Patent Application: US-201214236542-A

Abstract:
compositions and methods are disclosed for improving food safety . one or more lactic acid producing microorganisms are shown to inhibit pathogenic contaminations on food materials . the lactic acid producing microorganisms are capable of adhering to various surfaces and may serve as a bio - sanitizing agent . lactic acid producing microorganisms or cell free extract of these microorganisms may be used in an effective and natural method to prevent l . monocytogenes infection in food products , as well as in food processing facilities and equipments .

Description:
the present disclosure provides methods for reducing the pathogen content on a surface of food materials or a surface that is likely to be in contact with food . the methods include contacting the surface with a composition containing one or more lactic acid producing microorganisms . for purpose of this disclosure , the term “ food ” or “ food material ” may be used to refer to anything that are edible . in one embodiment , food may include but is not limited to meat , a meat product , seafood , a seafood product , a plant material , milk from an animal , milk derived from a plant material , or combination thereof . food can be in the form of a solid , a liquid , a paste , a powder , a suspension , or mixture thereof . the term “ food processing ” may refer to any process of harvesting , treating , milking , slaughtering , cutting , processing , dividing , packaging of food or food materials . enhanced inhibition and / or exclusion of pathogens may be achieved with the administration of multiple lab strains , or with the administration of one or more lab strains in combination with certain chemicals . similarly , advantageous effects may be achieved , for example , by multiple or repetitive contacts ( a chain of contacts ) of the surface with the lab microorganisms . while not limited by any scientific theory or mode of action , natural competition of certain microorganisms with pathogenic microorganisms may reduce or eliminate enterobacteria . microorganisms disclosed herein may act in various ways . for example , the microorganisms may act as a bacteriocin or may act by producing bacteriocins . they may inhibit one or more pathogens by competing for nutrients and / or attachment spaces with the pathogen . they may also interfere with biofilm formation by certain pathogens , such as listeria . as used herein , a method of contacting the surface with a composition may mean applying the composition directly or indirectly to the surface or bringing the surface to be in tough with the composition . in various aspects , a composition may be directly applied as a spray , a rinse , or a powder , or any combination thereof . as used herein , a spray refers to a mist of liquid particles that contain a composition of the present disclosure . a spray may be applied directly to the surface using items including , but not limited to , a spray can , a spray bottle , a spray gun . the composition of the present disclosure may be distributed initially as a liquid but may turn into a gas at room temperature and pressure . the terms “ lactic acid producing bacteria ( or microorganisms )” and “ lactic acid bacteria ( or microorganisms )” may be used interchangeably in this disclosure and are sometimes abbreviated as “ lab .” unless otherwise specified , the term cfu in this paragraph refers to the colony forming unit of a microorganism capable of forming colonies on solid media . a contacting step may occur while surface is being used , before or after use , while a food material is being processed , while a food material is being packaged , or while a food material is being stored in warehouse or on the shelf of a store . a composition as used herein may be in the form of a liquid , an aerosol , a heterogeneous mixture , a homogeneous mixture , a powder , or a solid dissolved in a solvent . as used herein , the term “ liquid ” means a substance in the fluid state of matter having no fixed shape . in a further aspect , the composition may be a solution . in a solution , a solute is dissolved in a second substance commonly known as a solvent . as described above , the term “ powder ” refers to a composition that is a dry or nearly dry bulk solid composed of a large number of very fine particles that may flow freely when shaken or tilted . a dry or nearly dry powder composition of the present invention preferably contains a low percentage of water , such as less than 5 %, less than 2 . 