it is the second most abundant transition metal ion in living organisms , including humans .
zinc is present in all the tissues , fluids , and organs within the human body , for a total body content of approximately 1.42.3  g. it is necessary for catalytic , structural , and regulatory functions in hundreds of enzymes and in thousands of protein domains .
enumerating and discussing the role of zinc in these functions is far beyond the aim of this study [ 25 ] . the recommended dietary intake for zinc varies with age and physiological status , ranging between 5 and 18  mg day .
severe zinc deficiency causes a number of adverse physiological consequences on the epidermal , gastrointestinal , central nervous , immune , skeletal , and reproductive systems [ 4 , 6 , 7 ] .
it has been demonstrated that the form of the trace elements affects the intake efficiency in animals .
several studies reported that certain organic compounds of trace elements ( including iron , zinc , magnesium , and selenium ) are more bioavailable than the inorganic forms , possibly because the mechanisms for absorption have adapted to these kinds of nutrients during species evolution [ 812 ] . moreover , in order to develop biotechnological sources of trace elements for diet supplementation , microorganisms ( e.g. , yeast , lactobacilli , and spirulina strains ) were proposed as organic matrixes for incorporation of minerals [ 10 , 13 , 14 ] . in fact , the addition of inorganic salts into cultivation media enables the biosorption of the mineral ions by the microbial biomass . as a consequence ,
the biomass becomes enriched with organic forms of trace elements , which are present as complexes with amino acids , proteins , lipids , and polysaccharides .
the present study investigated zinc biosorption by 16 strains of lactobacilli and bifidobacteria , in the perspective to evaluate whether they can function as organic matrixes for zinc incorporation .
sixteen human strains of lactobacillus and bifidobacterium ( table 1 ) were obtained from atcc , dsmz , and our own collection ( deptartment of life sciences , university of modena and reggio emilia ) .
the strains were cultured anaerobically in mrs broth ( difco laboratories ) containing 0.5  g  l l - cysteine  hcl . for the evaluation of zinc inhibitory concentration and metal uptake , solutions of znso4    7h2o or zncl2 were filter sterilized ( 0.22  m ) and added to sterile mrs - cysteine medium to obtain the appropriate concentration .
all chemicals were provided by sigma - aldrich ( steinheim , germany ) , unless otherwise specified .
the minimum inhibitory concentration ( mic ) of zinc salts was evaluated in mrs - cysteine agar plates containing znso4    7h2o or zncl2 at the following concentrations : 0.05 , 0.2 , 1 , 5 , 10 , 15 , 25 , 50 , 75 , and 100  mmol  l. overnight liquid cultures of lactobacilli or bifidobacteria were diluted with saline as appropriate ; 10 colony forming units were spread on the zn - containing agar plates .
plates were inspected for growth after anaerobic incubation at 37c for 48  h. the presence of a single colony or a faint residual haze was disregarded .
the lowest concentration which inhibited bacterial growth was recorded as the mic .   to determine zinc uptake ,
the strains of lactobacillus and bifidobacterium were cultured in mrs broth supplemented with 10  mmol  l znso4 .
the kinetics of zinc uptake by l. acidophilus wc 0203 was determined in ph - controlled batch cultures .
the strain was cultured in labfors bioreactor ( infors ag , bottmingen , switzerland ) containing 2  l of mrs - cysteine broth supplemented with 10  mmol  l znso4 .
the bioreactor was inoculated ( 10% v / v ) with an exponential - phase preculture grown in mrs - cysteine broth .
the ph was continuously measured ( 405-dpas probe , mettler toledo , switzerland ) and kept at ph 5.0 by automatic titration with 4  mol  l naoh .
anaerobic conditions were maintained by sparging the culture with 0.05  v / v / min filter - sterilized ( 0.22  m ) nitrogen .
samples were collected periodically to monitor growth and to quantify zinc uptake .   to investigate the mechanism of zinc uptake by l. acidophilus wc 0203 , ph controlled 48 h batch fermentations were carried out as follows ( figure 1 ) : the strain was cultured in mrs - cysteine broth for 48  h cultivation in the absence ( negative control ) and the presence of 10  mmol  l znso4 ( s0 and s1 , resp . ) ; the strain was cultured for 24  h in mrs - cysteine broth , then the culture was supplemented with 10  mmol l znso4 , and further incubated for 24  h ( s2 ) ; the strain was cultured for 24  h in mrs - cysteine broth , then the culture was pasteurized ( 70c for 15  min ) , supplemented with 10  mmol  l znso4 , and further incubated for 24  h ( s3 ) .
for all the treatments , bacterial biomass was harvested after 48  h for zinc quantification . to quantify zinc in microbial biomass ,
the cells contained in 50  ml of culture were collected by centrifugation ( 6,000  g for 10  min at 4c ) and repeatedly washed with d.d .
