during storage of grains , fruits , vegetables , silage , and processed foods , the contamination with molds and yeasts may cause spoilage , which can be associated with substantial economic losses and potential health hazards .   to extend the shelf life of susceptible food and feed , considerable amounts of chemical preservatives are used . the development of natural preservatives , consisting of microorganisms generally recognized as safe ( gras ) , might form an alternative to the chemicals . both lactic acid bacteria ( lab ) and propionibacteria are safe , well - characterized bacteria , commonly used in many industrial processes [ 1 , 2 ] . the antifungal activity of lab ( reviewed in ) is explored in both applied systems [ 46 ] and at substance level [ 79 ] . the use of propionibacteria as a biopreservative culture has been   tested alone [ 10 , 11 ] and also in combination with lab [ 1215 ] . glycerol is a colorless , odorless liquid , widely used in a great number of applications ( e.g. , skin care products and drug solvents ) . the rapid increase in biodiesel production seen in   recent years   provides an abundant and inexpensive source of glycerol as a residue . previously , studies on the antifungal activity of lab found that addition of glycerol enhanced the antifungal effect of certain species . in lab , the coenzyme b12-dependent glycerol / diol dehydratase is involved in the conversion of glycerol into 1,3-propanediol . an intermediate in the process is 3-hydroxypropionialdehyde , also known as reuterin , a potent antimicrobial compound . the presence of a diol dehydratase in propionibacterium freudenreichii has been demonstrated , but a comparison with a number of species belonging to the genera enterobacteriaceae indicated that the diol dehydratase of p.   freudenreichii is different from the enterobacteria enzyme . growth of propionibacteria on glycerol increases propionic acid production , but the effect on the antifungal activity of these bacteria when using glycerol as single carbon source has not been previously described . this study aimed at investigating the effect of glycerol on the antifungal activity of five different dairy propionibacteria species , as well as their metabolite profile when grown in the presence of glycerol . nmr spectroscopy was used to elucidate the conversion pathway and the end products , using c - labeled glycerol fed to cultures of propionibacteria . the propionibacteria used in this study were the type strains of the following dairy propionibacteria species , obtained from deutsche sammlung von mikroorganismen und zellkulturen gmbh ( dsmz ) : propionibacterium acidipropionici dsmz 4900 , p. freudenreichii subsp . the fungi used in the overlay assay and the microtitre plate assay were the yeast rhodotorula mucilaginosa j350 ( cfsqe 63 ) and the molds penicillium commune j236 ( ibt 10763 ) and penicillium roqueforti j268 ( ibt 6754 ) . all fungi are part of the culture collection of the department of microbiology , swedish university of agricultural sciences . cultures of propionibacteria were grown on modified sodium lactate ( sl ) medium ( 1% sodium lactate ; sigma - aldrich , steinheim , germany , 1% tryptone ; oxoid ltd . , hampshire , england , 0.5% yeast extract ; oxoid ltd . and 0.5% kh2po4 ) at 30c in anaerobic jars ( bbl , becton , dickinson and co. , sparks , md , usa ) under co2 + n2 atmosphere ( gaspak system , bbl , becton , dickinson and co ) . for growth experiments with glycerol , the same medium was used , but the sodium lactate was replaced by 1% glycerol ( merck , darmstadt , germany ) ( gly ) . the yeast and molds were kept on malt extract ( me ) agar ( oxoid ltd . ) the yeast was cultured in me broth ( becton , dickinson and co. ) on a rotary shaker ( infors ag , bottmingen - basel , switzerland ) at 120  rpm at 25c overnight , while the molds were surface spread on fresh me agar ( oxoid ltd . ) the conidia ( asexual spores ) were then harvested from the slants using sterile peptone water . yeast cells and mold conidia were counted using a brker counting chamber ( marienfeld , lauda - koenigshofen , germany ) . the propionibacteria were inoculated in two parallel 2-cm streaks on sl plates ( six per strain ) and incubated at 30c under anaerobic conditions for 72 hours . soft agar ( 0.15% malt extract ( becton , dickinson and co. ) and 1% agar base ( oxoid ltd . ) ) with different concentrations of glycerol ( 0 , 10 , 50 , 100 , 200 , and 500  mm ) was cooled to 45c before fungal cells or conidia were added to obtain a concentration of 10 spores*ml . after incubation at 30c for 48 to 72 hours under aerobic conditions , the plates were examined visually . the plates were prepared in almost the same way as for the overlay assay , but the overlaid soft agar contained either 0  mm or 100  mm glycerol and no fungal cells or conidia were added . after the second incubation ( 48 hours at 30c under aerobic conditions ) , the agar between the bacterial streaks was removed with a scalpel and mixed with sterile water to achieve a dilution of 1  :  10 . the fluid was homogenized in a laboratory blender ( stomacher 400 , seward ltd . , worthington , uk ) for 60 seconds , followed by a soaking period of 3060 minutes . the fluid was transferred to vials and centrifuged ( 4500  rpm , 10 minutes ) to obtain agar - free liquid . standard solutions of lactic , acetic , and propionic acids at concentrations of 0.1 , 0.5 , 1.0 , 5.0 , 10 , and 15  mm were included in the analysis , performed on a c-18 column ( zorbax sb - c18 , agilent technologies , waldbronn , germany ) at 30c . as mobile phase 20  mm h3po4 , at a flow rate of 1.0  ml min , was used and the eluate was monitored with a uv detector ( agilent 1100 series , agilent technologies ) at 210  nm . all experiments were repeated four times , and the values presented are the mean values . propionibacteria were counted using a petroff - hausser cell counter ( hausser scientific partnership , horsham , pa ) and inoculated to achieve a final concentration of 10 cells*ml in sl and gly broth ( four replicates for each combination ) . the cultures were incubated as still cultures at 30c for 72 hours . for viable bacterial count , one ml aliquots were serial diluted ( 1  :  10 ) six times with sterile peptone water , and 100  l of the two lowest dilutions were spread on sl agar plates and incubated at 30c under anaerobic conditions . the remainings of the fermented broths were centrifuged ( 4500  rpm , 10 minutes ) and filtered through 0.45  m filter ( nalgene nunc int . ) to obtain cell - free supernatant . all experiments were repeated four times , and the values presented are the mean values . the supernatants from the liquid cultures were also tested for antifungal activity using a microtitre plate assay . for each culture , two wells were prepared by adding 50  l of supernatant to 50  l of a cell or conidium suspension ( prepared as previously described ) to achieve a final concentration of 5  10  ml . uninoculated sl , gly , and me media were used as controls ( 50  l of broth + 50  l of cell or conidium suspension ) . the plates were incubated at 30c in plastic bags , supplemented with a moist paper tissue to maintain humidity , and visually examined for fungal growth inhibition after 48 to 72 hours . to correlate the growth stage and antifungal activity , freudenreichii and p. jensenii were washed in 0.8% nacl solution and counted using a petroff -hausser counting chamber . a concentration of 10 bacteria ml was inoculated in gly broth and incubated anaerobically as still cultures at 30c . after 2 , 8 , 12 , 24 , 36 , 48 , 56 , 72 , 80 , and 98 hours , 1  ml samples were withdrawn , and one aliquota was diluted and counted using a petroff -hausser counting chamber . the remaining of the sample was centrifuged ( 12 000  rpm , 10  min ) and the supernatant was collected and kept at 20c until further analysis . the supernatants were then diluted with sterile water two , four , and eight times . aliquots ( 50  l ) of all dilutions were added to two microtitre plate wells containing 50  l of a p. roqueforti conidial suspension . eight wells were also prepared with 50  l me broth and 50  l conidial suspension as growth controls . the final supernatant concentration in the wells was thus 50% ( for the pure supernatant ) , 25% , 12.5% , and 6.25% and the conidium concentration was 5  10  ml . plates were incubated at 30c in plastic bags containing a moist paper tissue to maintain humidity . after 48 hours , the plates were examined visually , and the degree of inhibition was compared to control wells and rated according to the following scale : 0 , no inhibition , 1 , weak visible inhibition , 2 , obvious inhibition , 3 , strong inhibition , but still visible growth , and 4 , complete inhibition . the glycerol conversion pathways in propionibacteria were studied by growing the cells in c - labeled glycerol , and analyzing the spent growth medium with nmr . based on results from the previous experiment , the setup was the same as in the correlation experiment above , except for the medium which contained c - labeled glycerol ( 50% 1,3-labeled and 50% 2-labeled ; larodan fine chemicals ab , malm , sweden ) instead of glycerol . the cell number was counted , and the remaining of the sample was centrifuged ( 12 000  rpm , 10  min ) , filtered ( 0.45  m ) , and frozen at 20c until further analysis . the supernatants were analyzed for organic acids and low - molecular - mass metabolites with hplc and c