the weissella species are gram - positive , non - spore - forming , heterofermentative , nonmotile ,   and irregular or coccoid rod - shaped organisms . members of the genus weissella have been isolated from a variety of sources , such as fresh vegetables , fermented silage , meat , or meat products .
several lactic acid bacteria belonging to the genera weissella have been introduced to wheat sourdough baking for in situ production of exopolysaccharides .
weissella kimchii pl9023 was selected as a prebiotic as it mostly produced hydrogen peroxide which inhibited the growth and adherence of vaginal isolates of candida albicans , escherichia coli , staphylococcus aureus , and streptococcus agalactiae .
lactic acid bacteria ( lab ) that produce dextrans include various leuconostoc , streptococcus , and lactobacillus species .
patel et al .   reported the dextran - production ability of pediococcus pentosaceus for the first time .
additionally , dextran production has typically served as a phenotypic test in the identification of bacteria classified in the genus weissella .
reported industrially useful dextran - producing weissella confusa e392 which could be a suitable alternative to the widely used l. mesenteroides b512f in the production of linear dextran .
first proposed by collins et al .   on the basis of the results of 16s rdna phylogenetic analyses ,
the genus weissella encompasses a phylogenetically coherent group of lactic acid bacteria and includes twelve validated leuconostoc - like species , currently , including w. confusa ( formerly lactobacillus confusus ) , w. minor ( formerly lactobacillus minor ) , w. kandleri ( formerly lactobacillus kandleri ) , w. halotolerans ( formerly lactobacillus halotolerans ) , w. viridescens ( formerly lactobacillus viridescens ) , weissella paramesenteroides ( formerly leuconostoc paramesenteroides ) , w. hellenica ,   w. thailandensis , w. cibaria ,   weissella kimchii ,   weissella soil , and weissella koreensis .
alternative molecular biological approaches have been developed in place of classical phenotypic methods for the identification of weissella species .
such methods include soluble whole - cell protein pattern analyses , restriction pattern of ribosomal dna   fatty acid analyses , random amplified polymorphic dna - pcr ( rapd - pcr ) , denaturing gradient gel electrophoresis ( dgge ) , and pcr targeting 16s/23s rrna gene spacer region . in the present study
, a novel weissella species isolated from fermented cabbage was characterised up to genus and species level using 16s rdna - based sequence analyses .
its glucan formation capacity has been explored in detail and compared with that of leuconostoc mesenteroides nrrl b-512f .
fresh chopped cabbage was subjected to fermentation in 2.5% ( w / v ) nacl solution .
one gram of fermented cabbage was ground to paste and mixed in 10  ml of saline ( 0.9% w / v ) homogeneously in test tubes .
one hundred microliter from all the dilutions of cabbage from 10 to 10 was taken and spread plated on petri plates of 1.7% ( w / v ) mrs agar with glucose as carbon source .
petri plates were incubated at 28c for 24  h. based on the higher - glucansucrase activity and glucan concentration determined ( as described later ) , a microbe cab3 ( named after its colony number ) was isolated .
the isolated bacterium was propagated as stab in mrs agar supplemented with sucrose as carbon source   at 25c and stored at 4c . for long - term preservation ,
the colony morphology of isolates grown on mrs medium for overnight at 25c was observed directly and by light microscopy .
cell mobility and gliding movement were assessed by phase - contrast microscopy ( 1000x ) using cells of mrs broth .
the cell morphology of the selected isolate was examined by scanning electron microscopy ( leo1430 vp , leo electron microscopy ltd . , cambridge , uk ) operated at 10.0  kv .
the catalase activity was determined by transferring fresh colonies from mrs agar to a drop of 5% ( v / v ) h2o2 on a glass slide .
for the temperature tolerance of the microorganism , the growth of selected isolate was carried out using mrs broth at different temperatures , that is , 4c , 10c , 15c , 20c , 30c , 37c , and 42c for 2  days . for salt tolerance of the isolate ,
the growth was observed using mrs broth in the presence of 4.0% and 6.5% ( w / v ) of nacl incubated at 25c for 2  d. the production of glucan ( slimy layer ) by isolate cab3 from sucrose was observed on mrs agar in which glucose was replaced by sucrose .  
the strain cab3 was tested for its ability to ferment various carbohydrates using the method of kandler and weiss . from the overnight - grown mrs broth containing 2% ( w / v ) glucose as carbohydrate source ,
50  l was inoculated in 5.0  ml liquid mrs medium lacking glucose but containing phenol red and other test carbohydrates to give a final inoculum to medium ratio of 1% ( v / v ) .
