By definition, resistant starch (RS) is that portion of the

Transcription

1 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, FOOD COMPOSITION AND ADDITIVES Mesurement of Resistnt Strch BARRY V. MCCLEARY nd DYMPNA A. MONAGHAN Megzyme Interntionl Irelnd Ltd., Bry Business Prk, Bry, County Wicklow, Irelnd A robust nd relible method ws developed to mesure resistnt strch (RS), i.e., strch tht enters the lrge intestine. In vivo conditions were reflected s much s possible while user-friendly formt ws mintined. Prmeters investigted included -mylse concentrtion, ph of incubtion, mltose inhibition of -mylse, the need for myloglucosidse inclusion, the effect of shking nd stirring on determined vlues, nd problems in recovering nd nlyzing the RS-contining pellet. The RS vlues obtined were in good greement with published in vivo dt. An interlbortory evlution of the method hs been completed (First Action Method ). By definition, resistnt strch (RS) is tht portion of the strch tht is not broken down by humn enzymes in the smll intestine. It enters the lrge intestine where it is prtilly or wholly fermented. RS is considered by mny to be prt of dietry fiber. The presence of strch frction resistnt to enzymic hydrolysis ws first recognized by Englyst et l. in 1982 during their reserch on the mesurement of nonstrch polyscchrides (1). This work ws extended by Berry (2) who developed procedure for the mesurement of RS incorporting the α-mylse/pullulnse tretment used by Englyst et l. (1), but omitting the initil heting step t 100 C, to more closely mimic physiologicl conditions. Under these conditions, the mesured RS contents of smples were much higher. This ws confirmed by Englyst nd Cummings (3 5) through studies with helthy ileostomy subjects. They showed tht the strch mesured by their method represented only portion of the strch tht cn resist digestion in the humn smll intestine. In 1992, Englyst et l. (6) divided resistnt strches into 3 clsses: RS 1 (physiclly trpped strch s found in corsely ground or chewed cerels, legumes, nd grins); RS 2 (RS grnules or nongeltinized strch grnules which re highly resistnt to digestion by α-mylse until geltinized, e.g., uncooked potto, green bnn, nd high mylose strch); nd RS 3 (retrogrded strch polymers, minly mylose, which re produced when strch is cooled fter geltiniztion). They developed method for the mesurement of redily digested strch (RDS), slowly digested strch (SDS), nd RS, nd the 3 RS frctions (6). In this method, RS Received July 24, Accepted by JL October 15, Corresponding uthor s e-mil: brry@megzyme.com. is clculted by subtrcting the sum of RDS plus SDS from totl strch. Although the method cn yield useful informtion, it is very lborious nd gives poor reproducibility without extensive trining of the nlyst (7). Furthermore, ccurcy is severely hmpered by the fct tht, with smples contining high levels of strch with low RS content, one lrge nlyticl vlue is subtrcted from nother lrge vlue; in fct, the errors in the mesurement my be s lrge s the RS vlue, for exmple, mterils with pproximtely 70% strch nd 2% RS. By the erly 1990s, the physiologicl significnce of RS ws fully relized. Severl new nd modified methods for its mesurement were developed during the Europen reserch progrm EURESTA (6, 8). The Chmp (8) method ws bsed on modifictions to the method of Berry (2), nd gve direct mesurement of RS. Bsiclly, smple size ws incresed from 10 to 100 mg; the smple ws digested with pncretic α-mylse only, tht is, not pncretic α-mylse plus pullulnse, s used by Englyst et l. (1) nd Berry (2), nd incubtions were performed t ph 6.9 [ph 5.2 ws used by Englyst et l. (1) nd Berry (2)]. RS determintions were performed directly on the pellet. Muir nd O De (9) developed procedure in which smples were chewed, treted with pepsin, nd then with mixture of pncretic α-mylse nd myloglucosidse (AMG) in shking wter bth t ph 5.0 nd 37 C for 15 h. The residul pellet (contining RS) ws recovered by centrifugtion, wshed with cette buffer by centrifugtion, nd the RS ws digested by combintion of het, dimethyl sulfoxide DMSO, nd thermostble α-mylse tretments. More recently, these methods hve been modified by Fisnt et l. (10), Goni et l. (11), Akerberg et l. (12), nd Chmp et l. (7). These modifictions included chnges in enzyme concentrtions, types of enzymes used (ll used pncretic α-mylse, but pullulnse ws removed nd, in some cses, replced by myloglucosidse), smple pretretment (chewing), ph of incubtion, nd ddition (or not) of ethnol fter the α-mylse incubtion step. All of these modifictions will hve some effect on the determined level of RS in smple. In developing the current modified method for the mesurement of RS, our im ws to provide robust nd relible method tht (s much s fesible) reflects in vivo conditions nd yields vlues tht re physiologiclly significnt (i.e., mesure the strch tht enters the lrge intestine). To do this, we studied the effect of concentrtion of pncretic α-mylse, the ph of incubtion, the importnce of mltose inhibi-