nmr spectroscopy . the hplc analysis was performed on a cation exclusion column ( rezex roa - organic acid , phenomenex inc . , torrance , ca , usa ) at 25c , using 5  mm h2so4 as mobile phase at a flow rate of 0.6  ml min . the eluate was monitored with a refractive index detector ( agilent 1100 series , agilent technologies ) . standard solutions of lactic , acetic , and propionic acid at concentrations of 10 , 25 , 50 , and 100  mm were included in the analysis . the c nmr samples were prepared by mixing aliquots ( 600  l ) of each supernatant with d2o ( 100  l ) . the samples were analysed by c nmr spectroscopy ( 100  mhz ) at 30c on a bruker drx-400 nmr spectrometer ( bruker biospin gmbh , rheinstetten , germany ) equipped with a 5-mm qnp probe head . data from 128 scans were accumulated , and the spectral width was 240  ppm . the ph of the supernatants was recorded using a phm92 ph meter ( radiometer , copenhagen , denmark ) . differences in bacterial growth ( when monitored ) , ph , and acid production were analyzed for each propionibacteria when grown in different media , for both agar and broth methods , by two - way analysis of variance using bonferroni posttests ( prism4 , graph pad   software ) . all five type strains of propionibacteria were tested against three different target fungi ( two molds and one yeast ) at glycerol concentrations between 0  mm and 500  mm . freudenreichii ( figure 1 ) , as well as by the other tested propionibacteria ( results not shown ) when the bacteria was grown in the presence of glycerol , with increasing concentrations of glycerol enhancing the antifungal effect . the indicator mold penicillium commune was the most sensitive fungi tested , and it was completely inhibited in the presence of 500  mm glycerol by most propionibacteria strains . penicillium roqueforti was only slightly inhibited by p. acidipropionici and p. thoenii even at high glycerol concentrations , but with p. freudenreichii subsp . the yeast r. mucilaginosa was the most resistant organism to the inhibitory activity of propionibacteria . from the results obtained , only p. jensenii showed visible inhibitory effects against r. mucilaginosa in the presence of glycerol , at concentrations of 200 and 500  mm the results of the acid analysis and ph measurements from the agar extractions are shown in table 1 . to evaluate the extraction efficiency of propionic acid from the agar , the analysis was first performed with plates containing 50 and 100  mm pure propionic acid . the procedure proved itself to be adequate since similar amounts of propionic acid were quantified in the extracts analyzed ( data not shown ) . from plates without bacteria , only lactic acid ( 120  mm ) and/or glycerol ( 30  mm ) were extracted from the medium . the initial ph of the uncultured plates , with or without glycerol , was 5.5 . the comparison between plates with and without glycerol for each propionibacterium   revealed significant difference in the remaining amount of lactic acid only for p. jensenii ( p < .01 ) with a smaller amount remaining on the plates without addition of glycerol . p. thoenii also showed a tendency towards less lactic acid remaining on plates without glycerol , but since the results from glycerol overlaid plates represented only two measurements , no reliable statistical analysis could be done . for p. freudenreichii subsp . shermanii , there was a significant difference in production of   propionic acid , with higher amounts produced in the presence of glycerol ( p < .001 and p < .05 , resp . ) . the content of glycerol decreased in the medium after bacterial growth for all strains , but to a minor extent with p. acidipropionici . there was a significant difference in the ph for all of the strains when their growth in the presence of glycerol was compared ( p < .001 ) . production of acids and change in ph were recorded for the five propionibacteria strains grown in sl and gly broths ( table 2 ) . the initial ph of uncultured sl broth was 5.5 , while gly broth had a ph of 6.0 . lactic and acetic acid were only detected in cultures of sl medium and the production of propionic acid was significantly higher in these cultures ( p < .001 for p. freudenreichii , and p. jensenii and p < .01 for p. acidipropionici ) with the exception of p. thoenii ( p > .05 ) . final counts cfu ml did only differ significantly between sl and gly for p. freudenreichii subsp . the final ph was noticeably lower in the gly medium ( p < .001 for all strains ) ( table 2 ) . the supernatants from the liquid culture experiment were tested for antifungal activity in a microtitre plate assay ( table 2 ) . supernatants from cultures grown in sl medium , did not inhibit growth of any of the tested fungi , while   growth of propionibacteria in gly medium gave a clear inhibitory effect . the molds were inhibited by p. acidipropionici , p. thoenii , and p. jensenii , but not by p. freudenreichii subsp . all three fungi were able to grow in uncultured sl and gly broths ( data not shown ) . freudenreichii and p. jensenii against penicillium roqueforti was evaluated during bacterial growth , and the results can be seen in figure 2 . no antifungal activity was detected before the stationary growth phase ( 48  h ) was achieved . however , after 48  h of incubation , the effect increased with time , reaching the highest level at 80  h. to demonstrate the increasing antifungal activity of the supernatants with time , they were diluted in sterile water two , four , and eight times . ( 1 on the scale ) against penicillium roqueforti   after 48  h of incubation when twofold diluted . at this dilution , maximum inhibition was seen at 80  h. when the culture filtrate was diluted four times , inhibition of penicillium roqueforti was weak even at 56 h but increased after 72  h and was maintained at the higher level throughout the experiment . freudenreichii . when the culture filtrate of p. jensenii was diluted two times ,  complete inhibition ( 4 on the scale ) was seen already at 48  h. with a fourfold dilution of the same culture filtrate , complete inhibition was reached at 56  h. when it was diluted eight times , clear inhibition of penicillium roqueforti was recorded after 72  h , with maximum inhibition achieved at 80  h and maintained until the end of the experiment . no further increase in inhibitory activity was observed between 80 and 98  h. the experiment was repeated with the same results ( not shown ) . a fresh culture of p. jensenii was grown for 96 h in gly broth prepared with c - labeled glycerol . samples were withdrawn at 0 , 24 , 48 , and 96  h and analyzed with respect to ph , bacterial counts , glycerol , and production of acids ( table 3 ) . the ph of the broth at the beginning of the experiment was 6.2 , and after 24  h of incubation with p. jensenii only a slight reduction to 6.1 was observed . after 48  h of incubation , the ph dropped to 4.9 and at the last sampling point , 96  h , it was 4.6 . initially , hplc analysis only detected glycerol in the gly broth , but with each following sampling point , the level of propionic acid increased at the expense of the glycerol concentration . however , the decrease in glycerol concentration was faster than the production of propionic acid , indicating the utilization of glycerol for bacterial growth supporting pathways . each sample was also analyzed with c nmr spectroscopy to follow the glycerol conversion , and propionic acid was the only c - labeled metabolite detected ( figure 3 ) . the concentration of propionic acid increased over time , with the highest concentration detected in the sample harvested after 96  h.   figure 3(a ) shows c - labeled glycerol , where asterisks denote the labeled carbons . the labeling pattern of the propionic acid formed is also shown by asterisks in figure 3(d ) . the signals from c - labeled glycerol decreased over time , and at 48  h ( figure 3(c ) ) , small signals from c - labeled propionic acid appeared . figure 3(d ) shows signals for propionic acid produced after 96  h of incubation . it also presents magnified regions of the c nmr spectrum and schematic pictures of the propionic acid formed with labeling at c1 and c2 , c1 , and c3 , and a single label at c2 . labeling at both c1 and c2 resulted in doublet signals for both c1 and c2 , caused by spin - spin coupling ( jc , c   53  hz ) of the neighboring c nuclei . labeling at both c1 and c3 did not result in any observable signal splitting , due to the small values for jc , c in aliphatic carboxylic acids ( often ~1 hz ) . the antifungal activity of dairy propionibacteria type strains was evaluated in the presence of glycerol . studies were performed on both liquid and solid media and complemented with a glycerol conversion study using c - labeled glycerol . mold inhibition in the overlay assay was observed with all bacteria , and by increasing the glycerol concentration , the inhibitory effect was enhanced . however , with the overlay assay , the inhibitory activity of the different bacteria against the yeast r. mucilaginosa was hardly affected by the increasing concentration of glycerol . in broth , molds were only inhibited by the supernatant of gly medium cultured with p. acidipropionici , p. jensenii , and p. thoenii . in contrast to the solid matrix assay , the yeast under evaluation was completely inhibited by all propionibacteria when the supernatant of gly medium was used . there is one important difference between the two experimental systems that needs to be