AOAC 985.29 Total Dietary Fiber in Foods (1)pdf电子书下载.pdf

1、45 4 07 AOAC Of fi cial Method 985 29 To tal Di etary Fi ber in Foods Enzymatic Gravimetric Method First Ac tion 1985 Fi nal Ac tion 1986 AOAC AACC Method Co dex Adopted AOAC Method A Prin ci ple Du pli cate test por tions of dried foods fat extracted if con tain ing 10 fat are gelatinized with Term

2、amyl heat stable am y lase and then en zy mat i cally di gested with pro te ase and amyloglucosidase to re move pro tein and starch When an a lyz ing mixed di ets al ways ex tract fat prior to de ter min ing to tal di etary fi ber Four vol umes of ethyl al co hol are added to pre cip i tate sol u bl

3、e di etary fi ber To tal res i due is fil tered washed with 78 ethyl al co hol 95 ethyl al co hol and ac e tone Af ter dry ing res i due is weighed One du pli cate is an a lyzed for pro tein and other is in cin er ated at 525 C and ash is de ter mined To tal di etary fi ber weight res i due weight p

4、ro tein ash B Ap pa ra tus a Fritted cru ci ble Po ros ity No 2 Py rex No 32940 coarse ASTM 40 60 m or Corning No 36060 B chner fritted disk Py rex 60 mL ASTM 40 60 m Clean thor oughly heat 1 h at 525 C and soak and then rinse in H2O Add ca 0 5 g Celite to air dried cru ci bles and dry at 130 C to c

5、on stant weight 1 h Cool and store in des ic ca tor un til used b Vac uum source Vac uum pump or as pi ra tor equipped with in line dou ble vac uum flask to pre vent con tam i na tion in case of H2O backup c Vac uum oven 70 C Al ter na tively 105 C air oven can be used d Des ic ca tor e Muf fle fur

6、nace f Wa t e r b a t h s 1 B o i l i n g 2 Con stant tem per a ture Ad just able to 60 C with ei ther multistation shaker or multistation mag netic stir rer to pro vide con stant ag i ta tion of di ges tion flasks dur ing en zy matic hy dro ly sis g Beakers Tall form 400 or 600 mL h Bal ance An a l

7、yt i cal readability to 0 1 mg i pH me ter Stan dard ized with pH 7 and pH 4 buff ers C Re agents a 95 Eth a nol v v Technical grade b 78 Eth a nol Place 207 mL H2O into 1 L vol u met ric flask Di lute to vol ume with 95 ethyl al co hol Mix and di lute to vol ume again with 95 ethyl al co hol if nec

8、 es sary Mix One vol ume H2O mixed with 4 vol umes 95 ethyl al co hol will also give 78 ethyl al co hol fi nal con cen tra tion c Ac e tone d Phos phate buffer 0 08M pH 6 0 Dis solve 1 400 g so dium phos phate dibasic an hy drous Na2HPO4 or 1 753 g dihydrate and 9 68 g so dium phos phate monobasic m

9、onohydrate NaH2PO4 H2O or 10 94 g dihydrate in ca 700 mL H2O Di lute to 1 L with H2O Check pH with pH me ter e Al pha amylase heat sta ble Termamyl 1 Store in re frig er a tor Based on Nel son Somogyi re duc ing sugar with sol u ble starch as sub strate 10 000 1000 units mL 1 unit is de fined as the

10、 amount of en zyme re quired to re lease 1 mole re duc ing sugar equiv a lents min at pH 6 5 and 40 C 2 Based on Ceralpha method us ing p nitrophenyl maltosaccharide as sub strate in the pres ence of a thermostable al pha glucosidase 3000 300 Ceralpha units mL 1 unit of en zyme is re quired to re le

11、ase 1 mole 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 te ase unit is de fined as the amount of en zyme re quired to hy dro lyze and solubilize in TCA 1 mole ty ro sine equiv a lents min from sol u ble ca sein at pH 8 0 and 40 C 7 15

12、 units mg 1 unit will hy dro lyze ca sein to pro duce color equiv a lent to 1 0 mole ty ro sine min at pH 7 5 and 37 C Color by Folin Ciocalteau re agent 2 Azo casein as say 300 400 Units mL 1 unit endo peptidase ac tiv ity is de fined as the amount of en zyme re quired to hy dro lyze and solubilize

13、 in TCA 1 mole ty ro sine equiv a lents min from sol u ble ca sein at pH 8 0 and 40 C g Amyloglucosidase Keep re frig er ated 1 Starch glu cose oxidase peroxidase method 2000 3300 Units mL 1 unit en zyme ac tiv ity is de fined as the amount of en zyme re quired to re lease 1 mole glu cose min at pH

14、4 5 and 40 C 2 PNPBM p nitrophenyl beta maltosidase method 130 200 Units mL 1 unit en zyme ac tiv ity PNP unit is the amount of en zyme which in the pres ence of ex cess lev els of beta glucosidase will re lease 1 mole p nitrophenyl from p nitrophenyl beta maltosidase min at 40 C The only en zyme wh

15、ich has been found to be sig nif i cantly con tam i nated with in ter fer ing ac tiv i ties is amyloglucosidase Thermostable al pha amylase and pro te ase from com mer cial sources have been found to be gen er ally free of in ter fer ing en zymes Low lev els of beta glucanase have been de tected in

16、pro te ase prep a ra tions but at lev els well be low that which would in ter fere with to tal di etary fi ber anal y sis The ma jor con tam i nant in amyloglucosidase prep a ra tion was shown to be an endo cellulase and re sulted in endo depolymerization of mixed linkage beta glucan from bar ley an

17、d oats with re sul tant un der es ti ma tion 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 ter na tively there are kits con tain ing all 3 en zymes pre tested avail able from a num ber of com pa nies h So d

18、ium hy drox ide so lu tion 0 275M Dis solve 11 00 g NaOH ACS in ca 700 mL H2O in 1 L vol u met ric flask Di lute to vol ume with H2O i Hy dro chlo ric acid so lu tion 0 325M Di lute stock so lu tion of known ti ter e g 325 mL 1M HCl to 1 L with H2O j Celite Acid washed 2005 AOAC IN TER NA TIONAL Ta

19、ble 985 29 Test sam ples for en zyme pu rity Test sam ple Ac tiv ity tested Test por tion weight g Ex pected re cov ery Cit rus pec tinPectinase0 195 100 Stractan larch gum Hemicellulase0 195 100 Wheat starchAm y lase1 00 1 Corn starchAm y lase1 00 2 Ca seinPro te ase0 30 2 Glucan bar ley gum a Gluc

20、anase0 195 100 a Sigma Chem i cal Co or Megazyme In ter na tional Ire land Ltd D En zyme Pu rity To en sure ab sence of un de sir able en zy matic ac tiv ity in en zymes used in this pro ce dure run ma te ri als listed in Ta ble 985 29 through en tire pro ce dure each time lot of en zymes is changed

21、 or at max i mum in ter val of 6 months to en sure that en zymes have not de graded E Test Por tion Prep a ra tion De ter mine to tal di etary fi ber on dried test sam ple Ho mog e nize test sam ple and dry over night in 70 C vac uum oven cool in des ic ca tor and dry mill test sam ple to 0 3 0 5 mm

22、 mesh If test sam ple can not be heated freeze dry be fore mill ing If high fat con tent 10 pre vents proper mill ing defat with pe tro leum ether 3 times with 25 mL por tions g test sam ple be fore mill ing Re cord loss of weight due to fat re moval and make ap pro pri ate cor rec tion to fi nal di

23、 etary fi ber found in de ter mi na tion Store dry milled test sam ple in capped jar in des ic ca tor un til anal y sis is car ried out F De ter mi na tion Run blank through en tire pro ce dure along with test por tions to mea sure any con tri bu tion from re agents to res i due Weigh du pli cate 1

24、g test por tions ac cu rate to 0 1 mg into 400 mL tall form beak ers Test por tion weights should not dif fer 20 mg Add 50 mL pH 6 0 phos phate buffer to each beaker Check pH and ad just to pH 6 0 0 2 if nec es sary Add 0 1 mL Termamyl so lu tion Cover beaker with Al foil and place in boil ing water

25、 bath 15 min Shake gently at 5 min in ter vals In crease in cu ba tion time when num ber of beak ers in boil ing water bath makes it dif fi cult for beaker con tents to reach in ter nal tem per a ture of 95 100 C Use ther mom e ter to in di cate that 15 min at 95 100 C is at tained To tal of 30 min

26、in water bath should be suf fi cient Cool so lu tions to room tem per a ture Ad just to pH 7 5 0 2 by add ing 10 mL 0 275M NaOH so lu tion Add 5 mg pro te ase Pro te ase sticks to spat ula so it may be pref er a ble to pre pare en zyme so lu tion 50 mg in 1 mL phosphate buffer and pipet 0 1 mL to ea

27、ch sam ple just be fore use Cover beaker with Al foil In cu bate 30 min at 60 C with con tin u ous ag i ta tion Cool Add 10 mL 0 325M HCl so lu tion Mea sure pH and dropwise add acid if nec es sary Fi nal pH should be 4 0 4 6 Add 0 3 mL amyloglucosidase cover with Al foil and in cu bate 30 min at 60

28、 C with con tin u ous ag i ta tion Add 280 mL 95 ethyl al co hol pre heated to 60 C mea sure vol ume be fore heat ing Let pre cip i tate form at room tem per a ture for 60 min Weigh cru ci ble con tain ing Celite to near est 0 1 mg then wet and re dis trib ute bed of Celite in cru ci ble by us ing s

29、tream of 78 ethyl al co hol from wash bot tle Ap ply suc tion to draw Celite onto fritted glass as even mat Main tain suc tion and quan ti ta tively trans fer pre cip i tate from en zyme di gest to cru ci ble Wash res i due suc ces sively with three 20 mL por tions of 78 ethyl al co hol two 10 mL po

30、r tions of 95 ethyl al co hol and two 10 mL por tions of ac e tone Gum may form with some prod ucts trap ping liq uid If so break sur face film with spat ula to im prove fil tra tion Time for fil tra tion and wash ing will vary from 0 1 to 6 h av er ag ing 0 5 h per sam ple Long fil tra tion times c

31、an be avoided by care ful in ter mit tent suc tion through out fil tra tion Dry cru ci ble con tain ing res i due over night in 70 C vac uum oven or 105 C air oven Cool in des ic ca tor and weigh to near est 0 1 mg Sub tract cru ci ble and Celite weight to de ter mine weight of res i due An a lyze r

32、es i due from 1 test por tion of set of du pli cates for pro tein by 960 52 see 12 1 07 us ing N 6 25 as con ver sion fac tor ex cept in cases where N con tent in pro tein is known In cin er ate sec ond test por tion of du pli cate 5 h at 525 C Cool in des ic ca tor and weigh to near est 0 1 mg Sub

33、tract cru ci ble and Celite weight to de ter mine ash G Cal cu la tions De ter mi na tion of blank B blank mg weight res i due PB AB where weight res i due av er age of res i due weights mg for du pli cate blank de ter mi na tions and PB and AB weights mg of pro tein and ash re spec tively de ter mi

34、ned in first and sec ond blank res i dues Cal cu late TDF as fol lows TDF weight res i due P A B weight test por tion 100 where weight res i due av er age of weights mg for du pli cate blank de ter mi na tions and P and A weights mg of pro tein and ash re spec tively in first and sec ond test por tion res i dues and weight test por tion av er age of 2 test por tion weights mg taken Ref er ences JAOAC 68 677 1985 69 259 1986 Re vised June 2003 Adopted as a Co dex De fining Method for gravimetry en zy matic di gest of to tal di etary fi bre in spe cial foods 2005 AOAC IN TER NA TIONAL