the acid production was recorded between 24 and 48  h. the acid production was indicated by a change in the colour of the phenol red indicator dye from red to yellow .
the isolated strain cab3 was tested for susceptibility to 30 antibiotics using a standardized filter - paper disc - agar diffusion assay , to determine the drug susceptibility of microorganisms .  
the antibiotic tests were performed using commercially available antibiotic octadiscs containing amoxyclav ( ac ) , cephalexin ( cp ) , ciprofloxacin ( cf ) , clindamycin   ( cd ) , cloxacillin ( cx ) , erythromycin ( e ) , tetracycline ( t ) , ampicillin ( a ) , carbenicillin ( cb ) , cefotaxime ( ce ) , chloramphenicol ( c ) , co - trimazine ( cm ) , gentamicin ( g ) , norfloxacin ( nx ) , oxacillin ( ox ) , amikacin ( ak ) , amoxycillin ( am ) , bacitracin ( b ) , cephalothin ( ch ) , novobiocin ( nv ) , oxytetracycline ( o ) , vancomycin ( v ) , penicillin - g ( p ) , tobramycin ( tb ) , cephaloridine ( cr ) , kanamycin ( k ) , lincomycin ( l ) , methicillin ( m ) , norfloxacin ( nf ) , and oleandomycin ( ol ) from hi - media pvt . ltd .
the bacterial cell pellet was lysed using a solution containing guanidium thiocyanate ( a chaotropic agent ) and sds ( a detergent ) , to extract dna .
this lysis solution was used to disrupt the cell , remove proteins and polysaccharides , and partial hydrolysis of rna .
the genomic dna was extracted by geneitm genomic dna extraction kit ( bangalore genei pvt . ltd . ) .
dna was then precipitated using alcohol and washed with 70% ( v / v ) alcohol to remove contaminants .
dna pellet was solubilized in sodium acetate buffer at ph 5.5 added to a final concentration of 0.3  m in isopropanol at higher temperature ( 5055c ) , to increase the solubility of genomic dna .
the extracted dna was purified using ultrapure prep kit ( kt83b , bangalore genei , india ) .
genomic dna was purified in a gravity flow column using elution buffer , followed by treatment with lysozyme , proteinase k , and rnase a supplied with the kit .
the quality of purified dna was evaluated on 1.2% agarose gel , a single band of high - molecular - weight dna was observed .
the universal 16s rdna primers , forward primer 8f ( 5agttgatcctggctcag3 ) , and reverse primer 1492r ( 5accttgttacgactt3 ) were used for the polymerase chain reaction ( pcr ) .
the pcr amplification was carried out in a reaction mixture containing ~10  ng genomic dna as template , 1  l dntp mix ( 2.5  mm each ) , 100  ng/l each of forward and reverse primer , 1x taq dna polymerase assay buffer ( 10x ) , 3u taq dna polymerase enzyme ( bangalore genei ltd.,bangalore , india ) , and distilled water enough to make up the volume to 50  l reaction mixture .
amplification conditions were 5  min initial denaturation at 94c , 30  s denaturation at 94c , 30  s of primer annealing at 54c , 1  min elongation at 72c for 35 cycles , and a final extension of 10  min at 72c .
the reactions were carried out in a thermal cycler ( applied biosystems , model abi 2720 ) .
fifteen l of pcr amplified product was electrophoresed on 1.2% ( w / v ) low - eeo agarose gel in 1x tbe buffer ( 45  mm tris - borate , ph 8.3 , and 1  mm na 2 edta ) at 100  v for 2  h. the gel was stained with ethidium bromide in a final concentration of 0.5  g / ml , visualized , and photographed under uv light .
the amplified 16s rrna gene from the isolate was eluted and purified from the gel slice using the geneipure tm gel extraction kit ( qiagen ) . forward and
reverse dna sequencing reaction of pcr amplicon was carried out with 8f and 1492r primers using bdt v3.1 cycle sequencing kit on abi 3730xl genetic analyzer .
consensus sequence of 1288  bp of 16s rdna gene was generated from forward and reverse sequence data using aligner software .
the 16s rdna gene sequence of the isolate cab3 was used to carry out blast with the nr database of ncbi genbank database .
based on maximum identity score , first ten sequences were selected and aligned using multiple alignment software program clustalw .
distance matrix was generated using rdp database , and the phylogenetic tree was constructed using mega 4 .
the enzyme assay was carried out in 1  ml reaction mixture containing 5% ( w / v ) sucrose , 20  mm sodium acetate buffer ( ph 5.4 ) , and 20  l cell - free supernatant .