5 %, or less than 1 % by weight . in a further aspect , the composition of the present disclosure may be a suspension . a suspension is a heterogeneous mixture containing solid particles that are sufficiently large for sedimentation . particles in a suspension are visible under a microscope and will settle over time if left undisturbed . in a further aspect of the present disclosure , the composition may be an emulsion . as used herein , the term “ emulsion ” means a mixture of two immiscible liquids . in yet another aspect , the composition of the present disclosure may be a colloidal dispersion . a colloidal dispersion is a type of chemical mixture where one substance is dispersed evenly throughout another . particles of the dispersed substance are only suspended in the mixture , unlike a solution , where they are completely dissolved within . this occurs because the particles in a colloidal dispersion are larger than in a solution — small enough to be dispersed evenly and maintain a homogenous appearance , but large enough to scatter light and not dissolve . colloidal dispersions are an intermediate between homogeneous and heterogeneous mixtures and are sometimes classified as either “ homogeneous ” or “ heterogeneous ” based upon their appearance . the lactic acid producing microorganisms of the present invention include any microorganism capable of producing lactic acid . in one aspect , the lactic acid producing microorganism is selected from the group consisting of : bacillus subtilis , bifidobacterium adolescentis , bifidobacterium animalis , bifidobacterium bifudum , bifidobacterium infantis , bifidobacterium longum , bifidobacterium thermophilum , lactobacillus acidophilus , lactobacillus agilis , lactobacillus alactosus , lactobacillus alimentarius , lactobacillus amylophilus , lactobacillus amylovorans , lactobacillus amylovorus , lactobacillus animalis , lactobacillus batatas , lactobacillus bavaricus , lactobacillus bifermentans , lactobacillus bifidus , lactobacillus brevis , lactobacillus buchnerii , lactobacillus bulgaricus , lactobacillus catenaforme , lactobacillus casei , lactobacillus cellobiosus , lactobacillus collinoides , lactobacillus confusus , lactobacillus coprophilus , lactobacillus coryniformis , lactobacillus corynoides , lactobacillus crispatus , lactobacillus curvatus , lactobacillus delbrueckii , lactobacillus desidiosus , lactobacillus divergens , lactobacillus enterii , lactobacillus farciminis , lactobacillus fermentum , lactobacillus frigidus , lactobacillus fructivorans , lactobacillus fructosus , lactobacillus gasseri , lactobacillus halotolerans , lactobacillus helveticus , lactobacillus heterohiochii , lactobacillus hilgardii , lactobacillus hordniae , lactobacillus inulinus , lactobacillus jensenii , lactobacillus jugurti , lactobacillus kandleri , lactobacillus kefir , lactobacillus lactis , lactobacillus leichmannii , lactobacillus lindneri , lactobacillus malefermentans , lactobacillus mali , lactobacillus maltaromicus , lactobacillus minor , lactobacillus minutus , lactobacillus mobilis , lactobacillus murinus , lactobacillus pentosus , lactobacillus plantarum , lactobacillus pseudoplantarum , lactobacillus reuteri , lactobacillus rhamnosus , lactobacillus rogosae , lactobacillus tolerans , lactobacillus torquens , lactobacillus ruminis , lactobacillus sake , lactobacillus salivarius , lactobacillus sanfrancisco , lactobacillus sharpeae , lactobacillus trichodes , lactobacillus vaccinostercus , lactobacillus viridescens , lactobacillus vitulinus , lactobacillus xylosus , lactobacillus yamanashiensis , lactobacillus zeae , pediococcus acidlactici , pediococcus pentosaceus , streptococcus cremoris , streptococcus discetylactis , streptococcus faecium , streptococcus intermedius , streptococcus lactis , streptococcus thermophilus , and combinations thereof . in one aspect , the lactic acid producing microorganism is selected from the group consisting of lactobacillus acidophilus , lactococcus lactis , and pediococcus acidilactici . in another aspect , the lactic acid producing microorganism is lactobacillus acidophilus . np51 ( a . k . a . la51 ) may be referred to as lactobacillus acidophilus / animalis or lactobacillus animalis because when strain np51 was first isolated , it was identified as a lactobacillus acidophilus by using an identification method based on positive or negative reactions to an array of growth substrates and other compounds ( e . g ., api 50 - chl or biolog test ). using modern genetic methods , however , strain np51 has recently been identified as belonging to the species lactobacillus animalis ( unpublished results ). lactobacillus amylovorus m35 ( a . k . a . np35 ), la45 , and np51 were deposited with the american type culture collection ( atcc , manassas , va . 20110 - 2209 ) on may 26 , 2005 and have the deposit numbers of pta - 6751 , pta - 6749 , and pta - 6750 , respectively . lactobacillus acidophilus strain l411 was deposited with the american type culture collection ( atcc , manassas , va . 20110 - 2209 ) on jun . 30 , 2005 and has the deposit number of pta - 6820 . pediococcus acidilactici d3 ( a . k . a . np3 ) was deposited with the american type culture collection ( atcc , manassas , va . 20110 - 2209 ) on mar . 8 , 2006 and has the deposit number of pta - 7426 . these deposits were made in compliance with the budapest treaty requirements that the duration of the deposit should be for thirty ( 30 ) years from the date of deposit or for five ( 5 ) years after the last request for the deposit at the depository or for the enforceable life of a u . s . patent that matures from this application , whichever is longer . the strains will be replenished should it become non - viable at the depository . the various aspects of the present disclosure include application of a composition to a surface of an object or a space . the composition may contain different microorganisms , different strains , or a combination of any number of different microorganisms and different strains . for example , one , two , three , four , five , six , or more microorganisms may be used . in another aspect , one , two , three , four , five , six , or more strains of the same microorganism may be used . different microorganisms may be added sequentially to the same surface , or may be prepared as a “ cocktail ” before being applied to the surface . as used herein , the term “ one or more ” may mean any integer equal or greater than one , and may include one , two , three , four , and so on . in one embodiment , “ one or more ” may mean from one to ten . it is to be noted that , as used in this specification and the claims , the singular forms “ a ,” “ an ,” and “ the ” include plural referents unless the context clearly dictates otherwise . thus , for example , reference to “ a pathogen ” includes reference to a mixture of two or more pathogens , reference to “ a lactic acid producing bacterium ” includes reference to bacterial cells that are lactic acid producing bacteria . the terms “ from ,” “ to ,” “ between ,” and “ at least ” as used herein are inclusive . for example , a range of “ between 5 and 10 ” means any amount equal to or greater than 5 but equal to or smaller than 10 . as used herein , the term pathogen refers to a biological agent that causes disease or illness to a host . a pathogen may be a bacterium , a virus , or a fungus , and may be active or dormant . in one aspect of the present disclosure , a pathogen is a bacterium . in another aspect , a bacterium is an enteropathogenic bacterium , or enteropathogen . in one aspect , the pathogen may be a pathogenic e . coli , a pathogenic staphylococcus , a pathogenic listeria , a pathogenic shigella , a pathogenic campylobacter , a pathogenic clostridium , a pathogenic mycobacterium , a pathogenic yersinia , a pathogenic bacillus , a pathogenic vibrio , a pathogenic streptococcus , a pathogenic aeromonas , a pathogenic klebsiella , a pathogenic enterobacter , a pathogenic citrobacter , a pathogenic aerobacter , a pathogenic serratia , and a pathogenic salmonella . in another aspect , the pathogen can be and includes e . coli o157 : h7 , staphylococcus aureus , listeria monocytogenes , campylobacter jejuni , or salmonella typhimurium . in one embodiment , the pathogen may be e . coli o157 : h7 . in another embodiment , the pathogen may be a listeria . a method of the present disclosure may affect the pathogen content on a surface of living or non - living matters . in one aspect , pathogen content refers to the number of pathogens in a food material . in another aspect , pathogen content refers to the number of pathogens in a sample of a food material . in another aspect , pathogen content refers to the number of pathogens in a sub - sample of a food material . the terms “ in ” and “ on ” as used herein , for example , in the phrase “ in a food material ,” means that a subject such as a pathogen is located inside , or on the surface of another subject , such a food material . it is to be understood that because pathogen may grow and expand its territory , a pathogen that initially resides on the surface of a material may grow and expand into the inside of the material . in another aspect , the pathogen content of a surface after a contacting step is preferably less than the pathogen content of the same surface before the contacting step . in one aspect , the term “ less than ” may mean having fewer number of total pathogen cells on a surface . in another aspect , “ less than ” may mean having fewer number of pathogen species on a surface . in a further aspect , “ less than ” may mean having fewer number of viable pathogens on a surface . as used herein , a decrease is defined as having lower number of pathogens than were on the surface before treatment of the surface with the disclosed composition . in one aspect , the lower number of pathogens is a lower number of viable pathogens or pathogens capable of replicating . in another aspect , a decrease can be and includes a reduction of at least about 5 %, at least about 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 %, at least about 95 %, at least about 99 %, at least about 99 . 9 %, at least about 99 . 99 %, or ideally 100 %. in a further aspect , affecting the pathogen content results in inhibition of further pathogen growth . in one aspect , pathogen growth is defined as the division of one pathogen cell into two daughter cells . in another aspect , inhibition results in stopping the growth of pathogens on a surface so that the total number of pathogens on the surface remains the same . in another aspect , inhibition results in slowing the growth of pathogens on the surface . slowing of pathogen growth can occur during the exponential phase of growth and results in a lower number of cell divisions per unit time as compared to a surface not treated with the methods of the present disclosure . in one aspect , inhibition of pathogen growth occurs immediately . in another aspect , inhibition of pathogen growth occurs one minute after , 30 minutes after , 45 minutes after , one hour after , two hours after , four hours after , six hours after , twelve hours after , eighteen hours after , one day , 3 days , or one week after the disclosed composition is applied to the surface . for the methods described herein , a reduction in pathogen content or concentration on the surface is achieved relative to a control . reduction of pathogens may be measured using methodology commonly used in the art . in one aspect , pathogen concentrations are measured in colony forming units ( cfu ) obtained from a fixed quantity of material or from a fixed area of surface . for example , the reduction in the number of cfu can be at least about 5 %, at least about 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 %, at least about 95 %, at least about 99 %, at least about 99 . 