45.4.07AOAC Of f i c ial Method 985.29To t al Di e tary Fi b er in FoodsEnzymatic–Gravimetric MethodFirst Ac t ion 1985Fi nal Ac tion1986AOAC–AACC MethodCo d ex-Adopted–AOAC Method*A.Prin ci pleDu p li c ate test por t ions of dried foods, fat-extracted if con t ain i ng >10% fat, are gelatinized with Termamyl (heat-stable α-am y l ase), and then en z y m at i c ally di g ested with pro t e a se and amyloglucosidase to re m ove pro t ein and starch. (When an a l yz i ng mixed di e ts, al w ays ex t ract fat prior to de t er m in i ng to t al di e tary fi b er.) Four vol u mes of ethyl al c o h ol are added to pre c ip i t ate sol u b le di e tary fi b er. To t al res i d ue is fil t ered, washed with 78% ethyl al c o h ol, 95% ethyl al c o h ol, and ac e t one. Af t er dry i ng, res i d ue is weighed. One du p li c ate is an a l yzed for pro t ein, and other is in c in e r a ted at 525°C and ash is de t er m ined. To t al di e tary fi b er = weight res i d ue – weight (pro t ein + ash).B. Ap p a r a t us(a)Fritted cru c i b le.—Po r os i ty No. 2 (Py r ex No. 32940, coarse, ASTM 40-60 µm; or Corning No. 36060 Büchner, fritted disk, Py r ex, 60 mL, ASTM 40-60 µm). Clean thor o ughly, heat 1 h at 525°C, and soak and then rinse in H2O. Add ca 0.5 g Celite to air-dried cru c i b les and dry at 130°C to con s tant weight (≥ 1 h). Cool and store in des i c c a t or un t il used.(b) V ac u um source.—V ac u um pump or as p i r a t or equipped with in-line dou b le vac u um flask to pre v ent con t am i n a t ion in case of H2O backup.(c) Vac u um oven.—70°C.Al ter na tively,105°C air oven can be used.(d) Des ic ca tor.(e)Muf fle fur nace.(f)Wa t e r b a t h s.—(1)B o i l i n g.(2)C o n s t a n t tem p er a t ure.—Ad j ust a ble to 60°C, with ei t her multistation shaker or multistation mag n etic stir r er to pro v ide con s tant ag i t a t ion of di g es t ion flasks dur i ng en z y m atic hy d ro l y s is.(g) Beakers.—Tall-form, 400 or 600 mL.(h) Bal a nce.—An a lyt i cal,readability to0.1mg.(i)pH me t er.—Stan d ard i zed with pH 7 and pH 4 buff e rs.C. Re a gents(a) 95% Eth a n ol.—v/v. Technical grade.(b) 78% Eth a n ol.—Place 207 mL H2O into 1 L vol u m et r ic flask. Di l ute to vol u me with 95% ethyl al c o h ol. Mix and di l ute to vol u me again with 95% ethyl al c o h ol if nec e s s ary. Mix. One vol u me H2O mixed with 4 vol u mes 95% ethyl al c o h ol will also give 78% ethyl al c o h ol fi n al con c en t ra t ion.(c)Ac e tone.(d)Phos p hate buffer.—0.08M, pH 6.0. Dis s olve 1.400 g so d ium phos p hate dibasic, an h y d rous (Na2HPO4) (or 1.753 g dihydrate) and 9.68 g so d ium phos p hate monobasic monohydrate (NaH2PO4⋅H2O) (or 10.94 g dihydrate) in ca 700 mL H2O. Di l ute to 1 L with H2O. Check pH with pH me t er.(e) Al p ha-amylase (heat sta b le).—Termamyl. (1) Store in re frig er a tor.Based on Nel s on/Somogyi re d uc i ng sugar with sol u b le starch as sub s trate.—10 000 + 1000 units/mL (1 unit is de f ined as the amount of en z yme re q uired to re l ease 1 µmole re d uc i ng sugar equiv a l ents/min at pH 6.5 and 40°C). (2) Based on Ceralpha method us i ng p-nitrophenyl-maltosaccharide as sub s trate in the pres e nce of a thermostable al p ha-glucosidase.—3000 + 300 Ceralpha units/mL (1 unit of en z yme is re q uired to re l ease 1 µmole p-nitrophenyl/min at pH 6.5 and 40°C).(f) Pro te ase.—Keep re frig er ated.(1)Ca sein as say.—300–400 Units/mL. (1 pro t e a se unit is de f ined as the amount of en z yme re q uired to hy d ro l yze (and solubilize in TCA) 1 µmole ty ro sine equiv a l ents/min from sol u b le ca s ein at pH 8.0 and 40°C); 7–15 units/mg (1 unit will hy d ro l yze ca s ein to pro d uce color equiv a l ent to 1.0 µmole ty r o s ine/min at pH 7.5 and 37°C). Color by Folin-Ciocalteau re a gent. (2) Azo-casein as s ay.