considered . in broth cultures , the bacterial biomass is removed after 72 hours of incubation , resulting in a constant ph and metabolite concentration for the following assay , while the agar plate assay produces a continuous supply of metabolites from the growing bacteria , diffusing into both layers of agar and lowering the ph . while the continuous diffusion of propionic acid in the overlay method favored the inhibition of mold growth , the yeast was not affected to the same extent . it is known that propionic acid , as well as sodium , potassium , and calcium propionate , is an effective inhibitor of many molds but may show only weak activity against yeasts and bacteria . when comparing an agar diffusion method and a broth microdilution test for determining the minimal inhibitory concentration ( mic ) values of different antibiotics against brachyspira hyodysenteriae , rohde et al the mic values obtained were , on average , one dilution step lower for the broth dilution method . the difference observed was explained based on the divergent characteristics of the solid and liquid media , which provide different conditions for the diffusion of antibiotics or other substances . the specific growth characteristics of the unicellular yeasts and the filamentous molds may also lead to different responses to antifungal conditions in diverse environments . however , the levels of glycerol added to the agar in the plate assays gave no measurable reduction in water activity ( data not shown ) . indeed , the addition of 100  mm glycerol to water only decreased the water activity from 1.000 to 0.995 , so a much higher glycerol concentration would be needed to cause any fungal growth inhibition . the final ph of liquid bacterial cultures grown in the presence of glycerol was significantly lower which might have contributed to the enhanced effect observed with these cultures . the composition of the media plays an important role on the final ph of the cultures . addition of acid to this medium lowers the ph ( 4.7 after addition of 50  mm propionic acid ) but not as much as compared to acid addition to   pure water ( ph 2.9 with the same acid ) , due to the buffering capacity of sodium lactate . glycerol has no buffering capacity , so acids produced by propionibacteria in gly medium decrease the ph more efficiently . because propionic acid has a pka of 4.87 , at the final ph achieved in the cultured broths ( ph of 4.34.7 ) , more than 50% of the total propionic acid content occurs in undissociated form . the inhibitory effect of weak acids ( i.e. , propionic acid ) is often attributed to the undissociated molecules . however , the exact mode of action is still not fully understood and a number of theories exist . one suggests the prevention of growth by inhibition of the active transport into the cells , while another involves the negative effects of pumping protons from the acid by the plasma membrane h - atpase pump , blocking further growth - supporting action . previously , the sensitivity of eight spoilage fungi to three weak acids at different ph values has been reported . for propionic acid at ph 5 , the mic reported for r. mucilaginosa was 30  mm , which clearly explains the suppressed growth of this yeast by all propionibacteria tested in liquid gly cultures ( table 2 ) . for molds used in this study , lind et al .   reported that propionic acid had an mic of 40  mm at ph 5.0 , which is also in accordance with the results reported here for liquid cultures . shermanii produced only 26  mm propionic acid , which was insufficient for total growth inhibition of the molds tested at ph around 5.0 . the cultures grown in sl medium displayed a higher final ph , close to 6 , which requires increased propionic acid concentrations for inhibition of fungal growth , since only approximately 7% of the propionic acid is present in its undissociated form at this ph . when production of acids in the two systems used for bacterial growth ( liquid and overlay assay ) is considered , the two methods can not be directly compared . supernatants from broth cultures are homogenous and contain the total amount of the acids determined by analysis . in gel plugs , the two layers of different substrate are mixed together for acid extraction , and the extract contains the mean value from both agar substrates . in solid medium , the amount of propionic acid produced was significantly higher for three of the five strains tested when glycerol was present in the substrate . considering that the bacteria were grown on sl agar have grown on sl for 72 hours and then provided with an additional carbon source ( overlaid glycerol ) , the higher propionic acid yield was not surprising , also visible in table 1 . in contrast , growth in liquid