A AOAC Official Method Fructans in Food Products

Transcription

1 A AOAC Offiil Method Frutns in Food Produts Ion Exhnge Chromtogrphi Method First Ation 1997 Finl Ation 1999 (Applile to the determintion of dded frutns in proessed foods.) See Tle A for the results of the interlortory study supporting the eptne of the method. A. Priniple Frutns re extrted from the produt with oiling wter. Aliquot of extrt is hydrolyzed using lyophilized mylogluosidse to remove strh. Prt of tht hydrosylte is treted with inulinse followed y determintion of relesed sugrs. The initil test portion nd first nd seond hydrosyltes re nlyzed using high performne nion exhnge hromtogrphy with pulsed mperometri detetion (HPAEC PAD). In sugr nlysis 1, free frutose nd surose re determined in initil extrt. In sugr nlysis 2, sum of free gluose nd gluose from mltodextrins nd strh re determined in the first hydrosylte. In sugr nlysis 3, totl mount of gluose nd totl mount of frutose from the hydrosylte plus gluose nd frutose from the seond hydrosylte re determined. Frutns re lulted from onentrtions of gluose nd frutose. B. Apprtus () Anion exhnge hromtogrph (HPAEC). Liquid hromtogrph grdient pump with eluent degs module, miro-injetion vlve, pulsed eletrohemil detetor working in pulsed mperometri detetion mode (PAD), or equivlent, nd utomted smpler. HPAEC onditions: olumn temperture, 40 ± 0.5 C; flow rte, 1.0 ml/min; injetion volume, 50 µl; detetor sensitivity, nlog rnge 1 3 µc. See Tle B for eluent grdient nd Tle C for detetor time progrm. (Note: Vry prmeters to optimize hromtogrphy.) () HPAEC olumn mm id with pelliulr nion-exhnge resin with similr gurd olumn (50 4 mm id). () Dt integrtor. Crop PA1. (d) Anlytil lne. Aurtely weighing to 0.1 mg. (e) Memrne filters. 0.2 µm porosity. (f) Column oven. Mintining 40 ± 0.5 C. (g) Vuum oven. Mintining 55 ± 3 C. (h) Retion tues. 15 ml, equipped with srew ps. (i) ph meter. Temperture ompensted, stndrdized with ph 4.0, 7.0, nd 9.0 uffer solutions. (j) Glss mirofier filters. Retining 0.7 µm prtile size. (k) Filter holder. 25 mm dimeter, for low pressure syringe, or equivlent. (l) Syringes. Luer lok, 10 ml, low pressure. (m) Desitor. With SiO 2, or equivlent desint. Every 2 weeks dry desint overnight t 130 C. (n) Glss ottles. or 150 ml with srew ps. (o) Mgneti stirrers nd stir rs. (p) Wter ths. With shker, mintining 85 ± 2 C nd 60 ± 2 C. (q) Volumetri flsks. ml, 1 L, nd 2 L. (r) Mortr with pestle. (s) Blender. (t) Knife nd sissors. (u) Mixing rods. (v) Vortex mixer. (w) Psteur pipets. 3 ml plsti pipets with lrge tip, or equivlent. C. Regents For extrtion, nd for moile phse nd regents preprtion, use H 2 O, ASTM qulity Type 1, or ACS (regent grde), or equivlent. () Aette uffer. ph 4.5. Pipet 28.0 ml 0.2M eti id (11.46 ml/l) nd 22.0 ml 0.2M N ette (28.23 g trihydrte/l) into ml volumetri flsk. Dilute to volume with H 2 O. () HCl 0.05M. Dilute stok solution of known titer, e.g., 50 ml 1M HCl (86 ml/l) to 1 L with H 2 O. () KOH 0.05M. Dilute stok solution of known titer, e.g., 50 ml 1M KOH (56 g/l) to 1 L with H 2 O. (d) Lyophilized mylogluosidse. Contining <0.02% gluose, frutose, nd surose. Store t 4 C when not in use. (Sigm A7420, or equivlent.) (e) Inulinse Contining <0.005% gluose, frutose, nd surose (ville s Frutozyme SP 230 solution from Novo Nordisk A/S Novo Alle, 2880 Dgsverd, Denmrk). Store t 4 C. (f) NOH 50%, ronte-free, density NOH solution is stle indefinitely stored under He. (g) Moile phse A. 10mM NOH solution, ronte-free. Prepre ronte-free solution s follows: Degs 2LH 2 O t lest Tle A Interlortory study results for determintion of frutns in food nd food produts y ion exhnge hromtogrphy Smple Men, g/g initil produt s r s R RSD r, % RSD R,% r R Low-ft spred Cheese spred Choolte Wine gum Powder drink mix Bisuits Blind duplites. r = 2.8 s r. R = 2.8 s R.