9 %, at least about 99 . 99 %, or ideally about 100 %. the reduction can also be ranges between any two of these values . alternatively , the reduction can be measured in “ log cycles .” each log reduction ( also referred to as log cfu or log in cfu when referring to the reduction in cfu of a pathogen ) in concentration is equal to a ten - fold reduction ( e . g . a one log reduction is a ten - fold reduction ; a two log reduction is a 100 - fold reduction , and so on .). the log cycle reduction can be at least about 0 . 5 , at least about 1 , at least about 1 . 5 , at least about 2 , at least about 2 . 5 , at least about 3 , at least about 3 . 5 , at least about 4 , and ranges between any two of these values . log cycle reductions can be easily converted to percent reduction . a 1 log cycle reduction is equal to 90 %, a 2 log cycle reduction is equal to 99 %, a 3 log cycle reduction is equal to 99 . 9 %, and so on . viability of the pathogen can also be measured . in one aspect , viability can be measured by a physical measurement , a chemical measurement , a measurement of chemical activity , or a measurement of turbidity . in another aspect , viability is measured by quantifying colony forming units ( cfu ) obtained from a fixed quantity of a material or fixed area of the surface . one - way anova with tukey hsd means comparison was performed where appropriate to determine statistical significance . statistical analyses were performed using jmp 7 ( sas institute inc , cary , n . c .). the examples below are provided to illustrate but not to limit the present invention . those of skill in the art should , in light of the present disclosure , appreciate that many changes may be made in the specific aspects which are disclosed within the following examples and elsewhere and still obtain a like or similar result without departing from the scope of the invention . in order to evaluate the ability of lab s to competitively exclude l . monocytogenes by forming biofilm , the hydrophobicity of several lab strains was evaluated to determine the potential effect of bacterial physiochemical properties on attachment ( nira et al , 2010 ). previous studies have shown that more hydrophobic cells tend to attach better to various surfaces . it has also been shown that hydrophobicity of a bacterium may correlate with its capability to form stable attachment to the surface of food , such as deli meat ( bonaventura et al , 2007 ). each culture of the labs and listeria monocytogenes was grown from stock , streaked on solid media and incubated at 37 ° c . for 24 h for isolation of single colonies . tryptic soy agar with 0 . 6 % yeast extract ( tsaye ) was used for l . monocytogenes and de man , rogassa and sharp ( mrs ) agar was used for individual np3 , np35 and np51 strains . a single colony was placed in individual 5 ml tubes of tsaye or mrs and incubated at 37 ° c . for 18 to 20 h . a 3 - strain combination which contains all three strains , np3 , np35 and np51 , was thawed from a commercially available packet and used directly . about 2 ml of bacterial cells were harvested by centrifugation and washed twice with phosphate buffered saline ( pbs ). the cells were then resuspended in water to an absorbance ( wavelength of 620 nm ) of about 1 . 0 . then , 1 ml of n - hexadecane was added to 1 ml of bacterial cell suspension and incubated at 30 ° c . for 10 min . the solution was mixed by vortexing for 60 seconds and the suspension was incubated for 15 min until the hydrocarbon phase rose completely to the top . optical density of the aqueous phase of the suspension was measured at 620 nm and was compared to the bacterial suspension before mixing with hexadecane . the hydrophobicity of bacteria adhering to hexadecane was calculated by the equation : wherein od 620 i is the initial od 620 before mixing with hexadecane , and od 620 h is the od 620 of the aqueous phase after mixing with hexadecane . the results of the hydrophobicity tests are shown in fig1 . x axis is strain of bacteria , y axis is % hydrophobicity . each bar represents the average of 3 experiments . error bars indicate standard deviation from the mean . lm191 is l . monocytogenes , and lg and lgmix are mixture of the three lab np3 , np35 and np51 . lb . animalis np51 exhibited the highest hydrophobicity with about 26 % while lb . amylovorus np35 exhibited the lowest hydrophobicity among all strains tested at about 2 . 5 % hydrophobicity . total carbohydrate production : l . monocytogenes and lab strains were grown to stationary phase , washed by centrifugation three times and resuspended to an abs 600 nm = 0 . 32 ± 0 . 