water until zinc concentration of in the supernatant decreased below the limit of detection ( 0.1  mol  l ) .
the microbial pellet was resuspended 1  :  1.5 ( w / v ) with 1.44  mol  l hno3 and heated in a water bath at 100c for 40  min .
the supernatant was collected and the precipitate was washed twice with 1  ml of d.d .
the supernatant and washing waters were mixed and supplemented with 0.1  ml of 14.4  mol  l hno3 .
water , filtered through a 0.22  m cellulose acetate filter ( millex - gv , millipore ) , and analyzed for zinc concentration using icp - aes ( perkin elmer optima 4200 dv ) .
a calibration curve was prepared using standard solutions from 0.15 to 300  mol  l zn in 288  mmol  l hno3 .
cell bound zinc was expressed as mol per gram of dry biomass weight ( mol g ) .  
all values are means of three separate experiments and are expressed as mean  standard deviation ( sd ) . within each strain
, differences among treatments were evaluated using one - way anova with repeated measures , followed by bonferroni post hoc comparisons .
differences among strains were evaluated using one - way anova followed by tukey post hoc comparisons .
the minimum inhibitory concentration ( mic ) of zncl2 and znso4 was determined for 11 strains of lactobacillus and 5 of bifidobacterium ( table 1 ) .
the anion did not affect the mic of zinc salts ( p > 0.05 ) .
for all the strains of lactobacillus and bifidobacterium , the mic of zinc salts was never below 15  mmol  l. among the strains of lactobacillus , 6 grew abundantly on 100  mmol  l zinc ( l. acidophilus wc 0202 , l. acidophilus dsmz 20552 , l. brevis atcc
4006 , l. gasseri wc 0213 , l. plantarum wc 0214 , and l. reuteri wc 0215 ) . among the strains of bifidobacterium ,
the highest mic of zinc ( 100  mmol  l ) was observed for b. breve wc 0480 .
cell - bound zinc was determined after 48  h of growth in mrs versus mrs supplemented with 10  mmol  l zinc .
zinc concentration in the un - supplemented mrs broth was determined and was 0.015  mmol l. in this medium , all the strains accumulated less than 4.6  mol g. cell - bound zinc was always significantly higher ( p < 0.05 ) when the strains were cultured in zinc - supplemented mrs , even if wide differences were observed among the strains ( table 1 ) . in the strains of bifidobacterium , zinc uptake ranged between 15 and 32  mol g. in the strains of lactobacillus , zinc ranged between 11 and 135  mol g and was the highest in l. acidophilus wc 0203 .   the relationship between microbial growth and zinc uptake was investigated in l. acidophilus wc 0203 using ph controlled batch fermentations in zinc - supplemented mrs ( figure 2 ) .
the strain grew with a specific growth rate  of 0.39  h. after 6  h glucose was depleted ( data not shown ) and the culture entered into the stationary phase , giving a biomass / glucose yield of 0.037 .
cell - bound zinc was 20  mol  g at the end of the exponential growth .
zinc uptake continued throughout the stationary phase up to 134  mol  g after 48  h of incubation ( figure 2 ) .   in order to explore the mechanism of zinc uptake by l. acidophilus wc 0203 , cell - bound zinc was measured after 48  h of cultivation in 4 different trials ( figure 1 ) . when the strain was cultured without zn supplementation ( s0 ) , cell - bound zinc was 4  mol  g. when zinc was added at the inoculum ( s1 ) , to stationary cultures of 24  h ( s2 ) , or to pasteurized cultures of 24  h ( s3 ) , cell - bound zinc was 127 , 125 , and 131  mol  g , respectively . zinc level in s1 , s2 , and
s3 trials was significantly higher ( p < 0.05 ) than in s0 ; differences among s1 , s2 , and s3 were not statistically significant ( p > 0.05 ) .
some species of lactobacillus and bifidobacterium are extensively used as probiotics in fermented foods and nutraceutical products . among probiotics beneficial effects , lactobacilli and bifidobacteria acidify the large intestine restricting putrefactive and potentially pathogenic bacteria , cause immune - stimulation , exert antioxidant and anticarcinogenic activities , produce vitamins , and activate biologically active compounds [ 1518 ] .
an increasing number of complete genome sequences of lactic acid bacteria are being added to databases and plenty of information about physiology , metabolism , and probiotic properties is available . however ,
only recently few strains of lactobacilli have been investigated for zinc biosorption . in order to propose probiotic carriers of organic zinc , the present study investigated the ability of 16 human strains of lactobacillus and bifidobacterium to bind zinc .
results from our screen revealed a significant variation in both mic and metal uptake of zinc among the various strains tested .
