酶法测定膳食纤维的推荐方法(AOAC
酶法测定膳食纤维的推荐方法(AOAC
酶法测定膳食纤维的推荐方法：试剂：1. 0.1M PBS, PH=0.6.2. 4M HCl ; 4M NaOH3. 95%乙醇，78%乙醇4. 丙酮酶：淀粉酶，蛋白酶，胰酶步骤：1. 湿样品需要均质并冻干，所有样品都需要粉碎至粒径0.3mm。
2. 当脂肪含量大于6-8%时或者需要适当粉碎时，需要在室温下用石油醚抽脂15min。
3. 称取1g样品，精确到0.1mg，转移至锥形瓶。
向其中加入25ml 0.1M的PBS，PH=6，充分悬浮样品。
4. 加入100ul 淀粉酶。
用膜盖住锥形瓶顶部，沸水浴保温15min，偶尔摇晃一下。
5. 室温下放凉，加入20ml蒸馏水，用HCL调至PH=1.5，用少量蒸馏水冲洗电极。
6. 加入100mg 胃蛋白酶，顶部盖膜，40℃保温并搅拌60min.7. 加入20ml蒸馏水，用NaoH调PH至6.8，少许蒸馏水冲洗电极。
8. 加入100ml 胰酶，顶部盖膜，40℃保温并搅拌60min.9. 用HCl调PH至4.5.10. 用干燥的称量过的G2坩埚（含0.5g硅藻土）作为辅助过滤设施。
用20m蒸馏水分两次冲洗。
A. 滤液残留（不溶性膳食纤维）：11. 用20ml 95%乙醇和20ml 丙酮分两次冲洗。
12. 105℃干燥至恒重，干燥器内冷却后称重（D1）。
13. 550℃灰化5h，干燥器内冷却后称重（I1）B．滤液（可溶性膳食纤维）14. 将滤液可冲洗水合并定容至100ml.15. 加入微热（60℃）的95%乙醇400ml，沉淀1h（时间可以缩短）.16. 用含有0.5g硅藻土的G2坩埚过滤。
17. 用20ml 78%乙醇、20ml 95%乙醇和20ml丙酮分别分两次冲洗。
18. 105℃烘至恒重，在干燥器内冷却后称重（D2）19. 550℃至少灰化5h，干燥器内冷却后称重（I2）空白：水溶性膳食纤维和不溶性膳食纤维空白值（B1和B2）的测定都是在没有添加样品的情况下进行。
膳食纤维测定方法
膳食纤维的测定方法【摘要】膳食纤维被称为人体的第七营养素，对维持人体健康具有重要作用。
膳食纤维通过发酵产物短链脂肪酸和对肠道菌群的调节作用从而影响肠道健康，本文对膳食纤维的测定方法进行了综述。
【关键词】膳食纤维；定义；测定膳食纤维已被确认为与传统的六大营养素并列的“第七营养素”，对维持人体健康具有重要的生理作用。
膳食纤维的理化特性概括起来是膨胀作用、持水能力、胶体形成、离子交换、改善胃肠微生物菌落和产生低热量等。
这些特性产生的生理作用如下：使人产生饱腹感并抑制进食，从而预防肥胖；润肠通便，防治肠道疾病和便秘；调控血清胆固醇，降血压，防治冠状动脉硬化，胆石症和预防心脑血管疾病；降血糖，防治糖尿病等。
目前，结肠癌、炎症性肠炎和其他结肠紊乱疾病已经严重影响身体健康。
膳食纤维为肠道微生物生长提供均衡的能量和营养，这是维持结肠生态系统平衡所必需的，另外，膳食纤维的发酵，特别是丁酸发酵，有利于结肠健康。
目前国内外业已研究开发的膳食纤维共有6大类约30余种，其中实际生产和应用的不超过10种。
一、膳食纤维膳食纤维（dietary fiber，df）被认为是食物中不被人体胃肠道消化酶水解，但能被肠道微生物消化的物质，特别是植物成分。
膳食纤维包括非淀粉多糖，如纤维素、半纤维素、树胶、果胶，以及木质素、抗性糊精和抗性淀粉。
二、膳食纤维的测定世界卫生组织建议的总膳食纤维摄入量下限为每人每天27.0克，上限为每人每天40.0克。
由此可见：膳食纤维检测结果的表示及产品标签标示等方面的问题应该作为膳食纤维研究中的又一个重要方面，而检测结果是由膳食纤维的检测方法和检测标准决定的，因此有必要建立统一的检测方法和标准。
df的不同测定方法因其测定原理不同结果差异较大。
自20世纪60年代初以来，分析化学家们建立起大量的检测方法，具有代表性的几种方法为非酶重量法、酶-重量和酶-化学法。
（1）非酶重量法。