2 666 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Figure 1. Stbility of pncretic -mylse on storge t ph 5.2, 6.0, nd 6.9 t 4 nd 37 C for up to 20 h. Figure 2. The effect of concentrtion of pncretic -mylse nd incubtion time on determined RS vlue of regulr mize strch (RMS) nd high mylose mize strch (HAMS) under stirring conditions t ph 6.9 nd 37 C for up to 24 h. tion of α-mylse, the need for myloglucosidse inclusion, the effect of shking nd stirring on determined vlues, nd problems in recovering nd nlyzing the RS-contining pellet. Experimentl Mterils () Chemicls nd enzymes. DL-mleic cid (M-0375), BSA (A-2153), mltose (M-9171), sodium zide (S-8032), crude pncretic α-mylse (A-3176; EC ), crystlline pncretic α-mylse (A-2643), nd pepsin (P-6887; EC ) were obtined from Sigm Chemicl Co. (Sigm-Aldrich Irelnd Ltd., Dublin, Irelnd). Acetic cid (glcil) GR, sodium hydroxide nd clcium chloride (CCl 2 2H 2 O) were from Merck (Drmstdt, Germny). Amyloglucoside (E-AMGDF; EC ), fungl α-mylse (E-ANAAM; EC ), nd mltse (E-MALTS; EC ) were from Megzyme (Megzyme Interntionl Irelnd Ltd., County Wicklow, Irelnd). Industril methylted spirits (IMS) were obtined from Lennox Lb Supply (Dublin, Irelnd). This is mixture of ethnol nd methnol (95 + 5%) nd some butnol. In this study, IMS cn be replced by lbortory grde (95 99%) ethnol. (b) Substrtes nd regents. Cerlph; α-mylse ssy kit (K-CERA), myloglucosidse ssy regent (R-AMGR3), Amylzyme tblets (T-AMZ200), nd glucose ssy kit (K-GLUC; bsed on glucose oxidse/peroxidse/4-mino ntipyrine) were from Megzyme. p-nitrophenyl-αglucopyrnoside (N-1377) ws from Sigm. (c) Buffers. Buffer A. 0.1M sodium mlete (ph 6.0) contining clcium chloride (1mM) nd sodium zide (0.02%). Buffer B. 0.1M sodium mlete buffer (ph 6.9) contining clcium chloride (1mM) nd sodium zide (0.02%). Buffer C. 0.1M sodium cette (ph 4.5) contining clcium chloride (1mM). Buffer D. 0.1M sodium cette (ph 5.2) contining clcium chloride (1mM). Buffer E. 1.2M sodium cette (ph 3.8). (d) Strch smples. Regulr Mize Strch (Lot 60401; RMS), High Amylose Mize Strch Lot (Lot 60107; HAMS), Hi Mize 1043 (Btch No ), nd Hi Mize (CO-347) were from Penford Austrlsi (New South Wles, Austrli). Hylon VII (Ref. 98GH8401), Novelose 330 (Ref. AH17529), Figure 3. The effect of concentrtion of myloglucosidse (AMG) on the mesured level of RS, free glucose, nd free glucose plus soluble strch frgments on hydrolysis of () RMS nd (b) HAMS by pncretic -mylse under stirring conditions t ph 6.0 nd 37 C for 16 h.

3 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble 1. The effect of shking speed on the determined RS vlue for strch smples (Assy Formt D) Resistnt strch, % (men ± SD) Smple 140 strokes/min 200 strokes/min 260 strokes/min HAMS 47.6 ± ± ± 0.80 Hylon VII 57.1 ± ± ± 1.4 Potto strch 79.6 ± ± ± 1.4 Hi Mize ± ± ± 0.60 CrystLen 44.2 ± ± ± 0.37 All nlyses were performed in triplicte. Tble 2. Determined RS vlues for smples using Assy Formts A D Men resistnt strch, % (w/w) Smple Formt A, ph 6.9, no AMG, stirring Formt B, ph 6.0, 12 U AMG, stirring Formt C, ph 6.9, no AMG, shking Formt D, ph 6.0, 12 U AMG, shking Regulr mize strch (60401) Ntive potto strch (AVEBE) HAMS (60107) Hi Mize (CO-347) Hylon VII (Ref. 98GH8401) Novelose 240 (Ref. 96LF10063) Novelose 330 (Ref. AH17529) Hi Mize 1043 (0216 TDF 64.5%) CrystLen (Opt; ) Potto mylose (Sigm T2, A-9262) ActiStr CrystLen HT nneled 51.7 Btchelors kidney bens (wet) Btchelors kidney bens (freeze-dried powder) Btchelors butter bens (wet) Btchelors butter bens (freeze-dried powder) Rob Roy flgeolet bens (wet) Rob Roy flgeolet bens (freeze-dried powder) Bked bens (wet) Red lentils (milled) Hricot bens (milled) Pinto bens (milled) Rosted buckwhet (milled) Durum whet pst (milled) Green bnn (freeze-dried powder) Potto (cooked, cooled, nd freeze-dried) Corn flkes (milled) All vlues re the men of duplicte nlyses.

4 668 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Figure 4. The effect of concentrtion of AMG on the mesured level of RS, free glucose, nd free glucose plus soluble strch frgments on hydrolysis of () RMS nd (b) HAMS by pncretic -mylse under shking conditions t ph 6.0 nd 37 C for 16 h. Novelose 240 (Ref. 96LF10063), nd Flogel 60 (Ref. 971GT746) were from Ntionl Strch nd Chemicl Co. (Bridgewter, NJ). CrystLen nd CrystLen HT (nneled) were from Opt Food Ingredients Inc. (Bedford, MA), nd ntive potto strch ws from Avebe (Foxhol, The Netherlnds). ActiStr (enzyme modified tpioc/cssv strch, U.S. Ptent ) ws from Cerestr (Vilvoorde, Belgium), nd potto mylose (A-9262) ws from Sigm. (e) Dry food nd ben smples. These were obtined nd prepred, s follows: dried red lentils nd dried rosted buckwhet (Rinbow Wholefoods, Kylemore Industril Estte, Dublin, Irelnd); dried orgnic penne pst from durum whet (Meridin Foods, Denbighshire, Wles); dried bens (The Helth Store, Nottinghm, UK); nd corn flkes (Kellogg s, Bttle Creek, MI). All mterils were milled to pss 1.0 mm screen, using Fritsch pulverisette 14 mill (Fritsch, Idr Oberstein, Germny). Moisture contents were determined for ll smples by AOAC Method (13). (f) Wet food nd ben smples. These were obtined nd prepred, s follows: cnned butter bens nd bked bens (Btchelors Ltd., Cbr West, Dublin, Irelnd); cnned red kidney bens (Chivers Irelnd Ltd., Dublin, Irelnd); cnned flgeolot bens (Boyne Vlley Foods, Droghed, Irelnd); pottoes nd green bnn (locl fruit nd vegetble store). Cnned bens were recovered on food striner nd wshed with wter. They were then minced with stndrd mechnicl met mincer (Porkert, Czech Republic) with 4.5 mm screen. Prt of this mteril ws nlyzed directly, nd the reminder ws lyophilized before nlysis. The lyophilized mteril ws milled to pss 1.0 mm screen, using Fritsch pulverisette 14 mill before nlysis. The moisture content of ll smples ws determined by AOAC Method (13). Totl strch contents of smples Figure 5. A comprison of the effect of () stirring nd (b) shking on the time course of hydrolysis of HAMS nd RMS by pncretic -mylse (10 mg/ml) in the presence of AMG (12 U/ssy; ph 6.0) or bsence of AMG t ph 6.9.

5 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble 3. Effect of dded mltose on determined RS vlues for smples nlyzed by Assy Formt A Smple Added mltose, mg were determined using Megzyme Totl Strch Assy Kit (AOAC Method ; 13). Reducing sugr levels in enzyme ssys were mesured with the Somogyi procedure (14). Glucose ws routinely mesured with the glucose oxidse peroxidse 4-minontipyrine procedure. RS vlues re presented either s percentge (w/w) of dry smple weight or s percentge of totl strch content of the smple. Equipment nd Apprtus () Stirrer. Vriomg Telemodul 40S (H+P Lbortechnik GmbH, Munich, Germny); used for stirring experiments (Assy Formts A nd B). Tubes were immersed in wter bth t 37 C nd stirred. An electromgnetic stirring block ws immersed in the wter bth with 16 stirring points; setting 120, 260 (stndrd setting), or 500 rpm. (b) Shking wter bth. Grnt OLS 200 (Grnt Instruments Cmbridge Ltd., Shepreth, UK); used for shking experiments (Assy Formts C nd D). Screw-cp tubes (Corning, Inc., Acton, MA; culture tubes, mm) were secured horizontlly in wter bth nd incubted t 37 C; shke speed 200 strokes/min, 35 mm stroke length. (c) Spectrophotometer. Used to determine bsorbnce vlues; Hitchi U-1500 (Hitchi Ltd., Tokyo, Jpn). (d) Mill. Fritsch Pulverisette 14. Smples were milled to pss 1.0 mm screen. (e) Stndrd met mincer. Used to mince ll fresh smples (Porkert). Smples were nlyzed directly or following lyophiliztion. Mesurement of Enzyme Activities Determined RS, % (w/w) Hi Mize CrystLen Potto mylose Novelose Potto strch Regulr mize strch All nlyses were performed in duplicte. α-amylse ws routinely ssyed using Cerlph regent contining end-blocked p-nitrophenyl mltoheptoside in the presence of excess thermostble α-glucosidse (15; AOAC Method ). Unless otherwise stted, ll ctivities reported here re in Cerlph Units (CU). The crude pncretic α-mylse used hd n ctivity of 3.0 CU/mg. α-amylse ctivity ws lso mesured on soluble strch by modifiction (15) of the Nelson-Somogyi reducing sugr procedure (14). AMG ssys were routinely performed using AMG ssy regent (16; p-nitrophenyl β-mltoside in the presence of excess quntities of β-glucosidse). Activity ws lso mesured on soluble strch using the Nelson-Somogyi reducing sugr method (14) s previously described (15). One unit is the mount of enzyme required to relese 1 µmol glucose per min from soluble strch under the defined ssy conditions (ph 4.5, 40 C). Unless otherwise stted, ll ctivities re reported here s units (U)/mL on soluble strch. The ph ctivity of α-mylse ws determined by suitbly diluting pncretic α-mylse in pproprite buffer [ph 5.2 (buffer D), 6.0 (buffer A), or 6.9 (buffer B)] nd performing the ssy with Cerlph regent. The ph stbility ws determined by preincubting enzyme t 10-fold the required concentrtion for ssy t 4 or 37 C for up to 24 h. The enzyme ws then diluted 10-fold in buffer B nd ssyed with Cerlph regent. The ph ctivity of AMG ws determined by suitbly diluting the enzyme in buffers A D (ph ) nd ssying ctivity using AMG ssy regent (16). The ph stbility ws determined by preincubting enzyme t 10-fold the concentrtion required for ssy t 4 or 37 C for up to 24 h. The enzyme ws then diluted 10-fold in buffer C nd ssyed using AMG ssy regent. Protese ctivity ws ssyed with Azo-Csein (Megzyme Ct. No. S-AZCAS), nd ctivity ws determined by reference to stndrd curve (15, Megzyme Technicl Booklet AZCAS 2/99). Determintion of Resistnt Strch, Solubilized Strch, nd Totl Strch () Stirring method (Assy Formt B). Tubes ( mm) contining smple (100 mg, weighed ccurtely) plus mgnetic stirrer br (5 15 mm) were treted with 4.0 ml pncretic α-mylse (10 mg/ml; 30 CU/mL) in buffer A plus 0.5 ml AMG (24 U/mL) in buffer A, nd the tubes were incubted t 37 C with continuous gentle stirring (260 rev/min) on Vriomg mgnetic stirrer. Tubes were removed fter 16 h, treted with 4.5 ml IMS (99%), nd stirred vigorously. Stir brs were removed using n externl mgnet, Tble 4. Effect of dded mltse enzyme on the determined level of RS for RMS nd HAMS (Assy Formt A) RS, % (w/w) Smple No mltse dded With mltse b RMS HAMS (60107) b All nlyses were performed in duplicte. Mltse dded t level of 500 units/test.

