kefir is a probiotic containing lactic acid bacteria , acetic acid bacteria , and yeast ( guzel - seydim et al . , 2011 ) . during the fermentation process , organic acids such as lactic and acetic acid and alcohol are produced and play a physiological role ( gaware et al . , 2011 ) . many studies have investigated the beneficial effects of kefir , including its antitumorigenic and antistress properties and its immunomodulatory and hypocholesterolemic functions in animal models ( gaware et al . , 2011 ; meydani and ha , 2000 ; rodrigues et al . , 2005 ; saloff - coaste , 1996 ; vinderola , 2005 ; wheeler et al . , 1997 ) . additionally , several studies have demonstrated its inhibitory activities against gram - negative and gram - positive food - borne bacterial pathogens ( cevikbas et al . , 1994 ; garrote et al . , 2000 ; silva et al . , 2009 ; ulusoy et al . , 2007 ) . in northeast brazil , the community organization pastoral da carianca distributes kefir grains to mothers with children affected by gastrointestinal diseases ( silva et al . , 2009 ) . ( 2003 ) reported that kefir administration is effective in preventing campylobacter jejuni colonization in chicks . however , although kefir has been used in the treatment of various gastrointestinal infectious diseases in humans and animals anecdotally , there is only limited information on the relationship between fermentation time and the range , potency , and onset of antimicrobial activity of kefir . in addition , several studies showed conflicting results on the antimicrobial spectrum against various pathogens by using kefir from different origins ( anderson and gilliland , 1999 ; pintado et al . , 1996 ) . therefore , here , we investigated the antimicrobial activities of kefirs fermented for 24 , 36 , 48 , or 72 h against eight bacterial test strains and compare them with the activity of organic acid and ethyl alcohol to elucidate the key attributer of the antimicrobial activity . additionally , we compared the antimicrobial spectra of four different kefirs to investigate the differences in the antimicrobial spectrum of kefir from different origins . this study aimed to elucidate the optimal fermentation time and conditions for achieving the broadest and most potent antimicrobial activities of kefirs against eight of food - borne pathogens and spoilage bacteria . four types of kefir grains , i.e. , a , l , m , and s , were corrected from private households in korea . each kefir grain is different in the shape and size , and thus , regarded as a different kefir grain : round and 6 - 10 mm , kefir grain a ; oval and 4 - 7 mm , kefir grain l ; oval and 8 - 12 mm , kefir grain m ; round and 10 - 15 mm , kefir grain s ; round and 13 - 19 mm . a total of 100 g of viable kefir grains was inoculated in 1000 ml sterilized milk ( 10% w / v ) and cultured at 25 for 24 , 36 , 48 , or 72 h. at the end of the fermentation process , the grains and milk were separated using a sterilized plastic filter ( 2-mm pore size ) . bacillus cereus atcc14579 , staphylococcus aureus atcc6538 , listeria monocytogenes atcc51776 , enterococcus faecalis atcc19433 , escherichia coli atcc 25922 , salmonella enteritidis ( originally obtained from the food and drug administration [ fda ] , college park , usa ) , pseudomonas aeruginosa atcc15522 , and cronobacter sakazakii atcc29544 were used in antimicrobial activity tests . each strain was streaked onto columbia blood agar ( biomerieux , france ) for two passages and incubated in tryptic soy broth ( difco laboratories , usa ) for 24 h at 37 for antimicrobial activity tests . for antimicrobial activity tests , kefir milk was centrifuged at 3,134 g for 10 min , and the supernatant was sterilized by filtration using a 0.45-m pore - size syringe filter ( millipore co. , usa ) . the ph of the filtered kefir supernatant was determined with a ph meter model 205 ( testo , germany ) equipped with temperature compensation , and calibrated using ph 4.00 and 7.00 buffers . antibacterial activity was detected by the spot on lawn method with some modifications ( cadirci and citak , 2005 ) . all test bacteria were cultured on mueller - hinton broth ( mhb ; difco ) and incubated at 37 for 24 h. the culture broth was diluted using