04 . the cell suspensions were deposited on sterile stainless steel coupons and were incubated at 23 ° c . for 3 hr . the attached cells were removed using a swab that was subsequently placed into pbs . the total carbohydrate produced by the bacteria was determined from the supernatant of the attached cell , and the planktonic cultures using phenol - sulfuric acid method ( chae et al ., 2006 ). as shown in fig2 , the attached cells produced significantly higher carbohydrate than planktonic cells . inhibition or exclusion of listeria attachment on stainless steel by lab attachment of individual lab strains and the 3 - strain combination to stainless steel coupons was evaluated . the coupons were from a donated deli slicer which was cut into coupons measuring 2 × 2 . 5 cm using a flow waterjet cutting system ( flow international corporation , kent , wash .) at c . mayo sheet metal . the coupons were washed , and autoclaved at 121 ° c . for 15 min before use . attachment / adherence of individual lab strains ( 10 8 cfu / ml ) as well as the 3 - strain combination to stainless steel coupons were tested . the 3 - strain combination , at 10 8 cells / well , was prepared and incubated at 23 ° c . for three hours to allow the bacteria to attach to the coupons . bacterial cells were prepared as described previously in example 1 . individual lab strains , np35 , np51 and np3 , as well as the 3 - strain combination , were placed in separate wells of a 6 - well plate . the concentration of the bacteria was about 10 8 - 10 9 cfu / well . stainless steel coupons were added to the 6 well plates . tsbye were used as the culture media because both l . monocytogenes and the lab strains can grow in the media . tsbye was also used for the competitive exclusion assay . after incubation , the coupons were washed with 1 ml pbs three times , transferred to new 6 well plates containing fresh tsbye . the plates were incubated at 23 ° c . for 24 and 72 h , respectively , and at 7 ° c . for 72 h and 7 days , respectively . the coupons were then recovered aseptically and washed three times with 1 ml pbs . each coupon was transferred to different 50 ml sterile centrifuge tubes and 15 ml pbs was added to each tube . the attached cells were released by sonicating the coupons for 3 min ( 2sec pulse on / 1 sec off ) and plated on mrs agar by serial dilution . the plates were incubated at 37 ° c . for 48 hours and the numbers of colonies were enumerated for each plate . fig3 shows attachment of individual strains , l . monocytogenes ( 10 3 cfu / ml ), p . acidilactici , lb . amylovorus , lb animalis ( 10 8 cfu / ml each ), and the combination of the three strains ( lab cocktail , 10 8 cfu / ml ) on stainless steel coupons at 23 ° c . for 24 and 72 hr . x axis is name of bacterial strains . y axis is colony forming units ( cfu ) of bacteria per milliliter of culture . green bars are results after 24 hours incubation , blue bars after 72 hours . all three individual strains and the 3 - strain combination showed strong adherence to stainless steel . inhibition of the attachment of l . monocytogenes ( 10 3 cfu / ml ) by lab ( 10 8 cfu / ml ) on stainless steel is shown in fig4 . lab was allowed to attach to the coupons for 24 hr prior to the addition of l . monocytogenes . fig4 a shows the amount of l . monocytogenes remained after 24 and 72 hr incubation . lm ( lab → lm ) indicates the amount of l . monocytogenes when the coupons were pre - treated with lab for 24 hr . incubation time is considered from the time after l . monocytogenes inoculation . fig4 b shows the amount of lab remained after 24 and 72 hr incubation . lab ( lab → lm ) indicates the amount of lab when l . monocytogenes was added . fig4 c shows the limit of attachment inhibition by different concentration of lab . lm ( lab → lm ): l . monocytogenes attached to the coupons when pre - treated with lab ( 10 8 , 10 6 , 10 4 , 10 2 cfu / ml ). asterisk shows the significant difference ( p & lt ; 0 . 05 ). x axis = time , y axis = log cfu / ml of l . monocytogenes . blue bars are l . monocytogenes only ( control ), red bars l . monocytogenes + lab . for the competitive - exclusion assay , the steps described above for the incubation of the labs with the coupons were followed until the coupons were washed three times with 1 ml of pbs . the coupons were placed in a new 6 well plate with fresh tsbye and l . monocytogenes was added at 10 3 - 10 4 cells / well . the 6 - well plates were incubated for three hours at 23 ° c . to allow for the attachment of l . monocytogenes to the coupons . bacterial cells were washed again , transferred to new plates and incubate for 3 days at 23 ° c . then the coupons were washed and sonicated to release the cells . the cells were plated on mrs for labs and modified oxford agar ( mox ) for l . monocytogenes and the plates were incubated at 37 ° c . for 48 hours . fig5 shows the results of competitive attachment inhibition between l . monocytogenes ( 10 3 cfu / ml ) and lab ( 10 8 cfu / ml ) on stainless steel . a ) l . monocytogenes remained after 24 and 72 hr incubation . lm ( lm + lab ): l . monocytogenes remained when it was added to the coupons at the same time as lab . incubation time is considered from the time after inoculation of lab and l . monocytogenes b ) lab remained after 24 and 72 hr incubation . lab ( lab + lm ): lab remained on the coupons when lab and l . monocytogenes were added at the same time . asterisk shows the significant difference ( p & lt ; 0 . 