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2001.02 Determination trans-Galactooligosaccharides AOAC2001.02

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AOACOfficialMethod2001.02Determinationoftrans-Galactooligosaccharides(TGOS)inSelectedFoodProducts.rar,2001.02,Determination,trans-Galactooligosaccharides,AOAC2001.02

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45.4.12AOAC Official Method 2001.02Determination of trans-Galactooligosaccharides(TGOS) in Selected Food ProductsIon-Exchange ChromatographyFirst Action 2001(Applicable to the quantitative determination of addedtrans-galactooligosaccharides in selected food products.)See Table 2001.02A for the results of the interlaboratory studysupporting the acceptance of the method.A. Principletrans-Galactooligosaccharides (TGOS) and lactose are extractedfrom a test portion with hot phosphate buffer. The extract is treatedwith ? -galactosidase to hydrolyze TGOS and lactose. Both the ini-tialandthetreatedsolutionareanalyzedusinghigh-performancean-ion-exchange chromatography with pulsed amperometric detection(HPAECPAD).Inthefirstassay,freegalactoseandlactosearede-termined in the initial test solution. In the second assay, the totalamount of galactose released from TGOS and lactose is determinedin the treated solution. TGOS are calculated from concentrations oflactose and galactose.B. Apparatus and Materials(a) High-performance anion-exchange chromatograph(HPAEC).Liquid chromatograph gradient pump, de-gas module,microinjection valve, pulsed electrochemical detector (1.0 mm di-ameter gold working electrode and a pH-Ag|AgCl combination ref-erence electrode; the titanium body of the cell serves as the counterelectrode) working in pulsed amperometric detection mode (PAD),automated sampler (Dionex Corp., PO Box 3603, Sunnyvale, CA94088-3603, USA, +1-408-737-0700, Fax: +1-408-730-9403, orequivalent) and data integrator (Shimadzu, 1 NishinokyoKuwabaracho, Nakagyou-ku, Kyoto 604-8511, Japan,+81-75-823-1111, Fax: +81-75-823-1361, or equivalent).HPAEC conditions.Column temperature, constant ? 0.5?C be-tween2030?C,preferably20? 0.5?C;flowrate,1.0mL/min;injec-tion volume, 20 ? L; detector sensitivity, analog range 13 ? C. SeeTable2001.02BforeluentgradientandTable2001.02Cfordetectortime program. Parameters may be varied in order to optimize chro-matography.(b) Column.CarboPac PA-1 Pellicular anion-exchange resin,250 ? 4 mm id with 50 ? 4 mm id guard column of same resin com-posed of sulfonated ethylvinylbenzene-divinylbenzene particles ag-glomerated with 350 ? m of Micro Bead quaternaryamine-functionalized latex, or equivalent.(c) pH meter.(d) Plastic vials.50 mL, with screw caps, resistant to tempera-tures up to 100?C.(e) Water baths.With shaker, maintaining 60 ? 2?C and 80 ?2?C.(f) Helium.Instrument grade, purity 99.996%.(g) Syringes.10mL,lowpressure,forusewith25mm,0.2? mporosity membrane filters.(h) Pipets.(1) 20,100,and1000? Lpipetwithdisposabletips.(2) Disposable Pasteur pipets, 146 mm.(i) Laboratory centrifuge.To hold microtubes; operating at11 000 ? g.(j) Benchcentrifuge.Tohold30mLtubes;operatingat1000? g.(k) Dilutor.Repeatedlydispensingsolutionsat? 1%accuracy.(l) Microtubes.Polypropylene 1.5 mL with cap.(m) Test tubes.Plastic, 15 mL, with screw caps.(n) Ultrasonic cleaner.With degas capability.(o) Vortex mixer.C. ReagentsFortestsolutionpreparation,extraction,andformobilephaseandre-agents preparation, use 18 M? DI water throughout.(a) Phosphate buffer.0.2M, pH 6.0. Dissolve 22.0 g KH2PO4and 6.0 g K2HPO4?3H2O in water and dilute to 1 L. Sterilize 30 minat 120?C in the autoclave.(b) Hydrochloricacid.1M.Dilute8.3mLHClto1Lwithwater.(c) Sodium hydroxide solution.50%, carbonate-free, density1.54 kg/L. To 100 g NaOH, containing ? 1% Na2CO3, add 100 mLwater. Stopper and swirl until solution is complete. Let stand untilNa2CO3has settled, leaving a clear liquid (about 10 days). Keeptightly closed when not in use.(d) Sodiumhydroxidesolution.1M.Dilute54mLNaOHsolu-tion, (c), to 1 L with CO2-free water.(e) ?