach under refrigeration conditions fig1 and 11 show the effectiveness of strain cbs136973 against escherichia coli o157 : h7 and salmonella baa - 709 on peach (“ elegant lady ” and “ placido ” varieties ), at 5 ° c . and 10 ° c . the suspension that was inoculated had a concentration of strain cbs136973 of approximately 10 8 cfu / ml and 10 7 cfu / ml of pathogenic microorganisms . as shown in fig1 , strain cbs136973 reduces the pathogen concentration , the reduction being greater at 5 ° c . than at 10 ° c ., and it is maintained even below the detection limit after 6 days of storage . similar results were obtained when strain cbs136973 was used against salmonella on peach of the “ placido ” variety ( fig1 ). in this case , after 6 days of storage at 5 ° c ., no salmonella was detected in the pieces of cut peach , whereas the population was maintained in the treatment without an antagonist ( control ). effectiveness of strain cbs136973 against salmonella choleraesuis baa - 709 on melon under refrigeration conditions fig1 shows the effectiveness of strain cbs136973 against salmonella baa - 709 on melon stored at 10 ° c . the suspension that was inoculated had a concentration of strain cbs136973 of approximately 10 8 cfu / ml and 10 7 cfu / ml of pathogenic microorganisms . as shown in the figure , at 10 ° c . the pathogen grew in the control treatment ( without an antagonist ), whereas in the treatment wherein cbs136973 was applied the population remained lower , with a reduction of more than 1 . 5 logarithmic units from the sixth day of storage . at 5 ° c ., under the conditions assayed , salmonella was not capable of growing and the addition of strain cbs136973 did not entail any changes with respect to the control treatment . effectiveness of strain cbs136973 applied at different doses against different foodborne pathogens on golden delicious apple at 20 ° c . tables 4 , 5 and 6 show the effectiveness of strain cbs136973 applied at different doses , and against different concentrations of the pathogens escherichia coli o157 : h7 , salmonella choleraesuis baa - 709 and listeria innocua cect - 910 . this effectiveness was measured in log 10 units of growth reduction in accordance with the formula cited above . as may be observed in the attached tables , the results show a pathogen growth reduction for concentrations of strain cbs136973 equal to or greater than the inoculated pathogen concentration . of the doses assayed , the 10 8 cfu / ml dose is the one which shows the best results for the three pathogens ( reductions greater than 3 . 5 logarithmic units ). however , the 10 7 cfu / ml dose of strain cbs136973 is the one considered to be most adequate for commercial application , since it guarantees a minimum reduction of two logarithmic units for the three pathogens studied , regardless of the concentration of pathogenic bacteria on the fruit . effectiveness of strain cbs136973 applied at different doses against different foodborne pathogens on melon at 20 ° c . and 10 ° c . in this example , a cocktail of strains of salmonella choleraesuis ( baa - 707 , baa - 709 , baa - 710 and baa - 711 ) or listeria monocytogenes ( cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ) was used . tables 7 and 8 show the effectiveness of strain cbs136973 , applied at different doses , on melon stored at 20 ° c ., against different concentrations of the cocktail of pathogens of the genera salmonella and listeria monocytogenes . this effectiveness was measured in log 10 units of growth reduction in accordance with the formula cited above . as may be seen in the tables , in melon , the 10 7 cfu / ml dose of strain cbs136973 shows growth reduction values of salmonella and listeria monocytogenes , applied at 10 5 cfu / ml of melon , which is approximately equivalent to 10 3 cfu / g melon , equal to or greater than 3 . 7 logarithmic units . fig1 and 14 show the effectiveness of different concentrations of strain cbs136973 on melon stored at 10 ° c ., against the aforementioned cocktails of salmonella choleraesuis and listeria monocytogenes applied at a concentration of 10 5 cfu / ml . as shown in the figures , the reduction of listeria monocytogenes and salmonella is greater the higher the concentration of strain cbs136973 . assay designed to demonstrate the effectiveness of antagonistic strain cbs136973 against the major foodborne pathogens in fresh - cut fruit under conditions that simulate commercial production . below we describe an example of an assay on “ golden delicious ” apple under conditions that simulate commercial production . in this example , a cocktail of strains of salmonella choleraesuis ( baa - 707 , baa - 709 , baa - 710 and baa - 711 ) and a cocktail of strains of listeria monocytogenes ( cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ) were used . the whole apples were disinfected , the cores were removed and they were cut into ten slices . subsequently , they were submerged in a bath containing the antioxidant natureseal ® as1 ( 6 %, agricoat ltd ., great shefford , uk ), for 2 min , under stirring , and were allowed to dry . once they were treated with the antioxidant , the pieces of apple were submerged in a suspension that contained the cocktail of strains of the pathogen and the antagonist , for 1 min , under stirring , simulating the application that would take place in a fruit treatment line tank . in the control treatment , the pieces of apple were submerged in a suspension that contained the cocktail of strains of the pathogen without the antagonistic strain . subsequently , the pieces of apple were allowed to drain , packaged ( 200 g ) in polypropylene containers with a 500 - ml capacity and sealed with a polypropylene film of the type habitually used , with a thickness of 35 μm , a permeability to o 2 and co 2 of 3500 cm 3 / m 2 * day * atm at 23 ° c ., and a permeability to steam of 0 . 9 g / m 2 * day at 25 ° c . and 75 % relative humidity . due to respiration of the fruit and the permeability characteristics of the film to o 2 and co 2 , a passive modified atmosphere ( pma ) is created inside the container . antagonistic strain cbs136973 may be affected by this atmosphere , for which reason its effectiveness must also be demonstrated under these modified atmosphere conditions . the pieces of apples were stored at 5 ° c . and at 10 ° c . for 15 days ( estimated shelf life for this type of products ). periodically , microbiological counts were performed and different quality parameters ( colour , texture , ph , acidity , soluble solids content and visual quality ) were determined . in order to obtain the suspension of strain cbs136973 , production in tsb liquid medium was used and the percent transmittance ( λ = 420 nm ) was adjusted for a concentration of the strain of 10 7 cfu / ml . the pathogens were inoculated in tubes containing 10 ml of tsb medium ( salmonella choleraesuis baa - 707 , baa - 709 , baa - 710 and baa - 711 ) or tyseb medium ( listeria monocytogenes cect - 4031 , cect - 4032 , cect - 933 , cect - 940 and lm230 / 3 ), which were incubated at 37 ° c . for 20 - 24 h . subsequently , they were centrifuged at 8000 rpm for 10 min and the cellular precipitate was re - dissolved with 5 ml of saline solution ( 0 . 85 g / l of nacl ). by measuring the transmittance at 420 nm and a curve previously obtained in the laboratory for each of the pathogens , the estimated pathogen concentration was determined , which in the assay described was a suspension with a concentration of 10 5 cfu / ml . effectiveness of strain cbs136973 against different foodborne pathogens on “ golden delicious ” cut apple packaged at different temperatures under conditions that simulate commercial conditions fig1 and 16 show the results obtained against the aforementioned cocktails of strains of salmonella and listeria monocytogenes , on “ golden delicious ” apple at 5 ° c . and 10 ° c . the suspension that was inoculated by means of immersion of the pieces of apple contained a concentration of strain cbs136973 of 10 7 cfu / ml and a concentration of 10 5 cfu / ml of both pathogenic microorganisms . as may be observed in the figures , strain cbs136973 was effective against salmonella , especially at 10 ° c ., where growth was observed . in the case of listeria monocytogenes , growth reduction was observed at both 5 ° c . and 10 ° c . therefore , it may be concluded that strain cbs136973 is effective against different foodborne pathogens under conditions that simulate commercial conditions ( refrigeration and passive modified atmosphere , pma ). moreover , it is worth noting that the application of strain cbs136973 did not affect the colour , or the texture , or the soluble solids or the acidity of the apples . although specific examples of the present invention have been described and represented , it is evident that a person skilled in the art may introduce variants and modifications , or replace the details with technically equivalent ones , without going beyond the scope of protection defined by the attached claims . 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