sl medium resulted in more propionic acid than growth in liquid gly medium . strains belonging to p. freudenreichii species showed a more pronounced difference , producing less than half of the propionic acid amount in gly than in sl broth . since the buffering capacity of sl medium ( sodium lactate ) is absent in gly medium , the production of propionic acid immediately lowers the ph , which can promote an inhibiting environment even for the propionibacteria itself . the results indicate a tendency toward a homofermentative pathway producing only propionic acid when glycerol was the energy source ( table 2 ) . earlier studies have shown that the use of glycerol as carbon source results in higher yield of propionic acid and less diversity in end - product composition compared to the use of glucose or lactic acid . a similar observation was also made in a comparison of glycerol and glucose as carbon sources for two species of propionibacteria in batch cultures .   the observed c labeling pattern of propionic acid agrees with a previously suggested metabolic pathway for the formation of propionic acid from glycerol . in this pathway , glycerol is transformed to pyruvate , via dihydroxyacetone phosphate and phosphoenolpyruvate ( figure 4 ) . oxaloacetate is transformed to malate and then to succinate , and finally co2 is eliminated from succinate to produce propionic acid . thus , during growth on c glycerol , a portion of the co2 released by the bacteria is expected to be c - labeled . if c - labeled co2 , derived from a labeled glycerol molecule , is added to pyruvate , propionic acid c - labeled on both c1 and c2 may be formed ( figure 4 ) , as observed in the present study ( figure 3 ) . the conversion of glycerol into 3-hydroxypropionaldehyde , also known as reuterin , in lab , involves a coenzyme b12-dependent glycerol / diol dehydratase . this enzyme is found in species of enterobacteriaceae and propionibacteriaceae , but the enzymes are immunologically distinct from each other . to investigate the possibility that propionibacteria produce a reuterin - like antimicrobial compound similar to that found in lab shermanii digested with bamhi , with two adjacent p - labeled ecori - fragments derived from l. coryniformis si3 according to standard methods . the two fragments , together constituting approximately 5500  bp , contained the complete pdua , the two pdub , the pduc , pdud , and pdue - genes . however , no signal was obtained even at low , stringency conditions ( data not shown ) . combinations of antifungal propionibacteria and glycerol might be used as biopreservation systems by the food and feed industry , where fungal spoilage are of concern . in a recent study , suhr and nielsen   found that propionate was an effective mold inhibitor , except against p. roqueforti , p. commune , and eurotium rubrum , as long as the ph and aw were not too high . both glycerol and propionic acid are approved food additives , and propionibacteria are today commonly used as starter cultures in dairy products and bread . therefore , the use of glycerol as part of a fungal inhibitory system along with   propionibacteria could provide additional positive properties , since glycerol often is used as an additive to reduce the aw . besides , the biodiesel industry would also benefit from new uses of   the increasing surplus of glycerol . finding a qualified combination of propionibacteria and glycerol for the application as a biopreservation system in the food and feed industry is an interesting challenge for future research .
dairy propionibacteria are widely used in starter cultures for swiss type cheese . 
 these bacteria can ferment glucose , lactic acid , and glycerol into propionic acid , acetic acid , and carbon dioxide . 
 this research examined the antifungal effect of dairy propionibacteria when glycerol was used as carbon source for bacterial growth . 
 five type strains of propionibacteria were tested against the yeast rhodotorula mucilaginosa and the molds penicillium commune and penicillium roqueforti . 
 the conversion of 13c glycerol by propionibacterium jensenii was followed with nuclear magnetic resonance . in a dual culture assay , 
 the degree of inhibition of the molds was strongly enhanced by an increase in glycerol concentrations , while the yeast was less affected . in broth cultures , 
 decreased ph in glycerol medium was probably responsible for the complete inhibition of the indicator fungi . 
 nmr spectra of the glycerol conversion confirmed that propionic acid was the dominant metabolite . 
 based on the results obtained , the increased antifungal effect seen by glycerol addition to cultures of propionibacteria is due to the production of propionic acid and ph reduction of the medium .