05 ). fig6 shows displacement of l . monocytogenes ( 10 3 cfu / ml ) by lab ( 10 8 cfu / m1 ) on stainless steel . l . monocytogenes was pre - treated to the coupons for 24 hr prior to the addition of lab . a ) l . monocytogenes remained after 24 and 72 hr incubation . lm ( lm → lab ): l . monocytogenes when the coupons were pre - treated with l . monocytogenes for 24 hr prior to the addition of lab . incubation time is considered from the time after lab inoculation . b ) lab remained after 24 and 72 hr incubation . lab ( lm → lab ): lab when the coupons were pre - treated with l . monocytogenes 24 hr prior to the addition of lab . asterisk shows the significant difference ( p & lt ; 0 . 05 ). these results showed that lab cocktail ( 10 8 cfu / ml ) significantly reduced the attachment of l . monocytogenes ( 10 3 cfu / ml ) when both organisms were added at the same time on the coupons , and when lab was added 24 hr prior to the addition of l . monocytogenes . the inhibition was stronger when the coupons were pre - treated with lab cocktail for 24 hr prior to the addition of l . monocytogenes . the results showed that the inhibition increased over the incubation period . the pre - attached lab cocktail coated the surface , and effectively prevented l . monocytogenes attachment . lab was even able to displace l . monocytogenes that was initially allowed to attach on the coupons for 24 hr . on the other hand , lab cocktail was unaffected with presence of l . monocytogenes on the coupons . based on the tests described above , while the optimum lab concentration for inhibition of l . monocytogenes was about 10 8 cfu / ml to inhibit the attachment of 10 3 cfu / ml l . monocytogenes , about 10 4 cfu / ml of lab was able to inhibit the attachment of 10 3 cfu / ml l . monocytogenes . specific inhibition of listeria by select lactic acid bacterial strains bacteria culture and maintenance were performed according to standard techniques in the field . more particularly , all bacterial strains ( p . acidilactici d3 , lb . amylovorous m35 , lb . animalis la51 , and l . monocytogenes 10403s ) were stored at − 80 ° c . as glycerol stocks ( 15 % glycerol in either de man , rogosa , sharpe [ mrs , emd chemicals , gibbstown , n . j . ; lab strains ] or brain heart infusion [ bhi , emd chemicals ; l . monocytogenes ] broth ). for bacterial growth , mrs ( lab strains ) and bhi ( l . monocytogenes ) broth and agar were prepared according to the manufacturer &# 39 ; s instructions . all strains were grown to stationary phase ( approx . 22 h ) at 37 ° c . without aeration . for experiments using cell free preparations of the lab cultures , the desired lab culture ( pre - grown as described above ) was centrifuged and the resulting supernatant filter sterilized . cell free supernatants ( cfs ) were stored at − 20 ° c . spot - on - lawn assays were performed to determine the antimicrobial properties of the three lab strains against l . monocytogenes . spot - on - lawn assays were conducted using a modified method previously described ( palmer et al . 2009 ). briefly , l . monocytogenes were inoculated into bhi soft agar ( 0 . 7 %), which had been tempered to 50 ° c . after thorough mixing , the l . monocytogenes soft agar was poured into sterile petri plates containing a base layer of bhi agar and allowed to solidify in a laminar flow hood for 30 min . four μl aliquots of p . acidilactici d3 , lb . amylovorous m35 , and lb . animalis la51 ( whole cell cultures or cfs ) were then spotted onto the solidified l . monocytogenes soft agar . for control purposes , plates were spotted with 4 μl of mrs broth or 0 . 01 g / ml nisin ( 2 . 5 % w / w , prepared in water and filter sterilized immediately prior to use ). the diameters of the zones of inhibition were recorded in millimeters . three biological replicates of spot - on - lawn assays using the whole cell lab cultures were performed and spot - on - lawn assays using the lab cfs were performed in duplicate . the spot - on - lawn assay results indicated that the p . acidilactici strain had the highest anti - listerial activity ( average zone of inhibition diameter 16 mm , table 1 ) and there was no substantial difference in its activity across the 4 temperatures tested ( fig7 ). on occasion resistant l . monocytogenes colonies were observed growing within the zones , however , these colonies were rarely observed at the lower temperatures ( e . g . 4 and 7 ° c .) and were most often observed at the highest temperature tested ( e . g . 37 ° c .). furthermore , there was no substantial difference observed between the effectiveness of the p . acidilactici whole cell culture and the p . acidilactici cfs ( fig7 ). these results suggest that the antimicrobial agent produced by p . acidilactici d3 is secreted . mic assays were performed to evaluate the antimicrobial properties of the three lab strains using a method complimentary to the spot - on - lawn method described above . mic assays were performed as previously described ( milillo et al . 