cell - bound zinc ranged between 15 and 32  mol g in bifidobacteria and between 11 and 135  mol  g in lactobacilli .
any correlation between mic and cell - bound zinc seems to be excluded ( r = 0.064 ) .
the strains which presented both the highest and the lowest level of zinc uptake belonged to the species l. acidophilus .
l. acidophilus wc 0203 bound 135  mol g ; l. acidophilus dsmz 20552 bound 11  mol  g ; the corresponding mics were 15  mmol  l and higher than 100  mmol  l , respectively .
a deeper characterization of zinc uptake mechanism and kinetics of l. acidophilus wc 0203 revealed that the amount of cell - bound zinc increased progressively during a batch process .
however , most of the uptake occurred during the stationary phase , when growth ceased .
in fact , zinc content was similar in biomass collected from 48 h batch cultures where the salt was added at the beginning of the process and in biomass of cultures where zinc was supplied in the late stationary phase .
a passive mechanism is probably involved in zinc uptake by l. acidophilus wc 0203 , because pasteurized and viable cultures accumulated similar amount of zinc . under these conditions ,
biomass incorporated up to 131  mol  g zinc , corresponding to approximately 8.5  mg  g. since zinc uptake seems to occur through nonmetabolically mediated mechanism of uptake , it is conceivable that zinc incorporation could be further enhanced if stationary phase cells are exposed to zinc concentrations even higher than the mic .
our results are in agreement with the observation that , in lactobacillus mesenteroides , zinc was mostly bound to carboxylate groups of proteins and that the cell wall was the major site of zinc localization , even though active internalization progressively occurred too .
diets composed primarily of plant ingredients contain diverse antinutritional factors which inhibit mineral absorption . among them , phytate exerts the major inhibitory effects on zinc absorption .
the phosphate groups of phytate can form strong and insoluble complexes with zinc ions , causing them to escape absorption and to be excreted with feces .
low molecular weight ligands of zinc can form soluble complexes with zinc , thus counteracting the effect of phytate .
therefore , chelators ( e.g. , edta ) , amino acids ( e.g. , glycine , histidine , and methionine ) , and organic acids ( e.g. , citrate ) have been used to enhance zinc bioavailability [ 20 , 21 ] .
certain animal proteins of food were demonstrated to improve zinc absorption as well , likely due to the amino acids and peptides , which are released with digestion , that keep zinc in solution .  
similar to zinc , the organic forms of other trace elements ( e.g. , magnesium and iron ) are more bioavailable and tend to be more effective against deficiency than the mineral ones [ 812 ] .
for the same reason , the utilization of microbial biomass for the delivery of microelements has been increasingly explored . above all , the dietary supplementation with selenium - enriched biomass of yeast or lactobacilli resulted in higher concentrations of selenium in tissues and body fluids than supplementation with inorganic selenium did [ 13 , 14 ] .  
the ability of l. acidophilus wc 0203 to function as an organic matrix for zinc incorporation could be exploited to supply organic forms of this metal .
this approach for zinc supplementation could be of interest if zinc complexes are released from enriched biomass upon passing through the upper gastrointestinal tract , counteracting the effects of antinutritional factors .
if biosorbed zinc is fully bioavailable , 1  g of zinc - enriched biomass of l. acidophilus wc 0203 could provide the host with a zinc amount ( 8.5  mg ) staying within the recommended daily intake ( 518  mg ) . therefore , the bioavailability of lactobacillus - bound zinc may deserve deeper investigation .
the results of this study provide new perspectives on the specific use of probiotics , such as to combine the healthy probiotic effects of the genus lactobacillus with the delivery of organic zinc , preventing sequestration by antinutritional factors .
 the study aims to investigate zinc biosorption by strains of lactobacilli and bifidobacteria with a view to exploit them as organic matrixes for zinc dietary supplementation . sixteen human strains of lactobacillus and bifidobacterium 
 were assayed for zinc uptake . 
 the minimum inhibitory concentration of zinc salts differed among the strains , but was never below 15  mmol  l1 . when cultured in mrs broth containing 10  mmol  l1 znso4 , all the strains were capable of accumulating zinc in the range between 11 and 135  mol  g1 . 
 the highest amount of cell - bound zinc was obtained in l. acidophilus wc 0203 . 
 ph - controlled batch cultures of this strain revealed that zinc uptake started in the growth phase , but occurred mostly during the stationary phase . 
 pasteurized and viable cultures accumulated similar amount of zinc , suggesting that a nonmetabolically mediated mechanism is involved in zinc uptake . 
 these results provide new perspectives on the specific use of probiotics , since l. acidophilus wc 0203 could function as an organic matrix for zinc incorporation . 
 the bioavailability of lactobacillus - bound zinc deserves to be investigated to provide a future basis for optimization of zinc supplementation or fortification . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions