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Cereal Chem - 2019 - McCleary - Measurement of Available Carbohydrates Digestible and Resistant Starch in Food

This research article presents methods for measuring available carbohydrates, digestible starch, and resistant starch in food products, highlighting their significance in relation to health issues like Type II diabetes and obesity. The authors developed simple and reliable methodologies that allow for accurate measurement of these carbohydrates, including an updated resistant starch procedure. The findings aim to improve food labeling and consumer awareness regarding carbohydrate content in various food items.

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Cereal Chem - 2019 - McCleary - Measurement of Available Carbohydrates Digestible and Resistant Starch in Food

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Received: 14 July 2019

| Revised: 19 August 2019

| Accepted: 20 August 2019

DOI: 10.1002/cche.10208

RESEARCH ARTICLE

Measurement of available carbohydrates, digestible, and resistant

starch in food ingredients and products

Barry V. McCleary | Ciara McLoughlin | Lucie M. J. Charmier | Paraic McGeough

Megazyme, Bray, Ireland

Abstract
Correspondence Background and objectives: The importance of selectively measuring available and
Barry V. McCleary, Megazyme, Bray unavailable carbohydrates in the human diet has been recognized for over 100 years.
Business Park, Southern Cross Road, Bray,
County Wicklow, Ireland. The levels of available carbohydrates in diets can be directly linked to major dis-
Email: [email protected] eases of the Western world, namely Type II diabetes and obesity. Methodology for
measurement of total carbohydrates by difference was introduced in the 1880s, and
this forms the basis of carbohydrate determination in the United States. In the United
Kingdom, a method to directly measure available carbohydrates was introduced in
the 1920s to assist diabetic patients with food selection. The aim of the current work
was to develop simple, specific, and reliable methods for available carbohydrates and
digestible starch (and resistant starch). The major component of available carbohy-
drates in most foods is digestible starch.
Findings: Simple methods for the measurement of rapidly digested starch, slowly
digested starch, total digestible starch, resistant starch, and available carbohydrates
have been developed, and the digestibility of phosphate cross‐linked starch has been
studied in detail. The resistant starch procedure developed is an update of current
procedures and incorporates incubation conditions with pancreatic α‐amylase (PAA)
and amyloglucosidase (AMG) that parallel those used AOAC Method 2017.16 for
total dietary fiber. Available carbohydrates are measured as glucose, fructose, and
galactose, following complete and selective hydrolysis of digestible starch, malto-
dextrins, maltose, sucrose, and lactose to glucose, fructose, and galactose. Sucrose
is hydrolyzed with a specific sucrase enzyme that has no action on fructo‐oligosac-
charides (FOS).
Conclusions: The currently described “available carbohydrates” method together
with the total dietary fiber method (AOAC Method 2017.16) allows the measurement
of all carbohydrates in food products, including digestible starch.
Significance and novelty: This paper describes a simple and specific method for
measurement of available carbohydrates in cereal, food, and feed products. This is
the first method that provides the correct measurement of digestible starch and su-
crose in the presence of FOS. Such methodology is essential for accurate labeling
of food products, allowing consumers to make informed decisions in food selection.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2019 Megazyme. Cereal Chemistry published by AACC International, Inc.

114 |
wileyonlinelibrary.com/journal/cche Cereal Chemistry. 2019;97:114–137.
|

19433638, 2020, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10208 by Readcube (Labtiva Inc.), Wiley Online Library on [14/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
McCLEARY et al. 115

KEYWORDS
available carbohydrates, dietary fiber, digestible starch, Fibersym®, resistant starch

1 | IN T RO D U C T ION interlaboratory evaluation, was adopted as AOAC Methods

2009.01 and 2011.25, and AACCI Methods 32‐45.01 and
Available carbohydrate has been defined as the sum of free 32‐50.01 (McCleary et al., 2010). Subsequently, various lim-
sugars (glucose, fructose, galactose, sucrose, maltose, lac- itations of this method were identified, particularly the fact
tose, and oligosaccharides) and complex carbohydrates (dex- that an incubation time of 16 hr was employed, which was
trins, starch, and glycogen). These are carbohydrates that are quite correctly considered not to be physiologically relevant.
digested and absorbed, and are glucogenic in humans (Anon, In the development of AOAC Methods 2009.01 and 2011.25,
2003). In the “FAO/WHO scientific update on carbohydrates an incubation time of 16 hr was chosen to maintain consis-
in human nutrition” (Mann et al., 2007), it was proposed that tency with the Official Method for measuring resistant starch
the term “dietary fiber” should be limited to polysaccharides (AOAC Method 2002.01; AOAC, 2012) and several other
that are intrinsic to the plant cell wall, and the methods for published methods (Akerberg, Liljeberg, Granfeldt, Drews,
measuring dietary fiber are those which can reliably quantify & Bjorck, 1998; Champ, 1992, Champ, Martin, Noah, &
the component polysaccharides. Direct chemical measure- Gratas, 1999; Faisant et al., 1995; Goni, Garcia‐Diz, Manas,
ment was recommended over empirical gravimetric methods & Saura‐Calixto, 1996; Muir & O'Dea, 1992). In response to
for this purpose. Resistant starch (RS) was not considered to this limitation, the integrated TDF method was modified by
be dietary fiber. In the food composition tables amassed in reducing incubation time with PAA/AMG from 16 to 4 hr,
McCance and Widdowson's “The Composition of Foods” and the enzyme concentrations appropriately increased to
(Anon, 2003), dietary fiber is determined as nonstarch ensure that the resistant starch values obtained for a number
polysaccharides (NSP), as defined by Englyst, Wiggins & of reference materials were in line with those obtained with
Cummings (1982), Englyst and Hudson (1987) and Englyst AOAC Methods 2002.02 (McCleary, McNally, & Rossiter,
and Cummings (1988). However, in the definition for di- 2002) and 2009.01 as well as ileostomy data (Champ et al.,
etary fiber adopted in June 2009 by the Codex Alimentarius 1999). This update (McCleary, Sloane & Draga, 2015) was
Commission (CAC) (2010), the definition includes carbohy- successfully subjected to interlaboratory evaluation under the
drate polymers that are not hydrolyzed by the endogenous en- auspices of AOAC International and ICC to become AOAC
zymes in the small intestine of humans and thus includes RS. Method 2017.16 and ICC Method 185 (McCleary 2018;
In most plant‐based foods, the major contributor to avail- McCleary, Cox, Ivory, & Delaney, 2018).
able carbohydrates is digestible starch. Digestible starch was In the current paper, the methodology employed in AOAC
subcategorized by Englyst, Kingman, and Cummings (1992) Method 2017.16 has been adapted to allow the specific mea-
as rapidly digested starch (RDS; that starch hydrolyzed by surement of digestible and resistant starch and available
saturating levels of pancreatic α‐amylase [PAA] and amy- carbohydrates. Special reference has been directed to the
loglucosidase [AMG] in 20 min) and slowly digested starch measurement of phosphate cross‐linked starch (RS4). In this
(SDS, starch digested by PAA and AMG between 20 and methodology, available carbohydrates are defined as glucose,
120 min). The remainder, the nonhydrolyzed starch, was fructose, galactose, maltose, lactose, sucrose, maltodextrins,
termed RS. However, several studies (Deiteren et al., 2010; and total digestible starch. Note that starch is measured as
Geboes, Luypaerts, Rutgeerts, & Verbeke, 2003; Geypens et digestible starch rather than total starch, and sucrose is mea-
al., 1999; Miller et al., 1997; Sadik, Abrahamsson, Bjornsson, sured specifically in the presence of fructo‐oligosaccharides
Gunnarsdottir, & Stotzer, 2003; Stotzer & Abrahamsson, (FOS) by hydrolysis with a sucrase enzyme that has no action
2010; Zarate et al., 2010) indicate that the time of residence on FOS.
of food in the human small intestine is approximately 4 hr (not
2 hr). For this reason, we introduce the term “total digestible
starch, TDS,” being starch which is digested by saturating
2 | M ATERIAL S AND M ETHO D S
levels of PAA and AMG at 37°C and pH 6.0 within 4 hr. The
starch not hydrolyzed in 4 hr is termed RS. Consistent with
2.1 | Materials
the Codex Alimentarius definition of dietary fiber, this RS is
included as part of dietary fiber.
2.1.1 | Chemicals and reagents
A method for measurement of dietary fiber, generally d/l‐Maleic acid (M‐0375), bovine serum albumin (A‐2153),
consistent with the Codex definition, was published in 2007 dimethyl sulphoxide (D‐8779), and sodium azide (S‐8032)
(McCleary, 2007), and this method, following extensive were from Sigma‐Aldrich Ireland Ltd. Acetic acid (glacial)
|

GR, sodium hydroxide, and calcium chloride (CaCl2.2H2O) and milled to pass a 0.5‐mm screen. Dry breakfast cereals
were from Merck. Amyloglucosidase (AMG: E‐AMGFR were milled to pass a 0.5‐mm screen and stored in airtight
and E‐AMGDF), pancreatic α‐amylase (PAA: E‐PANAA), Duran® bottles.
PAA/AMG mixture (PAA 40 KU/g plus AMG 17 KU/g;
E‐PAAMG), heat‐stable α‐amylase (E‐BLAAM), thermo-
stable α‐amylase (E‐BSTAA), protease (E‐BSPRT), Total
2.2 | Analytical methods
Starch assay kit (K‐TSTA), Resistant Starch assay kit (K‐
RSTAR), Resistant Starch (Rapid) assay kit (K‐RAPRS),
2.2.1 | Preparation of test samples
Digestible Starch/Resistant Starch assay kit (K‐DSTRS), High‐moisture containing samples (>25%) were freeze‐dried.
Available Carbohydrates assay kit (K‐AVCHO), α‐Amylase Samples ca. 50 g were ground in a grinding mill, to pass a 0.5‐
assay kit (Ceralpha®; K‐CERA), Rapid Integrated TDF assay mm sieve. All materials were transferred to a wide‐mouthed
kit (K‐RINTDF), Amberlite FPA53 (OH−; G‐AMBOH), plastic jar, sealed, and mixed well by shaking and inverting
and Amberlite 200C (H+; G‐AMBH) were obtained from and then stored in the presence of a desiccant. Food samples
Megazyme. were collected and prepared as “intended to be eaten”; that
is, pasta and potatoes were cooked. High‐fat‐containing sam-
ples such as chocolate peanuts, chocolate cookies, and jam
2.1.2 | Pure starch samples
tarts were homogenized using a Nutri‐Bullet homogenizer.
Regular maize starch (RMS Lot 60401) was from Penford A sample of the homogenized material (approximately 2 g,
Australasia. Hylon VII® (Ref. 98GH8401), Novelose 330® weighed accurately) was transferred to an ANKOM® filter
(Ref. AH17529), and Novelose 240® (Ref. 96LF10063) were bag, and the bags were sealed. The filter bags were dried in
from National Starch and Chemical Company. These compa- an oven at 105°C before being placed into a desiccator to
nies are now part of Ingredion. Native potato starch was from cool. The weight of the bag plus sample was measured and
Avebe (Foxhol, The Netherlands). ActiStar® (enzyme‐modi- recorded. The samples were then defatted using the ANKOM
fied tapioca/cassava starch; US Patent 6,043,229) was from defatting apparatus at 60°C over 90 min with petroleum
Cerestar (now Cargill Belgium). Potato amylose (A‐9262), ether. The recovered bags were air‐dried for 15 min in a fume
wheat starch (S‐5127), and ACS Soluble starch (S‐9765) cupboard and then dried in an oven at 105°C for 30 min.
were from Sigma Chemical Company.
2.2.2 | Measurement of enzyme activities
2.1.3 | Processed food and breakfast cereals
α‐Amylase activity in PAA was measured using the
Uncle Ben's Ready Rice, white, extra was obtained from Ceralpha® assay procedure employing benzylidene blocked
Professor William Park, Texas A & M University, College p‐nitrophenyl maltoheptaoside in the presence of excess
Station, Texas, USA. Brennans wholemeal bread, Heinz® levels of thermostable α‐glucosidase. Incubations were per-
baked beans, Kellogg's® corn flakes, Kellogg's® All Bran®, formed in sodium maleate buffer at pH 6.9 and 40°C as de-
Weetabix®, Kellogg's® Special K®, Kellogg's® Sugar scribed in the Ceralpha® kit booklet (Megazyme K‐CERA;
Frosties®, tinned butter beans, tinned chickpeas, tinned gar- AOAC Official Method 2002.01). One unit (U) of enzyme
den peas, tinned kidney beans, semigreen banana, Ryvita® activity is defined as the amount of enzyme that releases one
crackers, and Roma® macaroni pasta were obtained from a µmole of p‐nitrophenol per minute under the defined assay
local supermarket. procedure. The α‐amylase activity reported is that measured
at the optimal pH of 6.9. However, incubations for the meas-
urement of digestible starch, resistant starch, and available
2.1.4 | Beans and fresh vegetables
carbohydrates were performed at pH 6.0. α‐Amylase activity
Sweet corn, potatoes, garden peas, red kidney beans, chick- at pH 6.0 is ~77% of that at pH 6.9 (McCleary & Monaghan,
peas, fresh cabbage, broccoli, cauliflower, swede, red pep- 2002). AMG was assayed by incubating 0.2 ml of suitably
per, mushroom, ripe banana, uncooked red kidney beans diluted enzyme in 100 mM sodium acetate buffer (pH 4.5)
and soybeans, red onion, celery, sweet potato, semiripe with 0.5 ml of soluble starch (10 mg/ml) in 100 mM sodium
banana, carrots, and potato were obtained from a local su- acetate buffer (pH 4.5) at 40°C. At various time intervals,
permarket. Potato was cooked in boiling water for 30 min, reaction tubes were heated to ~100°C in a boiling water bath
mashed, and freeze‐dried. All fresh vegetables were sliced to terminate the reaction and released glucose was measured
into thin sections, freeze‐dried, milled to pass a 0.5‐mm using GOPOD reagent (Glucose assay kit; Megazyme K‐
screen, and stored in Duran® airtight bottles at room tem- GLUC). One unit of AMG is defined as the amount of en-
perature. Canned beans and vegetables were poured onto a zyme required to release one µmole of glucose per minute at
strainer and washed with demineralized water, freeze‐dried, pH 4.5 and 40°C. When in admixture with PAA, AMG was
|

assayed using AMG Assay Reagent (Megazyme R‐AMGR3)

and units of activity on starch were calculated using a con-
version factor. The AMG activity reported is that measured
at the optimal pH of 4.5. However, incubations for the meas-
urement of digestible starch, resistant starch, and available
carbohydrates were performed at pH 6.0. AMG activity at pH
6.0 is ~20% of that at pH 4.5 (McCleary & Monaghan, 2002).

2.3 | Measurement of RS and DS using the

rapid RS method
Resistant starch was measured according to AOAC Method
2002.02/AACC Method 32‐40.01 and also using the rapid
RS method described here. In this latter procedure, samples
of finely milled (0.5 mm) cereal or food samples (~100 mg
weighed accurately) were weighed into 16.5 × 101 mm, 13‐
ml polypropylene tubes, and the tubes were tapped gently
to ensure that all samples fell to the bottom of the tube. For
wet samples such as minced canned beans or food product,
a sample size of approximately 0.5 g (weighed accurately)
was analyzed. An aliquot (3.5 ml) of sodium maleate buffer
(pH 6.0) containing 2 mM calcium chloride was added, and
the contents mixed thoroughly on a vortex mixer for 5 s and
the tube placed in a water bath at 37°C for 5 min to allow the
contents to equilibrate to temperature. An aliquot (0.5 ml) of F I G U R E 1 Attachment of 13‐ml polypropylene tubes to a
PAA/AMG solution (0.4 KU PAA plus 0.17 KU AMG) was polypropylene tube holder in a Grant OLS 200 water bath [Color
added to each tube, and the tubes were capped tightly and at- figure can be viewed at wileyonlinelibrary.com]
tached horizontally, aligned in the direction of motion (Figure
1) in a shaking water bath set at 37°C (Note: If an (NH4)2SO4 first two supernatant solutions. The tubes containing the resi-
suspension of this enzyme preparation [PAA, 2 KU/ml; AMG, due were inverted on absorbent paper to remove excess liquid
0.83 KU/ml in 50% w/v ammonium sulfate] was used, the while ensuring that the pellets were not dislodged.
sample was suspended in 3.8 ml of sodium maleate buffer and
0.2 ml of enzyme suspension was added.). Tubes were incu-
bated at 37°C with continuous shaking (200 strokes/min) for
2.3.1 | Determination of RS
exactly 4 hr. The tubes were removed from the water bath one A magnetic stirrer bar (6 × 12 mm) and 2 ml of ice‐cold 1.7 M
at a time, ethanol or IMS (industrial methylated spirits; 4.0 ml, NaOH were added to each tube, and the pellets were re‐sus-
95% v/v) added, the tubes were capped, and the contents were pended (and RS dissolved) by stirring for approx. 20 min in
stirred vigorously on a vortex mixer. After removal of caps, an ice/water bath over a magnetic stirrer (Figure 2). Sodium
the tubes were centrifuged at 3,250 g (approx. 3,250 relative acetate buffer (8 ml, 1.0 M, pH 3.8) containing calcium chlo-
centrifugal force; rcf) for 10 min. Immediately after the centri- ride (5 mM) was added to each tube while stirring. AMG
fuge stopped, the supernatant solution was carefully decanted (0.1 ml, 3,300 U/ml) was immediately added; the tubes were
(ensuring that the pellets were not disturbed) and stored for the mixed well and placed in a water bath at 50°C and incubated
determination of DS. The pellets were re‐suspended in 2 ml for 30 min with intermittent mixing on a vortex mixer. For
of 50% v/v aqueous ethanol or IMS and mixed vigorously on samples containing <10% RS content, centrifuge aliquots of
a vortex mixer. A further 6 ml of 50% v/v aqueous ethanol the undiluted solutions at 8,000 g for 5 min in a microfuge.
or IMS was then added to the tube, the tube was capped, and The final volume in the tube (before removal of an aliquot
the contents were mixed thoroughly by inversion. Tubes were for centrifugation) was approximately 10.3 ml. However, this
tapped so that all liquid was removed from the caps. Caps volume varied, particularly when wet samples were analyzed,
were then removed, and the tubes were centrifuged at 3,250 and appropriate allowances for the final volumes were made
g for 10 min. The supernatant solutions were then carefully in the calculations. For samples containing >10% RS content,
decanted and added to the original supernatant. The residue the contents of the tubes were quantitatively transferred to
was re‐suspended in 8 ml of 50% v/v aqueous ethanol or IMS 100‐ml volumetric flask using a water wash bottle. The vol-
and centrifuged, and the supernatant decanted and added to the ume was adjusted to 100 ml with distilled water and mixed
|

bottoms of two 16 × 100 mm tubes, 0.1 ml of AMG (100 U/

ml) in 100 mM sodium acetate buffer (pH 4.5) was added, and
the tubes were incubated at 50°C for 30 min. GOPOD rea-
gent (3.0 ml) was added, and the tubes were incubated at 50°C
for 20 min. A reagent blank solution was prepared by mixing
0.2 ml of 100 mM acetic acid (pH 4.5) with 3.0 ml of GOPOD
reagent. Glucose standards (in quadruplicate) were prepared
by mixing 0.1 ml of glucose solution (1 mg/ml) with 0.1 ml of
100 mM sodium acetate buffer (pH 4.5) and 3.0 ml of GOPOD
reagent and incubating at 50°C for 20 min. Absorbance of each
solution was measured at 510 nm against the reagent blank.
The total carbohydrate content of the solutions was determined
on 50 µl aliquots using the phenol‐sulfuric acid procedure of
F I G U R E 2 Arrangement of tubes in an ice‐water bath over a DuBois, Gilles, Hamilton, Rebers, and Smith (1956).
magnetic stirrer for dissolution of resistant starch with 1.7 M NaOH Calculate DS (% w/w, “as is” basis) in test samples as
[Color figure can be viewed at wileyonlinelibrary.com] follows:

well. Aliquots of all solutions were centrifuged at 8,000 g for DS (g∕100g sample)
5 min. Duplicate aliquots (0.1 ml) of all solutions were trans- = ΔA × F × EV∕0.1 × 1∕1000 × 100∕W × 162∕180
ferred to the bottoms of glass test tubes (16 × 100 mm), and = ΔA × F∕W × 0.9
3.0 ml of GOPOD reagent was added with mixing, and the
where ΔA = absorbance (reaction) read against the reagent
tubes were incubated at 50°C for 20 min. A reagent blank so-
blank, F = conversion from absorbance to µg (the absorbance
lution was prepared by mixing 0.1 ml of 100 mM acetic acid
obtained for 100 µg of glucose in the GOPOD reaction is de-
(pH 4.5) with 3.0 ml of GOPOD reagent. Glucose standards
termined), (F = 100 [µg of glucose] divided by the GOPOD
(in quadruplicate) were prepared by mixing 0.1 ml of glucose
absorbance for this 100 µg of glucose), EV = sample ex-
solution (1 mg/ml) with 3.0 ml of GOPOD reagent and incu-
traction volume (ml) = 100. 0.1 = volume of sample analyzed,
bating at 50°C for 20 min. Absorbance of each solution was
1/1,000 = conversion from µg to mg, 100/W = conversion to
measured at 510 nm against the reagent blank.
100 mg of sample, W = sample weight in mg; 162/180 = factor
Content of RS was calculated as follows:
to convert from free glucose, as determined, to anhydro‐glucose
RS (g∕100g) as occurs in starch.
= ΔA × F × EV∕0.1 × 1∕1000 × 100∕W × 162∕180
= ΔA × F × EV∕W × 0.9 2.4 | Measurement of phosphate cross‐
where ΔA = absorbance of sample solution read against reagent
linked starch (RS4)
blank, F = factor to convert absorbance values to µg glucose Phosphate cross‐linked starch was measured by several
(100 µg glucose divided by the absorbance value obtained for methods, the method of Shukri, Zhu, Seib, Maningat, and Shi
100 µg of glucose), EV = sample extraction volume (10.3 or (2015) and Shi, Sun, and Shi (2019) and other methods as
100 ml), 0.1 = volume of sample analyzed, 1/1,000 = conver- described here. In the modified Shukri et al. (2015) and Shi
sion from µg to mg, 100/W = conversion to 100 mg sample, et al. (2019) methods as described here, the initial incuba-
W = sample weight in mg, 162/180 = factor to convert from tion with PAA and AMG was performed under the conditions
free glucose, as determined, to anhydro‐glucose, as occurs in of AOAC Method 2017.16 (PAA 100 U/ml; AMG 42 U/ml;
starch. Table 1) for 4 hr, instead of that reported by Shukri et al.
Calculations are simplified using a Megazyme (2015) and Shi et al. (2019) (PAA 50 U/ml; AMG 3 U/ml)
MegaCalc™ Excel® based calculator (RAPRS) in Supporting for 2 hr. The RS fraction was then recovered and digested ac-
Information. cording to the particular procedures described by Shukri et al.
(2015) or Shi et al. (2019). In the Shukri et al. (2015) method,
the RS fraction (e.g., from Fibersym® [RS4]) was recovered
2.3.2 | Determination of DS
by precipitation with ethanol, centrifugation, washing the
The combined supernatants were adjusted to 100 ml with residue with ethanol, and incubated with protease followed
100 mM sodium acetate buffer (pH 4.5), and an aliquot (2.0 ml) by thermostable α‐amylase (Bacillus sp.; 200 U) at 100°C
was centrifuged at 8,000 g for 5 min. To determine digest- for 30 min in 8 ml of 100 mM sodium acetate buffer, pH
ible starch, duplicate aliquots (0.1 ml) were transferred to the 5 containing 5 mM CaCl2. A second amount of α‐amylase
|

TABLE 1 A comparison of incubation conditions used in the resistant starch, digestible starch, available carbohydrates, and dietary fiber
assay proceduresa

Sample Buffer Pancreatic

weight volume α‐amylase Amyloglucosidase

U/ml of K Units K Units

Method, incubation time, and assay per Units/ml of per
Component measured catalogue number mg ml solution assay assay solution assay
Resistant starchb AOAC Method 2002.02 ~100 4 50 0.2 3 0.014
16 hr (K‐RSTAR)
Total dietary fiberb AOAC Method 2009.01/AACC ~1,000 40 50 2.0 3 0.14
Method 32‐45.01
16 hr (K‐INTDF)c
Total dietary fiberb AOAC Method 2011.25 ~1,000 40 50 2.0 3 0.14
AACC Method 32‐50.01
16 hr (K‐INTDF)
Resistant starch Rapid resistant starch ~100 4 100 0.4 42 0.17
4 hr (K‐RAPRS)
Digestible/resistant Digestible/resistant starch method ~500 20 100 2.0 42 0.85
starch 4 hr (K‐DSTRS)
Available Available carbohydrates ~500 20 100 2.0 42 0.85
carbohydrates 4 hr (K‐AVCHO)
Total dietary fiber AOAC Method 2017.16 ~1,000 40 100 4.0 42 1.7
4 hr (K‐RINTDF)
a
In all cases, incubations were performed in 50 mM sodium maleate buffer (pH 6.0) containing 2 mM calcium chloride.
b
Sodium azide (0.02% w/v) was included in the buffer in these procedures.
c
Megazyme catalogue numbers.

