»Lactase (b-D-galactoside galactohydrolase)is a hydrolytic enzyme derived from the mold Aspergillus oryzae.It contains not less than 30,000USP Lactase Units in each g.
NOTEOne USP Lactase Unit is the lactase activity contained in the amount of enzyme that hydrolyses one microequivalent of galactosidic linkage per minute at a pHof 4.5and at 37as directed in the Assay for lactase activity.
Packaging and storage Preserve in tight containers at room temperature.
Labeling Label it to indicate lactase activity in USP Units.
Microbial limits á61ñ It meets the requirements of the test for absence of Salmonellaspecies and Escherichia coli.
Loss on drying á731ñ Dry it in a vacuum at 60for 4hours:it loses not more than 6.0%of its weight.
Arsenic á211ñ: not more than 3µg per g.
Lead á251ñ: not more than 5µg per g.
Heavy metals á231ñ: not more than 30µg per g.
Assay for lactase activity
pH4.5Acetate buffer Dilute 57.5mLof glacial acetic acid with sufficient water to make 500mLof solution.Transfer 50mLof the glacial acetic acid solution into a 1000-mLvolumetric flask,add 11.3mLof 4Nsodium hydroxide,and dilute with water to volume.If necessary,adjust with the addition of either the glacial acetic acid solution or 4Nsodium hydroxide to a pHof 4.50±0.05.
Substrate solution On the day of use,weigh 370.0mg of o-nitrophenyl-b-D-galactopyranoside,and place in a 100-mLvolumetric flask.Add about 50mLof pH4.5Acetate buffer,swirl to dissolve,then dilute with pH4.5Acetate bufferto volume.
Standard preparation Transfer about 0.4g of USP Lactase RS,accurately weighed,to a 1000-mLvolumetric flask.Add about 600mLof water,allow to stand for 15minutes,swirl gently,and dilute with water to volume.Pipet 3.0mLof this solution into a 200-mLvolumetric flask,and dilute with water to volume.
Assay preparation Transfer an accurately weighed quantity of about 0.4g of Lactase to a 1000-mLvolumetric flask.Add about 600mLof water,allow to stand for 15minutes,swirl gently,and dilute with water to volume.Pipet 3.0mLof this solution into a 200-mLvolumetric flask,and dilute with water to volume.
Procedure Transfer 2.0mLof the Substrate solutionto 3separate test tubes labeled S,U,and B.Transfer the tubes to a thermostated water bath maintained at 37.0±0.1,and incubate for 10minutes.Following the incubation,add rapidly to tube S,0.5mLof the Standard preparation,to tube U,0.5mLof the Assay preparation,and to tube B(the reagent blank),0.5mLof water.Mix each tube on a vortex mixer for 1second,and immediately return the tubes to the water bath,which has been maintained at 37.0±0.1.After 15minutes of incubation,rapidly add 2.5mLof a 10%sodium carbonate solution to each test tube to stop the enzyme reaction.Add 20.0mLof water to each test tube,and mix.Concomitantly determine the absorbances of the three solutions at 420nm in a 1-cm cell using a suitable spectrophotometer (see Spectrophotometry á851ñ).Calculate the number of USP Lactase Units of the Lactase taken by the formula:
(P)(WS/WU)(AU-AB)/(AS-AB),in which Pis the potency of USP Lactase RSin USP Units per g;WSis the amount,in g,of USP Lactase RStaken;WUis the amount,in g,of the Lactase taken;and AUis the absorbance reading of tube U,ABis the absorbance reading of tube B,and ASis the absorbance reading of tube S.
Auxiliary Information Staff Liaison:Larry N.Callahan,Ph.D.,Scientist
Expert Committee:(BNT)Biotechnology and Natural Therapeutics/Diagnostics