
 lactobacillus strains are extensively used as probiotics in the food industry , and certain lactobacillus species are also of importance in general health , providing a beneficial microflora in the oral cavity , intestinal tract [ 2 , 3 ] , and vagina .
the genus lactobacillus contains a diverse assemble of gram - positive , catalase negative , and nonsporulating , rod - shaped organisms and includes more than 140 species [ 5 , 6 ] . among those ,
lactobacillus casei group , especially l. casei , l. paracasei , and l. rhamnosus , has attracted a lot of attention over the last 20 years .
this is linked to the considerable economic importance of the l. ( para)casei species , which is used in many food and feed applications such as dairy products and has a proven record in human and animal health . however , the taxonomy among the species in lactobacillus casei group is still vague .
the uncertain identification leads to confusion in phenotypic characters . in recent years , the identification of lactobacilli evolving along with molecular methods based upon the 16s rrna gene has been widely used .
many attempts to discriminate these species yielded inaccurate results , and only limited success could be achieved , largely due to the failure of differentiation between l. paracasei and l. casei strains [ 7  -10 ] .
for example , most of the strains described as l. casei much more differ from the species type strain of atcc 393 than from l. zeae strains [ 11 , 12 ] .
the technique of pcr - denaturing gradient gel electrophoresis ( dgge ) has been introduced recently .
this technique provides the information of variations in target genes within bacterial population which allows differentiating among the species that the dna sequences differs in at least one base pair . in this study
, it revealed that the dgge method with the designed primers of the v1 and region of v2-v3 region of 16s rrna genes enabled to distinguish among the species of l. casei , l. paracasei , l. rhamnosus , and l. zeae .
in addition , the phenotypic characters of acid production and inhibitory effect on oral pathogen were analyzed .
four reference strains of lactobacillus casei atcc 393 , l. paracasei ccug 32212 , lactobacillus rhamnosus atcc 7469 , and l. zeae ccug 35515 and 46 clinical strains , 10 l. casei isolates , 21 l. paracasei isolates and 15 l. rhamnosus isolates , were included in this study .
all clinical strains were previously identified according to 16s - rrna gene profiles by restriction fragment length polymorphism analysis ( pcr - rflp ) and protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .
two set of primers were used in this study , the first set of primers was hda-1-gc ( 5-cgcccggggcgcgccccgggcggggcgggggcacggggggactcctacgggaggcagcagt-3 ) and hda-2 ( 5-gtattaccgcggctgctggcac-3 ) according to walter et al . , which was designed targeting 200  bp of the v2-v3 region of 16s - rrna gene .
the second set of primers was carp-1-gc ( 5-cgcccggggcgcgccccgggcggggcgggggcacggggggggcgtgcctaatacatgcaa-3 ) and carp-2 ( 5-ggcaggttacccacgtgtt-3 ) , which was designed in this study targeting 112  bp of the v1 region .  
all dna samples were extracted using a genomic dna extraction kit ( rbc bioscience , taipei , taiwan ) , following the manufacturer 's protocol for gram - positive bacteria .
the condition of pcr was that each 50  l pcr reaction contained 10  mm tris - hcl ph 8.3 , 50  mm kcl ( geneamp pcr buffer ii from applied biosystems , foster city , ca , usa ) , 1  mm mgcl2 ( applied biosystems ) , 0.1  mm each dntp , 0.4 m both primers , 5  g / ml template , and 0.5  u ampli - taq dna polymerase ( applied biosystems ) .
the pcr reactions were run using a geneamp pcr system 2400 ( applied biosystems , foster , ca , usa ) . for primers carp-1-gc and carp-2 , reactions were run with an initial touchdown step in which the annealing temperature was lowered from 61 to 57c in intervals of 2c every 3 cycles , and 20 additional cycles were done with annealing at 55c .
denaturation was performed at 95c for 1  min , and extension was performed at 72c for 1  min and 30  s. for primers hda-1-gc and hda-2 , reactions were run for 35 cycles of denaturation at 95c for 60  s , annealing at 56c for 45  s , and extension at 72c for 60  s. for both amplification cycles , an initial denaturation at 94c for 5  min and a final extension at 72c for 7  min were carried out .
the dcode universal mutation detection system ( biorad , hercules , ca , usa ) was used for the sequence - specific separation of the pcr products .
electrophoresis was performed in a 8% polyacrylamide gel with gradient of 35 to 60% urea - formamide denaturant , and electrophoresis running time was adjusted to 6  h at 120  v. after electrophoresis , they were stained for 10  min in a sybr green solution ( molecular probes , or , usa ) and analyzed under uv illumination .  