6 670 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Tble 5. Effect of myloglucosidse (AMG) nd mltse on the determined level of RS in HAMS using Assy Formt C Pncretic α-mylse AMG or mltse RS, % (w/w) Pure (Sigm Ct. No. A-2643) Neither 58.2/59.6 Mltse (500 U) 47.6/49.4 AMG (12 U) 39.0/41.1 Crude (pncretin) AMG (12 U) 42.2/43.4 Routinely used s the source of α-mylse in the RS methods described here. nd the tubes were centrifuged t 3000 rpm (c 1000 g) for 10 min. Superntnts were crefully decnted nd the pellets were suspended in 2 ml 50% IMS with vigorous mixing on Vortex mixer. Another 6 ml 50% IMS ws dded with mixing, nd the tubes were gin centrifuged t 3000 rpm for 10 min. This suspension nd centrifugtion step ws repeted once more. The superntnts were crefully decnted nd the tubes were inverted on bsorbent pper to drin excess liquid. All tubes were covered with Prfilm nd stored t 4 C until redy for nlysis of RS content. This bsic formt ws modified to include vritions in (1) concentrtion of pncretic α-mylse (5 20 mg/ml); (2) incubtion time (1 30 h); (3) stirring speed (120, 260, nd 500 rpm); (4) incubtions in buffer B (ph 6.9) with exclusion of AMG, nd replcement by n equl volume of buffer B (Assy Formt A); nd, (5) with wet smples (e.g., minced bens), the smple weight incresed to 500 mg. (b) Shking method (Assy Formt D). Screw-cp tubes (Corning culture tubes, mm) contining smple ( mg, weighed ccurtely) were treted with 4.0 ml pncretic α-mylse (10 mg/ml) contining AMG (3 U/mL) in buffer A (ph 6.0), tightly cpped, mixed on Vortex mixer nd incubted t 37 C with continuous shking (200 strokes/min) in Grnt shking wter bth OLS 200 for 16 h. (Note: For liner motion, setting of 100 on the wter bth is equivlent to 200 strokes/min; 100 forwrd nd 100 reverse.) Tube cps were removed nd contents were treted with 4.0 ml IMS (99%) with vigorous mixing. (It is essentil tht ll smple ttched to the side of the tube is dislodged in these stirring nd wshing steps.) Tubes were centrifuged t 3000 rpm (c 1000 g) for 10 min with cps removed. Superntnts were crefully decnted nd the pellets were suspended in 2 ml 50% IMS with vigorous mixing on Vortex mixer. Another 6 ml 50% IMS ws dded with mixing nd the tubes were gin centrifuged t 3000 rpm for 10 min. This suspension nd centrifugtion step ws repeted once more. Superntnts were crefully decnted nd tubes were inverted on bsorbent pper to drin excess liquid. All tubes were recpped nd stored t 4 C until redy for nlysis of RS content. This bsic formt ws modified to include vritions in (1) concentrtion of pncretic α-mylse (5 20 mg/ml); (2) incubtion time (1 30 h); (3) shking speed (140, 200, nd 260 strokes/min); (4) incubtions in buffer B (ph 6.9) contining pncretic α-mylse (5 20 mg/ml) but no AMG (Assy Formt C); nd (5) with wet smples (e.g., minced bens), smple weight incresed to 500 mg. When pepsin pretretment step ws included, smples were treted with 2 ml 0.2M KCl/HCl buffer (ph 1.5) contining pepsin (2 mg/ml) t 37 C for 1 h. The smple ws then treted with 4 ml buffer A contining pncretic α-mylse (10 mg/ml) nd AMG (3 U/mL) s per the stndrd formt. The rection ws terminted with 6 ml IMS. (c) Mesurement of RS. A mgnetic stirrer br (5 15 mm) ws dded to ech tube nd the tubes were plced in test tube rck in n ice-wter bth over mgnetic stirrer. With the stirrer turned on, 2 ml 2M KOH ws dded to ech tube nd the slurry ws stirred for 20 min. Ech tube (one t time) ws treted with 8 ml 1.2M sodium cette buffer (ph 3.8; buffer E) nd then immeditely with 0.1 ml AMG (3200 U/mL) with vigorous stirring on Vortex mixer. After ddition of the cette buffer, the ph should be c The tubes were then plced in wter bth t 50 C for 30 min. If smples contined >10% RS, the solution ws quntittively trnsferred to 100 ml volumetric flsk (using wter wsh bottle) nd diluted to volume with wter, nd n liquot ws centrifuged t 3000 rpm for 10 min. If the RS content ws <10%, tubes were directly centrifuged t 3000 rpm for 10 min (no dilution). For such smples, the finl volume in the tube ws 10.3 ml; however, this volume will vry depending on the smple being nlyzed. Aliquots (0.1 ml, in duplicte) of either diluted or undiluted superntnts were treted with 3.0 ml glucose oxidse/peroxidse (GOPOD) regent nd incubted t 50 C for 20 min. Regent blnk solutions contined 0.1 ml 0.1M sodium cette buffer, ph 4.5 (buffer C) nd 3.0 ml GOPOD regent. Glucose stndrds (prepred in qudruplicte) contined 0.1 ml glucose (1 mg/ml) nd 3.0 ml GOPOD regent. After incubtion t 50 C for 20 min, the bsorbnce of ech solution ws mesured t 510 nm ginst the regent blnk. (d) Mesurement of solubilized strch. Totl strch content of smples ws routinely determined using AOAC Method (13). However, it ws lso determined s the sum of RS nd the strch tht is solubilized nd hydrolyzed to glucose in Assy Formts B nd D. The quntity of solubilized strch ws determined by pooling the superntnts (originl nd wshes) obtined by centrifugtion, diluting to 100 ml with wter, nd nlyzing for glucose on 0.1 ml liquots, s bove. For determintion of solubilized strch in mteril other thn pure strch smples, the smple must first be prewshed (twice) with 8 ml queous IMS (80%, v/v) before the pncretic α-mylse/amg tretment step to remove glucose, sucrose, nd other sugrs. The smple is suspended in queous IMS t room temperture, stirred on Vortex mixer, nd recovered by centrifugtion t 3000 rpm for 10 min. After the second centrifugtion, ll free liquid is crefully removed, nd the smple is then subjected to tretment by pncretic α-mylse/amg, s per the stndrd RS ssy formts.

7 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble 6. Comprison of RS vlues obtined using severl in vitro nlyticl methods nd in vivo results RS (in vitro method/results) Source of strch Englyst Fisnt Chmp McClery (Formt D) Goni b RS (in vivo results) Potto strch (ntive) Amylomize strch (ntive) Amylomize strch (retrogrded) b 30.1 Ben flkes c Corn flkes c Cnned bens ActiStr d 63 d 57 d d 59 d Vlues re presented s percentge of totl strch content of smple. All dt except tht of McClery (Formt D), Goni et l. (11), nd vlues for ActiStr re from Chmp et l. (18). In vivo results were obtined using either ileostomy ptients or the incubtion technique. b From Goni et l. (11). c From Goni et l. (11), clculting RS s percentge of totl strch, ssuming strch content for ben flkes of 40%, nd for corn flkes of 70% (bsed on in-house results for similr mterils). d Results kindly provided by Bernd Kettlitz, Cerestr (Vilvoorde, Belgium) except for vlues by McClery (Formt D), which were produced in-house. The Englyst dt were produced by Englyst Crbohydrte Services, Chmp dt t INRA (Nntes, Frnce) nd Goni dt t Cerestr (Vilvoorde, Belgium). (e) Clcultions. Clculte RS, solubilized strch, nd totl strch content (%, on dry weight bsis) in test smples, s follows: RS, g/100 g smple (smples contining > 10% RS): = E F 100/0.1 1/ /W 162/180 = E F/W 90 RS, g/100 g smple (smples contining < 10% RS): = E F 10.3/0.1 1/ /W 162/180 = E F/W 9.27 Solubilized strch, g/100 g smple: = E F 100/0.1 1/ /W 162/180 = E F/W 90 Totl strch = RS + solubilized strch where E = bsorbnce (rection) red ginst the regent blnk; F = conversion from bsorbnce to microgrms = 100 (µg glucose)/bsorbnce of 100 µg glucose; 100/0.1 = volume correction (0.1 ml tken from 100 ml); 1/1000 = conversion from microgrms to milligrms; W = dry weight of smple nlyzed [= s is weight (100-moisture content)/100]; 100/W = fctor to present strch s percentge of smple weight; 162/180 = fctor to convert from free glucose, s determined, to nhydroglucose s occurs in strch; 10.3/0.1 = volume correction (0.1 ml tken from 10.3 ml) for smples contining 0 10% RS where the incubtion solution is not diluted nd the finl volume is 10.3 ml. Results nd Discussion Numerous methods hve been developed for the in vitro mesurement of RS. In mny cses, but not ll, there hs been n ttempt to relte the in vitro results to those obtined from in vivo studies on humn or nonhumn subjects. In the development of these procedures, severl key prmeters hve been considered, such s smple preprtion (milling, mincing, or chewing); smple pretretment (pepsin pretretment t ph 2.0 to simulte stomch conditions); incubtion conditions (stirring/shking, ph, temperture, time, nd enzyme mixtures). In the current study, we criticlly evluted most of the bove prmeters nd estimted their effect on the determined level of RS in smples. Much of the preliminry work ws performed on 2 smples: high mylose mize strch (HAMS) nd regulr mize strch (RMS). Prototype methods were then pplied to rnge of commercilly vilble RS smples nd food mterils contining RS. The ultimte im ws to develop robust, user-friendly method tht will yield results in line with those obtined from in vivo studies. Mny methods developed for the mesurement of RS re bsed on incubtion of smples with pncretic α-mylse t ph From physiologicl point of view, the resons for using such low ph re not cler. The ph in the ileum is bout 7.0, which lso is the optiml ph for ctivity of pncretic α-mylse. Our studies show tht the reltive ctivity of pncretic α-mylse t ph 6.9, 6.0, nd 5.2 is 100, 77, nd 8.3%, respectively. Of even greter relevnce is the stbility of this enzyme t these ph vlues. As shown in Figure 1, pncretic α-mylse t 37 C is quite unstble t ph 5.2, with >95% inctivtion within 1 h. At ph 6.9 nd 6.0, the enzyme is much more stble, with 40% inctivtion in 6 h t ph 6.9 (37 C) nd 78% t ph 6.0. Even t 4 C, the enzyme is quite

8 672 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 unstble t ph 5.2. In methods where ph of ws used, this ws obviously done to fcilitte the ction of AMG, which hs n optiml ph for ctivity of bout 4.5. At ph 6.9, this enzyme hs insignificnt ctivity, nd thus it serves no purpose to dd it to the rection mixture, for exmple, in the methods of Chmp (8) or Goni et l. (11). The reltive ctivity of AMG t ph 5.2, 6.0, nd 6.9 is 100, 20, nd 4%, respectively. AMG shows no loss in ctivity on storge t ph 6.0 for 16 h t 37 C nd only 30% loss t ph 4.5. Therefore, in the current experiments when AMG ws dded to the incubtion mixtures, the incubtion ph used ws 6.0. At this ph, both AMG nd pncretic α-mylse re quite ctive nd reltively stble. AMG is included in the incubtion mixtures to ctlyze the hydrolysis to glucose of the soluble strch frgments nd mltodextrins produced on hydrolysis of strch by pncretic α-mylse. This removes the potentil inhibitory effect of mltose on pncretic α-mylse nd ensures tht soluble strch frgments re not precipitted by lcohol if this is used to terminte the incubtion. If lcohol is not used to terminte the rection, for exmple, the procedure of Goni et l. (11), then precipittion of soluble strch frctions is not considertion, s these re removed by centrifugtion nd decnttion. However, problems ssocited with the inhibition of pncretic α-mylse by mltose my still be relevnt. It is generlly considered tht, in performing RS nlyses, incubtion slurries should be shken, not stirred. However, experimentl dt confirming this ssertion re not redily vilble. To study this effect in detil, we performed prllel stirring nd shking experiments. For the stirring experiment, tube contents were stirred with mgnetic stirrer br on Vriomg Telemodul 40S t rnge of settings (120, 260, nd 500 rpm). Shking experiments were performed in Grnt Instruments shking wter bth OLS 200, with tubes ttched horizontlly in the bth nd ligned in the direction of motion. Shking speeds vried from 140 to 260 strokes/min (instrument settings of revs/min), but speed of 200 strokes/min ws routinely used. Figure 2 shows the effect of the concentrtion of pncretic α-mylse nd incubtion time on the determined RS vlue of RMS nd HAMS s obtined using the stirred-tube formt (Formt B). Clerly, the vlues obtined t the 3 concentrtions of enzyme used (5, 10, nd 20 mg/ml) re very similr for both strch smples nd t ech incubtion time. Thus, concentrtion of 10 mg/ml ws chosen for ll further experiments. This preprtion of pncretic α-mylse hd n ctivity of 3 CU/mg t ph 6.9 nd 40 C. Most published methods for RS mesurement use AMG in combintion with pncretic α-mylse; AMG is dded to ensure complete hydrolysis of soluble strch frgments nd mltoscchrides to glucose. Becuse the AMG cn theoreticlly ttck RS nd result in underestimtion of this component, experiments were performed to study this effect nd to optimize the concentrtion of the enzyme used in the ssys. The effect of dded AMG on the mesured level of RS in RMS nd HAMS is shown in Figure 3. It is evident tht concentrtion of AMG of pproximtely 8 U/test is required for complete hydrolysis of soluble strch frgments to glucose. With HAMS, the ddition of 8 U AMG per test resulted in drop in the mesured RS level from 47 to bout 37%. As the level of AMG is incresed to 30 U/test, the mesured RS level remined pproximtely the sme. The drop in the mesured level of RS on ddition of 8 U/test of AMG could be due to ny combintion of 3 effects, nmely, the hydrolysis of mltose nd thus the removl of its inhibitory effect on pncretic Tble 7. Repetbility of RS ssy, Formt D Resistnt strch, % (w/w), men b ± 2SD(CV c, %) Smple Dy 1 Dy 2 Dy 3 Dy 4 Interdy men ± 2 SD (CV, %) Hylon VII 52.7 ± ± ± ± ± 3.1 (3.7%) (1.7%) (0.6%) (0.9%) (3.0%) CrystLen 41.5 ± ± ± ± ± 2.5 (1.1%) (2.5%) (0.9%) (1.1%) (3.0%) Ntive potto strch (AVEBE) 78.1 ± ± ± ± ± 3.1 (1.1%) (4.2%) (2.4%) (0.8%) (2.0%) Green bnn (freeze-dried) 52.7 ± ± ± ± ± 4.4 (2.3%) (2.6%) (1.8%) (0.1%) (4.2%) Btchelors kidney bens (freeze-dried) 5.1 ± ± ± ± ± 0.2 (1.1%) (1.9%) (0.0%) (1.1%) (1.9%) Corn flkes 2.6 ± ± ± ± ± 0.1 (2.2%) (3.7%) (2.2%) (2.2%) (1.9%) b c All results re presented s RS s percentge of dry weight of smple. On ech dy, smples of ech mteril were nlyzed in triplicte. CV = coefficient of vrition.

9 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble 8. Effect of pepsin pretretment on determined RS content of smples (Assy Formt D) Smple type Pepsin pretretment RS, % (w/w) Btchelors kidney bens (cnned, lyophilized) Yes 5.10 ± 0.1 No 5.00 ± 0.1 Rob Roy flgeolot bens (cnned, lyophilized) Yes 4.55 ± 0.05 No 4.40 ± 0.10 Ntive potto strch Yes 73.9 ± 0.06 No 75.6 ± 1.05 Green bnn (lyophilized) Yes 50.8 ± 0.51 No 48.0 ± 0.20 Anlyses were performed in duplicte. α-mylse; hydrolysis of high degree of polymeriztion (DP) soluble strch frgments which would otherwise precipitte on ddition of IMS; or direct ction of AMG on RS. This spect is discussed lter. Incubtions in which the tubes were shken, rther thn stirred, were performed using Grnt Instruments shking wter bth OLS 200. Incubtion tubes were ttched horizontlly in the bth nd ligned in the direction of motion. Speed settings were such tht the smple ws continully suspended. The effect of vrying the shking speed on the determined level of RS of number of smples is shown in Tble 1. In generl, incresing the shking speed from 140 through 200, to 260 strokes/min resulted in smll decrese in the RS vlues, e.g., bout 5% for potto strch. The chnge in RS vlue for potto strch is consistent with the reported frgility of potto strch grnules nd is consistent with the very low RS vlues obtined for potto strch when the smples were stirred rther thn shken (Tble 2). The effect of dded AMG on the determined levels of RS, soluble strch frgments (plus glucose) nd free glucose, on hydrolysis of HAMS nd RMS by pncretic α-mylse in shking-tube experiments, is shown in Figure 4. Consistent with results obtined from stirred-tube experiments, level of AMG of 8 U/test ws sufficient to ensure complete hydrolysis of soluble strch frgments to glucose. As the concentrtion of AMG incresed from 0 to 8 U/test, the mesured RS for HAMS reduced from 64 to 43%. Incresing the AMG concentrtion from 8 to 30 U/test gve only slight further reduction in the mesured RS level, to bout 42%. This result is in generl greement with tht obtined in the stirred-tube experiments, where the mesured RS vlue lso showed little chnge over the AMG concentrtion rnge of 8 30 U/test. A comprison of the effect of stirring nd shking on the time course of hydrolysis of HAMS nd RMS by pncretic α-mylse (10 mg/ml) in the presence nd bsence of AMG (12 U/ssy) is shown in Figure 5. Rections were terminted by ddition of IMS. In ll cses, consistently lower RS vlues were obtined for smples when AMG ws included in the incubtion. Furthermore, higher vlues were obtined when smple tubes were shken rther thn stirred. RS is crystlline nd difficult to dissolve. Solvents used to dissolve this mteril include DMSO nd 2 4M KOH. We criticlly evluted methods nd solvents for the dissolution of the RS tht occurs in the wshed pellets obtined in Assy Formts A D. In most cses, DMSO completely dissolved RS. For 2 mterils, potto mylose nd Novelose 330 (17), this ws not the cse. In contrst, the RS in ll smples studied ws completely dissolved by stirring the pellet in 2M KOH in n ice-wter bth for 20 min (conditions used by severl uthors). On neutrliztion, it ws essentil to rpidly hydrolyze this strch to void recrystlliztion, which would gin render the strch resistnt to hydrolysis by AMG. To chieve this nd to simplify neutrliztion, concentrted sodium cette buffer (1.2M, ph 3.8) ws dded, followed immeditely by AMG (320 U/test). To determine the effect of mltose inhibition on the hydrolysis of strch by pncretic α-mylse, nd thus on the determined level of RS, severl experiments were performed. In n experiment using Assy Formt A, the mesured level of RS in RMS incresed >100%, from 9% dry weight bsis (dwb) to 20% dwb, in the presence of dded mltose (100 mg/test; Tble 3). The increse with HAMS nd other smples with high levels of RS were much smller, but still significnt (bout 2 3% dwb). This pprent mltose inhibition effect ws further studied using specific yest mltse (α-glucosidse) enzyme. This enzyme is highly ctive on mltose nd mltotriose, but hs no ction on soluble strch. In preliminry experiments, this enzyme could not be used in conjunction with the crude pncretic α-mylse tht is used routinely in the RS ssys (pncretin preprtion), s this preprtion contins n ctive protese which rpidly hydrolyzes nd inctivtes the mltse. This problem ws resolved by using pure (crystlline) pncretic α-mylse (Sigm Ct. No. A-2643), in the presence of which the mltse ws very stble for t lest 6 h (ctivity loss of <40% on incubtion t 37 C for 6 h). RMS nd HAMS were incubted with stirring (Formt A) with this pure pncretic α-mylse, with nd without dded mltse, nd the RS vlues were determined. RS vlues were lower (bout 5 8% dwb) when mltse ws included in the incubtion mixture (Tble 4). These results confirm tht mltose cuses some inhibition of pncretic α-mylse nd results in higher mesured RS vlues, prticulrly for RMS. However, the results from the mltse experiments only prtilly ccount for the significnt differences in mesured RS vlues in the presence or bsence of AMG t 8 U/test. This point is clerly demonstrted from the results in Tble 5, where RS vlues for HAMS were determined with Formt C (shking) using pure or crude pncretic α-mylse with AMG or mltse. Higher RS vlues were obtined with the shking formt (Tble 5) thn with the stirring formt (Tble 4), consistent with other results. Using either pure nd crude pncretic α-mylse in the presence of AMG, similr RS vlues were obtined, confirming tht the purity of the α-mylse ws not n issue. With pure α-mylse, the mesured RS vlue ws lower if mltse ws present in the incubtion mixture, demonstrting

10 674 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 some inhibitory effect of mltose on pncretic α-mylse. However, this effect ws much lower thn tht with dded AMG. This clerly demonstrtes tht the lower determined RS in the presence of AMG is due, in lrge prt, to the ction of AMG on strch mterils, possibly soluble strch s well s RS, s well s the removl of the inhibitory effect of mltose. The in vivo significnce of this is not immeditely pprent, becuse in the in vivo sitution, strch mterils re lso susceptible to hydrolysis by the α-glucosidse nd debrnching enzymes ttched to the brush border lining of the smll intestine. RS vlues obtined for rnge of strch smples, seed, nd food mterils, nlyzed with stirring or shking nd with or without dded AMG (Assy Formts A D), re shown in Tble 2. RS vlues were consistently lower when AMG ws dded to the incubtion mixture. When incubtion tubes were shken, the determined levels of RS were consistently higher thn when tube contents were stirred. The most drmtic differences observed between shking- nd stirred-tube experiments were for ntive potto strch. When incubtions were performed in shking wter bth, RS vlues of 73 79% were obtined, wheres vlues of just 4 8% were observed for smples stirred with mgnetic stirrer br. This is consistent with the reported frgility of potto strch grnules. This behvior is prticulrly pronounced for potto strch, but is lso significnt for bnn strch (Tble 2). With other ntive strch grnule mterils, nmely HAMS, HAMS (CO-347), nd Hylon VII, significntly different RS vlues were lso obtined between the shking- nd stirred-tube formts (Tble 2, Formts B nd D). This ws lso observed for Novelose 240, consistent with scnning electron microgrphs of this strch (15) which showed very similr fetures to Hylon VII. For retrogrded high mylose contining strch mterils (e.g., Novelose 330, Hi Mize 1043, nd CrystLen), the differences obtined with the 2 formts were not so mrked, yet still significnt (Tble 2). Formt C, s described here, is quite similr to the method of Goni et l. (11). The mjor difference is tht, in Formt C, the rection is terminted with IMS, nd the pellet obtined by centrifugtion is wshed with 50% IMS, insted of wter, s used in the Goni et l. method (11). The mjor dvntge of using IMS precipittion nd the inclusion of IMS in the wsh solution is tht firm pellet is obtined by centrifugtion, nd the centrifugtion time cn be sfely reduced to 10 min. With wter wshing, centrifugtion time of 30 min is required to ensure no loss of the pellet when the superntnt is decnted. The results obtined with the 4 ssy formts show tht the dt obtined with Assy Formt D re in best greement with currently vilble in vivo dt (Tble 6). These results re lso in good generl greement with other published dt on RS vlues (18). This is not surprising, s most methods re now stndrdizing the use of pncretic α-mylse, inclusion of AMG, nd shking-tube formt. In Tble 6, RS vlues for strch mteril supplied by Cerestr (ActiStr) re shown. All dt shown for ActiStr ws supplied by Bernd Kettlitz of Cerestr (Vilvoorde, Belgium) except for the vlue with Formt D, which ws obtined in-house. There is good greement mong ll methods nd the vlues re in close greement with in vivo dt. The reproducibility of Assy Formt D ws studied using 6 smples nlyzed in triplicte on 4 seprte dys. Results demonstrte tht within-dy repetbility nd between-dy reproducibility is very good (Tble 7). The mjor dvntge of the currently recommended formt (Formt D) is tht ll potentil vribles hve been evluted in detil, resulting in well understood, esy to use, nd robust ssy method. In some published methods for the ssy of RS, fresh smples were subjected to chewing before nlysis. This step ws excluded from the current formts becuse chewing is very subjective procedure nd vries significntly from person to person nd, possibly, for single person throughout dy. For fresh smples, we used commercilly vilble, hnd-operted met mincer with 4.5 mm screen. Fresh smples were either nlyzed directly, or lyophilized before