the enzyme activity was determined by estimating the released reducing sugar by nelson   and somogyi   method .
the glucansucrase was produced in the enzyme production medium as described by tsuchiya et al . .
fermentations were carried out in triplicate sets of 60  ml enzyme production medium in a 250  ml erlenmeyer flask incubated at 25c under shaking condition at 180  rpm .
the samples ( 1  ml ) were withdrawn at indicated time intervals and centrifuged at 8000  g for 10  min at 4c to separate the cells .
weissella confusa was grown at various temperatures : 22c , 25c , 28c , 30c , 32c , 37c , and 40c in the flasks as described previously in an orbital shaking incubator at 180  rpm to determine the optimum incubation temperature for the maximum enzyme production .
the culture ( 1.0  ml ) was withdrawn at the indicated time intervals and centrifuged at 8000  g for 10  min at 4c to pellet out the cells .
the production glucansucrase was compared under shaken - flask condition with the static flask culture at 25c in triplicate sets of 60  ml enzyme production medium in 250  ml erlenmeyer flasks .
the samples ( 1.0  ml ) were withdrawn at indicated time intervals and centrifuged at 8000  g for 10  min at 4c to pellet out the cells .
the cell - free extract was analysed for enzyme activity and protein concentration as described earlier .
the polysaccharide content of the isolated strain ( cab3 ) was determined by phenol - sulphuric acid method   in a microtitre plate .
the isolated strain was grown in 60  ml liquid medium described by tsuchiya et al .   at 25c and 180  rpm up to 32  h.
the samples ( 1  ml ) were withdrawn at regular intervals . to 200  l of the culture supernatant , three volumes of the prechilled ethanol were added and centrifuged at 12,000  g. the supernatant was discarded , and the precipitate was resuspended in 200  l distilled water .
the crude 50  ml cell - free supernatant with enzyme activity 6  u / ml and specific activity 1  u / mg was subjected to purification by fractionation using peg-400 ( 33% , w / v ) .
to confirm the existence of glucansucrase , the pas staining of the sucrose- and raffinose - incubated gel was carried out on 7% nondenaturing sds - page .
the electrophoresis was performed on a mini gel unit ( biorad ) using 1.5  mm thick gels , following the method of .
after run , the gel was treated thrice with a solution containing 20  mm sodium acetate buffer , ph 5.4 , 0.1% ( w / v ) triton x-100 , and 0.005% ( w / v ) calcium chloride for 20  min to remove sds .
then , the gel was incubated with 5% ( w / v ) sucrose / raffinose solution in 20  mm sodium acetate buffer , ph 5.4 for 1012  h. following incubation , the gel was washed twice with 75% ( v / v ) ethanol for 20  min and incubated in solution with 0.7% ( w / v ) periodic acid in 5% ( v / v ) acetic acid for 20  min at room temperature .
the gel was then washed thrice with 0.2% ( w / v ) sodium bisulfate in 5% ( v / v ) acetic acid solution and finally stained with schiff 's reagent ( 0.5% w / v basic fuchsin , 1% ( w / v ) sodium bisulfate , and 0.1  n hcl ) until the discrete magenta bands appeared within the gel .
the purified enzyme having 10.4  u / mg specific activity ( 1.0  mg / ml ) was used for further optimization of assay conditions .
the ionic strength of the buffer was varied from 10  mm to 400  mm .
the assay was carried out in 1  ml reaction mixture containing 5% ( w / v ) final sucrose concentration , in sodium acetate buffer ( ph 5.4 ) at 30c for 15  min . to study the effect of varied ph on enzyme activity ,
the ph of buffer was varied from 4 to 7 in 1  ml reaction mixture containing 5% ( w / v ) final sucrose concentration in 20  mm sodium acetate buffer at 30c for 15  min .
the enzyme assay was done in 5% ( w / v ) sucrose solution in 20  mm sodium acetate buffer ( ph 5.4 ) for 15  min .
based on higher glucansucrase activity and glucan concentration determined , a microbe cab3 was isolated from the fermented cabbage .
the isolated culture was identified according to their morphological , cultural , physiological , and biochemical characteristics [ 37 , 38 ] .
the tests used were gram reaction , production of catalase and growth at different temperatures and salt concentrations , and acid production from carbohydrates ( 2% w / v ) .
lactococcus spp . , and leuconostoc spp . by its biochemical and physiological properties like arginine deamination , esculin hydrolysis ,
the microorganism was identified by comparing the results with those from the literature ( table 1 ) .  