the inhibitory effect of the lactobacillus strains against streptococcus mutans atcc 25175 was assessed by an agar overlay method . in brief ,
lactobacillus strains ( producer strains ) were inoculated on the surface of the brain heart infusion agar and incubated anaerobically ( 80% n2 , 10% h2 , and 10% co2 ) for 2448  h at 37c to develop visible macrocolonies .
the indicator strain was precultivated in the brain heart infusion broth ( bhi ) , and the suspension of cells was adjusted to an optical density ( od ) 0.25 at 600  nm .
thereafter , 5  ml of bhi soft agar ( 7  g / l agar ) was seeded with 100  ul of an overnight culture of the indicator strain and immediately poured over the macrocolonies of lactobacillus .
the plates were incubated anaerobically at 37c for 24  h to generate an inhibitory zone .
the acid production of the lactobacillus strains was assessed according to piwat et al . .
lactobacillus strains were initially grown anaerobically ( 80% n2 , 10% h2 , and 10% co2 ) to exponential phase in filter sterilized ( pore size 0.22  m , nalgene , ny , usa ) de man , rogosa and sharpe ( mrs ) broth ( lab m , bury , uk ) at 37c for 1618  h. cells were harvested by centrifugation at 3000  rpm for 5  min at 4c , washed twice in phosphate buffered saline ( pbs ; oxoid , basingstoke , uk ) , and inoculated into 50  ml fresh , prewarmed mrs broth containing 2% ( w / v ) glucose , ph 7.0 , to give an optical density of 1.0 at 650  nm ( corresponding to 10  cells / ml ) .
the cultures were then incubated in an anaerobic chamber ( minimacs anaerobic workstation , don whitley scientific ltd , uk ) at 37c .
two milliliters of each sample was collected and analyzed for the growth and acid production at the start ( 0 ) and after 1.5 , 3 , 5 , 7 , and 24  h. bacterial growth was determined by measuring the final od reached at 650  nm , and the change in od was calculated .
in addition , the number of viable cells was also counted as cfu / ml on mrs agar following anaerobic incubation for 48  h.   acid production was studied by ph measurements using a ph electrode and ph - meter ( hanna ph 211 , hanna instrument , uk ) .
the amount of hydrogen ion [ h ] was obtained from ph values according to the formula : [ h ] = ( 10 ) . the rate of acidification by each strain ( acid production rate ) was determined from the change in h ( h ) divided by the average number of bacterial cells per hour in the logarithmic growth phase , as shown in the following equation : 
 ( 1)acid production rate=(h+)[((n2n1)/2)t2t1 ] , 
 where n1 and n2 are number of bacterial cells at time point 1 ( t1 ) and time point 2 ( t2 ) , respectively .
the data presented are means of triplicate measurements , and all experiments were performed twice .
  the dgge patterns of v1 region of 16s rrna sequences are shown in figure 1 , and each type species strain and identified clinical isolate showed its own unique dgge pattern .
l. rhamnosus atcc 7469 and identified clinical isolates revealed a different band from l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 . it was noted that those of clinical strains identified as l. casei gave the different dgge patterns from the type strain l. casei atcc 393 .
all clinical strains identified as l. casei  had a major band with the same distance as l. paracasei strains ; however , l. paracasei ccug 32212 and most of clinical isolates identified l. paracasei showed the extraminor bands .
when hda1 and hda2 primers were used to produce the dgge patterns of v2-v3 region of 16s rrna sequences , l. casei atcc 393 showed the same dgge pattern as all clinical strains identified as l. casei .
l. paracasei ccug 32212 and all clinical isolates revealed the same multiple bands which clearly differed from all l. casei strains ( figure 2 ) .  
the ability for growth inhibition of l. casei group against s. mutans atcc 25175 is shown in figure 3 . a statistically significant difference among the species
was found ( kruskal - wallis test , p < 0.05 ) after l. paracasei strains were identified separately .
had a significantly higher inhibitory effect than either l. casei or l. rhamnosus ( mann - whitney u test , p < 0.01 ) .   acid - production rate of clinical strains of l. casei group in the exponential growth phase was calculated from the 1.5 to 5  h incubation period .
also , a statistically significant difference among the species was found ( kruskal - wallis test , p
< 0.05 ) after l. paracasei strains were identified separately . l. paracasei strains had a significantly lower acid - production rate than either l. casei or l. rhamnosus ( mann - whitney u test , p < 0.05 ) ( figure 4 ) .
