Wheat Bran »Wheat Bran is the outer fraction of the cereal grain,comprising the pericarp,seed coat (testa),nucellar tissue,and aleurone layer,and is derived from Triticum aestivumLinné,T.compactumHost,T.durumDesf.,and other common einkorn and emmer wheat cultivars.It is obtained by the milling and processing of the whole wheat grain meeting U.S.Standards for Number 1wheat (7CFR810.2201).It contains not less than 36.0percent of dietary fiber. Packaging and storage— Preserve in well-closed containers,secured against insect attack (see Preservationunder Vegetable and Animal Substancesin the General Notices). Identification— When examined microscopically,the following components of Wheat Bran are visible.Fragments of aleurone and nucellar layers (about 60%of the components)and fragments of seed coat and pericarp (about 40%).Aleurone and nucellar tissues composed of a usually single layer of thick-walled,isodiametric,translucent cells having conspicuous protoplasm and a single,inconspicuous layer of thick-walled,nearly transparent cells.Inconspicuous seed coat,consisting of two layers of thin-walled cells crossing at roughly right angles to each other.Pericarp composed of an inconspicuous endocarp layer of elongated,thick-walled tube cells;a cross layer with cells longer than wide,arranged side-by-side in rows,having thick,highly pitted side and end walls;and epicarp and hypoderm layers with cells longer than wide,arranged alternately in rows and having thick,highly pitted side and end walls.Epicarp and hypoderm cells larger than and crossing at right angles to the cells of the cross layer.Afew trichomes also present,with lumens narrower than the thickness of their cell walls and originating from isodiametric-polygonal epicarp cells.If micronized,the original structures are mostly destroyed. Microbial limits á61ñ The total aerobic microbial count does not exceed 10,000cfu per g,and it meets the requirements of the test for the absence of Salmonellaspecies and Escherichia coli. Water,Method III,Procedure for Articles of Botanical Origin,á921ñ It loses not more than 12%of its weight. Total ash á561ñ: not more than 8%. Heavy metals,Method IIá231ñ: 0.004%. Absence of peroxidase activity— Transfer about 1g of Wheat Bran to a test tube,and add 50mLof water.Add,in the order specified,2mLof 5.68mMerythorbic acid,3mLof 0.69mMdichloroindophenol,and 0.1mLof 1.2%hydrogen peroxide,each freshly prepared.Stopper the test tube tightly,and shake until the sample is dissolved.Place into a water bath at 38for 5minutes:no color change is observed,indicating the absence of peroxidase activity. Limit of fat— Transfer about 2g of Wheat Bran,previously dried in a vacuum oven at 100for 5hours and accurately weighed,to an extraction thimble,and mix with an equivalent quantity of dry,clean sand.Place a fat-free cotton or glass wool plug on top of the thimble.Place the thimble in a continuous-extraction apparatus provided with a tared collection flask.Pour about 75mLof solvent hexane through the sample into the collection flask.Extract at a condensation rate of 5to 6drops per second for 4hours,then at a rate of 2to 3drops per second for the next 16hours.Detach the collection flask,carefully evaporate the solvent,and dry the collection flask and its contents in a drying oven at 100for 30minutes to constant weight.Calculate the percentage of the extract (crude fat)in the portion of Wheat Bran taken:not more than 6%is found. Limit of insect infestation— Prepare a smooth slurry by transferring about 50g of Wheat Bran to a 1Lbeaker and adding 500mLof 1.5Nhydrochloric acid.Add 50mLof light mineral oil,and carefully heat to boiling on a hot plate.Boil for 10minutes to digest,stirring occasionally to prevent scorching.Remove from the hot plate,and stir for 5minutes with a magnetic stirrer,increasing the stirring speed until a vortex is formed without visible splashing.Quantitatively transfer the contents of the beaker to a separatory funnel with the aid of hot water.Allow to stand for 30minutes,stirring gently with a glass rod several times during the first 10minutes.Drain the lower layer to about 2.5cm from the layer interface.Wash the funnel with hot water,and allow 5minutes for the layers to separate.Drain the lower layer and wash with cold water several times until the lower phase is clear.Filter the contents of the funnel through ruled filter paper with the aid of a Büchner funnel and suction.Thoroughly rinse the separatory funnel with water and a detergent solution,filtering each rinse through the same paper.Examine the ruled filter paper under a microscope at 30×magnification:not more than 25insect fragments are seen. Limit of protein— Place about 1g of Wheat Bran,accurately weighed,in a 500-mL Kjeldahl flask,and proceed as directed for Method Iunder Nitrogen Determination á461ñ.Multiply the percent of nitrogen found by 6.31:not more than 18.5%is found. Content of total dietary fiber— Phosphate buffer— Prepare a pH6.0phosphate buffer (see Buffer Solutionsunder Solutionsin the section Reagents,Indicators,and Solutions). Protease solution— Dissolve 5mg of protease in 0.1mLof Phosphate buffer. Sample preparation— Prepare two samples in parallel.In order to correct for any contribution from reagents,also perform examinations of blanks,which are treated similarly to the samples.Proceed as directed for Limit of fat.Mill to a coarse powder,and store in a desiccator. Procedure— Transfer about 1.0g of each Sample preparation,accurately weighed,into separate 400-mL,tall-form beakers.Add 50mLof Phosphate buffer,and adjust the pH,if necessary,to 6.0±0.1.Add 0.2mLof heat-stable a-amylase solution.Cover the beaker with aluminum foil,place in a boiling water bath for 15minutes at 100,shaking gently every 5minutes,and cool to room temperature.Adjust with about 10mLof 0.275Nsodium hydroxide solution to a pHof 7.5±0.1.Add freshly prepared Protease solution,cover the beaker with aluminum foil,and incubate for 30minutes at 60with continuous agitation.Cool,and add about 10mLof 0.325Nhydrochloric acid and adjust to a pHof 4.5±0.2.Add 0.3mLof amyloglucosidase,cover with aluminum foil,and incubate for 20minutes at 60with continuous agitation.Heat 280mLof alcohol to 60,add to the digest,and allow the precipitate to form at room temperature for 60minutes.Place 0.5g of chromatographic siliceous earth in a crucible with fritted disk,dry at 130to constant weight,and weigh.Wet the chromatographic siliceous earth in the crucible by using a stream of 78%alcohol from a washing bottle,and apply suction to evenly distribute the chromatographic siliceous earth over the fritted disk.Maintain suction,and quantitatively transfer the enzyme digest precipitate to the crucible.Wash the residue successively with three 20-mLportions of 78%alcohol,two 10-mLportions of alcohol,and two 10-mLportions of acetone.In some cases,gums may form during filtration,trapping liquid in residue.If so,break the surface film with a spatula to improve filtration.Dry the crucible containing the residue at 105in an air oven for 16hours,cool in a desiccator,and determine the weight of the residue.Determine the percentage of protein in the first Sample preparationas directed for Limit of protein.Incinerate the residue from the second Sample preparationas directed for Total Ashunder Articles of Botanical Origin á561ñ.Calculate the corrected weight,W,of the sample residue by the formula: WU(1-PU/100-AU/100)-WB(1-PB/100-AB/100), in which WUand WBare the average weights of the sample residues and the blank residues,respectively;PUand PBare percentages of protein present in the sample and in the blank,respectively;and AUand ABare percentages of ash found in the sample and in the blank,respectively.Then calculate the percentage of the total dietary fiber in the portion of Wheat Bran taken by the formula: 100W/WI, in which WIis the weight of the Sample preparationtaken.Correct the final percentage of the total dietary fiber for fat and for water:not less than 36.0%is found. Auxiliary Information— Staff Liaison:Gabriel I.Giancaspro,Ph.D.,Senior Scientist and Latin American Specialist Expert Committee:(DSB)Dietary Supplements:Botanicals USP28–NF23Page 2036 Phone Number:1-301-816-8343