2 Tle B Eluent grde for determintion of frutns in foods y ion exhnge hromtogrphy % Moile phse Time, min A B min with N or He in ottle of de-gs module. Without shking or mixing 50% NOH solution, (f), nd while lowing He on liquid surfe, pipet 1.04 ml from the middle of ottle nd dd gently to degssed H 2 O. Continue degssing solution 30 min efore use. (h) Moile phse B. 1M NOH, ronte-free. Degs H 2 Os in (g). Without shking or mixing 50% NOH solution, (f), nd while lowing He on liquid surfe, pipet ml from the middle of ottle nd dd gently to degssed H 2 O. Continue degssing solution 30 min efore use. (i) Gluose. D(+)-Dextrose nhydrous, regent grde. (j) Frutose. Levulose, regent grde. (k) Surose. Regent grde. (l) Gluoheptose. D-Gluoheptose (Pfnstiehl Lortories, In., 1219 Glen Rok Ave, Wukegn, IL 60085, USA, or equivlent). (m) Ltose. Monohydrte, regent grde. (n) Gltose. Regent grde. (o) Mltitol. 98%. Store t 4 C when not in use (Sigm Chemil Co., 3050 Sprue St, St. Louis, MO 63103, USA, or equivlent). (p) Sugrs stndrd Dry gluose, frutose, surose, gluoheptose, nd gltose referene sugr stndrds in vuum oven 48 h t 55 ± 3 C. (Note: Do not dry ltose nd mltitol.) Weigh mg mltitol, mg dextrose, mg gltose, mg frutose, mg ltose, nd mg surose nd trnsfer into single ml volumetri flsk. Dissolve sugrs ompletely in H 2 O using mgneti stirrer r nd plte. Add H 2 O to weight of g. (q) Gluoheptose internl stndrd Weigh mg gluoheptose (l) nd trnsfer into ml volumetri flsk. Dissolve sugr ompletely in H 2 O using mgneti stirrer. Add H 2 O to weight of g. Tle C Detetor time progrm for determintion of frutns in foods y ion exhnge hromtogrphy Time, s Tension, V (r) Sugrs working stndrd solutions. (1) S mg of eh sugr/kg (gluoheptose, mltitol, dextrose, gltose, ltose, frutose, nd surose). Prepre y diluting 5.0 g gluoheptose internl stndrd solution, (q), nd 5.0 g sugr stndrd solution, (p), to g with H 2 O. (2) S mg gluoheptose/kg nd 25 mg/kg of eh sugr: dextrose, mltitol, gltose, ltose, frutose nd surose. Prepre y diluting 5.0 g gluoheptose internl stndrds solution, (q), nd 2.5 g sugr stndrd solution, (p), to g with H 2 O. (3) S mg gluoheptose/kg nd 5 mg/kg of eh sugr: dextrose, mltose, gltose, ltose, frutose nd surose. Prepre y diluting 5.0 g gluoheptose internl stndrd solution, (q), nd 0.5 g sugr stndrd solution, (p), to g with H 2 O. D. Preprtion of Test Produts Homogenize test smples immeditely efore nlysis s follows: Before mixing in lender, freeze stiky or ftty produts (e.g., hoolte nd rs tht form pste-like mss in lender). Cut gummy, stiky, nd pste-like produts tht nnot e mixed in lender into smll piees with knife or sissors so tht prtile size is 5 mm dimeter. Shtter hrd produts in mortr (e.g., hrd ndies), so prtile size is 5 mm dimeter. E. Extrtion See Figure for flow digrm of extrtion nd hydrolysis. Aurtely weigh to the nerest 0.1 mg test portion (M 1 ) from D ontining 1 g frutn (ut not exeeding 30 g nd/or 5 g strh); ple in ml eker ontining mixing rod. [Note: For strhy produts (e.g., isuits, kes, other kery produts nd erels) weigh 5 g test portion (M 1 ) in 250 ml eker ontining mixing rod.] See Tle D for guide vlues. Add 40 ml oiling H 2 O (dd ml to strhy produts) nd immeditely hek ph (Meotrode, Hmilton P/H /03, or equivlent) with mild gittion. ph should e etween 6.5 nd 8.0. If neessry, djust ph immeditely with 0.05M KOH or 0.05M HCl. (Note: Continue ph mesurement nd djustment until test portion is ompletely dissolved.) Rinse eletrode with oiling H 2 O. Trnsfer solution quntittively into weighed ml volumetri flsk (for strhy produts trnsfer solution to 250 ml flsk), rinsing eker with oiling H 2 O. Ple flsk in wter th for 10 min t 85 ± 2 C with ontinuous stirring. Let solution ool to room temperture. Dilute to ml, weigh, nd homogenize. (Weight of solution is M 2 ). From this step on, ensure tht solutions re mintined t or ove room temperture. Beuse of possile presene of insolule mtter nd/or ft in some extrts, it my e diffiult to homogenize them well. To ensure proper homogeniztion, proeed s follows: Shke volumetri flsk with extrt very vigorously. Trnsfer extrt into eker nd ontinue very vigorous mixing using mgneti stirrer. While mixing vigorously, withdrw 2 liquots using Psteur pipet: 50 g liquot (for diret nlysis, A 0 ), nd 15 g liquot (for hydrolysis, M 3 ). Dilute liquots ontining frutn to 1% frutn without ny heting. F. Enzymti Hydrolysis Weigh 50 g homogenized mixture from E nd set side for diret nlysis (ssy A 0 ). (1) First hydrolyztion. Trnsfer 15 g (weighed to nerest 10 mg) homogenized mixture (M 3 ) into tred glss ottle with srew-p nd dd the sme mount of ette uffer. ph of solution

3 enzyme for 50 mg strh, sine 1 Unit liertes 1 mg gluose from strh). If mount of strh nd mltodextrins is unknown, onsider unknown prt of test portion s % strh. For pste-like produts use 35 mg mylogluosidse (with tivity of 51 Units/mg); for other produts nd strhy produts use 10 mg. Inute mixture 30 min in wter th t 60 ± 2 C with onstnt, mild gittion. Strt timing 30 min from the moment retion mixture rehes 60 C. Ensure tht during gittion no fom forms nd no ir ules re rought into suspension. Let ool to room temperture nd weigh (net weight is M 4 ). Weigh 10 g first hydrolyzte nd set side for nlysis (ssy A 1 ). (2) Seond hydrolyztion. To the remining prt of the first hydrolyzte (net weight is M 5 ), dd suffiient mount of inulinse solution, tking into ount mount of frutn present in test portion nd enzyme onentrtion (e.g., for Frutozyme SP 230 with tivity of 1.8 Units/mg, use 56 mg enzyme for mg frutn, sine 1 Unit hydrolyzes 1 mg frutn; guide vlue 150 mg Frutozyme). If mount of frutns is unknown, onsider unknown prt of test portion s % frutn. Inute gin 30 min in wter th t 60 ± 2 C with onstnt, mild gittion. Strt timing 30 min from the moment retion mixture rehes 60 C. Ensure tht during gittion no fom forms nd no ir ules re rought into suspension. Let ool to room temperture nd weigh (net weight is M 6 ; ssy A 2 ) Figure Flow digrm of extrtion nd hydrolysis for determintion of frutns in foods y ion exhnge hromtogrphy. should e 4.5 ± If neessry, djust with 0.05M KOH or 0.05M HCl. Add suffiient mount of mylogluosidse, tking into ount mount of strh nd mltodextrins present, nd enzyme onentrtion (e.g., for mylogluosidse with tivity of 51 Units/mg, use 1 mg G. Determintion of Mono- nd Dishrides () Preprtion of extrt nd hydrolyztes for HPAEC PAD nlysis. Dilute homogenized mixture from E, nd first nd seond hydrolyztes from