AOAC Starch(total)in Cereal Products amyloglucosidase-a-amylase method

32.2.05A

AOAC Official Method996.11

Starch(Total)in Cereal Products

Amyloglucosidase–a-Amylase Method

First Action1996

AOAC–AACC Method

(Applicable to determination of total starch in cereal products.) See Table996.11for the results of the interlaboratory study sup-porting the acceptance of the method.

Caution:See Appendix B,safety notes.Glucose oxidase-

peroxidase-aminoantipyrine buffer mixture,MOPS,

and acetate buffers contain sodium azide.Avoid con-

tact with skin and eyes.In case of contact,immediately

flush contact surfaces with plenty of water.Disposal of

these reagents into sinks with copper or lead plumbing

should be followed immediately with large quantities

of water to prevent potential explosive hazards.

Dimethyl sulfoxide is a skin irritant and should be used

with caution.

A.Principle

Test samples are hydrated and starch is hydrolyzed to maltodextrins with thermostableα-amylase at95–100°C.Tempera-ture and pH are adjusted and maltosaccharides are quantitatively hy-drolyzed to glucose with highly purified amyloglucosidase.Glucose is determined with high purity glucose oxidase–peroxidase reagent. Products containing high-amylose starches or resistant starch are pre-treated with dimethyl sulfoxide at95°C before treatment with α-amylase.Starch content is reported on“as is”basis.

B.Apparatus

(a)Grinding mill.—Centrifugal,with12-tooth rotor and0.5mm sieve,or similar device.Alternatively,cyclone mill can be used for small quantities.

(b)Bench centrifuge.—Holding16×120mm glass test tubes, with rating of ca1000×g.

(c)Water bath.—Maintaining50±0.1°C.

(d)Boiling water bath.—Boiling H2O at95–100°C(e.g.,fryer filled with H2O).

(e)Vortex mixer.

(f)pH Meter.

(g)Stopclock timer(digital).

(h)Top-loading balance.

(i)Analytical balance.

(j)Laboratory oven.—With forced-convection;maintaining 103±1°C;used for determining dry weight of test sample.

(k)Spectrophotometer.—Operating at510nm.

(l)Pipets.—Delivering100and200μL;with disposable tips.Al-ternatively,motorized hand-held dispenser can be used.

(m)Positive displacement pipetter.—With5.0mL tips accu-rately delivering100and200μL;and50mL tips delivering3.0mL. (n)Dispenser.—500mL,to https://www.360docs.net/doc/849837224.html,ed for glucose oxidase–peroxidase–aminoantipyrine buffer mixture.

(o)Glass test tubes.—16×120mm,17mL,for centrifugation at ca1000×g;and18×150mm.

(p)Test tube racks.—48place,holding16×120and 18×150mm tubes.

(q)Thermometer.—Reading103±1°C.

(r)Filter paper.—Fast,ashless.

C.Reagents

(a)3-(N-morpholino)propanesulfonic acid(MOPS) buffer.—pH7.0.Contains50mM MOPS,5mM calcium chloride, and0.02%sodium azide.In1L volumetric flask dissolve11.55g MOPS in900mL H2O and adjust pH to7.0with1M HCl(ca17mL). Add0.74g CaCl2.2H2O and0.2g sodium azide.Dilute to volume with H2O.Buffer is stable at room temperature.

(b)Thermostable a-amylase solution.—10mL;3000U/mL.Di-lute1mLα-amylase solution(in50%glycerol)to30mL with MOPS buffer,(a).Thermostable a-amylase solution is stable up to

Table996.11Interlaboratory study results for determination of total starch in processed cereal products by amyloglucosidase–a-amylase method

Sample Mean total

starch a,%Moisture,%No.of labs s r a s R a RSD r,%RSD R,%r b R c

Chicken feed pellets44.911.432 1.4 2.1 3.1 4.7 3.9 5.9 White bread60.910.632 1.6 3.0 2.6 4.9 4.58.4 Green pea38.512.431 1.3 1.9 3.4 4.9 3.6 5.3 High amylose maize starch74.813.426 2.2 3.6 2.9 4.8 6.2

White wheat flour68.012.831 2.0 2.9 2.9 4.3 5.68.1 Wheat starch85.312.226 2.8 3.3 3.3 3.97.89.2 Oat bran38.58.831 1.5 1.9 3.9 4.9 4.2 5.3 Spaghetti67.511.831 2.6 3.2 3.9 4.77.39.0 High amylose-maize starch

(DMSO method)

84.213.431 1.8 2.4 2.1 2.9 5.0 6.7 Wheat starch(DMSO method)84.712.231 2.6 3.9 3.1 4.67.310.9

a Calculated on“as is”basis.

b r=2.8×s

r

.

c R=2.8×s

R

.

3years when frozen.(Note:One unit[U]ofα-amylase activity is amount of enzyme required to release1μmole p-nitrophenol from “end-blocked”p-nitrophenyl maltoheptaoside in presence of satu-rating levels ofα-glucosidase and amyloglucosidase[i.e.,Ceralpha α-amylase assay reagent]at40°C and pH6.0.)Thermostableα-am-ylase solution should be free of detectable levels of free glucose.

