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AOAC_2000[1].11_Polydextrose_in_Foods(食品中聚葡萄糖的测定)

45.4.06C

AOAC Official Method2000.11

Polydextrose in Foods

Ion Chromotography

First Action2000

[Applicable to the determination of2–95%(w/w)polydextrose in foods.]

See Table2000.11A for the results of the interlaboratory study supporting acceptance of the method.

A.Principle

Polydextrose is extracted from food with hot water and centri-fuged.The supernatant passes through a centrifugal ultrafilter to re-move high molecular weight interferences.The filtrate is treated with an enzyme mixture(isoamylase,amyloglucosidase,and fructanase)to remove any oligosaccharide interferences,mainly malto-oligomers and fructans.Polydextrose standards undergo the same treatment.High-pressure anion exchange chromatography with electrochemical detection(HPAEC–ED)is used to quantitate a high molecular weight fraction of polydextrose.This polydextrose method can be used as an adjunct to the current AOAC methods for measuring total dietary fiber.

B.Apparatus

(a)Water baths.—Set at80±2°C(increased to boiling);and50±2°C.

(b)High speed centrifuge and rotors.—6000×g,preferably up to38000×g with50mL(holds ca38mL)centrifuge tubes;9200×g with1.7mL microcentrifuge tubes;and6400×g with15mL centri-fuge tubes,e.g.,Beckman(Beckman Coulter,Inc.,4300N Harbor Blvd,PO Box3100,Fullerton,CA92834-3100,USA)J2-21centri-fuge with JA-17,JA18.1,and JA-25.15rotors,or equivalent.

(c)Analytical balance.—0.1mg accuracy.

(d)Bottles and tubes.—Containers with screw caps(250mL), withstanding80°C water.Centrifuge tubes50mL(holds ca38mL), withstanding38000×g(e.g.,Nalgene),and microcentrifuge tubes

(1.7mL),withstanding10000×g.

(e)Vortex mixer.

(f)Pipets.—Adjustable pipet,20–200and200–1000μL.

(g)Filters.—PTFE syringe filter,0.2μm pore size,13mm diam-eter.

(h)Centrifugal ultrafiltration devices.—100000NMWL(nom-inal molecular weight limit)polyethersulfone membrane,2mL ca-pacity, e.g.,Millipore(Bedford,MA)Ultrafree-CL PBHK BIOMAX-100,or equivalent.

(i)High-performance anion-exchange chromatograph (HPAEC–ED).—LC capable of producing ca3000psi(200bar); gradient pump(capable of handling NaOH eluents),and pulsed inte-grated electrochemical detector(with gold electrode),e.g.,Dionex Corp.(PO Box3603,Sunnyvale,CA94088-3603,USA)DX500 basic gradient carbohydrate system,or equivalent.See Ta-ble2000.11B for detector voltage settings for Dionex ED40. (j)LC column.—Microporous substrate(10μm)agglomerated with microbead quaternary ammonium functionalized latex,250×4mm column[e.g.,CarboPak PA1(Dionex,or equivalent)]with guard column(e.g.,CarboPak PA1Guard,23×3mm,or equivalent).

C.Reagents

(a)Solvents.—Deionized water(resistance≥18M S-cm).

(b)Sodium hydroxide.—50%;carbonate free,density1.54kg/L. To100g NaOH,containing≤1%Na2CO3,add100mL H2O.Stop-per and swirl until solution is complete.Let stand until Na2CO3has settled,leaving clear liquid(about10days).Keep tightly closed when not in use.

(c)Acetic acid.—0.2M.Dilute1.20g acetic acid to100mL with water.

(d)Sodium acetate solution.—0.2M.Dissolve2.72g Na ace-tate?3H2O in water and dilute to100mL.

(e)Acetate buffer.—pH4.5.Mix28mL acetic acid,(c),with 22mL sodium acetate,(d),and dilute to100mL with water.