(200 U) was added, and the sample was incubated for a fur- to equilibrate to temperature. An aliquot (0.5 ml) of PAA/
ther 30 min. The solution was cooled to 50°C, 132 U of AMG AMG solution (0.4 KU PAA plus 0.17 KU AMG) was added
added, and the solutions were incubated for 1 hr. Released to each tube, and the tubes were capped tightly and attached
glucose was analyzed with GOPOD reagent and RS calcu- horizontally, aligned in the direction of motion (Figure 1) in a
lated according to Shukri et al. (2015). In the “improved” in shaking water bath set at 37°C. Tubes were incubated at 37°C
vitro assay of Shi et al. (2019), the RS residue was suspended with continuous shaking (200 strokes/min) for exactly 4 hr.
in 2 M KOH and stirred at room temperature for 4 hr. The Ethanol or IMS (4.0 ml, 95% v/v) was added to each tube, and
solution was neutralized with HCl, ethanol was added, and the contents were stirred vigorously on a vortex mixer. Tubes
the residue was recovered by centrifugation (the supernatant were centrifuged, and the initial supernatant was stored in a
was recovered for determination of DS.). The residue was sealed tube for determination of digestible starch. The resi-
suspended in 8 ml of 100 mM sodium acetate buffer (pH 5.0), due containing the RS was recovered and washed as in the
thermostable α‐amylase (200 U) was added, and the solution standard rapid RS method, and the supernatants from the two
was incubated at 100°C for 30 min with intermittent vigorous washings with aqueous ethanol were added to the initial su-
stirring. This α‐amylase treatment was then repeated a sec- pernatant and used for the measurement of digestible starch.
ond time. The solution was cooled to 50°C, 132 U of AMG Several procedures were evaluated for the dissolution and hy-
added, and the solution incubated at 50°C for 1 hr. Released drolysis of the starch in the residue fraction, namely.
glucose was analyzed with GOPOD reagent and RS calcu-
lated according to Shi et al. (2019). a. the residue was suspended in 8 ml of 100 mM sodium ac-
In the current procedure, samples (~100 mg weighed etate buffer (pH 5.0), 0.1 ml of thermostable α‐amylase
accurately) were weighed into 16.5 × 101 mm, 13‐ml poly- (200 U, E‐BSTAA) was added, and the tubes were incu-
propylene tubes, and an aliquot (3.5 ml) of sodium maleate bated at 100°C for 30 min. The tubes were then cooled to
buffer (pH 6.0) plus 2 mM CaCl2 was added, and the con- 50°C, 0.1 ml of AMG (330 U) was added, and the tubes
tents mixed thoroughly on a vortex mixer for 5 s and the tube were incubated at 50°C for 30 min. Volumes were adjusted
placed in a water bath at 37°C for 5 min to allow the contents to 100 ml with distilled water and the contents thoroughly
|

mixed. Aliquots (2.0 ml) were centrifuged at 8,000 g for

2.5 | Chromatographic separation of the
5 min in a microfuge, and 0.1 ml aliquots removed for the
carbohydrates present in the hydrolysate of
determination of glucose using GOPOD reagent and deter-
Fibersym®
mination of RS according to the procedure used in the RS
(rapid) method. Digestible starch was determined using the Twenty separate samples of Fibersym® (1.00 g each) were
same procedure for the determination of digestible starch incubated with PAA/AMG according to AOAC Method
in the RS (rapid) method. 2017.16. After 4 hr, the pH was adjusted to ~8.2 according
b. The residue was suspended in 2 ml of sodium hydroxide to the procedure, and the solutions heated to ~95°C to in-
(1.7 M) and vigorously mixed on a vortex mixer, and the activate PAA and AMG. Four volumes of ethanol (160 ml)
tubes were incubated at 50°C for 15 min (incubation times was added to each of the twenty containers and mixed thor-
of 30, 60, and 120 min were also evaluated). The solutions oughly. After standing at room temperature overnight, the
were neutralized by addition of 8 ml of sodium acetate buf- contents of all containers were pooled and centrifuged at
fer (600 mM, pH 3.8), 0.1 ml of thermostable α‐amylase 24,000 g for 20 min. The supernatants (original superna-
(200 U, E‐BSTAA) was added, and the tubes were incu- tants) were carefully decanted, combined, and concentrated
bated at 100°C for 30 min. The tubes were then cooled to by rotary evaporation. The residues were pooled into two
50°C and 0.1 ml of AMG (330 U) was added, and the solu- 400‐ml centrifuge containers and suspended in ~200 ml
tions incubated at 50°C for 30 min. Volumes were adjusted of 80% v/v ethanol in water (in each container) and recov-
to 100 ml with distilled water and the contents thoroughly ered by centrifugation. This procedure was repeated once
mixed. Aliquots were removed for the measurement of glu- more. Each of the supernatants was pooled with the original
cose and determination of RS according to example “a.” supernatant and concentrated. Carbohydrate in the super-
Digestible starch was determined using the same procedure natant fraction (DS) was concentrated to ~30 mg/ml, and
for the determination of digestible starch in the RS (rapid) aliquots (16 ml) fractionated on a column (5 × 95 cm) of
method. Bio‐Gel P‐2, Extra Fine (Bio‐Rad Laboratories) in distilled
c. The residue was suspended in 2 ml of ice‐cold sodium hy- water at 60°C. Fractions of 20 ml were collected, and ali-
droxide (1.7 M) and vigorously mixed on a vortex mixer, quots analyzed for total carbohydrate using the phenol‐sul-
and the tubes were incubated at ~4°C with stirring for furic acid procedure (DuBois et al., 1956). The fractions
30 min. The solutions were neutralized by addition of 8 ml shown in Figure 5 were collected and concentrated (where
of sodium acetate buffer (600 mM, pH 3.8), and all other necessary) by rotary evaporation to a carbohydrate concen-
steps were as described in example “b.” tration of ~0.5 mg/ml. These solutions were analyzed for
d. The residue was suspended in 2 ml of sodium hydroxide free glucose by incubating aliquots (0.1 ml) plus 0.1 ml of
(1.7 M) and vigorously mixed on a vortex mixer, and the sodium acetate buffer with 3.0 ml of GOPOD reagent at
tubes were incubated at 4°C for 20 min. The solutions 50°C for 20 min. The absorbance was measured against a
were neutralized by addition of 8 ml of sodium acetate blank solution containing 0.2 ml of 100 mM sodium ac-
buffer (1.2 M, pH 3.8), 0.1 ml of AMG (330 U) was etate buffer (4.5) plus 3.0 ml of GOPOD reagent. Glucose
added, and the tubes were incubated at 50°C for 30 min. standard solutions were prepared by incubating glucose
Volumes were adjusted to 100 ml with distilled water and (0.1 ml, 1.0 mg/ml) plus 0.1 ml of 100 mM sodium acetate
the contents thoroughly mixed. Aliquots were removed buffer (pH 4.5) with 3.0 ml of GOPOD reagent at 50°C for
for the measurement of glucose and determination of RS 20 min concurrently with the sample solutions. Hydrolysis
according to example “a.” Digestible starch was deter- by AMG was determined by incubating sample aliquots
mined using the same procedure for the determination of (0.1 ml, ~0.5 mg/ml) in 100 mM sodium acetate buffer (pH
digestible starch in the RS (rapid) method. This is the 4.5) with 0.1 ml of AMG (10 U) for 30 min at 50°C with
procedure employed to dissolve RS in the rapid RS assay 3.0 ml of GOPOD reagent. Total carbohydrate concentra-
procedure. tion of the samples was determined on 0.1 ml aliquots using
the phenol‐sulfuric acid procedure (DuBois et al., 1956).
In other experiments, the total starch content of phosphate The combined residues from alcohol precipitation of
cross‐linked and other starches was determined by directly the PAA/AMG incubation mixtures (the RS fractions)
using the dissolution and hydrolysis conditions for the RS resi- were suspended in 40 ml of 1.7 M NaOH, heated at 50°C
due described in examples “a”–“d” above. for 30 min, and then neutralized by addition of 160 ml of
The total carbohydrate content of the digestible starch and 600 mM sodium acetate buffer (pH 3.8) plus 5 mM CaCl2;
hydrolyzed resistant starch fractions was determined by ana- the pH was ~5.3. Thermostable α‐amylase (2 ml, 2,000 U/
lyzing an aliquot (50 µl) with the phenol‐sulfuric acid proce- ml) was added, and the solution incubated at ~100°C for
dure of DuBois et al. (1956). 30 min. The temperature was reduced to 50°C and 2 ml of
|

AMG (3,300 U/ml) added, and the solutions incubated for Aliquots (2 ml) of each solution were transferred to 2.0‐ml
30 min at 50°C and then at ~100°C for 10 min to inactivate polypropylene microfuge tubes and centrifuged at 8,000 g for
the AMG. The solution was concentrated to ~20 mg carbo- 5 min. Duplicate aliquots (0.1 ml) were then transferred to
hydrate/ml, centrifuged at 29,000 g for 15 min to remove the bottoms of 16 × 100 mm glass test tubes, 0.1 ml of AMG
a very light precipitate (which on recovery represented (10 U) in 200 mM sodium acetate buffer (pH 4.5) was added,
0.16% of total carbohydrate in the Fibersym® sample), and and the tubes were incubated at 50°C for 30 min. GOPOD rea-
aliquots (16 ml) fractionated on a column (5 × 95 cm) of gent (3.0 ml) was added, and the tubes were incubated at 50°C
Bio‐Gel P‐2, Extra Fine (Bio‐Rad Laboratories) in distilled for 20 min. A reagent blank solution was prepared by mixing
water at 60°C. Fractions of 20 ml were collected, and al- 0.2 ml of 200 mM acetic acid (pH 4.5) with 3.0 ml of GOPOD
iquots analyzed for total carbohydrate using the phenol‐ reagent and incubating at 50°C for 20 min. Glucose standards
sulfuric acid procedure (DuBois et al., 1956). Individual (in quadruplicate) were prepared by mixing 0.1 ml of glucose
fractions were concentrated (where necessary) to ~0.5 mg/ solution (1 mg/ml) with 0.1 ml of 200 mM sodium acetate buffer
ml, and duplicate aliquots (0.1 ml) were analyzed for total (pH 4.5) and 3.0 ml of GOPOD reagent and incubating at 50°C
carbohydrate by the phenol‐sulfuric acid method. Separate for 20 min. The absorbance of each solution was measured at
aliquots (0.1 ml) were analyzed for free glucose by incu- 510 nm against the reagent blank.
bation with GOPOD reagent (3.0 ml) at 50°C for 20 min Calculate DS (RDS, SDS & TDS; % w/w, “as is” basis) in
or were incubated with AMG (0.1 ml, 10 U) in 100 mM test samples as follows:
sodium acetate buffer (pH 4.5) for 30 min at 50°C and then
with GOPOD reagent (3.0 ml) at 50°C for 20 min, and the DS (RDS, SDS or TDS) (g∕100g sample) = ΔA × F × EV∕W
absorbance at 510 nm measured against a reagent blank. × D∕0.1 × 100 × 1∕1,000,000 × 100∕W × 162∕180
Glucose standard solutions and blank solutions were run = ΔA × F∕W × 0.38745.
concurrently.
where ΔA = absorbance (reaction) read against the reagent
blank after 20 min (RDS); after 120 min − 20 min (SDS);
2.6 | Measurement of digestible (RDS, SDS, after 240 min (TDS), F = conversion from absorbance to
TDS) and resistant starch µg (the absorbance obtained for 100 µg of glucose in the
Samples of finely milled (<0.5 mm) cereal or food samples GOPOD reaction is determined; F = 100 [µg of glucose] di-
(~0.5 g weighed accurately) were weighed into 30 × 84 mm vided by the GOPOD absorbance for this 100 µg of glucose),
(40 ml) polypropylene tubes, and the weight recorded. and EV = extraction volume (ml) = 20.5.
A 20 × 6 mm stirrer bar was added to each tube, the sam- W = “as is” weight of sample analyzed in g, that is, ~0.50 g
ple was wet with 0.5 ml of ethanol (95% v/v), and 17.5 ml (weighed accurately).
of maleate buffer was added to each tube. The tubes were
capped and placed in a special polypropylene holder (Figure
3) on a 2mag Mixdrive 15® submersible magnetic stirrer in a
water bath and allowed to equilibrate to 37°C over 5 min with
stirring at 170 rpm. An aliquot (2.5 ml) of PAA/AMG solu-
tion (PAA, 2 KU; AMG, 0.85 KU) was added, and the tubes
were capped and incubated at 37°C with stirring at 170 rpm
on the 2mag Mixdrive 15® submersible magnetic stirrer. If
an (NH4)2SO4 suspension of this enzyme preparation (PAA,
2 KU/ml; AMG, 0.83 KU/ml) was used, the sample was sus-
pended in 19 ml of sodium maleate buffer and 1.0 ml of en-
zyme suspension was added.

2.6.1 | Determination of DS
Aliquots (1.0 ml) of the stirred reaction solution were removed
F I G U R E 3 Samples (~0.5 g) in 40 ml, 30 × 84 mm
using a positive displacement dispenser at 20 min (for determina-
polypropylene tubes in a designed polypropylene tube holder
tion of RDS), at 120 min (for determination of SDS; SDS = DS (Megazyme cat. no. D‐PPTH) [C (w)] on a 2mag Mixdrive 15®
at 120 min − DS at 20 min), and at 240 min (for determina- submersible magnetic stirrer in a custom‐made water bath (Megazyme
tion of TDS). These aliquots were immediately added to 20 ml cat. no. D‐TDFBTH). This arrangement allows stirring of 15 samples
of 50 mM acetic acid solution, and the tubes were capped and at controlled speed (170 rpm) and 37°C [Color figure can be viewed at
mixed thoroughly. These were stored at 4°C awaiting analysis. wileyonlinelibrary.com]
|

D = dilution of sample (21; 1.0 ml of sample added to from µg to g, 100/W = conversion to g/100 g, W = “as is”
20 ml of dilute acetic acid). weight of sample analyzed in g (~0.50 g weighed accurately),
0.1 = volume of sample analyzed. 100 = conver- and 162/180 = factor to convert from free glucose, as deter-
sion to g/100 g. 1/1,000,000 = conversion from µg to g. mined, to anhydro‐glucose as occurs in starch.
162/180 = factor to convert from free glucose, as determined, Calculations are simplified using a Megazyme
to anhydro‐glucose as occurs in starch. MegaCalcTM Excel®‐based calculator (RAPRS‐RS) in
Calculations are simplified using a Megazyme Supporting Information.
MegaCalc™ Excel®‐based calculator (DSTRS‐DS) in
Supporting Information.
2.7 | Measurement of available
carbohydrates
2.6.2 | Determination of RS
Aliquots (4 ml) were removed from the stirring reaction solu-
2.7.1 | Method
tions using a positive displacement dispenser after 240 min Samples of finely milled cereal or food samples (~0.5 g
(4 hr) of incubation, and transferred to a 16.5 × 101 mm, 13‐ weighed accurately) were weighed into 30 × 84 mm (40 ml)
ml polypropylene tubes containing 4.0 ml of ethanol (95% polypropylene tubes and the weight recorded. Incubation
v/v) or IMS. The tubes were capped, and the contents thor- with PAA/AMG for 4 hr was then performed exactly as de-
oughly mixed by repeated inversion. Tubes were centrifuged scribed for “digestible and resistant starch” above. Aliquots
at 3,250 g for 10 min in a bench centrifuge, and the superna- (1.0 ml) were removed and added to 25 ml of 50 mM acetic
tant carefully decanted immediately after the centrifuge had acid as described and mixed thoroughly, and samples (2 ml)
stopped. Each pellet was re‐suspended in 2 ml of 50% v/v were centrifuged at 8,000 g for 5 min. Aliquots (0.1 ml)
aqueous IMS in water by stirring on a vortex mixer. Another of this solution were analyzed for available carbohydrates
6 ml of 50% v/v aqueous IMS was then added to the tube, (total digestible starch, maltodextrins, sucrose, lactose, glu-
which was then capped, and the contents mixed on a vortex cose, and fructose). All incubations were performed as de-
mixer. The tubes were centrifuged, and the pellets recovered scribed in Figure 4. Reagents required for this determination
by centrifugation. This process of suspension and centrifu- are available in the Available Carbohydrates assay kit from
gation was repeated, and the supernatant again carefully de- Megazyme (cat. no. K‐AVCHO). Aliquots (0.1 ml) of centri-
canted. Free liquid in the tube was removed by inverting the fuged solution in a spectrophotometer cuvette were incubated
tubes on absorbent paper while ensuring that the pellet was with 0.1 ml of a solution containing β‐galactosidase (800 U/
not dislodged. A magnetic stirrer bar (6 × 12 mm) and 2 ml ml), sucrase (20 U/ml), and maltase (100 U/ml) in 50 mM so-
of cold 1.7 M NaOH were added to each tube, and the pel- dium maleate buffer (pH 6.5) containing BSA (0.5 mg/ml) at
lets were re‐suspended (and the RS dissolved) by stirring the 25°C for 20 min to hydrolyze lactose, sucrose, and maltose to
tube contents for approx. 20 min in an ice/water bath over a monosaccharides. Distilled water (2.0 ml), imidazole buffer
magnetic stirrer (Figure 2). The solutions were neutralized (0.1 ml, 2 M, pH 7.6) containing MgCl2 (100 mM), and a
with 8 ml of 1.0 M sodium acetate buffer (pH 3.8) containing solution (0.1 ml) of NADP+ (20 mg/ml) plus ATP (40 mg/
5 mM CaCl2, starch hydrolyzed, and glucose measured as ml) were added and mixed, and the absorbance (A1) meas-
described for rapid RS method. ured at 340 nm after 3 min. An aliquot (20 µl) of hexokinase
Calculate RS (% w/w, on an “as is” basis) in test samples (420 U/ml) and glucose 6‐phosphate dehydrogenase (110 U/
as follows: ml) was then added, the solution mixed and incubated for
5 min at 25°C, and the absorbance (A2) measured. An ali-
RS (g∕100g sample) quot (20 µl) of phosphoglucose isomerase (1,000 U/ml) was
= ΔA × F × EV∕4 × FV∕0.1 × 1∕1,000,000 × 100∕W × 162∕180 then added, the solution mixed and incubated for 10 min at
= ΔA × F∕W × FV × 0.004613 25°C, and the absorbance (A3) measured. Finally, an aliquot
(20 µl) of a mixture of galactose dehydrogenase (200 U/
where ΔA = absorbance (reaction) read against the reagent ml) and galactose mutarotase (4.1 mg/ml) was added and
blank, F = conversion from absorbance to µg (the absorbance the solution mixed and incubated for 10 min at 25°C and
obtained for 100 µg of glucose in the GOPOD reaction is de- the absorbance (A4) measured. The difference (A2 − A1) for
termined; F = 100 [µg of glucose] divided by the GOPOD ab- both blank (see Figure 4) and sample was determined and
sorbance for this 100 µg of glucose), EV = extraction volume the absorbance difference of the blank was subtracted from
(ml) = 20.5, 4 = volume of solution taken from the reaction the absorbance difference of the sample, thereby obtaining
mixture for RS analysis, FV/0.1 = 0.1 ml aliquots taken from ΔAglucose. The absorbance difference (A3 − A2) for both blank
final volume (FV, either 100 or 10.3 ml) for the determination and sample was determined and the absorbance difference of
of glucose using GOPOD reagent, 1/1,000,000 = conversion the blank was subtracted from the absorbance difference of
|

F I G U R E 4 Procedure for the

sequential measurement of glucose, fructose,
and galactose in a spectrophotometer cuvette

the sample, thereby obtaining ΔAfructose. The absorbance dif- For fructose (g/L):
ference (A4 − A3) for both blank and sample was determined
2.44 × 180.16
and the absorbance difference of the blank was subtracted c= × ΔAfructose × 26
6300 × 1 × 0.1
from the absorbance difference of the sample, thereby obtain-
= 18.124 × ΔAfructose
ing ΔAgalactose.
The concentration of glucose, fructose, and galactose For galactose (g/L):
(g/L) was calculated as follows: 2.46 × 180.16
c= × ΔAgalactose × 26
V × MW 6300 × 1 × 0.1
c= × ΔA × D. = 18.290 × ΔAgalactose
𝜀×d×v
where V = final volume (ml); MW = molecular weight of When analyzing solid and semisolid samples that are
glucose, fructose, or galactose (g/mol); ε = extinction coef- weighed out for sample preparation, the content (g/100 g) is
ficient of NADPH at 340 nm = 6,300 (L × mol−1 × cm−1); calculated from the amount weighed as follows:
d = light path (cm); v = sample volume (ml); D = dilution
factor (26‐fold). Content of D − glucose (g∕100g) :
It follows for glucose (g/L): = cD−glucose (g∕L) × EV∕1000 × 1∕W × 100
2.42 × 180.16
c= × ΔAglucose × 26 Content of D − fructose (g∕100g) :
6300 × 1 × 0.1
= 17.993 × ΔAglucose = cD−fructose (g∕L) × EV∕1000 × 1∕W × 100
|

Content of D − galactose (g∕100g) : 1996; Muir & O'Dea, 1992), most involved a ~16‐hr incu-
= cD−galactose (g∕L) × EV∕1000 × 1∕W × 100 bation with pancreatic α‐amylase (PAA) with shaking at
37°C. AOAC Method 2002.02, which combines many of
where cD‐ glucose (g/L)= concentration of D‐ glucose per liter the attributes of these methods, was subjected to an inter-
of undiluted extraction solution, cD‐fructose (g/L) = concen- laboratory evaluation involving 39 laboratories worldwide.
tration of D‐fructose per liter of undiluted extraction solu- The incubation conditions employed in AOAC Method
tion, cD‐galactose (g/L) = concentration of D‐galactose per 2009.01 (the integrated TDF method) are based on those
liter of undiluted extraction solution, EV = volume of solu- used in AOAC Method 2002.02; however, the scale of the
tion used in the initial extraction (20.5), EV/1,000 = ad- assay was increased 10‐fold to ensure that sufficient resi-
justment from g/L of undiluted extraction solution to g/ due was recovered for accurate gravimetric measurement.
volume of extraction solution actually used, W = weight of A major criticism of AOAC Method 2009.01 was that the
sample analyzed in g, and 100 = conversion of results to incubation time with PAA/AMG of 16 hr is not physi-
g/100 g. ologically relevant. In response, a modified method was
Available carbohydraytes (g∕100g) developed involving an incubation time with PAA/AMG of
4 hr. The concentrations of both PAA and AMG were in-
= D − glucose (g∕100g) + D − fructose (g∕100g)
creased to ensure that RS values obtained with this method
+ D − galactose (g∕100g) (the rapid integrated TDF method) were in line with those
Calculations are simplified using a Megazyme obtained with AOAC Methods 2002.02 and 2009.01 and
MegaCalcTM Excel®‐based calculator (AVCHO) in ileostomy studies. This method was successfully evalu-
Supporting Information. ated under the auspices of AOAC International and ICC to
become AOAC Method 2017.16 and ICC Method 185. In
the current work, these assay modifications were applied
2.8 | Hydrolysis of sucrose,
to the original resistant starch method (AOAC Method
Raftilose® and Raftaline® (FOS) by invertase
2002.02) in developing a rapid RS method. Incubations
(β‐fructofuranosidase) and sucrase
are performed in leak‐proof, disposable polypropylene
Aliquots of sucrose, Raftaline® or Raftilose® (1.0 ml, tubes, and sample amount (~100 mg) and buffer volume
10 mg/ml), in distilled water were incubated with either (4 ml) are one‐tenth of that employed in AOAC Method
invertase (1.0 ml, 200 U on sucrose) in 100 mM sodium ac- 2017.16. Enzyme concentrations (PAA 0.4 KU/4 ml; AMG
etate buffer (pH 4.5) or sucrase (1.0 ml, 40 U on sucrose) in 0.17 KU/4 ml; Table 1), buffer pH (6.0), incubation time
100 mM sodium maleate buffer (pH 6.5) containing BSA (4 hr), and incubation temperature (37°C) are the same as
(0.5 mg/ml) at 30°C. Incubations were terminated at 10, 30, that employed in AOAC Method 2017.16. Incubations were
or 60 min by placing the tubes into a boiling water bath for performed in a shaking water bath (200 linear strokes per
5 min. A zero time incubation was performed by incubat- minute) with tubes aligned in the direction of shaking to
ing the enzyme in the boiling water bath for 5 min before ensure complete sample suspension during the period of in-
adding the sucrose, Raftaline® or Raftilose®. All samples cubation (Figure 1). Resistant starch values obtained for a
were transferred to microfuge tubes and centrifuged at range of samples using AOAC Method 2002.02 and the new
8,000 g for 3 min. Aliquots of the supernatant solutions rapid RS method are shown in Table 2. Very similar values
were analyzed by HPLC using two TSKgel® G2500PWXL were obtained for a broad range of samples, but a slightly
columns, 30 cm × 7.8 mm, connected in series. The col- higher value was obtained for the native, high amylose
umns were operated at 80°C with distilled water mobile maize starch, Hylon VII, in line with results obtained with
phase at 0.5 ml/min. A Bio‐Rad Laboratories, cation/anion AOAC Method 2017.16 for dietary fiber. The repeatability
guard column (cat. no. 125‐0118) was employed to deion- of the rapid RS method was determined by analyzing seven
ize the samples. samples in duplicate over 4 days, and the results are shown
in Table 3. Interday repeatability values ranged from 2.17%
to 4.84% across a broad range of resistant starch levels, in
3 | R ES U LTS A N D D IS C U S S ION
line with values obtained in previous studies (McCleary et
al., 2002) where repeatability values ranged from 1.9% to
3.1 | Measurement of RS (rapid method)
3.0%. The very high interday repeatability standard devia-
and DS
tion for wheat starch relates to the very low level of RS
Through the European EURESTA research program, a in this sample. These results demonstrate that the rapid RS
range of methods for the measurement of RS were devel- method is as repeatable as AOAC Method 2002.02 and that
oped (Akerberg et al., 1998; Champ, 1992; Champ et al., very similar values are obtained for most samples. The sig-
1999; Englyst et al., 1992; Faisant et al., 1995; Goni et al., nificant advantage is that the incubation time is reduced to
|

TABLE 2 A comparison of the values

AOAC Rapid RS (100 mg Digestible starch
obtained for resistant starch content of a
2002.02 samples) procedurea
range of samples using AOAC Method
2002.02, the rapid resistant starch (RS) Regular maize starch (Lot 60401) 1.0 1.2 1.6
method, and RS measured as part of the High amylose maize starch (Lot 43.0 47.3 40.6
digestible starch/resistant starch procedure 60107)
described in this study Hi Maize 1043® (Lot 02161) 45.7 45.0 44.5
Hylon VII® (Ref 98GH8401) 48.6 52.3 48.7
Wheat starch (Sigma Lot S512L) 0.4 0.3 0.2
®
Novelose 330 42.0 37.5 37.0
Novelose 240® 42.9 42.6 43.1
®
Crystalline 40.9 36.7 37.6
Native potato starch (Avebe) 63.4 63.9 30.8
Potato amylose 35.6 35.3 32.9
®
Actistar 49.2 49.3 47.0
Heinz® baked beans (freeze‐dried) 3.6 3.8 4.3
®
Brennans whole meal bread 0.9 0.8 0.7
Canned bachelors butter beans 3.1 3.3 3.1
®
Kellogg's cornflakes 2.2 2.1 1.9
UB express boiled rice 2.4 2.4 2.3
Ryvita® dark rye crackers 1.7 1.9 1.9
a
In this procedure, an aliquot (4 ml) of the stirred reaction suspension was removed and added to 4 ml of etha-
nol and RS recovered and washed as in the rapid RS method, hydrolyzed, and analyzed.

the physiologically relevant time of 4 hr, and with this re- Fibersym® using either the Shukri et al. (2015), Shi et al.
duced time, there is no requirement for sodium azide pre- (2019), or the current 50°C‐NaOH procedure are shown. In
servative in the incubation buffer. all cases, Fibersym® was first incubated with PAA/AMG
according to the rapid RS method, and then, the residues
were dissolved and hydrolyzed according to Shukri et al.
3.2 | Measurement of phosphate cross‐
(2015), Shi et al. (2019), or the 50°C‐NaOH procedure. For
linked starch (RS4)
Fibersym®, the DS fraction was ~33% w/w (as is) (average
It is well known that the resistant starch component of of all samples) and the RS fraction for all three methods was
Fibersym® (RS4) is not quantitatively measured under the very similar at ~39.5%–42.0% (as is). On direct analysis of
conditions described for the dissolution and hydrolysis of the total starch content of Fibersym® with the Shukri et al.
other resistant starch fractions. RS4 does not dissolve in (2015) method, a value of 81.3% w/w (dwb) was obtained,
DMSO at ~100°C and is only partially soluble in 2 M KOH and the Shi method gave ~82.3% w/w (dwb), while for the
or 1.7 M NaOH at 0–4°C. Shukri et al. (2015) and Shi et al. 50°C‐NaOH procedure a value of ~84.0% w/w (dwb) was
(2019) have described two quite protracted methods for the obtained. In no case, values were higher than 84.0% (dwb)
dissolution and measurement of RS4. obtained. Total starch values obtained for wheat starch
In the current studies, a range of solvents and incubation using the 50°C‐NaOH procedure were ~94.9% w/w (dwb)
conditions were evaluated. Samples were suspended in ei- and that for Hylon VII were ~95.2% w/w (dwb) consistent
ther 100 mM sodium acetate buffer (pH 4.5) plus CaCl2 at with values obtained with other procedures. In an attempt
100°C, 1.7 M NaOH (at 4 or 50°C), or 2 M KOH (at 4°C, to further understand the key steps involved in the hydroly-
as in the rapid RS method). Following pH adjustment of the sis of Fibersym®, the effect of temperature of incubation in
alkaline solutions to ~pH 5.3, the solutions were incubated the presence or absence of 1.7 M NaOH, the role of ther-
with thermostable α‐amylase at 100°C followed by AMG mostable α‐amylase, and the time of incubation with AMG
at 50°C. In other options, the incubation with thermo- were studied and the results are summarized in Table 5. In
stable α‐amylase at 100°C was deleted (as per the rapid RS each case, DS was hydrolyzed and removed according to
method). In all cases, volumes were adjusted to 100 ml and the rapid RS method and an average value of 32.8% w/w
samples were removed for the determination of glucose. In (as is basis) was obtained. Values obtained for the RS frac-
Table 4, the starch values obtained for the RS fraction of tion varied significantly. Under the incubation conditions
|

TABLE 3 Repeatability study of the rapid resistant starch (RS) method for the measurement of RS in a range of food materials

Resistant starch, % (w/w)a, meanb ± 2 SD, (%RSDr)

Interday mean,
Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr)
Heinz® beans [InterLab 13.08.13] 3.4 ± 0 3.7 ± 0 3.6 ± 0.2 3.7 ± 0 3.6 ± 0.3
0.04 0.44 3.10 0.63 3.91
Starch wheat, unmodified, Sigma 0.3 ± 0 0.4 ± 0.1 0.3 ± 0 0.3 ± 0 0.3 ± 0.1
S5127, Lot 0490048 2.21 12.47 2.24 2.15 18.01
Kellogg's® corn flakes 20.12.10 2 ± 0.1 2.2 ± 0.1 2.1 ± 0 2.1 ± 0 2.1 ± 0.2
1.61 1.82 0.54 0.23 3.78
Tinned chick peas 20/7/11 4.3 ± 0.1 4.7 ± 0 4.6 ± 0.1 4.6 ± 0.1 4.5 ± 0.3
1.28 0.39 1.56 1.26 3.40
Semigreen banana 18.6 ± 0.3 18.7 ± 0.6 20 ± 1.1 20.6 ± 0.4 19.5 ± 1.9
0.81 1.72 2.65 0.95 4.84
Native potato starch Sigma S‐4851 70 ± 0.5 69 ± 1.3 71.6 ± 0.5 72.7 ± 0.2 70.8 ± 3.1
Lot 49H04211 0.36 0.91 0.32 0.11 2.21
Hylon VII® Lot 60901 47.3 ± 1.6 48.4 ± 0.5 49.5 ± 0.3 49.2 ± 2.1 48.6 ± 2.1
1.69 0.50 0.30 2.17 2.17
®
ActiStar (before purification) 46.7 ± 0.6 48.6 ± 0.1 49.7 ± 0.9 51.9 ± 0 49.2 ± 4
0.69 0.10 0.89 0.03 4.07
Abbreviations: %RSDr, repeatability standard deviation; SD, standard deviation.
a
All results are presented as starch on an “as is” basis.
b
On each day, samples of each material were analyzed in duplicate.

TABLE 4 A comparison of methods employed to measure total starch in Fibersym® and other starches, either directly, or as the combined
values of digestible starch and resistant starch

Digestible starch Resistant starch Total starch

Sample Method (as is) (as is) Total starch (as is) (dwb)
Fibersym® Shukri et al. (2015)a — — 72.4 ± 0.4 81.3 ± 0.7
b
Shukri et al. (2015) 32.8 ± 0.2 39.5 ± 0.1 72.3 ± 0.1 81.1 ± 0.1
Shi et al. (2019)a — — 73.3 ± 0.2 82.3 ± 0.2
Shi et al. (2019)b 33.0 ± 0.1 39.9 ± 0.1 72.9 ± 0.1 81.8 ± 0.1
a
NaOH 15 min at 50°C — — 73.8 ± 0.7 82.8 ± 0.8
NaOH 15 min at 50°Cb 32.9 ± 0.2 42.0 ± 0.2 74.9 ± 0.2 84.0 ± 0.3
NaOH 30 min at 4°Ca — — 69.7 ± 0.4 78.2 ± 0.4
b
NaOH 30 min at 4°C 33.0 ± 0.1 37.1 ± 0.9 70.1 ± 0.8 78.7 ± 0.8
Hylon VII® Shukri et al. (2015)a — — 76.1 ± 0.3 87.2 ± 0.3
Shukri et al. (2015)b 38.7 ± 0.9 38.2 ± 2.0 76.9 ± 1.5 88.1 ± 1.6
a
Shi et al. (2019) — — 75.0 ± 0.2 86.0 ± 0.2
Shi et al. (2019)b 38.6 ± 0.5 40.3 ± 0.7 78.9 ± 0.6 90.5 ± 0.7
NaOH 15 min at 50°Ca — — 83.0 ± 0.3 95.2 ± 0.4
b
NaOH 15 min at 50°C 38.6 ± 0.5 43.1 ± 0.6 81.7 ± 0.5 93.7 ± 0.6
NaOH 30 min at 4°Ca — — 83.8 ± 0.6 96.2 ± 0.7
NaOH, 30 min at 4°Cb 38.6 ± 0.1 43.3 ± 0.6 81.9 ± 0.5 93.9 ± 0.6
a
Wheat starch Shukri et al. (2015) — — 82.3 ± 0.1 93.0 ± 0.1
Shi et al. (2019)a — — 78.1 ± 1.2 88.2 ± 1.3
NaOH 15 min at 50°Ca — — 84.0 ± 1.2 94.9 ± 1.3
b
NaOH 15 min at 50°C 84.7 ± 0.7 0.2 ± 0.1 85.2 ± 0.7 96.3 ± 0.8
NaOH 15 min at 4°Ca — — 85.3/83.7 95.5/93.7
Note: The Shukri et al. (2015) and Shi et al. (2019) methods were performed as described by the authors except that the initial incubation with PAA/AMG was for 4 hr
according to AOAC Method 2017.16. The NaOH/50°C procedure was performed as described in this study.
a
Total starch determined directly.
b
Total starch determined as the combined values of digestible and resistant starch.
|

TABLE 5 The effect of digestion and enzyme treatments conditions on the measurement of the starch content of Fibersym®

α‐amylase Incubation Total

NaOH (15 min) (280 U, 100°C) time with Digestible starch Resistant Total starch starch
Temperature of Incubation 300 U g/100 g starch (g/100 g) (g/100 g) “as (g/100 g)
Samples incubation time, min AMG (min) “as is”a “as is” is” “dwb”
A Not included 15 30 32.8 ± 0.4 40.0 ± 0.6 72.8 ± 0.5 81.7 ± 0.5
B Not included 30 30 32.8 ± 0.4 40.5 ± 0.6 73.3 ± 0.5 82.3 ± 0.5
C Not included 60 30 32.8 ± 0.4 40.2 ± 0.5 73.0 ± 0.5 81.9 ± 0.5
D 4°C 0 30 32.8 ± 0.4 1.6 ± 0.2 34.4 ± 0.3 38.6 ± 0.4
E 4°C 30 30 32.8 ± 0.4 39.9 ± 1.0 72.7 ± 0.8 81.6 ± 0.9
F 50°C 0 30 32.8 ± 0.4 5.4 ± 0.6 37.2 ± 0.5 41.7 ± 0.6
G 50°C 30 30 32.8 ± 0.4 41.2 ± 0.5 74.0 ± 0.5 83.1 ± 0.5
H 50°C 30 60 32.8 ± 0.4 41.5 ± 0.6 74.3 ± 0.5 83.4 ± 0.6
Note: A. The RS containing residue was suspended in 8 ml of 100 mM sodium acetate buffer (pH 5.0) and incubated with thermostable α‐amylase for 15 min and with
AMG as shown in the table for 15 min.
B. Incubations performed as for example A, but incubation with thermostable α‐amylase for 30 min.
C. Incubations performed as for example A, but incubation with thermostable α‐amylase for 60 min.
D. The RS containing residue was suspended in NaOH at 4°C and stirred for 15 min. The solution was then neutralized with acetate buffer and AMG (330 U) was
added and incubated at 50°C for 30 min.
E. The RS containing residue was suspended in NaOH at 4°C and stirred for 15 min. The solution was then neutralized with acetate buffer; α‐amylase (280 U) was
added and incubated at 100°C for 30 min. Temperature was lowered to 50°C, and AMG (330 U) was added and incubated for 30 min.
F. The RS containing residue was suspended in NaOH at 50°C according to the rapid RS method. The solution was then neutralized with acetate buffer, and AMG
(330 U) was added and incubated at 50°C for 30 min.
G. The RS containing residue was suspended in NaOH at 50°C according to the rapid RS method. The solution was then neutralized with acetate buffer, and α‐amylase
(280 U) was added and incubated at 100°C for 30 min. Temperature was lowered to 50°C, and AMG (330 U) was added and incubated for 30 min.
H. Incubations were the same as for “G”, except that the incubation with AMG was for 60 min.
a
The value shown is an average of the determinations for all samples.

employed in the standard rapid RS method (1.7 M NaOH 120 min (values not shown) gave no change in the deter-
at 4°C) (Example D), little of the RS in Fibersym® was mined starch values for the RS fraction of Fibersym®. In
hydrolyzed (~1.6% w/w). Interestingly, very little of the no case did the combined value of the digestible starch plus
resistant fraction was hydrolyzed (~5.4% w/w) even if the starch from the residue (RS) fraction for Fibersym® (mea-
NaOH incubation was performed at 50°C (Example F). In sured as glucose with GOPOD reagent) exceed 84% w/w
all cases where thermostable α‐amylase was employed, the (dwb). The starch values determined using either DMSO
highest starch values were obtained. Similar values (~84% or NaOH to dissolve Fibersym® are compared in Table 6.
w/w) were obtained on incubation with thermostable α‐ With the DMSO format (AOAC Method 996.11), the starch
amylase, whether (examples G and H) or not (examples value obtained (66.0%) is approximately 80% of that ob-
A–C) the Fibersym® was pre‐treated with 1.7 NaOH at tained by the Shukri et al. (2015) procedure (Table 4), con-
50°C for 15 min. Increasing the incubation time with AMG sistent with the reports by these authors.
from 30 to 60 min (examples G and H) gave no increase In an attempt to try to understand why quantitative recovery
in the determined values. Increasing the incubation time of Fibersym® as glucose in starch‐type assays is not achieved,
with 1.7 M NaOH at 50°C from 15 min to either 30, 60, or the digested samples (total starch, digestible starch [DS], and

TABLE 6 Effect of dissolution solvent and temperature of dissolution on measurement of the starch content of Fibersym® and Hylon VII

Incubation time with

Incubation with solvent α‐amylase Total starch, % w/w dwb

Solvent Time Temperature Time at 100°C Fibersym® Hylon VII

DMSO (2 ml) 5 min 100°C 6 min in MOPS (pH 6.5) 66.0 ± 0.8 94.5 ± 0.9
KOH (2 ml, 2 M) 20 min 4°C 30 min in acetate (pH 5) 81.3 ± 0.4 95.1 ± 1.5
NaOH (2 ml, 1.7 M) 15 min 50°C 30 min in acetate (pH 5) 83.7 ± 0.2 95.6 ± 0.6
Sodium acetate buffer — 100°C 30 min in acetate (pH 5) 80.5 ± 0.1 87.6 ± 0.5
(10 ml, 100 mM)
|

digested resistant starch [DRS] fractions) were analyzed for and this represents 5 × 32.8/100% (i.e., 1.6%) of the total
both total carbohydrate by the phenol‐sulfuric acid procedure Fibersym® sample. The DRS fraction consisted of 87% mono-
and glucose by the GOPOD method. For reference, Hylon saccharide (shown to be exclusively glucose using GOPOD
VII® was analyzed concurrently and the determined glucose reagent), ~7% of disaccharides, 1% trisaccharide, and ~5%
was ~98% of the total carbohydrate value. For Fibersym®, glu- higher DP oligosaccharides. The di‐ and trisaccharide fractions
cose was ~92% of the total carbohydrate value, clearly demon- (~8%) were resistant to hydrolysis by AMG, and the higher DP
strating that the hydrolyzate still contained ~8% of chemically fraction (~ 5%) was hydrolyzed to an extent of <5% by AMG
modified glucose/gluco‐oligosaccharides that is resistant to (i.e., 4.8% resistant oligosaccharides). Thus, the combined re-
hydrolysis to glucose by AMG and thus not measured by the sistant fractions in DRS were ~13% of the total hydrolyzate.
glucose‐specific GOPOD reagent. To further identify the na- From Table 5, Example “B,” the DS fraction is 32.8% “as is”
ture of the resistant fractions, Fibersym® was hydrolyzed with and the resistant starch fraction is 42.8% “as is” determined
PAA/AMG according to the rapid resistant starch procedure as glucose. In allowing for the ratio of determined glucose to
(for 4 hr at 37°C). The DS fractions from 20 incubations were total carbohydrate contents (phenol‐sulfuric) of these two frac-
pooled, concentrated, and chromatographed on Bio‐Gel P‐2. tions, the total recovery of the sample is 32.8 × 100/95 (DS
The residues fractions in the PAA/AMG incubations were also fraction) + 42.8 × 100/87 (DRS fraction) = 34.5 + 49.2, or
pooled, washed with 50% aqueous ethanol, stirred in 1.7 M 83.7% “as is” (i.e., 93.7% “dwb”). The resistant starch fraction
NaOH at 50°C for 30 min, neutralized, and hydrolyzed with is ~55% “dwb” (from the DRS fraction) plus ~2% (from the
thermostable α‐amylase for 30 min at 100°C followed by AMG DS fraction), that is, 57% dwb, consistent with values obtained
for 30 min at 50°C. Under these conditions, the residue was by gravimetry in AOAC Method 2017.16.
essentially completely solubilized. The DRS fraction was also Hydrolysis of starch by PAA/AMG mixtures is critically
concentrated and chromatographed on Bio‐Gel P‐2. Fractions dependent on both the concentrations of the enzymes used and
were collected and analyzed for carbohydrate (Figure 5) using the time of incubation. In AOAC Method 2002.02, 0.2 KU of
the phenol‐sulfuric acid procedure (DuBois et al., 1956). The PAA and 0.014 KU of AMG are employed per assay (in 4.0 ml
DS fraction consisted of 89% monosaccharide (shown to be of buffer) and incubations are performed at 37°C for 16 hr.
exclusively glucose using GOPOD reagent), 4% disaccharide, This incubation time was chosen on the basis of previously
2% trisaccharide, and 5% higher DP oligosaccharides. The di‐ published work (Akerberg et al., 1998; Champ, 1992; Champ
and trisaccharides were completely hydrolyzed to glucose by et al., 1999; Faisant et al., 1995; Goni et al., 1996; Muir &
AMG, showing that they were maltose and maltotriose that es- O'Dea, 1992) and particularly because the RS value obtained
caped hydrolysis by AMG in the initial incubation with PAA/ for a set of reference samples was in line with results from
AMG. The higher DP fraction was hydrolyzed to an extent ileostomy studies (Champ et al., 1999). The time course hydro-
of <5% by AMG. Thus, the digestible starch fraction con- lysis of a range of starches under these conditions is shown in
tained ~5% of oligosaccharides resistant to digestion by AMG, Figure 6. In their studies on measurement of the RS content of

5
Digestible starch (DS) fraction of Fibersym
Glucose
Carbohydrate, mg/ml

4
Glucose = 89%
3 DP 2 =4%
DP 3 =2%
Higher DP =5%
2

1 Higher DP 2
3
0
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Tube number (20 ml/tube)

2.5
Digested resistant starch (DRS) fraction of Fibersym Glucose F I G U R E 5 Chromatography on
Carbohydrate, mg/ml

2.0 Bio‐Gel P‐2 of the “digestible starch” and

Glucose = 87 % hydrolysed “resistant starch” fractions
1.5 DP 2 =7%
DP3 =1% of Fibersym®. Samples (20 ml, 20, or
Higher DP =5%
1.0 30 mg/ml) were applied and eluted
with distilled water at 60°C. Fractions
0.5 Higher DP
2 (20 ml) were collected and analyzed for
3
0.0 carbohydrate using the phenol‐sulfuric acid
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 procedure [Color figure can be viewed at
Tube number (20 ml/tube) wileyonlinelibrary.com]
|

100 Wheat starch measured directly rather than by difference as in the Englyst
et al. (1992) procedure. A further difference is that highly pu-
Digestible starch g/100 g (dwb)

FiberRite
80 Cornflakes rified, standardized enzymes are employed and incubations
Fibersym
are performed in tubes that allow sample removal while the
Brennans WMB
60 incubation is continuing (Figure 3). Incubation conditions
Hylon VII
are identical to those in AOAC Method 2017.16 except that
40 Baked Beans
sample size, buffer volumes, and amounts of PAA and AMG
are all reduced twofold. Enzyme concentration, incubation
20 pH and temperature, and stirring conditions are the same.
Consistent with the method of Englyst et al. (1992), sample
0
aliquots are removed at 20 min to measure rapidly digested
0 2 4 6 8 10 12 14 16 starch (RDS) and 120 min to measure slowly digested starch
Time of incubation, hr (SDS). However, a third sample is removed at 240 min (4 hr)
to measure total digestible starch (TDS); [see “Measurement
F I G U R E 6 Time course hydrolysis of a range of starches and
of digestible (RDS, SDS, TDS) and resistant starch”—
starch‐containing foods by a mixture of pancreatic α‐amylase and
amyloglucosidase under the incubation conditions of the integrated
Methods]. This time is in line with the reported time of resi-
TDF procedure (AOAC Method 2009.01). Samples (0.5 ml) were dence of food in the human small intestine (Camalilleri et al.,
removed at various time intervals, the enzymes inactivated, and 2010; Deiteren et al., 2010; Geboes et al., 2003; Geypens et
glucose measured according to Materials and Methods [Color figure al., 1999; Miller et al., 1997; Sadik et al., 2003; Stotzer &
can be viewed at wileyonlinelibrary.com] Abrahamsson, 2010; Zarate et al., 2010). After 240 min (4 hr)
of incubation, a sample (4 ml) is removed to measure resistant
starch. The sample is added to 4 ml of ethanol, and all fur-
Fibersym®, both Shukri et al. (2015) and Shi et al. (2019) used ther washing, dissolution, and hydrolysis steps are the same as
the incubation conditions of AOAC Method 2002.02, except those employed in the rapid RS method. The repeatability of
that the incubation time with PAA/AMG was reduced from this method for the measurement of RDS, SDS, TDS, and RS
16 to 2 hr. Not surprisingly, much less of the Fibersym® was is shown in Tables 7‒10. Seven samples with a broad range
hydrolyzed, and thus, much higher RS values were obtained. of digestible and resistant starch values were analyzed, and
From Figure 6, it can be seen that under the incubation condi- excellent repeatability was obtained for each value for all of
tions of AOAC Method 2002.02, Fibersym® is hydrolyzed to the samples. Resistant starch values determined for a range of
an extent of ~70% after 16 hr (i.e., ~30% RS), but only 13% samples with the digestible/resistant starch method are com-
after 2 hr. With an incubation time of 2 hr, even wheat starch pared to values obtained with AOAC Method 2002.02 and
has a RS value of ~60% w/w, which clearly shows that using the rapid RS method in Table 2. Similar values were obtained
the PAA/AMG content employed in AOAC method 2002.02 with each of the procedures with the clear exception of native
with this shortened incubation time generates nonsensical potato starch, which is known to be a fragile starch and read-
results. AOAC Method 2009.01 employs the incubation con- ily damaged under incubation conditions that involve stirring
ditions of AOAC Method 2002.02, and a DF value of ~30% (McCleary & Monaghan, 2002).
w/w was obtained for Fibersym®. This method was recently
updated (AOAC Method 2017.16) by reducing the incubation
time to 4 hr while increasing the concentrations of both PAA
3.4 | Measurement of available
and AMG to ensure that the RS values obtained with a set of
carbohydrates
reference samples still aligned with ileostomy data. Under Available carbohydrates have been defined as the sum of sug-
these conditions, a RS value of ~60% w/w was obtained for ars (glucose, fructose, galactose, sucrose, maltose, lactose,
Fibersym®, still significantly less than the value of ~86% w/w and oligosaccharides) and complex carbohydrates (“malto”
(dwb) reported by Maningat, Seib, and Bassi (2013). dextrins, starch, and glycogen; Anon, 2003). Historically,
these have been measured individually by a combination
of enzymatic and HPLC procedures and the values pooled.
3.3 | Measurement of digestible starch and
The method described here involves the complete and spe-
resistant starch
cific hydrolysis of each carbohydrate to the component
The method described here for the measurement of digestible monosaccharides, glucose, fructose, and galactose and spe-
starch and resistant starch is aligned closely to the method cific enzymatic measurement of these in a single reaction
of Englyst et al. (1992), with the modification that samples cuvette. With the increased knowledge of starch hydrolysis
are removed for analysis at 20, 120 min, and also 240 min. in the human small intestine and the recognition of the im-
Furthermore, the resistant starch content of the sample is portance of resistant starch as a component of dietary fiber,
|

