Patent Application: US-24339105-A

Abstract:
a novel strain of lactic acid bacteria was found to be heat resistant and able to grow in a sulfur - limiting medium containing a high concentration of sodium selenite . the microorganism is a non - spore forming and gram - positive coccus , which is identified with & gt ; 90 % confidence using the api biochemical and sugar fermentation tests , ribotyoing and 16s rrna sequencing as pediococcus pentosaceus sp80 . in the current study , p . pentosaceus sp80 grown on slm containing 250 ppm sodium selenite produced both organic and inorganic forms of selenium . these selenium compounds can be separated using an anion exchange chromatography technique . the concentrations of selenium detected in the organic and inorganic fractions were 4 . 34 and 21 . 7 ppm , respectively . selenium - enriched bacteria are useful as a source of selenium for supplementing the diets of animals and humans . animals fed efficacious amounts of the selenium - enriched bacteria show improved feed conversion rates and higher levels of glutathione peroxidase activity in heart , kidney and liver tissues indicating an increased absorption and retention of selenium over control diets .

Description:
inorganic selenium compounds , as used in this disclosure , include salts of selenium , and preferably alkali - metal salts of selenite and selenate , and more preferably sodium selenite . organic selenium compounds include seleno - amino acid compounds and complexes , and preferably selenomethionine , selenocystine , selenocysteine , se - methylselenocysteine , gamma - glutamyl - se - methylselenocysteine , gamma - glutamyl - selenomethionine , selenocystathionine and se - adenosyl selenohomocysteine . lactic acid bacteria is a group of gram positive , non - pathogenic bacteria that can ferment carbohydrates to produce lactic acid . genera of lab include lactococcus , lactobacillus , streptococcus and pediococcus . isolation and identification of lactic acid bacteria from the gi tract of chickens gastrointestinal tracts from three healthy chickens were obtained from a local market certified by the agri - food & amp ; veterinary authority of singapore . sections comprising the duodenum , jejunum and ileum of each intestinal tract were macerated and their contents were inoculated into de man rogosa sharpe broth ph 6 . 3 ( mrs ) ( becton , dickinson , usa ) and incubated in an incubator set at 30 ° c . under 5 % co 2 for 24 h . overnight cultures were streaked for isolation of pure colonies on mrs agar , ph 6 . 3 before sub - culturing in mrs broth using the same conditions and kept in 40 % glycerol at − 80 ° c . for long - term storage . gram staining and biochemical tests ( api ® 50 ch test kit ; biomerieux , usa ) were performed to identify the strains of lactic acid bacteria isolated from the gastrointestinal tract of healthy chickens . a total of 120 strains of microaerophilic and anaerobic bacteria were isolated from the duodenum , jejunum and ileum portions of the intestinal tract of healthy chicken using the mrs media , ph 6 . 5 . using the gram - staining technique and biochemical tests , the morphologies and identities of these bacteria were identified . it was found that 97 strains of the bacteria isolated were gram - positive bacteria with the majority showing a rod - shape morphology ( table 1 ). a strain of lab , identified herein as sp80 was selected out of the 97 strains previously isolated and screened for growth in media containing sodium selenite ( 25 , 50 , 100 and 200 ppm ). the bacterium was selected based on the growth rate and ability of the cells to convert sodium selenite to amorphous selenium , indicated by the formation of red - pigmented colonies . sp80 was grown in mrs broth at 30 ° c . under 5 % co 2 for 24 h . five - ml portions of the culture were centrifuged at 5700 × g for 10 min and the supernatant discarded . the pellet was then resuspended in chl media ( biomérieux , usa ) before dispensing into the cupules of the api ® 50 ch test strips . the strips were incubated at 37 ° c . for 48 h . after 48 h , the results of the biochemical tests were analyzed using the automated identification software , api ® lab plus ™ ( biomérieux , usa ). this strain was also found to be heat resistant at 60 ° c . for 30 min ( data not shown ). results of the characterization study indicated that the bacterium has 97 . 3 - 99 . 