(f)Fructanase(exo-inulinase).—667U/mL.Contents of bottle (8000U)dissolved in12mL acetate buffer,(e).One unit (Megazyme International Ireland Ltd.,Bray Business Park,Bray, Co.Wicklow,Republic of Ireland,or equivalent)is amount of en-zyme required to release1μmol fructose reducing sugar equivalents from kestose per minute under standard assay conditions(10mM kestose,pH4.5,40°C).Fructanase contains approximately1% endo-inulinase.

(g)Amyloglucosidase.—3260U/mL(soluble starch),200U/mL (p-NP-β-maltoside).One unit(soluble starch)is amount of enzyme required to release1μmol glucose from starch per minute under stan-dard assay conditions(starch substrate;10mg/mL starch,pH4.5, 40°C).One unit(p-NP-β-maltoside)is amount of enzyme required to release1μmol p-nitrophenol(pNP)from p-nitrophenol-β-maltoside (in presence of excess-β-glucosidase)per minute under standard as-say conditions(10mM p-NP-β-maltoside,pH4.5,40°C);Megazyme E-AMGDF,or equivalent.

(h)Isoamylase.—200U/mL.(Megazyme E-ISAMY,or equiva-lent.)One unit is amount of enzyme required to release1μmol glu-

Table2000.11A.Interlaboratory study results for polydextrose in foods by ion chromatography

Test sample0,%No.of labs a(b)s r RSD r,%s R RSD R,%HORRAT Rec.,% Milk chocolate candy19.878 1.10 5.52 2.0110.11 4.099.4 Iced tea 1.8680.12 6.370.2010.70 2.993.0 Sugar cookie8.117(1)0.41 5.100.8610.64 3.681.1 Grape jelly 5.147(1)0.469.040.7214.06 4.5102.8 Polydextrose92.578 3.64 3.93 4.15 4.48 2.292.6 Soft jellied candy33.148 2.557.71 3.5810.81 4.687.2 Powdered drink mix 1.847(1)0.147.710.189.88 2.792.0 a(b)a=Number of collaborating laboratories retained after outliers were eliminated;(b)=number of outlier laboratories.

cose reducing sugar equivalents from oyster glycogen per minute under standard assay conditions(10mg/mL oyster glycogen, pH3.5,40°C).

(i)Buffered enzyme mix.—Combine fructanase(f;2mL,1324 U),amyloglucosidase(g;84μL,274U),and isoamylase(h;84μL, 16.8U).Dilute in acetate buffer,(e),to20mL.Prepare fresh each day,and store at4°C.

(j)Mobile phase A.—0.15M NaOH.Dilute8.10mL50%NaOH, b,to1L with degassed water and store under inert gas.

(k)Mobile phase B.—0.15M NaOH and0.5M sodium acetate. Dissolve41.015g sodium acetate(68.04g3H2O)in1L0.15M NaOH.Degas mobile phases before use and store under inert gas. See Table2000.11C for eluent gradient program at flow rate of

1.2mL/min.

D.Preparation of Standards

(a)Polydextrose.—FCC grade;moisture content determined by the Karl Fischer method.Available from Danisco Cultor(440Saw Mill River Rd,Ardsley,NY10502,USA).

(b)Polydextrose stock standard solution.—5000mg/L.(Direc-tions are for Dionex DX500system;other systems may require a different range.)Weigh500mg polydextrose(accurate to10mg) into preweighed screw cap glass container,B(d).Add ca100g wa-ter,preheated to80°C,cap tightly,and vortex30s.Place container in 80°C water bath for10min,and vortex for30s at5and10min to solubilize the polydextrose.Remove container from water bath,let cool to room temperature,and reweigh.

(c)Intermediate standards.—Serially dilute5000mg/L stan-dard,(b),to make2500,2000,1500,1250,1000,750,500,and 250mg/L polydextrose standards in water.These standards will be diluted5-fold during procedure described in section F and are con-sidered the working standards(500,400,300,250,200,150,100, and50mg/L polydextrose).An8-point calibration curve should give a polynomial regression with a correlation coefficient≥0.995. Stability.—Standards are stable at4°C for1month.