the results of   the comparison showed that the isolated strain cab3 has the same characteristics as w. confusa ( table 1 , column 5 , which is , 4 ) . from scanning electron microscopy ,
the isolate cab3 was found to be phenotypically homogeneous with irregular short rod cells in pairs or chains with a width and length of 0.5 - 0.6  m and 1.21.4  m , respectively ( figure 1 ) .
the colony produced slimy layer when grown on mrs agar plate was supplemented with sucrose .
the strain could grow in presence of 4.0% , 5.0% , 6.0% , and 6.5% ( w / v ) nacl , but not beyond 8.0% nacl .
the strain very efficiently utilised cellobiose , galactose , maltose , ribose , sucrose , and xylose but poorly fermented mellibiose , raffinose , and trehalose which was in accordance with fermentation pattern of weissella confusa reported earlier .
the cab3 did not utilize rhamnose which is also characteristic of weissella sp . .  
the isolate cab3 was sensitive to chloramphenicol , erythromycin , and tetracycline and moderately resistant to gentamicin , kanamycin , norfloxacin , and vancomycin which is characteristic of weissella confusa .
16s rrna gene sequence analyses after pcr amplification was performed for identifying the isolate cab3 .
the profiles obtained by pcr amplification allowed the identification of the isolate at both genus and species levels .
the amplified product of 16s rrna showed a single band of 1,500  bp ( figure 2 ) . forward and
reverse dna sequencing reaction of pcr amplicon was carried out with 8f and 1492r primers using bdt v3.1 cycle sequencing kit on abi 3730xl genetic analyzer .
the consensus sequence of 1288  bp of 16s rdna gene was generated from forward and reverse sequence data using aligner software ( figure 3 ) .
the 16s rrna sequence was compared with reference bacteria from the national centre for biotechnological information ( ncbi ) genbank ( http://www.ncbi.nlm.nih.gov/ ) and ribosomal database project ( rdp ) .
the homology in sequences identified by the above methods was used to find out common ancestry .
the bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the taxa analyzed .
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 500 replicates ) is shown next to the branches .
the evolutionary distances were computed using the kimura 2-parameter method   and are in the units of the number of base substitutions per site .
all positions containing gaps and missing data were eliminated from the dataset ( complete - deletion option ) .
the isolate cab3 and the strain weissella confusa imau:10190 ( genbank accession number gu 138518.1 ) clustered together ( figure 4 ) which confirmed the isolate to be weissella confusa .   hence , the isolate cab3 was identified as weissella confusa and assigned the genbank accession number gu138518.1 .
a bioinformatic tool , genedoc software , version 2.7.1 , was used for more 16s rrna gene sequence investigation of the other closely related weissella sp . the partial sequence ( 1288 nucleotides ) of 16s rdna of weissella confusa ( cab3 ) was 100% similar to 16s rrna genes from other related strains of weissella sp . in ncbi (
temperatures ranging from 22c to 40c were studied for the production of enzyme under shaking at 180  rpm ( figure 5 ) . at 25c ,
the activity achieved was maximum ( 6.2  u / ml ) , at a temperature lower than 25c , the cell growth was slow which might be the cause for lower enzyme activity as shown in figure 5 . at higher temperature ,
the enzyme activity decreased which might be due to the deactivation of the enzyme at higher temperatures . shaking condition significantly favoured the enzyme production giving 6.1  u / ml enzyme activity which was 1.5 times higher than the enzyme activity observed under static condition ( 4.1  u / ml ) as shown in figure 6 .
the maximum enzyme activity was achieved 4  h later in case of static flask culture .  
figure 7 shows a complete fermentation profile of the new strain of weissella confusa ( cab3 ) .
the fermentation profile of the new strain of weissella confusa ( cab3 ) was compared with other lactic acid bacterial strains using the same medium composition and the parameters compared are listed in table 2 .
it was reported that 23c temperature under static flask culture condition was optimum for the production of dextransucrase from l. mesenteroides nrrl b-512f , and 25c temperature under shaking condition was found to be optimum for l. mesenteroides
nrrl b-640 , whereas , for weissella confuse , the temperature 25c and shaking conditions were optimum . from the results , it was clear that the enzyme activity ( 6.1  u / ml ) from weissella confusa was 50% , 27% , and 65% higher than the enzyme activity from l. mesenteroides nrrl b-512f ( 4.1  u / ml ) , l. mesenteroides nrrl b-640 ( 4.8  u / ml ) , and p. pentosaceus , respectively , as shown in table 2 . the cell growth obtained in all strains after 12  h was similar .
there was a sharp decrease in the ph of the broth with weissella confusa after 12  h ( figure 7 ) , when the maximum activity was achieved , whereas the ph drop was much lower from the point where the maximum enzyme activity was observed for l. mesenteroides nrrl b-512f .
the ph range for maximum enzyme production for weissella confusa was narrow 5.25.6 , whereas for l. mesenteroides nrrl b-512f , l. mesenteroides nrrl b-640 , and p. pentosaceus , it was 6.06.5 , 4.45.9 , and 4.0 - 5.0 , respectively .
the cell - free supernatant of isolated strain weissella confusa ( cab3 ) gave glucan concentration of 34  mg / ml ( figure 7 ) .
this was very high as compared to other lactic acid bacteria such as from l. mesenteroides nrrl b-512f ( 6.9  mg / ml ) , l. mesenteroides nrrl b-640 ( 7.0  mg / ml ) , and pediococcus pentosaceus ( 10.2  mg / ml ) . the glucan concentration of the strain can be further increased by optimizing the medium composition and culture conditions .
the enzyme was purified from cell - free supernatant with 1.0  u / mg specific activity .
the fractionation by 33% ( w / v ) peg-400 gave 10.4  u / mg specific activity with 10-fold purification and 26% overall yield .
the purified enzyme showed , approximately , 180  kda molecular size on the nondenaturing sds - page gel when stained with coomassie brilliant blue ( figure 8 ; lane 2 ) .
the pas staining of the gel incubated in sucrose ( figure 8 ; lane 3 ) showed that the activity band corresponded to the presence of active form of the purified glucansucrase ( figure 8 ; lane 2 ) .
no activity band after pas staining appeared after the incubation of the nondenaturing sds - page gel in raffinose ( figure 8 ; lane 4 ) , thus excluding the presence of fructosyltransferase .
the partially purified enzyme was maximally active at the temperature 35c , at ph 5.4 in 0.2  mm sodium acetate buffer ph 5.4 ( figure 9 ) .
the results showed that the glucansucrase activity was stable within the range of 1020  mm ( figure 9 ) .
the enzyme lost 41% of its activity at 50  mm . beyond 50  mm ,
the decrease in the enzyme activity was not significant with the increase of ionic strength .
at ph below and above 5.4 , a sharp decrease in enzyme activity was observed , which has been observed with other glucansucrases [ 16 , 17 ] .
the isolated bacterium ( cab3 ) was identified at species and genus levels based on 16s rdna sequencing to be weissella confusa imau:10190 ( genbank accession number gu 138518.1 ) .
the isolated strain could be very promising candidate for glucan production at industrial level as it has high glucan concentration ( 34  mg / ml ) .
the physiological and biochemical characterisation was carried out to distinguish the isolate from other closely related lactic acid bacteria .
based on biochemical and physiological studies , the strain showed its relation with other weissella sp .
the isolate weissella confusa ( cab3 ) could not ferment mellibiose , raffinose , and trehalose , a common trait of weissella confusa .
the partially purified enzyme was confirmed as glucansucrase by pas staining of nondenaturing sds - page gel incubated with sucrose .
the presence of fructosyltransferase was ruled out based on the absence of the band in raffinose - incubated nondenaturing sds - page gel .
the partially purified enzyme showed highest activity at 35c , when assayed in 20  mm sodium acetate buffer ( ph 5.4 ) .
the wide applications of glucan in food , pharmaceutical , and cosmetics industries emphasize the importance of exploration of the new strains and characterization of their traits .
this study reveals a novel high glucan producing bacterial isolate which could be a good alternative for glucan - production in the future .
 a gram - positive , nonmotile , irregular , short , rod - shaped new strain of weissella confusa bacterium was isolated from fermented cabbage . 
 the isolate was physiologically and biochemically characterised . 
 the 16s rdna was amplified by polymerase chain reaction ( pcr ) . 
 the isolate was identified as weissella confusa ( genbank accession number : gu138518.1 ) based on nucleotide homology and phylogenetic analysis . 
 the isolate produces glucansucrase when grown in sucrose - supplemented culture medium which catalyses glucan formation . 
 this novel isolate possesses high capacity of industrial use due to its high productivity of glucan ( 34  mg / ml ) as compared to other strains reported . 
 the optimum temperature for glucansucrase production was 25c . 
 the shaking condition gave an enzyme activity of 6.1  u / ml which was 1.5 times higher than that given by static condition ( 4.1  u / ml ) . 
 the temperature   35c , ph 5.4 , and ionic strength 1020  mm were optimum for enzyme assay . 
 this investigation unraveled the abundance of industrially valuable microflora of the north east india . 