2011 ) with the following modifications . briefly , 50 μl of sterile bhi broth was added to all test wells in a 96 well plate . next , 50 μl of each lab cfs was added to the first well ( row a ) of each column ; then , serial 1 : 2 dilutions were performed down to the last row ( h ). this arrangement allowed for the testing of cfs dilutions of 1 : 4 ( row a ), 1 : 8 ( b ), 1 : 16 ( c ), 1 : 32 ( d ), 1 : 64 ( e ), 1 : 128 ( f ), 1 : 256 ( g ), and 1 : 512 ( h ). at this point , for the 7 ° c . mic experiments , the 96 well plate was transferred to 4 ° c . to pre - chill the dilutions . once the plate has been completely chilled , 50 μl of stationary phase l . monocytogenes ( 5 × 10 5 cfu ) freshly re - suspended in pre - chilled bhi broth was added to all test wells . for 23 ° c . mic experiments , all solutions and cultures were utilized at room temperature . mrs broth , bhi broth , 0 . 01 g / ml nisin ( prepared as described above ), and mrs broth adjusted to ph 4 ( using conc . lactic acid ) served as controls . the 96 well plate was then incubated at 7 ° c . or 23 ° c ., as appropriate , and the optical density results were recorded at 595 nm using a plate reading spectrophotometer with the automatic mixing function enabled ( biorad model 3550 microplate reader ; biorad , hercules , calif .). the lab cfs mic was defined as the lowest concentration of lab cfs that restricted l . monocytognes growth to an optical density & lt ; 0 . 05 . mic assays were performed in duplicate . cfs for all three lab strains was tested , and only the p . acidilactici cfs was effective at inhibiting l . monocytogenes growth over a wide range of concentrations ( up to a 1 : 512 dilution when exposure occurred for a short time [ i . e . for 24 h at 23 ° c . or 12 days at 7 ° c .]). however , over a longer course of exposure ( i . e . 48 h at 23 ° c . or 32 days at 7 ° c .) l . monocytogenes inhibition was limited to only the highest concentration of p . acidilactici cfs tested for either temperature ( i . e . 1 : 4 ). even though no l . monocytogenes inhibition was observed for lb . animalis and lb . amylovorous with the spot - on - lawn assay , some l . monocytogenes inhibition was observed in the mic assay . the amount of inhibition was similar to that observed for the control ( ph 4 mrs ), suggesting that the low ph of the lab cfs itself may be inhibitory to l . monocytogenes . this mechanism is likely because ph 4 . 3 has been shown to be the minimum ph for l . monocytogenes growth ( george et al . 1988 ). with the mic established , l . monocytogenes survival assays were performed at 7 ° c . for 21 days to determine if the lab cfs simply inhibited l . monocytogenes growth ( e . g . was bacteriostatic ) or was bacteriocidal . the lab strain / cfs concentration found most effective at inhibiting l . monocytogenes in the mic assays ( i . e . 1 : 4 ) was further examined for l . monocytogenes cell survival to determine whether the lab cfs was bacteriocidal . l . monocytogenes ( approximately 1 × 10 6 cfu / ml ) incubating in lab cfs ( prepared as described above ) at 7 ° c . was serially diluted in sterile phosphate buffered saline ( pbs ) and plated onto bhi agar for the duration of the experiment . three biological replicates of the survival assays were performed . consistent with the mild inhibition observed in the mic assay , the survival assay results show that when exposed to lb . animalis and lb . amylovorous cfs l . monocytogenes may initially decrease slightly but over time its numbers were stable ( fig8 ). l . monocytogenes exposed to the p . acidilactici cfs was reduced by nearly 5 logs within 24 h with a low level of l . monocytogenes surviving for the duration of the experiment . for control purposes , glucose ( 20 g / l , the same amount as in mrs broth ) was added to the lab cfs in order to compensate for the glucose consumed by the lab during growth . the addition of glucose was not observed to alleviate l . monocytogenes inhibition ( data not shown ), indicating that its inhibition during exposure to lab cfs was not due to a lack of nutrients available for growth . interestingly , these data also demonstrate that though resistance of l . monocytogenes to p . acidilactici cfs had previously been observed in the spot - on - lawn assays , resistance was not observed during the survival assay . l . monocytogenes is known to be able to develop resistance to bacteriocins produced by p . acidilactici ( gravesen et al . 2002 ) but studies have shown it to be effective against l . monocytogenes at 4 ° c . ( nielsen et al . 1990 , uhart et al . 2004 , youssef et al . 1991 ). thus , it is possible that the observed lack of resistance was due to the low temperature at which the survival assays were conducted as well as the significant contribution of the acidic ph ( see ph 4 mrs control ; fig8 ). this is consistent with results from the spot - on - lawn assay which indicated that spontaneous resistance occurred much less frequently for the spot - on - lawn assays conducted at 4 and 7 ° c . as compared to assays at higher temperature . experiments were performed to determine factors that may contribute to the lab inhibition of l . monocytogenes . the following conditions were tested to evaluate their contribution to l . monocytogenes inhibition : ( i ) ph ; ( ii ) heat stability ; and ( iii ) protease sensitivity . to determine the effect of ph on l . monocytogenes inhibition , the lab cfs ph ( routinely measured at approx . 4 ) was adjusted to 7 using 1 n naoh and the cfs re - filter sterilized prior to use . the effects of the ph - adjusted lab cfs on l . monocytogenes inhibition were measured using the mic experimental design described above . heat stability and protease sensitivity treatments were performed as described previously ( rea et al . 2010 ) and with the following changes . for heat treatment , p . acidilactici cfs was exposed to temperatures ranging from 40 to 100 ° c . for 15 to 90 min in a circulating water bath , as well as 121 ° c . for 15 to 90 min at 15 psi ( standard autoclave temperature and pressure ). for protease treatment , p . acidilactici cfs was exposed to 25 mg / ml proteinase k for 1 h at 37 ° c . the effects of the heat and protease treatments on l . monocytogenes inhibition were subsequently evaluated using the spot - on - lawn method described above . in addition , lactic acid production by the 3 lab strains was measured using their corresponding cfs and a colorimetric lactate dehydrogenase activity kit according to the manufacturer &# 39 ; s instructions ( biovision , inc ., san francisco , calif .). while ph may be one factor that contributes to the anti - listerial activity , it may not be the main contributing factor of p . acidilactici &# 39 ; s anti - listerial activity . although the three lab strains have very different effects on l . monocytogenes in the spot - on - lawn and mic assays , all three lab strains were found to produce approximately the same amount of lactate ( p & gt ; 0 . 05 , fig9 ). to lend support to the hypothesis that ph is not the only antilisterial mechanism , the lab cfs ph ( normally approximately 4 ) were adjusted with 1n naoh to approximately 7 and l . monocytogenes was subsequently exposed to the ph neutral cfs using the mic experimental method described above . after 24 h of incubation at 23 ° c ., a 1 : 4 dilution of cfs from lb . amylovorous and p . acidilactici , but not lb . animalis were inhibitory to l . monocytogenes . however , unlike the non - ph adjusted cfs , none of the ph adjusted lab cfs were inhibitory after 48 h of exposure . these data confirm that ph is not the only antilisterial mechanism of action for at least lb . amylovorous and p . acidilactici . as p . acidilactici was by far the most antilisterial of the three lab , the source of its antimicrobial activity was further investigated by examining its heat and protease stability using spot - on - lawn assays with heat and protease treated cfs . p . acidilactici cfs maintained full activity following all thermal treatments tested ( data not shown ). relative to the room temperature control , it maintained some activity following autoclaving ( 121 ° c . and 15 psi ) for 15 min ( fig1 a & amp ; 10b ), and activity was undetectable after autoclaving for 90 min ( data not shown ). in addition , p . acidilactici cfs lost activity following exposure to proteinase k ( fig1 c & amp ; 10d ). heat stability in combination with protease sensitivity is a common characteristic of class ii bacteriocins , which p . acidilactici has been shown to sometimes possess ( miller et al . 2005 ). analysis with pcr to detect the papa gene , which encodes the pediocin ach / pa - 1 preprotein , confirmed that p . acidilactici d3 contains this gene ( unpublished data ). p . acidilactici and pediocin ach / pa - 1 , the bacteriocin commonly produced by p . acidilactici , has been studied for use against l . monocytogenes but typically studies have only examined using p . acidilactici as a whole cell culture or the purified pediocin ( allende et al . 2007 , bari et al . 2005 , buyong et al . 1998 , foegeding et al . 1992 ). however , there are several drawbacks to using either of these approaches , including : reliance on p . acidilactici survival and in situ pediocin production ; lack of commercially available pediocin ; confirmation of pediocin activity in situ ; and labeling requirements for purified chemicals . for use in food products at high risk for l . monocytogenes contamination , especially those where in situ pediocin production by p . acidilactici might not be possible ( e . g . non - fermented foods ) and those where in situ pediocin activity might be effected by the surrounding food matrix , using p . acidilactici cfs represents a natural alternative . moreover , scattered reports exist documenting the potential for use of p . acidilactici cfs for control of l . monocytogenes in dairy products and ground beef ( hartman et al . 2011 , pucci et al . 1988 ). in conclusion , of the three lab strains tested , the p . acidilactici strain was the most effective at inhibiting l . monocytogenes growth and produced up to a 4 . 5 log reduction in l . monocytogenes numbers in vitro . the source of p . acidilactici cfs effectiveness against l . monocytogenes was shown to be a combination of properties that specifically target l . monocytogenes ( i . e . bacteriocin production ) and are generally antimicrobial ( i . e . acidic ph ). together , these data indicate that p . acidilactici cfs may represent an effective , natural control method for l . monocytogenes in food products . all references cited throughout the text or listed below are hereby incorporated into this disclosure as if fully reproduced herein . amezquita , a . ; 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