TABLE 7 Repeatability study on the

Rapidly digested starch, % (w/w)a, meanb ± 2 SD,
measurement of “rapidly digested starch” in
(%RSDr)
Interday mean, the digestible starch‐resistant starch assay
Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr) procedure
Regular maize 21 ± 1.4 19.9 ± 0.4 19.2 ± 1.3 19.7 ± 0.2 19.9 ± 1.6
starch 3.29 0.91 3.32 0.46 3.95
®
Hylon VII 7.4 ± 0.3 6.8 ± 0.5 6.5 ± 0.3 7 ± 0.4 6.9 ± 0.8
2.35 3.61 2.60 2.78 5.52
UB express 58.9 ± 2.7 59.3 ± 3.9 57.3 ± 0.7 57.2 ± 2.6 58.2 ± 2.9
boiled rice 2.29 3.32 0.60 2.24 2.48
ActiStar® 21.2 ± 0.2 20.6 ± 0.9 20.3 ± 0.8 22.5 ± 0.7 21.2 ± 1.9
0.43 2.11 1.91 1.65 4.38
Garden peas 13 ± 0.1 12.7 ± 0.3 12.8 ± 0 12.7 ± 0.1 12.8 ± 0.4
0.38 1.23 0.11 0.56 1.37
All bran 23.6 ± 0.3 24 ± 0.3 24.1 ± 0.2 23.9 ± 0 23.9 ± 0.4
0.56 0.62 0.44 0.03 0.83
Butter beans 17.9 ± 0.5 18.5 ± 0.2 19.3 ± 1.8 19.1 ± 1.4 18.7 ± 1.4
1.46 0.63 4.75 3.55 3.81
Abbreviations: %RSDr, repeatability standard deviation; SD, standard deviation.
a
All results are presented as starch on an “as is” basis.
b
On each day, samples of each material were analyzed in duplicate.

TABLE 8 Repeatability study on the

Slowly digested starch, % (w/w)a, meanb ± 2 SD,
measurement of “slowly digested starch” in
(%RSDr)
Interday mean, the digestible starch‐resistant starch assay
Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr) procedure
Regular maize 51.1 ± 2.5 48.4 ± 1.7 49.8 ± 1.2 47.3 ± 1.3 49.2 ± 3.3
starch 2.47 1.78 1.24 1.33 3.39
Hylon VII® 14.3 ± 0.1 16.5 ± 0.2 16.5 ± 0.5 17.3 ± 0.1 16.2 ± 2.4
0.31 0.75 1.62 0.42 7.42
UB express 13.6 ± 3.8 12.3 ± 1.4 11.7 ± 1.3 11.4 ± 0.4 12.2 ± 2.4
boiled rice 13.85 5.61 5.43 1.69 9.77
ActiStar® 5.3 ± 1.1 6 ± 0.2 5.7 ± 0 7 ± 0.2 6 ± 1.4
10.44 1.78 0.22 1.62 11.63
Garden peas 2.6 ± 0.3 2.7 ± 0.6 2.6 ± 0.1 2.5 ± 0.1 2.6 ± 0.3
4.97 11.28 2.44 1.15 5.97
All bran 1.4 ± 1.1 1.2 ± 0.7 0.7 ± 0.3 0.6 ± 0.3 1 ± 0.9
40.84 30.81 19.00 22.52 45.75
Butter beans 14.1 ± 2 13.3 ± 0.4 12.7 ± 0.3 12 ± 1.8 13 ± 2
7.21 1.62 1.35 7.44 7.55
Abbreviations: %RSDr, repeatability standard deviation; SD, standard deviation.
a
All results are presented as starch on an “as is” basis.
b
On each day, samples of each material were analyzed in duplicate.

it has become important to specifically measure digestible conditions described for the digestible starch/resistant starch
starch, rather than total starch, for the calculation of avail- procedure. A sample of the incubation solution is removed
able carbohydrates. Consequently, the procedure described and added to dilute acetic acid, and this is used in the deter-
here for the measurement of total digestible starch (TDS) mination of available carbohydrates. In the incubation with
forms the basis of the current method for available carbohy- PAA and AMG, maltose, maltodextrins, glycogen, and di-
drates. Samples are incubated with PAA and AMG under the gestible starch are hydrolyzed to glucose (with trace levels
|

TABLE 9 Repeatability study on the

Total digested starch, % (w/w)a, meanb ± 2 SD,
measurement of “total digestible starch” in
(%RSDr)
the digestible starch‐resistant starch assay Interday mean,
procedure Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr)
Regular maize 82.1 ± 0.4 79 ± 0.8 79.5 ± 0.4 79.8 ± 1.7 80.1 ± 2.7
starch 0.25 0.54 0.23 1.07 1.67
®
Hylon VII 33.2 ± 0.9 36.5 ± 1.7 35.7 ± 0.2 38.2 ± 0.6 35.9 ± 3.9
1.34 2.39 0.32 0.84 5.47
UB express 72.5 ± 1.3 70.2 ± 0.9 70.8 ± 0.4 71.8 ± 0.1 71.3 ± 2
boiled rice 0.93 0.66 0.29 0.06 1.40
ActiStar® 34.6 ± 0.4 34.1 ± 4.1 33.1 ± 0.8 35.6 ± 2.3 34.4 ± 2.7
0.55 6.05 1.21 3.21 3.89
Garden peas 16.9 ± 0 16.6 ± 0.4 16.3 ± 0.5 16.4 ± 0.1 16.5 ± 0.6
0.04 1.11 1.61 0.17 1.77
All bran 24.9 ± 1.5 25.2 ± 0.2 25.1 ± 0.1 25.1 ± 0.2 25.1 ± 0.6
2.93 0.32 0.22 0.45 1.27
Butter beans 34.4 ± 0.6 34.5 ± 0.2 34.7 ± 0 34.9 ± 1.8 34.6 ± 0.8
0.87 0.28 0.05 2.54 1.20
Abbreviations: %RSDr, repeatability standard deviation; SD, standard deviation.
a
All results are presented as starch on an “as is” basis.
b
On each day, samples of each material were analyzed in duplicate.

of maltose). On subsequent incubation with sucrase, maltase, invertase (β‐fructofuranosidase), but this enzyme also acts
and β‐galactosidase, lactose is hydrolyzed to glucose and on fructo‐oligosaccharides resulting in overestimation of the
galactose, sucrose is specifically hydrolyzed to glucose and sucrose. Hydrolysis of sucrose and Raftilose® (a commercial
fructose, and maltose is hydrolyzed to glucose (Figure 4). fructo‐oligosaccharide mixture) by invertase and sucrase is
Measurement of glucose, fructose, and galactose is shown in shown in Figure 8. Incubation conditions with invertase are in
Figure 7. Traditionally, sucrose has been hydrolyzed using line with those used in commercial sucrose assay kits. Under

T A B L E 1 0 Repeatability study on
Resistant starch, % (w/w)a, meanb ± 2 SD, (%RSDr)
the measurement of “resistant starch” in Interday mean,
the digestible starch‐resistant starch assay Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr)
procedure
Regular maize 1.9 ± 0.1 2 ± 0.1 1.9 ± 0 2 ± 0.1 2 ± 0.1
starch 1.52 2.77 0.07 2.77 2.47
Hylon VII 48.2 ± 0.1 47.7 ± 1.9 47.1 ± 0.2 46.6 ± 0.6 47.4 ± 1.5
0.14 1.95 0.17 0.60 1.60
UB express 2.5 ± 0.4 2.6 ± 0.3 2.6 ± 0.2 2.4 ± 0.2 2.5 ± 0.3
boiled rice 8.06 5.22 4.58 3.28 6.33
ActiStar 52 ± 0.1 52.3 ± 0.3 51 ± 0.1 51.8 ± 0.9 51.8 ± 1.1
0.07 0.25 0.13 0.88 1.05
Garden peas 7.9 ± 0.4 7.9 ± 0.3 7.6 ± 0.7 8 ± 0.6 7.9 ± 0.5
2.36 1.69 4.55 3.42 3.09
All bran 0.3 ± 0 0.3 ± 0 0.3 ± 0 0.3 ± 0 0.3 ± 0
4.36 0.95 0.14 3.69 3.96
Butter beans 3.5 ± 0.1 3.4 ± 0.1 3.5 ± 0.2 3.3 ± 0 3.4 ± 0.2
1.43 1.16 2.24 0.38 3.22
Abbreviations: %RSDr, repeatability standard deviation; SD, standard deviation.
a
All results are presented as starch on an “as is” basis.
b
On each day, samples of each material were analyzed in duplicate.
|

1.5 F I G U R E 7 Time course measurement

GalDH/GalM
of glucose, fructose, and galactose under
Glu (20 µg) + Fru (20 µg)
the incubation conditions described in
+ Gal (40 µg)
Absorbance, 340 nm

Figure 4 [Color figure can be viewed at

PGI
1.0 wileyonlinelibrary.com]
Galactose 80 µg

HK/G6PDH

0.5

Blank
0.0
0 5 10 15
Incubation time (min)

these incubation conditions, sucrase gives complete hydroly- completely hydrolyses sucrose to glucose and fructose within
sis of sucrose after 60 min (Sucrose + Sucrase/60) but has 10 min and also gives near‐complete hydrolysis of Raftilose®
no action on Raftilose (Raftilose + Sucrase/60) as required in the same time (Raftilose + Invertase/10). Clearly, in-
in the available carbohydrates method. In contrast, invertase vertase is unsuitable for the specific hydrolysis of sucrose in

FIGURE 8 HPLC chromatography on two TSKgel® G2500PWXL columns of the products of hydrolysis of sucrose and Raftilose® by either
invertase or sucrase enzymes as described in Materials and Methods [Color figure can be viewed at wileyonlinelibrary.com]
|

the presence of fructo‐oligosaccharides. The β‐galactosidase 13). Interday repeatability is excellent, with RSDr values
employed here is from Aspergillus niger and is known to ranging from 1.6 to 3.58.
give complete hydrolysis of galacto‐oligosaccharides (GOS)
as well as lactose. More specific hydrolysis of lactose can
be achieved with β‐galactosidase from Escherichia coli, but
4 | CONCLUSIONS
different incubation conditions (pH ~ 7.5) would be required.
Since GOS are rarely used in food products other than specific
4.1 | Digestible and resistant starch
baby formulations, the A. niger β‐galactosidase was chosen In this paper, we describe simple, reliable, and accurate meth-
for this methodology. The incubation format employed in the ods for the measurement of resistant starch (RS), digestible
measurement of available carbohydrates is shown in Figure starch (RDS, SDS, and TDS), and available carbohydrates
4. This differs from previously published formats in that ga- and highlight key considerations in each determination.
lactose is also measured. Accurate hydrolysis of digestible (nonresistant) starch is
The content of glucose, fructose, and available carbohy- a critical element of all of these determinations. The incu-
drates content of a number of food products is shown in Table bation conditions employed in each of these assays mimic
11. These products contained no lactose or galactose. In Table those used in the rapid integrated total dietary fiber method
12, the available carbohydrates content of three food prod- (AOAC Method 2017.16, ICC Method 185); the ratio of sam-
ucts, two of which contain lactose, is shown. Repeatability of ple weight, buffer volume, and concentration of enzymes are
the available carbohydrates method has been determined by the same. Under incubation conditions designed to measure
analyzing eight food products in duplicate over 4 days (Table total dietary fiber (AOAC Method 2017.16), available carbo-
hydrate values can be obtained simply by removing aliquots

T A B L E 1 1 Available carbohydrate
D‐glucose D‐fructose Available carbohy-
contentsa of a range of samples (“as is”
Samples (g/100 g) (g/100 g) drates (g/100 g)
basis, or freeze‐dried as stated)
Kellogg's® cornflakes 76.1 2.8 78.9
®
Kellogg's all bran 37.9 7.1 45.0
®
Weetabix 68.1 1.1 69.2
Kellogg's® Special K® 69.4 4.2 73.6
® ®
Kellogg's Frosties 67.1 13.4 80.5
Roma® macaroni pasta 69.1 0.0 69.1
Rooster potato (freeze‐dried) 50.9 0.8 51.7
Sweet potato (freeze‐dried) 46.6 17.6 64.2
Red onion (freeze‐dried) 28.2 24.2 52.4
Cauliflower (freeze‐dried) 11.8 11.4 23.2
Celery (freeze‐dried) 12.1 9.5 21.6
Broccoli (freeze‐dried) 7.1 6.2 13.3
Carrot (freeze‐dried) 26.8 18.3 45.1
Swede (freeze‐dried) 33.8 17.4 51.2
Red pepper (freeze‐dried) 24.3 37.9 62.2
Mushroom (freeze‐dried) 3.1 0 3.1
Ripe banana (freeze‐dried) 39.8 29.4 69.2
Red kidney beans (dry) 12.1 2.6 14.7
Soya bean (dry) 3.9 3.2 7.1
®
Heinz baked beans (freeze‐dried) 43.2 3.5 48.7
Ryvita® dark rye crackers 68.7 4.8 73.5
Wheat starch 86.3 0.0 86.3
Hylon VII® 33.4 0.0 33.4
Potato amylose 59.4 0.0 59.4
Regular maize starch 83.7 0.0 83.7
a
All values are the average of duplicate determinations.
|

T A B L E 1 2 Glucose, fructose,
Glucose Fructose Galactose Available carbohy-
galactose, and available carbohydrate
Sample (g/100 g) (g/100 g) (g/100 g) drates (g/100 g)
contentsa of three defatted and freeze‐dried
Chocolate chip cookies 43.9 1.78 0.75 45.45 food samples
Chocolate chip cookies 43.17 1.78 0.67 45.63
Chocolate peanuts 11.22 2.34 6.34 19.90
Chocolate peanuts 11.26 2.35 6.34 19.95
Jam tarts 57.80 10.24 0.00 68.04
a
All values are the average of duplicate determinations and on a dry weight basis.