(c)Amyloglucosidase solution.—10mL;200U/https://www.360docs.net/doc/849837224.html,e directly without dilution.Solution is viscous;for dispensing,use positive displacement dispenser.Amyloglucosidase solution is stable up to 3years at4°C.(Note:One unit[U]of enzyme activity is amount of enzyme required to release1μmole p-nitrophenol from p-nitrophenylβ-maltoside in the presence of saturating levels of β-glucosidase[i.e.,amyloglucosidase assay reagent]at40°C and pH4.5.)Amyloglucosidase solution should be free of detectable levels of free glucose.

(d)Glucose oxidase–peroxidase–aminoantipyrine buffer mix-ture.—Mixture of glucose oxidase,12000U/L;peroxidase, 650U/L;and4-aminoantipyrine,0.4mM.

Prepare buffer concentrate by dissolving13.6g KH2PO4,4.2g NaOH,and3.0g4-hydroxybenzoic acid in90mL distilled H2O.Ad-just to pH7.4with either2M HCl(16.7mL HCl/100mL)or 2M NaOH(8.0g NaOH/100mL).Dilute solution to100mL,add 0.4g sodium azide,and mix until dissolved.Buffer concentrate is stable up to3years at4°C.

To prepare glucose oxidase–peroxidase–aminoantipyrine buffer mixture,dilute50mL buffer concentrate https://www.360docs.net/doc/849837224.html,e part of diluted buffer to dissolve the entire contents of vial containing freeze-dried glucose oxidase–peroxidase mixture.Transfer contents of vial to1L volumetric flask containing diluted buffer.Reagent is stable 2–3months at4°C and2–3years at–20°C.Color formed with glu-cose is stable several hours.(Note:Glucose oxidase must not be con-taminated withβ-and/orα-glucosidase and chromogen color complex must be stable at least60min.)

C h e c k c o l o r f o r m a t i o n a n d s t a b i l i t y o f g l u c o s e oxidase–peroxidase–aminoantipyrine buffer mixture by incubating (in duplicate)3.0mL glucose oxidase-peroxidase-aminoantipyrine buffer mixture with glucose standard(100μg dried crystalline glu-cose in0.2mL0.2%sodium benzoate solution).After15,20,30, and60min incubation,read absorbance,A,of solution at510nm. Maximum color formation should be achieved within20min,and color should be stable at least60min at50°C.

(e)Aqueous ethanol.—About80%(v/v).Dilute80mL95%etha-nol(laboratory grade)to95mL with H2O.

(f)Sodium acetate buffer.—(1)200mM,pH4.5.—Pipet11.8mL glacial acetic acid(1.05g/mL)to900mL H2O.Adjust pH to4.5with 1M NaOH solution(ca60mL is required).Add0.2g sodium azide and dilute to1L with H2O.(Caution:Sodium azide should not be added until pH is adjusted.Acidification of sodium azide releases poisonous hydrazoic acid.)Buffer is stable12months at room tem-perature.(2)50mM,pH4.5.—Dilute200mM acetate buffer1+3 with H2O.

(g)Dimethyl sulfoxide(DMSO).—Laboratory grade.

(h)Glucose standard stock solution.—1mg/mL.Before prepar-ing solution,dry powdered crystalline glucose(purity≥97%)16h at 60°C under vacuum.Dissolve0.1g dried glucose,weighed to near-est mg,in100mL distilled water.

(i)Corn starch.—Containing known content of starch(e.g.,ca 98%dry weight).

Items(a)–(c),(h),and(i)are supplied in Total Starch Assay kit available from Megazyme International Ireland Ltd,Bray Business Park,Bray,Co.Wicklow,Ireland.

D.Preparation of Test Samples,Standards,and Reagent Blank (a)Test sample.—Grind ca50g laboratory sample in grinding mill to pass0.5mm sieve.Transfer all material into wide-mouthed plastic jar and mix well by shaking and inversion.

(b)D-Glucose standard working solutions.—50and100μg.Add 50and100μL D-glucose standard stock solution,C(h),to separate test tubes,and adjust volume in each tube to100μL with H2O.Pre-pare solutions immediately before use.

(c)Reagent blank.—Transfer0.1mL H2O into test tubes and pro-ceed with total starch determination using standard assay procedure starting from step E(a)(7).

E.Determination of Total Starch

(a)Standard procedureα-amylase/amyloglucosidase (AA/AMG).—Run D-glucose working standard solutions(in qua-druplicate),reagent blank(in duplicate),and corn starch with each set of https://www.360docs.net/doc/849837224.html,e reagent blank to zero spectrophotometer.(1)Ac-curately weigh90–100mg ground test portion directly into glass test tube.Tap tube gently on laboratory bench to ensure that all par-ticles drop to bottom of tube.(Note:When analyzing cereal prod-ucts containing high levels of glucose[processed cereal products {e.g.,breakfast cereals}and all products of unknown or uncertain composition{i.e.,products containing free glucose or maltodextrins}],pre-extract90–100mg of weighed,ground test sample2×with10mL80%aqueous ethanol,C(e),at ca80°C over 10min/extraction.Centrifuge slurry at1000×g and discard https://www.360docs.net/doc/849837224.html,e sediment for analysis.)(2)Add0.2mL80% aqueous ethanol to tube and stir on Vortex mixer to ensure that test portion is wet.Add3.0mL thermostableα-amylase,C(b),and mix contents of tube on Vortex mixer to ensure complete dispersion.