9 percent identity with pediococcus pentosaceus ( table 2 ). the strain of pediococcus pentosaceus sp80 isolated in our laboratory fermented nine different types of sugars , including amydalin , arbutin , maltose and trehalose . consistent with the report by facklam et al ., ( 1989 ) p . pentosaceus was shown to react with salicin , maltose and trehalose ( 32 ). our study indicated that p . pentosaceus sp80 is a gram - positive non - spore forming coccus that grows well under microaerophilic conditions at 30 ° c . samples of p . pentosaceus sp80 have been deposited with the american type culture collection ( atcc ) under accession number pta 6736 . culture of pediococcus pentosaceus sp80 on media containing sodium selenate and selenite . pediococcus pentosaceus sp80 obtained from − 80 ° c . freezer was resuscitated and grown in mrs broth at 30 ° c . under 5 % co 2 for 24 h . the cells were then inoculated into sulfur - limiting media ( slm ; ph 6 . 0 ) containing 40 g / l of buffered peptone water ( bpw ; ph 7 . 4 ), 32 g / l lab - lemco powder , 16 g / l yeast extract ( oxoid , uk ), 80 g / l glucose , 20 g / l sodium acetate trihydrant , 8 g / l di - potassium hydrogen phosphate anhydrous , 0 . 8 g / l magnesium chloride , 0 . 2 g / l manganese ( ii ) chloride ( merck , germany ), 8 g / l citric acid and 4 g / l tween ® 80 . the medium was then supplemented with sodium selenate ( 0 , 250 ppm ) or selenite ( 0 , 1 , 10 , 50 , 100 , 250 , 500 and 1000 ppm ) ( sigma chemicals , usa ). the cultures were incubated at 30 ° c . under 5 % co 2 for 24 h . serial dilutions and plate count were performed using bpw and mrs agar , respectively . separation of organic and inorganic selenium by anion exchange chromatography . overnight cultures containing pediococcus pentosaceus sp80 were centrifuged at 20 , 000 × g for 15 min and washed with phosphate buffered saline ( pbs - 1 ×; ph 7 . 4 ). the cell pellet containing sp80 was then resuspended in pbs - 1 × before disruption using a cell - sonicator ( misonix , usa ) set at 6 w with 30 - sec intervals for 15 cycles . cell debris was treated with 1 . 5 m nitric acid ( merck , germany ) at 80 ° c . for 20 min . cell free extracts were then obtained after being spun at 1000 × g for 15 min . the ph of cell free extract was adjusted to 5 . 0 - 5 . 5 using 1 m sodium hydroxide ( merck , germany ). one - ml portion of the cell free extract was dispensed into a 1 - cm ( diameter )× 20 cm ( height ) anion exchanger column ( bio - rad , usa ) packed with dowex ® 1 - 8 x ( sigma chemicals , st louis ), with particle size of 100 - 200 mesh , to a height of 10 cm . elution was performed with 0 . 01 m of sodium chloride and hydrochloric acid at 0 . 2 and 0 . 5 m , respectively . five - ml fractions of the eluant were collected for se analysis using icp - ms . detection of total selenium by icp - ms . an elan 6100 icp - ms ( perkin elmer , usa ) at psb corporation , singapore was stabilized for one hour prior to injection of samples . a 3 - point standard calibration was performed using the atomic spectroscopy standard at 10 , 20 and 50 ppb , respectively . the atomic spectroscopy standard contains 10 ppb each of manganese , copper , rhodium , cadmium , indium , barium , lead and uranium . the accepted correction coefficient was set at less than 0 . 995 . five - ml portions of samples were then subjected to the elan 6100 for icp - ms analysis . putative strains of lab were grown on mrs agar plates containing various concentrations of selenate and selenite . strains that grew on mrs agar plates containing sodium selenate ( 25 , 50 , 100 and 200 ppm ) formed white colonies , similar to those grown on control medium . in contrast , red - pigmented colonies were observed on agar media containing sodium selenite of up to 200 ppm . generally , the color intensity of the colonies ranged from pink to dark red , corresponding with the increase in concentrations of sodium selenite used in the media . the coloration of colonies observed in the lab cells grown on media containing sodium selenite may be due to the presence of amorphous selenium deposited by the bacteria . gharieb ( 1995 ) has demonstrated that the reduction of selenite to elemental selenium on czapek - dox agar has resulted in the red - pigmented fungi colonies ( 33 ). candida albicans was shown to rapidly reduce selenite but this reduction was inhibited by methionine , formate , fluoride , dinitrophenol and certain sulfhydryl poisons present in the medium ( 34 ). our screening experiments have demonstrated that some strains of the isolated 97 lab were not inhibited by sodium selenite of up to 200 ppm . in addition , a number of these strains were found to be fast growers , with their colonies appearing only after 12 h of incubation ( table 3 ). the strain of pediococcus pentosaceus identified herein as sp80 also grows well in a sulfur - limiting medium ( slm ) containing up to 1000 ppm of selenite and produces a