T A B L E 1 3 Repeatability study on
Available carbohydrates, % (w/w)a , meanb ± 2 SD,
the measurement of available carbohydrates
(%RSDrc)
Interday mean, (glucose plus fructose) in a range of food
Sample Day 1 Day 2 Day 3 Day 4 ±2 SD, (%RSDr) products
Wheat starch 87.3 ± 2.1 90.2 ± 2.2 88.2 ± 0.6 90 ± 1.1 88.9 ± 2.8
1.21 1.21 0.34 0.61 1.60
All bran 43.2 ± 2.2 45.4 ± 0.4 43.2 ± 1 44.5 ± 0.2 44.1 ± 2.2
2.55 0.40 1.20 0.27 2.53
Sweet potato 59.6 ± 0.2 60.7 ± 1.3 58.2 ± 2.1 60.4 ± 1 59.7 ± 2.3
0.14 1.10 1.80 0.81 1.92
Ripe banana 65.4 ± 0.4 70 ± 0.3 67.1 ± 1 66.8 ± 0.6 67.3 ± 3.6
0.28 0.19 0.77 0.46 2.68
Carrot 53.7 ± 0.9 57.4 ± 0.6 55.1 ± 1.5 55.3 ± 0.3 55.4 ± 2.9
0.87 0.54 1.36 0.23 2.58
Red pepper 51 ± 0.5 55.4 ± 2.5 53.8 ± 3.1 52.9 ± 1.9 53.2 ± 3.8
0.49 2.25 2.92 1.83 3.58
®
Ryvita 60.6 ± 1.3 61.5 ± 1.3 61 ± 2.4 62.6 ± 0.7 61.4 ± 2
1.07 1.04 1.96 0.60 1.61
Swede 55 ± 4.3 53.6 ± 0.4 54.2 ± 1.3 54.1 ± 2 54.2 ± 2.1
3.92 0.34 1.19 1.82 1.96
Note: The repeatability (%RSDr) of the available carbohydrates assay method was assessed using eight milled
samples. For each sample, duplicate extractions were processed and applied to the assay on each day across
four separate days.
The available carbohydrate content of the samples tested covered a working range of 44.1%–88.9% (w/w) on
a dry weight basis. The repeatability (%RSDr) across this sample data set was excellent, less than or equal to
3.58% for all samples.
a
All results are presented on a dry weight basis.
b
On each day samples of each material were analyzed in duplicate.
c
SD = standard deviation. %RSDr = repeatability standard deviation.

(e.g., 0.2 ml) from the incubation solutions for available car- rapidly available glucose (RAG) and insulin response. RAG
bohydrate analysis. The digestible starch methodology de- is derived from free glucose, maltodextrins, and starch that
scribed here allows a comparison of the rates of digestion of are digested within 20 min. In the current method, separate
various starches and should be useful in monitoring food pro- measurement of the glucose, fructose, and galactose derived
cessing conditions aimed at producing slowly digested and from rapidly digested carbohydrates, together with the known
resistant starch. As detailed here, “available carbohydrates” glycemic index for each sugar, makes it possible to determine
are determined on samples removed at an incubation time the glycemic index of the food.
of 4 hr. However, a measure of “glycemic carbohydrates,”
that is, the carbohydrates which affect the glycemic index,
can be obtained by removing samples from the incubation
4.2 | Phosphate cross‐linked starch (RS4)
mixture at 20 min. Englyst, Englyst, Hudson, Cole, and Digestible starch is the major contributor to available carbo-
Cummings (1999) have shown a good correlation between hydrates in many foods. Starch has been modified in several
|

Dietary fiber or Digested starch, % w/w (dwb)

100
Wheat starch Hydrolyzed
(measured as glucose)
80
Fibersym as DF

20
Fibersym Hydrolyzed
(measured as glucose)

0
Wheat starch as DF
70 75 80 85 90 95 100
Temperature of incubation with -amylase, oC

92 % 75 % 17 % 5%

-Amylase activity left after 30 min at stated temperature

FIGURE 9 Hydrolysis of wheat starch and Fibersym® under conditions exactly as described for AOAC Method 985.29, but with incubations
performed at 70, 80, 90, 95, and 100°C. Incubations with protease and AMG were as described in AOAC Method 985.29. A sample of the
incubation solution (10 ml) was removed and immediately filtered. A sample (1 ml) was added to 25 ml of distilled water and mixed. An aliquot
(0.1 ml) of this was incubated with 30 U of AMG at 40°C for 15 min and measured for glucose using a glucose oxidase/peroxidase reagent. DS
was calculated from glucose value, and the dietary fiber value (RS) was determined by subtracting the DS value from the total sample weight.
The α‐amylase activity remaining in the incubation mixtures was determined using the Ceralpha® method [Color figure can be viewed at
wileyonlinelibrary.com]

ways to reduce digestibility and contribute positively to the the incubation time with PAA/AMG is 4 hr, and concentra-
dietary fiber content of the food. One of the most widely tions of both PAA and AMG were adjusted to ensure that
used, chemically modified starches is phosphate cross‐linked the measured RS values obtained for a number of reference
starch (e.g., Fibersym®). Measurement of this, either directly samples were in line with ileostomy data. Under the incuba-
or indirectly, is a matter of ongoing debate. Maningat et al. tion conditions of AOAC Method 2017.16, the DF values ob-
(2013) have reported fiber values for phosphate cross‐linked tained for most RS containing samples were similar to those
starch (e.g., Fibersym®) in excess of 85% w/w (dwb) using obtained with AOAC Method 2009.01. One major exception
the Prosky et al. (1985) dietary fiber method (AOAC Method is Fibersym®, for which a DF value of ~60% was obtained
985.29/AACC Method 32‐05.01). To obtain these values, as compared to a value of ~30% w/w obtained under the ex-
the incubation step with heat‐stable α‐amylase must be per- tended incubation conditions of AOAC Method 2009.01.
formed at 98–100°C. At incubation temperature below 98°C, In a recent report on the in vivo digestibility of cross‐linked
the determined dietary fiber value plummets (Figure 9). For phosphorylated (CLP) starch in ileostomy subjects (Iacovoua
example, at an incubation temperature of 95°C, a DF value of et al., 2017), DF output values of 40% w/w (average over 10
just 43% is obtained and this drops to 28% at 80°C. Clearly, patients) were obtained for Fibersym® using the Prosky et al.
the values obtained are method dependent and the method (1985) method. The authors state that “the 40% in vivo RS for
employed has no relationship to digestion conditions in the CLP wheat starch determined by the Prosky assay to quantitate
human small intestine, and therefore, the Prosky dietary fiber outgoing fiber in ileostomy subjects is erroneously low” and
method is simply unsuitable for use with samples containing that the “main cause of the low recovery is due to α‐amylase
RS4. AOAC Method 2009.01 was developed as an alterna- damage to granules of CLP wheat starch in a subject's small
tive method for the measurement of total dietary fiber with intestine.” These results and the authors' conclusions are, in
an attempt to obtain physiologically relevant measurement fact, consistent with the results obtained with the rapid inte-
of RS. However, since an incubation time with PAA/AMG grated TDF procedure (AOAC Method 2017.16). Starch gran-
of 16 hr was employed, the method was not considered to ules, including RS4, are digested to different extents by PAA
be physiologically relevant. Literature reports indicate that during passage through the human small intestine, highlighting
the time of residence of food in the small intestine is ~4 hr the importance of simulating in vivo digestion conditions in in
(±1 hr); consequently, a modified integrated TDF method vitro assays of RS and DF. However, the authors, not satisfied
(AOAC Method 2017.16) was developed. In this method, with the experimental results obtained, introduced a “recovery
|

correction factor” to adjust the experimentally determined ORCID

fiber values to values in line with what they obtained for na-
Barry V. McCleary https://orcid.
tive CLP using the Prosky et al. (1985) procedure; a physio-
org/0000-0002-7103-5759
logically nonrelevant in vitro assay. The “recovery correction
factor” was based on the ratio (~80%) they obtained for starch
measured with AOAC Method 996.11 (DMSO format) and R E F E R E NC E S
the Shukri et al. (2015) method for six 2‐hr ileostomy effluent
samples apparently chosen at random from their study. It is Akerberg, A. K. E., Liljeberg, H. G. M., Granfeldt, Y. E., Drews,
A. W., & Bjorck, I. M. E. (1998). An in vitro method, based on
not clear why the authors simply did not analyze the starch
chewing, to predict resistant starch content in foods allows paral-
content of the total effluents with the “quantitative” Shukri et
lel determination of potentially available starch and dietary fiber.
al. (2015) method. In contrast to the starch values of ~100% Journal of Nutrition, 128, 651–660. https ://doi.org/10.1093/
w/w obtained by Shukri et al. (2015) and Shi et al. (2019) v for jn/128.3.651
Fibersym®, we were able to obtain values no higher than 84% Anon (2003). McCance and Widdowson's, The composition of foods,
dwb, independent of which assay format was used. In the re- sixth summary edition, Food Standards Agency, RSC.
ported ileostomy study of Iacovoua et al. (2017), the average in AOAC International (2012). Official methods of analysis of AOAC
vivo recovery of wheat starch was 10.8% w/w, which is much International (19th ed.). Methods 925.10, 985.29, 991.42, 991.43,
993.19, 994.13, 996.01, 2001.03, 2002.01, 2002.02, 2009.01, and
higher than the <1% w/w values obtained in other ileostomy
2011.25. Rockville, MD: AOAC International.
studies and in vitro assays. The authors suggest that this high Camilleri, M., Thorne, N. K., Ringel, Y., Hasler, W. L., Kuo,
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last amended 2010. Rome, Italy: FAO.
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by gas‐liquid chromatography of constituent sugars as alditol ac- McCleary, B. V., McNally, M., & Rossiter, P. (2002). Measurement of
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Additional supporting information may be found online in
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Cereal Chem - 2019 - McCleary - Measurement of Available Carbohydrates Digestible and Resistant Starch in Food

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Cereal Chem - 2019 - McCleary - Measurement of Available Carbohydrates Digestible and Resistant Starch in Food

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Received: 14 July 2019

| Revised: 19 August 2019

Measurement of available carbohydrates, digestible, and resistant

Barry V. McCleary | Ciara McLoughlin | Lucie M. J. Charmier | Paraic McGeough

Megazyme, Bray, Ireland

1 | IN T RO D U C T ION interlaboratory evaluation, was adopted as AOAC Methods

assayed using AMG Assay Reagent (Megazyme R‐AMGR3)

2.3 | Measurement of RS and DS using the

bottoms of two 16 × 100 mm tubes, 0.1 ml of AMG (100 U/

Sample Buffer Pancreatic

U/ml of K Units K Units

mixed. Aliquots (2.0 ml) were centrifuged at 8,000 g for

F I G U R E 4 Procedure for the

TABLE 2 A comparison of the values

Resistant starch, % (w/w)a, meanb ± 2 SD, (%RSDr)

Digestible starch Resistant starch Total starch

α‐amylase Incubation Total

Incubation time with

Solvent Time Temperature Time at 100°C Fibersym® Hylon VII

2.0 Bio‐Gel P‐2 of the “digestible starch” and

TABLE 7 Repeatability study on the

TABLE 8 Repeatability study on the

TABLE 9 Repeatability study on the

1.5 F I G U R E 7 Time course measurement

Figure 4 [Color figure can be viewed at

Dietary fiber or Digested starch, % w/w (dwb)

-Amylase activity left after 30 min at stated temperature

correction factor” to adjust the experimentally determined ORCID

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