F, so tht gluose, frutose, nd surose ontents re within onentrtion rnge of sugrs working stndrd solutions, nd dd gluoheptose internl stndrd solution s follows: (1) Dilute 2.0 g gluoheptose internl stndrd solution, C(q), nd mount of homogenized mixture (M 7 ), within rnge of gluose stndrd solution, to g with H 2 O (ssy A 0 ). (2) Dilute 2.0 g gluoheptose internl stndrd solution, C(q), nd mount of first hydrolyzte (M 8 ), within rnge of frutose stndrd solution, to g with H 2 O (ssy A 1 ). Tle D Guide vlues for dilutions for HPAEC-PAD frutn nlysis for mounts of A 0,A 1, nd A 2 to e diluted to g with H 2O Produt g M 1 ga 0 (M 7 ) g A 1 (M 8 ) g A 2 (M 3 ) Low-ft spred (8% inulin) Cheese or heese spred (5% inulin) nd 5 Choolte (10% inulin) 10 1 nd Choolte pste (10% inulin) nd Bkery produts (5% oligofrutn) 5/ nd Dry ie-mix (15% oligofrutn) nd 1 Vegetles (5% inulin) Diry produts (3% oligofrutn) nd Fruit preprtes (20% oligofrutn) Cerels (10% inulin) 5/ Confetionery, sweet (35% oligofrutn) A powder for ie rem preprtion. Mrmlde nd frutn.

4 (3) Dilute 2.0 g gluoheptose internl stndrd solution, C(q), nd mount of seond hydrolyzte (M 9 ), within rnge of surose stndrd solution, to g with H 2 O (ssy A 2 ). Lote guide vlues in Tle D. (Note: Prepre 2 different dilutions of the sme test solution if lrge differene etween onentrtions of different sugr ompounds to e nlyzed is expeted.) For foods with ft ontent of more thn 5 frutn ontent, deft diluted, filtered solutions efore injetion s follows: Trnsfer 4 g diluted solution in retion tue nd dd 4 ml hexne. Agitte 5 min on vortex mixture nd entrifuge to seprte hexne phse. Disrd hexne phse. Filter queous phse through glss mirofier filters, B(j), nd then through 0.2 µm memrne filter efore injetion. (Note: Use of internl stndrdiztion method is optionl. Alterntively, externl stndrdiztion method n e pplied. Use the sme proedure s internl stndrdiztion method ut without using internl stndrd. The response ftor is then relted to the response of stndrd ompound itself.) () Determintion. Ensure tht the sme type of integrtion is used on unknown test nd stndrd working solutions y hoosing pek width, threshhold settings, nd other integrtion prmeters. Crefully ontrol seline seletion. Use pek height or pek re for quntifition. Estlish system s linerity y running sugrs working stndrd solutions S 1,S 2, nd S 3. Run nlysis y rketing duplite test solutions with sugrs working stndrd solutions (e.g., S 1, test 1A 0, test 1A 1,S 2, test 1A 2, test 2A 0,S 3, test 2A 1, test 2A 2,S 1, test 3A 0, test 3A 1,S 2, et.). Continue until ll solutions hve een nlyzed. Use verge response ftors from sugrs working stndrd solutions rketing tests to lulte sugr onentrtions for eh test () Possile interferenes. For produts ontining mltitol nd/or ltose (e.g., some hoolte, hoolte pste, heese, nd rekfst drinks), gluose results fter hydrolysis my e overestimted. Therefore, lulte mltitol ontent in produts nd orret gluose results for tht prt of mltitol tht hs een hydrolyzed y inulinse Corret gluose results lso for tht prt of gltose tht hs een formed y hydrolysis of ltose y inulinse H. Clultions Clulte response ftors, R, of frutose, gluose, surose, mltitol, nd gltose s follows: R=(C/C ) (P /P) where C = onentrtion of sugr stndrd in working stndrd solution, mg/kg; C = onentrtion of gluoheptose (internl stndrd), mg/kg; P = height of sugr pek; P = height of gluoheptose pek. Using Tle E lulte frutose nd surose ontent in diluted solutions from ssy A 1, nd frutose nd gluose ontent in diluted solutions from ssy A 2. Clulte mltitol nd gltose ontents in diluted solutions from ssys A 1 nd A 2 s follows (use orresponding response ftors nd pek height [or pek re] of the omponents): C s = R C s (P s / P s ) Tle E Clultions of sugr ontents in test smples Assy Sugr A 0 A 1 A 3 Gluose G 1 G t Frutose F f d F t Surose S e Mltitol f Ml 1 g Ml 2 Gltose h Gl 1 i Gl 2 G 1 = Sum of free gluose (G f ) nd gluose from mltodextrins nd strh (G m ). G t = Totl mount of gluose. F f = Amount of free frutose in g/ g initil test portion. d F t = Totl mount of frutose. e S = Amount of surose in g/ g initil test portion. f Ml 1 = Amount of mltitol efore inulinse hydrolysis. g Ml 2 = Amount of mltitol fter inulinse hydrolysis. h Gl 1 = Amount of gltose efore inulinse hydrolysis. i Gl 2 = Amount of gltose fter inulinse hydrolysis. where C s = onentrtion of sugr in diluted test solution, mg/kg; R = response ftor; C s = onentrtion of glooheptose (internl stndrd) in diluted test solution, mg/kg; P s = height of sugr pek in diluted test solution; P s = height of gluoheptose pek in diluted test Clulte free frutose (F f ) nd surose (S) ontents; gluose (G 1 ), mltitol (Ml 1 ), nd gltose (Gl 1 ) ontents fter hydrolysis with mylogluosidse; nd totl gluose (G t ) nd totl frutose (F t ) ontents; nd mltitol (Ml 2 ) nd gltose (Gl 2 ) ontents fter hydrolysis with inulinse solution in perent of initil test portion s follows: CFf M2 F f = M1 M7 where C Ff = mg frutose/kg diluted solution ssy A 0. C M S = S 2 M1 M7 where C S = mg surose/kg diluted solution ssy A 0. C M M G 1 = G1 where C G1 = mg gluose/kg diluted solution ssy A 1. Ml 1 = C M M Ml1 where C Ml1 = mg mltitol/kg diluted solution ssy A 1. Gl 1 = C M M Gl1 where C Gl1 = mg gltose/kg diluted solution ssy A 1.

5 CGt M M M G t = where C Gt = mg gluose/kg diluted solution ssy A 2. CFt M M M F t = where C Ft = mg frutose/kg diluted solution ssy A 2. Ml 2 = C M M M Ml2 where C Ml2 = mg mltitol/kg diluted solution ssy A 2. Gl 2 = C M M M Gl2 where C Gl2 = mg gltose/kg diluted solution ssy A 2. Clulte gluose relted frutns (G i ) nd frutose relesed from frutns (F i ) s follows: G i = G t G s G 1 G Ml G l F i = F t F s F f where G s = gluose relesed from surose = S/1.9; G Ml = gluose relesed from mltitol = [(Ml 1 Ml 2 )/1.9]; G L = gluose relesed from ltose = (Gl 2 Gl 1 ); F s = frutose relesed from surose = S/1.9. Clulte frutn ontent (i) in % s follows: i = k (G i + F i ) ( n 1) k = 180n where n = verge degree of polymeriztion = [(F i /G i ) + 1] for pure GF n mixtures. For inulin from hiory n = 10 n e used (k = 0.91). For oligofrutose, n = 4 n e used (k = 0.925). Amount of gluose formed y hydrolysis of ltose my e lulted y dividing differene in ltose ontent efore nd fter inulinse hydrolysis y 1.9. Amount of gluose formed y hydrolysis of mltitol is the sme s the mount of soritol formed y hydrolysis of mltitol y inulinse hydrolysis. Referene: J.AOAC Int. 80, 1029(1997). Revised: June 2000