mhb to 0.5 mcf and spread onto mueller - hinton agar ( mha ; difco ) using sterilized cotton swabs . twenty microliters of each kefir supernatant was directly dropped onto the surface of the mha . the plates were incubated for 24 h at 37 , and the inhibition zone was observed . the presence of a clear zone at the site of kefir supernatant inoculation was considered as total inhibition , while a decrease in cell density was considered as partial inhibition . as a control , lactic acid solution ( sigma - aldrich , usa ) , acetic acid solution ( sigma - aldrich ) , and ethyl alcohol ( duksan pure chemicals , korea ) were diluted with sterilized distilled water . all solutions were sterilized by filtration using a 0.45-m poresize syringe filter ( millipore ) before use . four types of kefir grains , i.e. , a , l , m , and s , were corrected from private households in korea . each kefir grain is different in the shape and size , and thus , regarded as a different kefir grain : round and 6 - 10 mm , kefir grain a ; oval and 4 - 7 mm , kefir grain l ; oval and 8 - 12 mm , kefir grain m ; round and 10 - 15 mm , kefir grain s ; round and 13 - 19 mm . a total of 100 g of viable kefir grains was inoculated in 1000 ml sterilized milk ( 10% w / v ) and cultured at 25 for 24 , 36 , 48 , or 72 h. at the end of the fermentation process , the grains and milk were separated using a sterilized plastic filter ( 2-mm pore size ) . bacillus cereus atcc14579 , staphylococcus aureus atcc6538 , listeria monocytogenes atcc51776 , enterococcus faecalis atcc19433 , escherichia coli atcc 25922 , salmonella enteritidis ( originally obtained from the food and drug administration [ fda ] , college park , usa ) , pseudomonas aeruginosa atcc15522 , and cronobacter sakazakii atcc29544 were used in antimicrobial activity tests . each strain was streaked onto columbia blood agar ( biomerieux , france ) for two passages and incubated in tryptic soy broth ( difco laboratories , usa ) for 24 h at 37 for antimicrobial activity tests . for antimicrobial activity tests , kefir milk was centrifuged at 3,134 g for 10 min , and the supernatant was sterilized by filtration using a 0.45-m pore - size syringe filter ( millipore co. , usa ) . the ph of the filtered kefir supernatant was determined with a ph meter model 205 ( testo , germany ) equipped with temperature compensation , and calibrated using ph 4.00 and 7.00 buffers . antibacterial activity was detected by the spot on lawn method with some modifications ( cadirci and citak , 2005 ) . all test bacteria were cultured on mueller - hinton broth ( mhb ; difco ) and incubated at 37 for 24 h. the culture broth was diluted using mhb to 0.5 mcf and spread onto mueller - hinton agar ( mha ; difco ) using sterilized cotton swabs . twenty microliters of each kefir supernatant was directly dropped onto the surface of the mha . the plates were incubated for 24 h at 37 , and the inhibition zone was observed . the presence of a clear zone at the site of kefir supernatant inoculation was considered as total inhibition , while a decrease in cell density was considered as partial inhibition . as a control , lactic acid solution ( sigma - aldrich , usa ) , acetic acid solution ( sigma - aldrich ) , and ethyl alcohol ( duksan pure chemicals , korea ) were diluted with sterilized distilled water . all solutions were sterilized by filtration using a 0.45-m poresize syringe filter ( millipore ) before use . the ph values of the kefirs during the fermentation process are shown in table 1 . during the fermentation process temporal changes in the antimicrobial spectra of the kefirs against the food pathogens and spoilage bacteria are also presented in table 1 . antimicrobial activity generally increased along with fermentation time in all types of kefirs ; this effect may be related to the decreased ph observed during the fermentation process . ( 2007 ) , in which there were no differences in the antimicrobial activities of kefirs fermented for 24 or 48 h. silva et al . ( 2009 ) , however , reported results that were consistent with our observations that the antimicrobial activities of kefirs generally increased with prolonged fermentation times . the traditional method for producing kefir is to