brick red pigmented culture when compared to those grown in selenate - enriched media . we suspected that sp80 may have metabolized sodium selenite and portions of which were converted to elemental se . it is possible that excess elemental se may then be deposited to the exterior of the cells and in the media as brick - red pigment . the reduction of selenite or selenate into elemental se by microorganisms that causes the appearance of brick red pigments in the culture media were also observed by various researchers ( 14 , 30 , 31 ). in our current study , selenium was detected in the cell free extract of the hno 3 - digested sp80 cells grown in slm supplemented with 250 ppm of sodium selenite . using icp - ms technique , 32 . 20 ppm of total selenium was detected from hydrolyzed cells of sp80 . no selenium was detected in the cell free extract of sp80 grown in the control media . when the cell free extract of selenium - enriched sp80 was passed through an anion exchanger , two organic and inorganic selenium fractions were eluted separately and collected . the fractions were subjected to analysis by icp - ms and the results of this study indicated that the organic and inorganic fractions contained 4 . 34 and 21 . 7 ppm of selenium , respectively ( fig1 ). zhang and frankenberger had demonstrated the separation of selenium species from plant water extracts using the anion exchange chromatography ( 21 ). at ph 5 - 5 . 5 , semet exists as zwitterionic ions and could be easily eluted out from the column packed with dowex ® by 0 . 01 m of nacl . in contrast , the inorganic selenium remained bound to the resin and could only be eluted using acidic ( 0 . 2 and 0 . 5m of hcl , respectively ) solutions . accordingly , a novel strain of lactic acid bacteria , strain sp80 was able to grow in a sulfur - limiting medium containing high concentrations of selenate and selenite . the strain has been identified using biochemical and sugar fermentation tests as pediococcus pentosaceus . pediococcus pentosaceus sp80 when cultured in a sulfur - limiting medium was found to take up selenite , convert or reduce it to elemental se , forming a brick - red precipitation . the organic and inorganic selenium present in p . pentosaceus sp80 were fractionated using an anion exchange chromatography technique . the selenium content in both organic and inorganic fractions was analyzed and quantified using icp - ms analysis . the selenium concentrations in the organic and inorganic fractions were found to be 4 . 34 and 21 . 7 ppm , respectively . bacterial culture and culture conditions . the pure culture of pediococcus pentosaceus sp80 was resuscitated and grown in a sulfur - limiting medium ( slm ; ph 6 ) supplemented with 10 ppm of sodium selenite ( sigma chemicals , usa ). the bacteria were grown at 30 ° c . under 5 % co 2 condition for 24 h . scale - up fermentation and freeze drying of bacterial culture . a 2 - l b - braun biostat ® b - dcu stirred tank fermenter ( braun , germany ) was used to produce 10 liters of fermented culture containing approximately 10 8 cpu per ml of p . pentosaceus sp80 . the medium was sterilized in the fermenter by autoclaving at 121 ° c . for 20 min . after the medium has cooled to room temperature , a 1 - percent overnight seed culture of p . pentosaceus sp80 was inoculated into the pre - sterilized sulfur - limiting medium containing 10 ppm of sodium selenite . the fermentation temperature was maintained at 30 ° c . the culture was allowed to grow in the fermenter for 24 h . the 24 - h culture was then freeze - dried at − 40 ° c . under vacuum . preparation of mice feed supplements and diets . the mice feed was obtained from a local feed importer . the composition of the mice feed is set out in table 4 . the mice pellets were ground and passed through a mesh - 20 size sieve . experimental feed a is the negative control diet containing ground feed alone ( table 5 ). experimental feeds b , c , and d are negative control diets supplemented with sodium selenite , freeze - dried sp80 prepared and described as before , and sel - plex ®, a commercial source of selenium - enriched yeast ( alltech , usa ), respectively ( table 5 ). except for the negative control diet , the final concentrations of total selenium in experimental feeds b , c and d were adjusted to 0 . 