nlysis. All results re reported on smple dry weight bsis. However, becuse the method llows mesurement of both RS nd totl strch (by summing RS nd soluble strch mterils), results cn lso be presented s percentge of totl strch (Tble 6). In the digestive process, foods re subjected to protese (pepsin) hydrolysis in the stomch t bout ph 2.0, before they rech the ileum where strch is hydrolyzed. Some uthors hve simulted this step in in vitro ssys by including pepsin pretretment step becuse, in certin smples high in protein, such s bens, the protein my prtilly encpsulte the strch, restricting its hydrolysis by mylolytic enzymes. To evlute the significnce of this effect, we nlyzed severl high protein smples using Formt D, with or without pepsin pretretment step (Tble 8). Results clerly demonstrted tht the protese pretretment step hd n insignificnt effect on the determined RS vlues, perhps becuse the pncretic α-mylse preprtion used in these ssys contins n ctive protese (trypsin, 50 mu/mg; chymotrypsin, 325 mu/mg pncretic α-mylse preprtion), which is probbly dequte to relese strch from the protein mtrix over the incubtion period. Conclusions In the current study, we criticlly evluted mny of the prmeters tht re likely to ffect the determined levels of RS in smples. Our im ws to develop simple, relible, nd robust procedure tht would yield RS vlues in line with published in vivo dt. We ttempted to imitte in vivo conditions s much s possible, while mintining user-friendly formt. Steps included in previously published methods, if found to hve no significnt effect on results, were removed. One such step is pepsin tretment of the smple. This tretment is included in the methods of Muir nd O De (9) nd Goni et l. (11), but our studies (Tble 8) showed tht such n incubtion step hd no effect on the determined RS vlues, thus it ws not included. It hs been suggested tht such tretment my help relese strch tht is physiclly encpsulted in protein (11), but even those results showed tht the effect of pepsin tretment on the mesured RS ws, t most, mrginl. We believe tht the pepsin tretment hd little effect on the determined RS vl-

11 MCCLEARY & MONAGHAN: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, ues (even for legume seeds) becuse the crude pncretic α-mylse used in the test contins ctive proteses. The current procedure yields RS vlues tht re generlly in good greement with those obtined by Goni et l. (11); however, in our hnds, the reproducibility ws better. We ttribute this to the use of lcohol precipittion, nd inclusion of lcohol in the wshing steps, which resulted in firm pellet on centrifugtion, thus minimizing losses while removing the superntnt. AMG ws included in the incubtion mixture to more closely simulte in vivo conditions, wherein mltoscchrides nd soluble strch re hydrolyzed to glucose nd bsorbed. Inclusion of AMG required the choice of n incubtion ph of 6.0 insted of ph 6.9, which is closer to the physiologicl vlue, to ensure tht the AMG ws ctive (bout 20% of the ctivity t ph 5.2). This ph chnge hd miniml effect on ctivity of pncretic α-mylse, or on its stbility t 37 C. Mny of the published ssy formts for RS involve incubtions t ph (6, 7). However, t this ph, pncretic α-mylse hs low ctivity (8% of mximum) nd is rpidly inctivted (95% in1ht37 C). The importnce of RS s dietry fiber component hs been recognized for over 20 yers. However, for inclusion in nutrient tbles, there is need for simple, relible, nd robust ssy tht yields results in line with in vivo dt. We believe tht the method described here meets these requirements. To verify this, n interlbortory evlution of the method under the uspices of AOAC INTERNATIONAL hs been performed (First Action Method ). Current AOAC INTERNATIONAL procedures for the mesurement of totl dietry fiber mesure some RS in smple. This is precisely why specific nd quntittive test is required. However, if specific test for RS is dopted, some modifictions to AOAC totl dietry fiber methods will be needed to ensure complete removl of ll strch. This modifiction will most probbly involve pretretment of the smple with DMSO or KOH to ensure complete solubiliztion of the strch before α-mylse tretment (19, 20). The choice of the pretretment step will be dictted by the effect of this step on other dietry fiber components. References (1) Englyst, H.N., Wiggins, H.L., & Cummings, J.H. (1982) Anlyst 107, (2) Berry, C.S. (1986) J. Cerel Sci. 4, (3) Englyst, H.N., & Cummings, J.H. (1985) Am. J. Clin. Nutr. 42, (4) Englyst, H.N., & Cummings, J.H. (1986) Am. J. Clin. Nutr. 44, (5) Englyst, H.N., & Cummings, J.H. (1987) Am. J. Clin. Nutr. 45, (6) Englyst, H.N., Kingmn, S.M., & Cummings, J.H. (1992) Eur. J. Clin. Nutr. 46 (Suppl. 2), S33 S50 (7) Chmp, M., Mrtin, L., Noh, L., & Grts, M. (1999) in Complex Crbohydrtes in Foods, S.S. Cho, L. Prosky, & M. Dreher (Eds), Mrcel Dekker, Inc., New York, NY, pp (8) Chmp, M. (1992) Eur. J. Clin. Nutr. 46 (Suppl. 2), S51 S62 (9) Muir, J.G., & O De, K. (1992) Am. J. Clin. Nutr. 56, (10) Fisnt, N., Plnchot, V., Kozlowski, F., Pcouret, M.-P., Colonn, P., & Chmp, M. (1995) Sci. Aliment. 15, (11) Goni, I., Grci-Diz, E., Mns, E., & Sur-Clixto, F. (1996) Food Chem. 56, (12) Akerberg, A.K.E., Liljberg, G.M., Grnfeldt, Y.E., Drews, A.W., & Bjorck, M.E. (1998) Am. Soc. Nutr. Sci. 128, (13) Officil Methods of Anlysis (2000) 17th Ed., AOAC IN- TERNATIONAL, Githersburg, MD, Methods nd (14) Somogyi, M. (1952) J. Biol Chem. 195, (15) McClery, B.V. (1999) Cerel Food World 44, (16) McClery, B.V., Bouhet, F., & Driguez, H. (1991) Biotech. Tech. 5, (17) Shi, Y.-C., & Jeffcot, R. (2000) in Advnced Dietry Fibre Technology, B.V. McClery & L. Prosky (Eds), Blckwell Science Ltd., Oxford, UK, pp (18) Chmp, M., Kozlowski, F., & Lecnnu, G. (2000) in Advnced Dietry Fibre Technology, B.V. McClery & L. Prosky (Eds), Blckwell Science Ltd., Oxford, UK, pp (19) Delcour, J.A., & Eerlingen, R.C. (1996) Cerel Food World 41, (20) McClery, B.V. (2001) Cerel Food World 46,