ferment milk with kefir grain for 18 - 24 h at 20 - 25 before consumption ( beshkova et al . , 2002 ; farnworth and mainville , 2008 ; otles and cagindi , 2003 ) . however , here , the broadest antimicrobial spectra against eight of food pathogens and spoilage organisms were obtained after at least 36 - 48 h of fermentation for all types of kefirs used in this study . + + , total inhibition ; + , partial inhibition ;  , no inhibition . bc , bacillus cereus atcc14579 ; sa , staphylococcus aureus atcc6538 ; lm , listeria monocytogenes atcc51776 ; ef , enterococcus faecalis atcc19433 ; ec , escherichia coli atcc25922 ; se , salmonella enteritidis obtained from the fda ; pa , pseudomonas aeruginosa atcc15522 ; cs , cronobacter sakazakii atcc29544 . in addition , kefir a showed antimicrobial activity against b. cereus , e. coli , s. enteritidis , p. aeruginosa , and c. sakazakii . in contrast , kefir l , m , and s showed more broad - spectrum antimicrobial activity , inhibiting b. cereus , s. aureus , e. coli , s. enteritidis , p. aeruginosa , and c. sakazakii growth . those results suggest that the kefirs from different origins have different antimicrobial spectra , which is consistent with previous studies ( anderson and gilliland , 1999 ; pintado et al . , 1996 ) . ( 2011 ) reported that kefir inhibited b. subtilis , s. aureus , e. coli , e. faecalis , and s. enteritidis , but did not inhibit p. aeruginosa or candida albicans . santos et al . ( 2003 ) and ulusoy et al . ( 2007 ) reported that kefir could inhibit l. monocytogenes . remarkably , antimicrobial activity against s. aureus was only observed in kefirs l , m , and s after fermentation for 48 h. c. sakazakii was completely inhibited by kefir fermented for 36 or 72 h , and the activity decreased in kefir l fermented for 48 h. in kefir s , inhibitory activity against s. enteritidis was present at 36 and 72 h , but not at 48 h. considering that kefir supernatant contains various metabolites and inhibitory compounds such as organic acids , hydrogen peroxides , ethyl alcohol , diacetyl , peptides , and possibly bacteriocins , it could be postulated that these compounds interact each other to enhance or antagonize their antimicrobial effects ( kim et al . , 2015 ) . for instances , the antimicrobial activities of some bacteriocins could be inactivated by organic acids or enzymatic degradation ( joshi et al . , 2006 ) . it is thus inferred that the antimicrobial activity of kefir could be derived from different key compounds at each fermentation stage resulting in inconsistent antimicrobial pattern over time . moreover , the growth of l. monocytogenes was only inhibited by kefir m fermented for 24 h , suggesting kefir m could contain a microorganism which produces an anti - l . these data suggested that the antimicrobial activity of kefirs could be attributable to specific antimicrobial substances and not simply due to low ph values ( witthuhn et al . , 2005 ) . to demonstrate this , antimicrobial activity of lactic and acetic acid against the test strains were also investigated ( table 1 ) . we found that only b. cereus growth was partially inhibited by the lactic acid and acetic acid solutions at ph 3.5 ( table 1 ) . the growth of test strains was inhibited by kefir , although the ph values of kefirs in our study ranged from 4.05 to 3.64 . additionally , we conducted antimicrobial activity tests using ethyl alcohol solution because ethyl alcohol is an antimicrobial substance produced by yeast in kefir ( gaware et al . , 2011 ) . the growth of all test strains , however , was not affected by the ethyl alcohol solution ( table 1 ) . therefore , we concluded that the antimicrobial activity of kefir was attributable to the hurdle effect of antimicrobial substances tested above , or to the single unknown bioactive compounds such as antimicrobial peptides ( bacteriocins ) or polysaccharides ( exopolysaccharides ) ( moraes et al . , 2010 ) . it is well known that the presence of bacteriocins could be screened by ph neutralization , heat and enzyme treatment of the cell - free supernatants ( harris et al . thus , future studies should be followed to elucidate the key antimicrobial compounds against each pathogenic bacterium and its mechanism .