非酶重量法又称粗纤维测定法，由einhof于1801-1809年建立。
检测膳食纤维的方法
检测膳食纤维的方法膳食纤维是指不能被人体消化吸收的多种碳水化合物，在人体内部没有被完全吸收利用，而是在消化道内发挥一系列重要生理功能的物质。
对于人体健康来说，膳食纤维具有重要的保健作用，能够降低血脂和血糖水平、促进肠道蠕动、预防便秘、降低结肠癌的发生率等。
为了能够准确地检测膳食纤维含量，提供科学的衡量指标，目前有一些常用的方法。
1. Gravimetric method（重量法）重量法是一种基本的膳食纤维分析方法，通过测定样品在经过一系列消化和提取过程后，残留物的质量来计算膳食纤维的含量。
首先，将样品经过酶解和洗涤等处理，去除可消化的部分，然后通过烘干使其失重，最后计算失重的质量即为膳食纤维的含量。
2. Chemical method（化学法）化学法是通过化学反应来测定膳食纤维的含量。
常用的化学方法有酚硫酸法、酶解法和高压液相色谱法等。
其中，酶解法是将样品暴露在特定的酶中，通过酶的作用降解多糖，然后通过化学分析方法确定被酶降解的物质的含量，从而计算膳食纤维的含量。
3. Enzymatic-gravimetric method（酶重法）酶重法结合了重量法和酶解法，通过测量提取液中的纤维残留物的质量以及可被酶解的非纤维物质的质量，从而计算出纤维的含量。
与传统的重量法相比，酶重法可以更加准确地测定纤维的含量。
4. Near Infrared Reflectance (NIR) Spectroscopy（近红外反射光谱法）近红外反射光谱法是一种无损检测方法，通过测量样品在近红外波段内的光谱反射，通过与已知含量的样品进行比对，从而确定膳食纤维的含量。
这种方法具有快速、无需样品处理的优点，但需要建立可靠的模型来实现准确的测量。
总结起来，目前常用的检测膳食纤维的方法有重量法、化学法、酶重法和近红外反射光谱法。
这些方法各有优势和局限性，需要根据实际需要选择适合的方法。
随着科学技术的发展，对膳食纤维的检测方法也将不断改进和完善，为人们提供更加准确和可靠的数据。
AOAC 985.29 食物中总膳食纤维 酶-重量法
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。
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AOAC婴幼儿食品营养素检测 国际标准研讨会
受国际配方食品咨询委员会委托，AOAC(国际官定分析检测协会) 计划在今后 两年半时间内，对婴幼儿配方食品及成人营养品中优先考虑的至少20种营养素制 定AOAC国际标准。为此，在2010年9月和11月，AOAC分别举行了两次SPIFAN会议 (Stakeholder Panel on Infant Formula and Adult Nutritional)，讨论了最 初的五个营养素检测方法，并指定了标准主持实验室和协同验证实验室。为推动 中国对婴幼儿食品营养检测国家标准的制修订能力建设，部分中国专家和企业代 表赴美国参加了工作会议。
分部是由AOAC总部批准成立的专门设在中国的分部组织， 旨在帮助来自分析化学、微生物及其他专业领域的中国会员更 多地了解AOAC；积极参加AOAC的各项活动；协助AOAC总部联系 中国会员；组织各项专业培训及教育活动等。
AOAC Official Methods SM
AOAC 逐步认可新的检测技术作为官方方法，如 LC-MS（液 质联用技术） 和 SPR(表面等离子共振技术)。 2007-至今，SPADA (PCR 检测微生物会议）
液相色谱-质谱联用仪：它结合了液相色谱仪有效分离热不稳性及高沸点 化合物的分离能力与质谱仪很强的组分鉴定能力。是一种分离分析复杂有 机混合物的有效手段。实现对复杂混合物更准确的定量和定性分析。而且 也简化了样品的前处理过程，使样品分析更简便。
质谱仪由以下几部分组成