E.Preparation of Test Solution

Grind or cut food into small particles.Store in sealed containers to prevent moisture changes.If approximate amount of polydextrose in food is known,weigh quantity of food to yield amount of polydextrose within range of the calibration curve.If amount of polydextrose in the food is not known,estimate amount of polydextrose and choose a test portion weight based on that estimate.

Extraction of polydextrose from food.—Weigh appropriate amount of food test sample(accurate to10mg)and add to preweighed container,B(d),with screw cap.Add ca100g hot water preheated to80°C,and immediately replace cap tightly.Vortex mix for30s to disperse food.Place container in water bath at80°C for 10min and vortex mix for30s at5and10min to solubilize the polydextrose.Remove container from water bath,let cool to room temperature,and reweigh.Record total weight,g,and calculate weight,g,of water.Transfer ca25g aliquot into a35mL centrifuge tube,B(b).Centrifuge mixture38000×g for10min to separate sol-ids from supernatant.

F.Treatment of Polydextrose Standards and Food

Take2mL aliquot of polydextrose intermediate standard,D(c),or 2mL aliquot of supernatant from E(freeze remaining solution for possible concentration adjustment).Transfer into centrifugal ultrafiltration device,B(h).Centrifuge at5000×g(6400rpm)for 45min.Record weight to0.1mg of1.7mL microcentrifuge tube, B(b).Take0.2mL aliquot of ultrafiltrate,place in the weighed tube and record weight to0.1mg.Add0.8mL buffered enzyme mix,C(i). Record final weight to0.1mg.Calculate weight of0.2mL aliquot and weight of0.8mL buffer enzyme mix.Secure cap and Vortex mix thoroughly.Incubate at50°C for60min.Then place in boiling water bath for10min to denature enzymes.Cool in ice bath or freezer until enzyme precipitates(ca5min in ice).Centrifuge at 9200×g(10000rpm)https://www.wendangku.net/doc/4a12423717.html,e supernatant within72h of preparation.

G.Determination

Filter supernatant from F through0.2μm syringe filter,B(g),into autosampler vial.Inject25μL through HPAEC.Analyze each test supernatant in triplicate and determine mean detector response.De-termine peak area for high molecular weight component of polydextrose at retention time ca12min;RSD should be≤5%.If not, repeat HPAEC https://www.wendangku.net/doc/4a12423717.html,pare this area with that of the calibra-tion curve.The polydextrose concentration should be in range of the calibration curve.If it is not,rerun the assay,adjusting concentration of initial test sample supernatant(E).

H.Calculations

(1)Stock standard concentration(μg/g):

Stock standard,μg/g=

mg polydextrose(dry basis)

g water

×1000

where1000is the conversion factor of mg toμg.

(2)Working standard concentration(μg/g):

Working standard,μg/g=

A A

intermediate stock concentration

Table2000.11B.Detector voltage settings

Time(s)Potential(V)integration

0.000.05

0.200.05Begin

0.400.05End

0.410.75

0.600.75

0.61–0.15

1.00–0.15

Table2000.11C.Gradient elution for HPAEC–PAD analysis

Time,min Mobile phase A,%Mobile phase B,%

00:007030

10:000100

15:007030

20:00End

where A1=weight of aliquot(0.2mL)of intermediate standard,g; A2=weight of aliquot(0.8mL)of buffered enzyme mix,g. (3)Polydextrose(%polydextrose)determination:

%Polydextrose=P

A A

F W

×××00001

where P=polydextrose concentration(μg/g),obtained from the cal-

ibration curve–second order polynomial fit;F=weight of food,g;

W=weight of water,g;A1=weight of aliquot(0.2mL)of diluted

food,g;A2=weight of aliquot(0.8mL)of buffered enzyme mix,g.

The0.0001is the conversion factor fromμg/g to%.

Reference:J.AOAC Int.84,472(2001).

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