due to their bioactive properties , some vegetables or their extracts have been applied to a large number of human activities ( e.g. food preservation ) since ancient times ( medina et al . , 2006 ) .
in particular , a wide range of plant extracts showed certain levels of inhibition against bacterial growth . in most cases ,
the antibacterial activity is attributed to the presence of phenolic compounds that are metabolites involved in the resistance against parasites ( servili et al .
olives , virgin olive oil and its secondary products such as olive mill wastewater showed a high level of phenolic compounds .
the glucoside oleuropein is the main phenolic compound ( secoiridoids ) in fruit ; moreover in olive products the molecules of its hydrolysis exert a stronger antimicrobial activity ( medina et al . , 2006 ) .
however , most part of these compounds is lost in wastewater during the olive oil extraction process .
it was recognized that the presence of phenolic compounds reduces the microbial degradability of oil vegetation waters ( vws ) leading to pollution problems ( capasso et al .
, 1995 ; tafesh , et al . , 2011 ; saadi et al . ,
2007 ) however , vws could be considered as additional resources for the virgin olive oil ( voo ) industry .
in fact several phenolic compounds occurring in vw , having the same chemical characteristics of voo phenols , possess many biological properties that include antioxidant activity and , for this reason , may be used in food industry as natural preservatives or bio - active ingredients ( servili et al . , 2011b ) .
the aim of the study was to assess the in vitro bactericidal effect of purified phenols extracted from oil vegetation water ( peow ) obtained by membrane filtration techniques from vw on several food - borne strains ( spoilage bacteria , food - borne pathogens and starter cultures ) .
the assessment of bactericidal activity could define some threshold doses for a further application on real food models .
in figure 1 the protocol for the separation of the phenols extracted from oil vegetation water is reported .
a panel of 18 food - borne strains was investigated for the minimum bactericidal concentration ( mbc ; table 1 ) in a microtiter assay .
several two - fold dilutions of the peow ( figure 1 ) were performed in a 20% ethanol / water solution ( 12.0 ; 6.0 ; 3.0 ; 1.5 ; 0.75 ; 0.375 mg / ml ) , considering a 65% of total phenolic content on the initial extract ( carraro et al . , 2014 ) .
each well was added with 50 l of peow and 200 l of bacterial suspension in a 96 well plate with a final bacterial concentration of 10 ufc / ml . for each plate 3
wells without bacteria were performed according peow dilution ( negative control ) . moreover , for each strain 3 wells were added with 200 l of bacterial suspension and 50 l of a 20% ethanol / water solution ; an additional positive control was performed for each strain using 250 l of bacterial suspension .
microtiter plates were incubated at specific temperature / time for each strains and the mbc was evaluated on agar plate by spreading 10 l of each suspension .
the dose that kills the bacteria was recorded and the mbc was defined as the level that did not allowed the survival of all replicates . a visual examination of well bottoms was also applied in order to have a visible growth and define the minimum inhibitory concentration ( mic ) .
table 1 reported the mbc value for the food - borne bacteria tested . among pathogenic strains , s. aureus and l. monocytogens showed the lowest level of resistance to peow ( mbc=1.53 mg / ml ) . for gram positive strains , some events of heteroresistance were observed and a stringent evaluation of mbc was based on the highest level that did not allowed the growth on all replicates . on the opposite , in gram negative strains ( e.g s. typhimurium and pseudomonas spp . ) the mbcs ranged between 6 to 12 mg / ml with the exception of escherichia coli o:157 h7 ( 3 mg / ml ) .
starter cultures were dramatically reduced in growth ( e.g. s. xylosus ; mbc 0.75 - 1.5 mg / ml ) .
pediococcus pentosaceus seemed the more resistant species among actic acid bacteria ( lab ) , the strain was able to survive at higher concentrations of peow ( mbc 12 mg / ml ) .
the ethanol / water solution did not affect the survival of any tested strains , the results were in agreement with the previously observation on e. coli ( carraro et al . , 2014 ) .
the final concentration of ethanol was around 1/4 of the initial solution ( 5% ) , this level was ineffective to kill the pre - inoculum of the tested bacteria .
the mic is the lowest concentration of an antimicrobial that inhibit the growth of a microorganism .
the visual examination has resulted in some misinterpretation due to the turbidity of the extract after the incubation , especially at higher peow concentrations ( 6 - 12 mg / ml ) ; for this reason the evaluation of mic is not reported .
as reported by carraro et al . ( 2014 ) the composition of the extract showed that oleuropein - aglycone di - aldehyde was the major secoiridoid constituent ( 471.7  1.9 mg / g ) .
others chemical compounds are hydroxytyrosol ( 72.7  0.6 mg / g ) , tyrosol ( 17.8  0.1 mg / g ) and verbascoside ( 83.6  1.0 mg / g ) .
the in vitro tests suggested an interesting bactericidal effect of peow , especially on gram positive food - borne pathogens .
other studies reported that s. aureus and l. monocytogenes showed a higher sensitivity to phenols derived from several olive matrices ( e.g olive oil , olive leaf and purified compounds ) ( medina et al .
, 2006 ; pereira et al . , 2007 ) . moreover , thafesh et al .
( 2011 ) suggested that the use of a combination of polyphenols extracted by olive mill wastewater is effective against several human pathogens .
the present results confirmed the potential of peow , though the bioactivity could be related to the content of certain specific phenolic compounds ( thafesh et al . , 2011 ) .
this is probably due to the formation of a brown - coloured quinonic form due to the oxidative degradation of phenols .
the present results suggested two thresholds of bactericidal effect on gram positive food - borne pathogens .
however , other pertinent considerations need to be considered when these products are added to the food ( e.g organoleptic traits , diffusion , bonding with some food constituents ) .
taking into account mbc results , the lab and s. xylosus are among the most sensitive bacteria . in a functional milk beverage fortified with phenolic compounds
still , the level of inclusion was limited ( 100 and 200 mg / l ) .
in other fermented products , the supplementation of higher levels of peow , as a natural ingredient , could reduce the performance or the quality of ripening .
for further applications , a deep screening of starter cultures is required in order to select the species able to grow in presence of phenol during fermentation .
the mbc suggested that one strain of pediococcus pentosaceus was able to grow at 12 mg / ml : this species could be applied to some fermented food ( e.g salami ) .
this work is the first step before the use of these substances on the food models ( e.g. challenge test and storage test ) for the further application of this extract as an ingredient .
the two thresholds proposed for the pathogenic food - borne bacteria need to be considered together with other pertinent food aspects ( e.g. organoleptic traits , antioxidant effects ) .
 the aim of the study was to assess the in vitro effect of phenols extracted from oil vegetation water ( peow ) on several food - borne strains . 
 antibacterial activity of peow was based on the minimum bactericidal concentration ( mbc ) on microtitre assay . 
 the taxa tested were : staphylococcus ( n. 5 ) , listeria ( n. 4 ) , escherichia ( n. 2 ) , salmonella ( n. 1 ) , pseudomonas ( n. 3 ) , lactobacillus ( n. 2 ) and pediococcus ( n. 1 ) . 
 s. aureus and l. monocytogens showed the lowest level of resistance to peow ( mbc=1.5 - 3 mg / ml ) . 
 in contrast , the gram negative strains ( e.g. s. typhimurium and pseudomonas spp . ) were in some cases unaffected by the tested doses and the mbcs ranged between 6 to 12 mg / ml . starter cultures were dramatically reduced on growth ( e.g. staphylococcus xylosus ; 0.75 mg / ml mbc ) . 
 the thresholds for pathogenic strains could be considered for further applications of peow in food models ( e.g. shelf life or challenge test studies ) . 