(3)Immediately place tube in boiling water bath for2min,remove from water bath,and mix vigorously on Vortex mixer.Return tube to boiling water bath for additional3min and then mix contents vigorously on Vortex mixer.(Note:Some solids will adhere to side of test tube;however,this will not affect analysis since tube con-tents are treated with enzyme in this step.)(4)Place tubes in water bath set at50°C and let equilibrate5min.Add4.0mL200mM so-dium acetate buffer,C(f)(1),and0.1mL amyloglucosidase solu-tion,C(c),and vigorously mix contents on Vortex mixer.Cap tube with marble and incubate30min at50°C.(5)Quantitatively trans-fer the entire contents of test tube to100mL volumetric https://www.360docs.net/doc/849837224.html,e water wash bottle to rinse tube contents thoroughly.Dilute to100 mL with H2O.(Note:If product contains<10%starch,adjust vol-ume to10.0mL[instead of100mL].Make appropriate adjustments to calculations.)Thoroughly mix contents of flask.Centrifuge aliquot of suspension10min at1000×g.Alternatively,filter aliquot through filter paper.(6)Carefully and accurately transfer0.1 mL aliquot of each supernatant(or filtrate)to bottoms of separate test tubes;use2tubes/supernatant(or filtrate).(7)Add3.0mL glu-cose oxidase–peroxidase–aminoantipyrine buffer mixture,C(d),to each tube(reaction solutions from test portion and corn starch,re-

agent blank,and D-glucose standard working solutions),and incu-bate20min at50°C.(8)Measure and record absorbance,A,of each test solution at510nm against reagent blank.Average A values for each test and use in Calculations,G.

(b)Modified procedure(DMSO/AA/AMG).—For products con-taining enzyme-resistant starch.(1)Accurately weigh90–100mg ground test portions directly into glass test tube.Tap tube gently on laboratory bench to ensure that all particles drop to bottom of tube. (Note:When analyzing cereal products containing high levels of glucose,pre-extract90–100mg weighed,ground test sample2×with10mL aqueous ethanol,C(e),at ca80°C over10min/extrac-tion.Centrifuge slurry at1000×g and discard https://www.360docs.net/doc/849837224.html,e sedi-ment for analysis.)(2)Add0.2mL80%aqueous ethanol to tube and stir on Vortex mixer to ensure that test portion is wet.(3)Immedi-ately add2mL DMSO solution,C(g),and stir tube on Vortex mixer. Place tube in vigorously boiling water bath and remove after5min. Add3.0mL thermostableα-amylase solution,C(b),and mix con-tents on Vortex mixer to ensure complete dispersion.(4)Immedi-ately proceed according to standard procedure(AA/AMG)starting from step E(a)(3).

F.Determination of Enzyme-Resistant Starch(Optional) (Note:This part of method was not validated in collaborative study.) Level of enzyme-resistant starch in products depends on nature of original starch(e.g.,high amylose)and on processing conditions. This may vary from0.1–30%total starch in product.Determine level of enzyme resistant starch as follows:

(1)Analyze product according to standard procedure, E(a)(1)–(4).

(2)Centrifuge reaction solution10min at1000×g and carefully pour supernatant into100mL volumetric flask.

(3)Resuspend pellet in5mL50mM sodium acetate buffer, C(f)(2),by vigorous stirring on Vortex mixer.Add additional5mL 50mM sodium acetate buffer,mix,and centrifuge10min at 1000×g.

(4)Combine supernatant with that from step(2),and dilute to volume.Analyze for starch starting with step E(a)(6).

(5)To pellet from(3)add2mL DMSO and analyze for starch by modified procedure E(b)starting from step E(b)(3)[proceed to pro-cedure E(a)as stated].

(6)At step E(a)(5),adjust volume to10mL(instead of100mL).

(7)Proceed with standard procedure,E(a),starting from step E(a)(6).Make appropriate adjustments to calculations.

G.Calculations

Calculate total starch content(percent,on as is basis)in test sam-ple as follows:

Total starch,%=A×F×1000×

1

1000

×

100

W

×

162

180

=A×

F

W

×90

where A=absorbance of reaction solutions read against reagent blank;F=factor to convert absorbance values toμg glucose= 100μg glucose/absorbance value for100μg glucose;1000=vol-ume correction,i.e.,0.1mL taken from100mL;1/1000=conver-sion fromμg to mg;100/W=conversion to100mg test portion; 162/180=factor to convert from free glucose,as determined,to anhydroglucose,as occurs in starch.

Reference:J.AOAC Int.80,571(1997).

Revised:March1998