3 ppm , respectively . pre - treatment of study animals . thirty - two adult mice used in the experiment were divided into 4 treatment groups ( table 5 ). each group consisted of 4 male and 4 female mice . four mice of the same gender were kept in one cage . there was a short acclimatization period of 1 week before feeding them with the various diets . all mice received the control diet for 1 week before the intervention . animal management . all mice were kept at 25 ° c . in an incubator . feed and water were available ad libitum . the weight of all mice and the feed consumed was monitored on a weekly basis throughout the period of study . on the sixth week , mice were randomly selected from each treatment group per cage to be sacrificed and their serum , heart , liver and kidney were obtained for analysis . prior to dissection , tissues were perfused with 0 . 9 % nacl containing 0 . 16 mg / ml of heparin to remove red blood cells and clots . one - gram of tissue was then homogenized in 4 - 8 ml of cold buffer containing 50 mm tris - hcl ( ph 7 . 5 ), 5 mm edta , and 1 mm dtt . the suspension was then centrifuged at 10 , 000 × g for 20 min at 4 ° c . after 20 min , the supernatant was removed for analysis using the cellular calibiochem ®&# 39 ; s glutathione peroxidase assay kit ( cat # 354104 ) ( merck kgaa , germany ). the bioavailability of selenium was determined using a spectrophotometer at 340 nm to measure the glutathione peroxidase ( gpx ) activity in the serum , heart , liver and kidney of the mice from each treatment group . the calculation of gpx activity is determined by dividing the sum of the net rate of nadph and the reduction x sample factor by 0 . 0062 . results from the treated groups were - compared with the control groups and between treatments using the graphpad prism statistical package ( graphpad software , inc ., usa ) to test for significant difference at p & lt ; 0 . 05 . body weight gains , feed intakes , feed conversion ratios and gpx activities were also subjected to analyses of variance and the standard errors of difference tested for statistical significance . in the current study , mice are fed on different diets containing organic and inorganic selenium from various sources . regardless of their origins , the mineral , selenium has to be ingested , absorbed and retained within the mice in order for it to perform its function . glutathione peroxidase ( gpx ) is a tetrameric protein weighing approximately 85 , 000 d . gpx has 4 atoms of selenium bound as seleno - cysteine moieties that confer the catalytic activity . therefore , the glutathione peroxidase assay was performed to compare the glutathione peroxidase activities between different treatment groups to reflect the level of se absorbed and retained , in various tissues of mice fed with selenium from various sources . in our study , the gpx activity in various organs was consistently higher in male mice fed on diets containing se - enriched bacteria when compared to the negative control diets ( p & lt ; 0 . 05 ) ( tables 7 - 9 ). results from the mice trial also consistently showed that the gpx activity in livers , kidneys and hearts was higher in mice fed on diets containing se - enriched bacteria compared to those that fed on se - enriched yeast ( p & lt ; 0 . 05 ) ( tables 7 - 9 ). our data also indicated that the activities of gpx within the liver and heart of female mice fed on diets containing se - enriched bacteria were higher compared to the negative control diets ( p & lt ; 0 . 05 ) ( tables 8 , 9 ). in terms of feed intake efficiency , it is interesting to note that although a standard 5 - gram of the respective feed was given to each mouse per day , mice fed with se - enriched bacteria showed a significant enhancement in feed conversion rate ( fcr ) of the male mice compared to those fed on negative control diets ( p & lt ; 0 . 005 ) ( table 6 ). a , b values with different superscripts in the same column are significantly different [ p & lt ; 0 . 05 ]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding . a , b values with different superscripts in the same column are significantly different [ p & lt ; 0 . 05 ]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding . a , b values with different superscripts in the same column are significantly different [ p & lt ; 0 . 05 ]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding . the foregoing description and drawings comprise illustrative embodiments of the present inventions . the foregoing embodiments and the methods described herein may vary based on the ability , experience , and preference of those skilled in the art . merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method . the foregoing description and drawings merely explain and illustrate the invention , and the invention is not limited thereto , except insofar as the claims are so limited . those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention . 1 . arthur j r ., mckenzie c ., beckett g j . 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