-Galactosidase solution.2000 U/mL. Suspend approxi-mately 50 000 U/g ? -galactosidase originating from Aspergillusoryzae (available as Lactase F from Amano Enzyme, Inc., 2-7,1-chome, Nishiki Naka-ku, Nagoya, 460-8630 Japan;+81-52-211-3082; Fax: +81-52-211-3054, or equivalent) in phos-phate buffer solution, (a), to obtain a final activity of 2000 U/mL.One unit hydrolyzes 1.0 ? mol o-nitrophenyl-? -D-galactoside too-nitrophenol andD-galactose at pH 4.5, 25?C. Store suspension in 2002 AOAC INTERNATIONALTable 2001.02A.Interlaboratory study results for determination of trans-galactooligosaccharides in selected food products byion-exchange chromatographyMatrixLabsa(b)Mean, g/100 gsrsRRSDr, %RSDR, %rRRec., %Orange juice9 (0)3.60.290.220.8290Custard9 (0)4.80.300.4041.1396Cereal9 (0)43.910.90.541.5098Yogurt drink9 (0)5.60.410.461.3193Biscuits9 (0)7.90.920.9211.611.62.582.5888Lemonade syrup8 (1)13.80.400.631.7692a(b)Number of laboratories where a = number of laboratories retained after outliers removed and (b) = number of outlier laboratories.refrigeratorwhennotinuse.Stirsuspensionwellbeforeuse.Useen-zyme-suspension within 8 h from preparation.(f) Acetonitrile.LC grade.(g) Acetonitrile solution.20% (v/v). Dilute 200 mLacetonitrile, (f), with water to 1 L.(h) Acetonitrile solution.3% (v/v). Dilute 30 mL acetonitrile,(f), with water to 1 L.(i) Sodium acetate.Sodium acetate anhydrous, reagent grade.(j) Mobile phase A.12.5mM NaOH solution, carbonate-free.De-gas 2 L water in a bottle using the degas module for at least15 min with He or place a filtering flask filled with 2 L water for10mininanultrasonicbathundervacuum.Withoutshakingormix-ing, pipet 1.30 mL 50% NaOH solution, (c), to the de-gassed water.Continue de-gassing the resulting solution for 30 min before use.(k) Mobile phase B.125mM NaOH solution, carbonate-free.Prepare as in (j), but add 13.9 mL 50% NaOH solution into 2 Lde-gassed water instead of 1.30 mL. De-gas the solution for 30 minbefore use.(l) Mobile phase C.125mM NaOH solution, carbonate-free,and500mMsodiumacetate.Dissolve82.04gsodiumacetate,(i),in2 L de-gassed water and filter through a 0.2 m membrane filter.Add 13.9 mL 50% NaOH to the filtrate. De-gas the solution for30 min before use.(m) Galactose.Anhydrous.(n) Lactose.Monohydrate (stable at 103?C).(o) Sugar standard stock solution.Dry both referencegalactose and lactose monohydrate standards to constant weight at103?Cforapproximately4hinaconvectionoven.Accuratelyweighand transfer into 100 mL volumetric flask 80 mg galactose, (m),(S1). Dissolve in water and dilute to the mark with water (0.8 mggalactose/mL).Similarly prepare the lactose stock solution (1.425 mg anhydrouslactose/mL) by weighing 150 mg lactose monohydrate, (n), (S2?).Compensate for the water of crystallization of lactose by multiply-ing S2? by 0.95 = weight anhydrous lactose (S2).(p) Workingstandardsolutions.Dilute5.00mLbothstockso-lutions S1and S2to 1 L in a 1 L volumetric flask (WS1). Repeat thisoperation with 10.00 mL (WS2), 15.00 mL (WS3), and 20.00 mL(WS4) of both stock solutions, respectively, to make 1 L of eachworking standard combination solution. See Table 2001.02D.D. Preparation of Test SampleHomogenize liquid laboratory samples immediately before anal-ysis. Cut or shatter hard materials to pass through a 1 mm2sieve(No. 18).E. ExtractionNote: Accurately weigh to nearest 1 mg. See Figure 2001.02 forflow diagram of extraction and hydrolysis.Weighanempty50mLplasticvialwithscrewcap(M1).Weighanamount of test sample, corresponding to ca 0.10.3 g of total TGOSandlactose,butnotexceedinga10gtestportion,intothis50mLvial(M2,netweightofthetestportiononly).Addca40mLhot(ca80?C)phosphate buffer, C(a), close the vial with the screw cap, and mix.Keepthevialsat80? 2?Cinawaterbathwithcontinuousstirringfor30min.Letcooltoroomtemperatureinanicebath.MeasurethepHandadjust,ifnecessaryto5.76.3with1MNaOHor1MHCl.Dilutethetestsolutiontoca50mLwiththephosphatebuffer,C(a).Finallyweighthevial,includingthescrewcap,andsolution,anddeterminethe net weight of the test extract (M3).F. Enzymatic Hydrolysis(1) Treatedsolution.Weighanemptyplasticvialincludingthescrewcap,(M4).Transfer20goftestextractfromEintothisvialanddetermine the net weight of the test extract (M5). Pipet 1 mL of? -galactosidase solution, C(e), into the vial, close, and mix gently.(2) Initial test solution.Weigh an empty plastic vial includingthe screw cap, (M7). Pipet 1 mL ? -galactosidase solution, C(e), and1 mL phosphate buffer, C(a), into this vial. Deactivate the enzymeby heating for 10 min in a water bath at ca 100C. Cool, and weigh20 g of test extract from E into the vial (M8, net weight of test solu-tion only), close, and mix. Incubate both active and deactivated en-zyme containing extracts for 30 min at 60 ? 2?C with constant mildagitation, measuring heating time from the time the mixtures reach60?C. Avoid formation of foam or air bubbles during shaking. Letcool to room temperature using an ice bath. Add 5 mL 20%acetonitrile,C(g),totreatedsolutioncontainingactiveenzyme,mix, 2002 AOAC INTERNATIONALTable 2001.02B.Eluent gradient for HPAECPAD analysisTime (min)Mobile phase, %ABC0.00955020.10955035.000100036.000100036.100010046.000010046.10955061.009550Table 2001.02C.Detector program for HPAECPAD analysisTime (s)Potential (V)Integration0.000.050.200.05Begin0.400.05End0.410.750.600.750.610.151.000.15Table 2001.02D.Working standard solutionsDesignationmL S1mL S2Galactose+LactoseWS15.005.004 ? g/mL+7.125 ? g/mLWS210.0010.008 ? g/mL+ 14.25 ? g/mLWS315.0015.0012 ? g/mL+ 21.375 ? g/mLWS420.0020.0016 ? g/mL+ 28.5 ? g/mLandweightheclosedvial(M6).Add4mL20%acetonitrile,C(g),tothe initial test solution containing the deactivated enzyme, mix andweightheclosedvial(M9).Centrifugeallthesolutionsat10 000? gfor10minandfiltertheaqueoussupernatantthrougha0.2? mmem-brane filter. Use the filtrates for concentration measurements. Thedeactivatedenzyme-containingextractsaredesignatedasA1andthehydrolyzed extracts are designated as A2.G. Determination of Lactose and Galactose(1) Preparation of test solutions for HPAECPAD analy-sis.Dilute the centrifuged filtrates from F with 3% acetonitrile,C(h), so that galactose and lactose contents are within the workingstandardsconcentrationrange.Itmaybenecessarytouse3differentdilutionfactors,dependingonthenatureofthetestmaterials.Guidevalues and denotation for dilution factors can be found in Ta-ble 2001.02E.(2) Determination.Usethesametypeofintegrationforthetestsolutions and for the standard working solutions by choosing thesame peak width, threshold settings, and other integration parame-ters. Carefully control the baseline selection by extending the base-line next to the peak. Use peak area for quantification.First run the 4 calibration standards of each sugar to establish lin-earity. Then repeat the 4 standards. Between every 2 sets of stan-dards, run 9 test solutions e.g., WS1, WS2, WS3, WS4, initial testsolution 1A1(dilution factor D1), initial test solution 1A1(dilutionfactor D3), treated solution 1A2, initial test solution 2A1(dilutionfactor D1), initial test solution 2A1(D3), treated solution 2A2, initialtest solution 3A1(D1), initial test solution 3A1(D3), treated solution3A2, WS1, WS2, WS3, WS4, initial test solution 4A1(D1), initial testsolution 4A1(D3), treated solution 4A2, etc. Continue this processuntilalltestsolutionshavebeenanalyzed.Useaverageresponsefac-torfromthestandardsbracketingthetestsolutionstocalculatesugarconcentration for each test solution.(3) Possible interferences.Galactose is the most important pa-rameter for the assessment of TGOS content in the product. Inaccu-racies in the determination of galactose after enzymatic hydrolysisof test samples high in lactose like cereal and powdered milk basedproducts,caninfluencethemeasurementofgalactoseliberatedfromTGOS. Also nonselective hydrolysis of alpha-galactans (e.g., carobpowder) by ? -galactosidase may occur, leading to erratic results.Use fresh enzyme preparations only.H. CalculationsCalculate the initial (free) galactose, Gb, and (initial = final) lac-tose, Lb, contents in the buffered extracts assay A1and total (final)galactosecontent,Gt,ofthehydrolyzedsolutionA2ing/100gprod-uct of test sample using the following formula:Gb=CDMMFMGb?1978100()whereCGb=mggalactose/kgininitialtestsolutionA1andFisafac-tor calculated as:FMMM?231100Gt=CDMMFMGt?2645100()where CGt= mg g