—300–400 Units/mL [1 unit endo-peptidase ac t iv i ty is de f ined as the amount of en z yme re q uired to hy d ro l yze (and solubilize in TCA) 1 µmole ty r o s ine equiv a l ents/min from sol u b le ca s ein at pH 8.0 and 40°C].(g) Amyloglucosidase.—Keep re frig er ated.(1)Starch/glu c ose oxidase–peroxidase method.—2000–3300 Units/mL (1 unit en z yme ac t iv i ty is de f ined as the amount of en z yme re q uired to re lease1µmole glu c ose/min at pH 4.5 and 40°C). (2) PNPBM (p-nitrophenyl beta-maltosidase) method.—130–200 Units/mL (1 unit en z yme ac t iv i ty [PNP unit] is the amount of en z yme, which in the pres e nce of ex c ess lev e ls of beta-glucosidase, will re l ease 1 µmole p-nitrophenyl from p-nitrophenyl beta-maltosidase/min at 40°C).The only en z yme which has been found to be sig n if i c antly con tam i nated with in ter fer ing ac tiv i ties is amyloglucosidase. Thermostable al p ha-amylase and pro t e a se from com m er c ial sources have been found to be gen e r a lly free of in t er f er i ng en z ymes. Low lev e ls of beta-glucanase have been de t ected in pro t e a se prep a r a t ions, but at lev e ls well be l ow that which would in t er f ere with to tal di etary fi ber anal y sis.The ma jor con tam i nant in amyloglucosidase prep a r a t ion was shown to be an endo-cellulase and re s ulted in endo-depolymerization of mixed-linkage beta-glucan from bar l ey and oats, with re s ul t ant un d er e s t i m a t ion of this di etary fi ber com po nent.The con tam i na tion of amylogucosidase with endo-cellulase (beta-glucanase) can be eas ily de tected.Al t er n a t ively, there are kits con t ain i ng all 3 en z ymes (pre t ested) avail a ble from a num b er of com p a n ies.(h)So dium hy drox ide so lu tion.—0.275M. Dis s olve 11.00 g NaOH ACS in ca 700 mL H2O in 1 L vol u m et r ic flask. Di l ute to vol u me with H2O.(i) Hy d ro c hlo r ic acid so l u t ion.—0.325M. Di l ute stock so l u t ion of known ti t er, e.g., 325 mL 1M HCl, to 1 L with H2O.(j) Celite.—Acid-washed.© 2005 AOAC IN T ER N A T IONAL Ta b le 985.29. Test sam p les for en z yme pu r ityTest sam p leAc tiv itytestedTest por t ionweight, gEx pectedre cov ery,% Cit rus pec tin Pectinase0.195–100 Stractan (larch gum)Hemicellulase0.195–100 Wheat starch Am y lase 1.00–1Corn starch Am y lase 1.00–2Ca sein Pro te ase0.30–2β-Glucan (bar l ey gum)aβ-Glucanase0.195–100aSigma Chem i c al Co. or Megazyme In t er n a t ional Ire l and, Ltd.D.En zyme Pu rityTo en sure ab sence of un de sir able en zy matic ac tiv ity in en zymes used in this pro c e d ure, run ma t e r i a ls listed in Ta b le 985.29 through en t ire pro c e d ure each time lot of en z ymes is changed, or at max i m um in t er v al of 6 months to en s ure that en z ymes have not de g raded.E. Test Por t ion Prep a r a t ionDe t er m ine to t al di e tary fi b er on dried test sam p le. Ho m og e n ize test sam p le and dry over n ight in 70°C vac u um oven, cool in des i c c a t or, and dry-mill test sam p le to 0.3–0.5 mm mesh. If test sam p le can n ot be heated, freeze-dry be f ore mill i ng. If high fat con t ent (>10%) pre v ents proper mill i ng, defat with pe t ro l eum ether (3 times with 25 mL por t ions/g test sam p le) be f ore mill i ng. Re c ord loss of weight due to fat re m oval and make ap p ro p ri a te cor r ec t ion to fi n al % di e tary fi b er found in de t er m i n a t ion. Store dry-milled test sam p le in capped jar in des i c c a t or un t il anal y s is is car r ied out.F.De ter mi na tionRun blank through en t ire pro c e d ure along with test por t ions to mea s ure any con t ri b u t ion from re a gents to res i d ue.Weigh du p li c ate 1 g test por t ions, ac c u r ate to 0.1 mg, into 400 mL tall-form beak e rs. Test por t ion weights should not dif f er >20 mg. Add 50 mL pH 6.0 phos p hate buffer to each beaker. Check pH and ad j ust to pH 6.0 ± 0.2 if nec e s s ary. Add 0.1 mL Termamyl so l u t ion. Cover beaker with Al foil and place in boil i ng water bath 15 min. Shake gently at 5 min in t er v als. In c rease in c u b a t ion time when num b er of beak e rs in boil i ng water bath makes it dif f i c ult for beaker con tents to reach in ter nal tem per a ture of95°–100°C. Use ther mom e ter to in di cate that 15 min at 95°–100°C is at t ained. To t al of 30 min in water bath should be suf f i c ient.Cool so l u t ions to room tem p er a t ure. Ad j ust to pH 7.5 ± 0.2 by add i ng 10 mL 0.275M NaOH so l u t ion.Add 5 mg pro t e a se. (Pro t e a se sticks to spat u la, so it may be pref e r a b le to pre p are en z yme so l u t ion (50 mg in 1 mL phosphate buffer) and pipet 0.1 mL to each sam p le just be f ore use.Cover beaker with Al foil. In c u b ate 30 min at 60°C with con t in u o us ag i t a t ion. Cool. Add 10 mL 0.325M HCl so l u t ion. Mea s ure pH and dropwise add acid if nec e s s ary. Fi n al pH should be 4.0–4.6. Add 0.3 mL amyloglucosidase, cover with Al foil, and in c u b ate 30 min at 60°C with con tin u ous ag i ta tion.Add280mL 95% ethyl al c o h ol pre h eated to 60°C (mea s ure vol u me be f ore heat i ng). Let pre c ip i t ate form at room tem p er a t ure for 60 min. Weigh cru c i b le con t ain i ng Celite to near e st 0.1 mg, then wet and re d is t rib u te bed of Celite in cru c i b le by us i ng stream of 78% ethyl al c o h ol from wash bot t le. Ap p ly suc t ion to draw Celite onto fritted glass as even mat. Main t ain suc t ion and quan t i t a t ively trans f er pre cip i tate from en zyme di gest to cru ci ble.Wash res i d ue suc c es s ively with three 20 mL por t ions of 78% ethyl al c o h ol, two 10 mL por t ions of 95% ethyl al c o h ol, and two 10 mL por t ions of ac e t one. Gum may form with some prod u cts, trap p ing liq u id. If so, break sur f ace film with spat u la to im p rove fil t ra t ion. Time for fil t ra t ion and wash i ng will vary from 0.1 to 6 h, av e r a g i ng 0.5 h per sam p le. Long fil t ra t ion times can be avoided by care ful in ter mit tent suc tion through out fil tra tion.Dry cru c i b le con t ain i ng res i d ue over n ight in 70°C vac u um oven or 105°C air oven. Cool in des i c c a t or and weigh to near e st 0.1 mg. Sub t ract cru c i b le and Celite weight to de t er m ine weight of res i d ue. An a l yze res i d ue from 1 test por t ion of set of du p li c ates for pro t ein by 960.52 (see 12.1.07), us i ng N × 6.25 as con v er s ion fac t or, ex c ept in cases where N con t ent in pro t ein is known.In c in e r a te sec o nd test por t ion of du p li c ate 5 h at 525°C. Cool in des i c c a t or and weigh to near e st 0.1 mg. Sub t ract cru c i b le and Celite weight to de t er m ine ash.G.Cal cu la tionsDe t er m i n a t ion of blank:B = blank, mg = weight res i d ue − P B−A Bwhere weight res i d ue = av e r a ge of res i d ue weights (mg) for du p li c ate blank de t er m i n a t ions; and P B and A B = weights (mg) of pro t ein and ash, re s pec t ively, de t er m ined in first and sec o nd blank res i d ues.Cal c u l ate TDF as fol l ows:TDF, % =[(weight res i d ue −P−A−B) / weight test por t ion] × 100 where weight res i d ue = av e r a ge of weights (mg) for du p li c ate blank de t er m i n a t ions; and P and A = weights (mg) of pro t ein and ash, re s pec t ively, in first and sec o nd test por t ion res i d ues; and weight test por t ion = av e r a ge of 2 test por t ion weights (mg) taken.Ref er ences:JAOAC 68, 677(1985); 69, 259(1986).Re v ised: June 2003* Adopted as a Co d ex De f ining Method for gravimetry/en z y m atic di g est of to t al di e tary fi b re in spe c ial foods.© 2005 AOAC IN T ER N A T IONAL。