the lactobacillus casei group is one of special relevance for dairy food ( cheese , yoghurt , and other fermented milk products ) and pharmaceutical industry due to its important role in promoting human health .
based on their ability to inhibit the growth of various pathogens , they have been used as probiotics in the gut for decades .
previously , a single species with five subspecies has been reclassified into three species including l. casei , l. paracasei , and l. rhamnosus in 1989 .
however , this classification initiated a controversy , generally due to the failure of differentiation between l. paracasei and former l. casei strains even by molecular technique [ 19  -21 ] including our previous study .  
research has been focused on the application of molecular biology techniques for accurate differentiation among the strains .
we developed a dgge method with the designed primers of the v1 in combination with v2-v3 region of 16s rrna genes , which enabled to distinguish the close related strains in the l. casei group .
it was shown that carp1-gc and carp2 primers could clearly distinguish the type strain l. rhamnosus atcc 7469 , l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 from each other , and each individual revealed its own unique pattern ( figure 1 ) .
it was surprising that those of clinical strains identified as l. casei gave different dgge patterns from the type strain l. casei atcc 393 .
the results indicated that there were different sequences in v1 region of all the type strains , which confirmed the information obtained by comparing the published 16s rrna gene sequences of the type strain l. rhamnosus atcc 7469 , l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 .  
hda1-gc and hda2 primers could distinguish the type strain and identified clinical isolates of l. casei from the type strain and identified clinical isolates of l. paracasei ( figure 2 ) . by showing the multiple bands of all strains of l. paracasei
, it indicated that l. paracasei strains contained more than one copy of 16s rrna gene .
it is likely that the taxonomy of this group will undergo further changes with more extensive studies providing more evidence in the coming years .
thus , such method may assist in future taxonomic considerations of this group and could be a beneficial implementation for future research .  
in oral cavity , lactobacilli have frequently been isolated from carious lesions , and thus , they are thought to be associated with the development of dental caries . the reason was based on their acid production and aciduric characteristics [ 16 , 23 , 24 ] . as many lactobacillus species have similar nutritional and growth requirements , it is often difficult to use classical microbiological methods to identify close related species . consequently , in most of the dental literature , they are specified and are referred to only as lactobacilli .
however , it is important to understand the certain role of various lactobacilli , whether they are harmful , beneficial , or neutral for the development of dental caries .
the inhibitory activities of oral lactobacillus against oral pathogens , for example , cariogenic bacteria , periodontopathogens , and candida , have been reported [ 15 , 25 , 26 ] . however , the cariogenic characteristics of lactobacillus , especially acidogenic activity , have always been concerned . in most clinical studies , l. casei and l. paracasei
they have been reported for their association with dental caries in several studies [ 28  -30 ] .
however , less is known of how acidogenicity and inhibitory activity differ among these two species identified with current taxonomic methods . in this study , the heterogeneity of acid production and antimicrobial effect among the species of l. casei / l .
it was found that l. paracasei strains were stronger in growth inhibition against oral pathogens and were weaker in acid production .
the results indicated that l. paracasei may be of benefit as probiotics for the prevention of oral diseases than the others due to its low acidogenic and high inhibition effect . in conclusion ,
the technique has been proven to be able to differentiate between closely related species in l. casei group and thus provide reliable information of their phenotypic appearances .
 this study aimed to develop a 16s rrna pcr - denaturing gradient gel electrophoresis ( dgge ) to identify the species level of lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect . 
 pcr - dgge has been developed based on the 16s rrna gene , and a set of hda-1-gc and hda-2 , designed at v2-v3 region , and another set of carp-1-gc and carp-2 , designed at v1 region , have been used . 
 the bacterial strains included l. casei atcc 393 , l. paracasei ccug 32212 , l. rhamnosus atcc 7469 , l. zeae ccug 35515 , and 46 clinical strains of l. casei / paracasei / rhamnosus . 
 inhibitory effect against streptococcus mutans and acid production were examined . 
 results revealed that each type species strain and identified clinical isolate showed its own unique dgge pattern using carp1-gc and carp2 primers . hda1-gc and 
 hda2 primers could distinguish the strains of l. paracasei from l. casei . 
 it was found that inhibitory effect of l. paracasei was stronger than l. casei and l. rhamnosus . 
 the acid production of l. paracasei was lower than l. casei and l. rhamnosus . 
 in conclusion , the technique has been proven to be able to differentiate between closely related species in l. casei group and thus provide reliable information of their phenotypic appearances . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion