RIBOFLAVIN from BACILLUS SUBTILIS-枯草芽孢桿菌核黃素

RIBOFLAVINfromBACILLUSSUBTILIS

Preparedatthe53rdJECFA(1999)andpublishedinFNP52Add7(1999),supersedingspecificationspreparedatthe51stJECFA(1998),publishedinFNP52Add6.AgroupADI0-0.5mg/kgbwforriboflavinfromBacillussubtilis,syntheticriboflavinandriboflavin-5-phosphatewasestablishedatthe51stJECFA(1998).

SYNONYMS VitaminB2;lactoflavin;INSNo.101(i)

SOURCE PreparedbysubmergedfermentationbyBacillussubtilisgeneticallymodifiedforriboflavinoverproduction.Thestrainisnon-pathogenicandnon-toxicogenic.

DEFINITION

Chemicalnames Riboflavin;3,10-dihydro-7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo-[g]pteridine-2,4-dione;7,8-dimethyl-10-(1'-D-ribityl)isoalloxazine

C.A.S.number 83-88-5

Chemicalformula C17H20N4O6

Structuralformula

Formulaweight 376.37

Assay Notlessthan98.0%andnotmorethan101.0%,calculatedonthedriedbasis

DESCRIPTION Yellowtoorange-yellowcrystallinepowderFUNCTIONALUSESColour,nutrientsupplementCHARACTERISTICS

IDENTIFICATION

Solubility(Vol.4) Practicallyinsolubleinethanol,acetoneanddiethylether;verysolubleindilutealkalisolutions

Spectrophotometry(Vol.4)

UsingtheaqueoussolutionfromtheAssay,determinetheabsorbance(A)at267nm,375nmand444nm.TheratioA375/A267isbetween0.31and

0.33.TheratioA444/A267isbetween0.36and0.39.

D

Specificrotation [alpha]20:Between-120and-135o

Drythesampleat100ofor4h.Dissolve50.0mgin0.05Nsodiumhydroxidefreefromcarbonateanddiluteto10.0mlwiththesamesolvent.Measuretheopticalrotationwithin30minofdissolution.

Colourreaction Dissolveabout1mgofsamplein100mlofwater.Thesolutionhasapalegreenish-yellowcolourbytransmittedlight,andbyreflectedlighthasanintenseyellowish-greenfluorescence,whichdisappearsontheadditionofmineralacidsandalkalis.

PURITY

Lossondrying(Vol.4) Notmorethan2.0%(105o,4h)

Sulfatedash(Vol.4) Notmorethan0.1%

Test2gofthesample(MethodI)

Lumiflavin(Vol.4) Notmorethan0.025%

SeedescriptionunderTESTS

Primaryaromaticamines(Vol.4)

Notmorethan100mg/kgcalculatedasaniline

Lead(Vol.4) Notmorethan1mg/kg

Determineusinganatomicabsorptiontechniqueappropriatetothespecifiedlevel.TheselectionofsamplesizeandmethodofsamplepreparationmaybebasedontheprinciplesofthemethoddescribedinVolume4,“InstrumentalMethods.”

TESTS

PURITYTESTS

Lumiflavin(Vol.4) ReferenceSolution:Dissolve25mgoflumiflavinin50.0mlofchloroform.Dilute1.0mlofthissolutionwithchloroformto20.0ml,anddilute2.5mloftheresultantsolutionto100ml.Thissolutioncontains0.625μglumiflavinperml.

TestSolution:Shake25mgofthesamplewith10.0mlchloroformfor5minandfilter.

ThinLayerChromatography:

Stationaryphase:PrecoatedHPTLCplatesofsilicagelWRF254,10x20cm,layerthickness0.1mm(MerckCatNo1.12363)

Mobilephase:WaterRunlength:approx.6cm

Elutiontime:approx.20min

Applicationvolumes:10μlofReferenceSolutionand10μlofTestSolution

Detection:Drytheplateinacurrentofcoldairandevaluatethefluorescenceat366nm

AnyspotinthechromatogramoftheTestSolution,whichcorrespondstothemainspotoftheReferenceSolution,shallnotbelargerormoreintenselycolouredthantheReferenceSolutionspot.

METHODOFASSAY

Carryouttheassayinsubduedlight.Inabrown-glass500-mlvolumetricflask,suspend65.0mgofthesamplein5mlofwater,ensuringthatitiscompletelywetted,anddissolvein5mlof2Nsodiumhydroxidesolution.Assoonasdissolutioniscomplete,add100mlofwaterand2.5mlofglacialaceticacidanddiluteto500.0mlwithwater.Place20.0mlofthissolutioninabrownglass200-mlvolumetricflask,add3.5mlof