数据控制和采集及供电系统 ┏━━━━┳━━━━━╋━━━━━━┓ 进样系统 离子源 质量分析器 检测接收器
于250 mL三角瓶中，固体试样需用约50 mL 45 ℃～50 ℃水使其溶解，加 入维生素D3内标1 mL(1 ug/mL) ，混合均匀。
膳食纤维 标准方法
膳食纤维标准方法
膳食纤维是指人体无法消化吸收的碳水化合物类物质。
膳食纤维对人体健康具有重要的作用，包括促进消化系统健康、调节血糖和胆固醇水平、预防便秘以及控制体重等。
为了准确测量食物中的膳食纤维含量，需要进行标准方法的测定。
目前，国际通用的膳食纤维含量测定方法有两种：AOAC （Association of Official Analytical Chemists）方法和ISO （International Organization for Standardization）方法。
1. AOAC方法：AOAC方法是美国官方方法，也是国际上最常用的方法。
根据AOAC 991.43或AOAC 985.29方法，首先将食物样品经过一系列处理，如酶解、水解等，获得可溶性和不可溶性纤维。
然后，借助酶解、滴定、重量等技术手段，可以得到总纤维、不可溶性纤维和可溶性纤维的含量。
2. ISO方法：ISO方法是由国际标准化组织制定的方法，与AOAC方法相似。
ISO 13904和ISO 15954方法是常用的ISO 方法。
这些方法主要利用酶解、水解、甲弹法等技术，将膳食纤维分为不可溶性纤维和可溶性纤维，并使用滴定、重量等手段进行测定。
无论使用AOAC方法还是ISO方法，都需要进行样品的预处理、酶解、滴定等步骤，以获得准确的膳食纤维含量。
这些方法在实验室条件下进行，需要仪器设备和专业操作人员进行操作。
需要注意的是，虽然AOAC和ISO方法都是国际通用的标准方法，但在具体的实验操作过程中，可能会存在一些差异，因此在测定过程中应当依据相应的方法详细操作，并遵循实验室的操作规程。
AOAC985.29食物中总膳食纤维酶-重量法
AOAC985.29食物中总膳食纤维酶-重量法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 unitis 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 pH6.5 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-gl ucosidase, 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 sefrom 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 notdif 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 in1 mL phosphate buffer) and pipet 0.1 mL to each sam p le just bef 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 t in 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 hol, 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 testpor 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。
总膳食纤维国标测定方法-符合AOAC等
总膳食纤维测定的介绍1、在α-淀粉酶的作用下，PH为6的磷酸盐缓冲溶液，95—100度下加热15分钟。
2、用蛋白酶在PH为7.5时60度培养30分钟。
3、用淀粉葡(萄)糖苷酶在PH为4.0---4.6下60度培养30分钟。
4、4体积的95%的乙醇沉淀。
5、过滤。
6、用78%和95%的乙醇和丙酮清洗沉淀物。
7、烘干称重。
8、干样可以拿去做凯氏定氮，也可以在525度的马弗炉里灰份5个小时，然后去称重。
不溶的膳食纤维的定义为进行烘干前用乙醇进行清洗并用温水洗涤后残留物。
总膳食纤维（TDF）—不溶膳食纤维= 可溶膳食纤维（SDF）标准酶法测定食品和饲料中的总膳食纤维量1、研磨分级样品2、在105度的烘箱烘干并恒重，在干燥箱中冷却到室温。
3、如果样品脂肪含量高于10%，需要用石油醚进行脱脂，在最终结果中再进行校正。
4、称出0.5—1克的样品，并转移到400毫升的烧杯中。
5、用α-淀粉酶在50毫升的PH为6的磷酸盐缓冲溶液中培养15分钟，培养温度为95—100度，温度可以用温度计控制。
6、冷却到室温，并用0.275 N 浓度的氢氧化钠溶液调节PH到7.5。
7、将烧杯和样品一起转移到磁力搅拌培养器中（GDE）。
8、在搅拌的情况下，加入蛋白酶在60度的情况下培养30分钟。
9、冷却到室温，用0.325的盐酸调节PH值为4.0—4.6。
10、在搅拌的情况下，加淀粉葡(萄)糖苷酶，在60度时培养30分钟。
11、通过加4体积的95%的乙醇沉淀可溶性膳食纤维，并且在室温下沉淀大约1个小时。
12、称量已经添加了0.5克的硅藻土(作为助滤剂)玻璃坩埚.13、将坩埚放在CSF6 (或者FIWE6)上,倒入上述操作的沉淀物,并用V ACUUM进行吸液排空,用78%的乙醇溶液进行洗涤转移沉淀物。
14、用20毫升的78%的乙醇溶液洗涤玻璃坩埚中的沉淀物两次，再用10毫升95%的乙醇溶液洗涤两次，10毫升的丙酮溶液洗涤两次并排除废液。
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酶法测定膳食纤维的推荐方法：
试剂：1. 0.1M PBS, PH=0.6.
2. 4M HCl ; 4M NaOH
3. 95%乙醇，78%乙醇
4. 丙酮
酶：淀粉酶，蛋白酶，胰酶
步骤：1. 湿样品需要均质并冻干，所有样品都需要粉碎至粒径0.3mm。
2. 当脂肪含量大于6-8%时或者需要适当粉碎时，需要在室温下用石油醚抽脂15min。
3. 称取1g样品，精确到0.1mg，转移至锥形瓶。
向其中加入25ml 0.1M的PBS，PH=6，充分悬浮样品。
4. 加入100ul 淀粉酶。
用膜盖住锥形瓶顶部，沸水浴保温15min，偶尔摇晃一下。
5. 室温下放凉，加入20ml蒸馏水，用HCL调至PH=1.5，用少量蒸馏水冲洗电极。
6. 加入100mg 胃蛋白酶，顶部盖膜，40℃保温并搅拌60min.
7. 加入20ml蒸馏水，用NaoH调PH至6.8，少许蒸馏水冲洗电极。
8. 加入100ml 胰酶，顶部盖膜，40℃保温并搅拌60min.
9. 用HCl调PH至4.5.
10. 用干燥的称量过的G2坩埚（含0.5g硅藻土）作为辅助过滤设施。
用20m蒸馏水分两次冲洗。
A. 滤液残留（不溶性膳食纤维）：
11. 用20ml 95%乙醇和20ml 丙酮分两次冲洗。
12. 105℃干燥至恒重，干燥器内冷却后称重（D1）。
13. 550℃灰化5h，干燥器内冷却后称重（I1）
B．滤液（可溶性膳食纤维）
14. 将滤液可冲洗水合并定容至100ml.
15. 加入微热（60℃）的95%乙醇400ml，沉淀1h（时间可以缩短）.
16. 用含有0.5g硅藻土的G2坩埚过滤。
17. 用20ml 78%乙醇、20ml 95%乙醇和20ml丙酮分别分两次冲洗。
18. 105℃烘至恒重，在干燥器内冷却后称重（D2）
19. 550℃至少灰化5h，干燥器内冷却后称重（I2）
空白：水溶性膳食纤维和不溶性膳食纤维空白值（B1和B2）的测定都是在没有添加样品的情况下进行。
使用新的一批酶时，应该不定时检查空白值。
计算：W=样品质量（g）。
D=干燥后的质量（g）。
I=灰化后的质量（g）。
B=无灰空白质量（g）
不溶性膳食纤维=D1-I1-B1。