Measurement of resistant starch: factors affecting the amount of starch escaping digestion in vitro13 G 1.141/jr and

Jane

ABSTRACT

Kerin

We

developed

mimics

the physiological

system

all undigested

The

richest

peas. amounts

bananas

and

0.032

was

of RS were RS

termed

firm

assay

bananas oats

and

(0.161

wt,

respectively.

roasted

gig

(RS).

diet.

wt)

In resisting fiber,

and

to 0.028

potato

over-

night at 4 #{176}C produced RS. Whole rice contained

a 2.8-fold increase in the amounts of more RS than did ground rice whereas

grinding

amounts

did

Amounts

not

affect

of RS

dicating

that

caping

digestion.

in food

chewing

foods and processing escaping digestion 1992:56: KEY

can

This

of

decreased also

assay

RS

in roasted

with affect

may

techniques in the small

the

amount

of starch

be useful

in predicting

result in high intestine.

amounts

Am

Resistant

chewing,

in vitro

starch,

starch

measurement

digestion,

food

the

assay

was

pro-

used

techniques

Introduction

escaping

Materials

contain variable small intestine

shown

amounts and pass

that

many

carbohydrate-rich

ofstarch into the

that colon

escapes (1-4).

foods

for many are

reasons.

physically

ray diffraction, have

starches

possess

starch

inaccessible

come accessible ifthe pattern of the starch starches

First,

also been an

affects

granules

(6-9). granule,

digestibility

classified

into

A pattern,

the

enzymes

tuber

and

be-

The

categories: (potato)

oats)

the B-type

is digested starch

without

(eg, potato

cooking and

common cereal

starches

have

is poorly

until

cooked (gelatinized) (10). Finally, the two major polymeric forms of starch found in food, amylose and amylopectin, can recrystallize (ie, retrograde) when cooled after cooking, in which form they amylose

are

resistant

to enzymic

retrogrades

.4,;? J Cliii Nuir

to a form

1992:56:123-7.

digestion

(4,

1 1). In

that

is highly

resistant

Printed

in USA.

particular, to redis-

© 1992 American

were Best

effect

dietary

diabetes (15). the

fiber

(1 3), cardio-

of RS in the

diet

amount

can

be

of RS in

in vitro assay that mimics for starch digestion. This of various

and

Society

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purchased

(Kellogg,

food-processing

roasting)

on the amount

roasted

Torquay.

Victoria,

obtained

from

work

Botany,

local

supermarkets.

local

Australia),

long-grain

boiled

with

samples

and

The

mouth

was

at the

firm tips

at 4 #{176}C), and

rinsed

for

prepared

10-1

(eg, boiled

ie, to the with

it was carefully the number

stage

water

before

removed of times

Council of Australia. 3 Reprints not available. Received June 3. 1991. Accepted for publication

October

were

bananas only.

used

Rice

until

in

[whole

potatoes were

were cooled

firm

bananas

very

soft.

or boiled just

Foods,

bananas

cooked,

5 mm

before

and

cooled)

swallowing.

chewing,

and

from the a particular

mouth. food

I From the Department of Human Nutrition, Geelong. Australia. 2 Supported by a grant from the National Health

Nutrition

& Asian firm

was boiled for 10- 1 5 mm, (cooked and cooled potatoes

were normally,

and The

green

in water

chewed

Gourmet

potatoes,

supermarket.

yellow

and

for Clinical

Rolled

Victoria,

Australia),

(Health,

in a refrigerator

g) were

NSW,

Australia),

and ground ( 1 mm)] boiled for 20-30 mm overnight

from Wahgunyah,

chickpeas

the

were

Foods,

rice (Sunrice Australia, Leeton, NSW, Aus(Naytura Health Food Co. Mulgrave, Victoria,

chewing the food some experiments

whereas digested

used Bull,

Australia),

Food

the

(ungelatinized)

banana)

in which

digestion.

short-grain white tralia), chickpeas

(220

a B pattern, whereas pea and bean starches are intermediate between A and B and have been designated as type C (10). These differences in the crystalline nature ofstarch dictate susceptibility ofthe starch to enzymic digestion, for example the A-type starch (eg,

(John

this

in whole

Second, the crystalline as determined by xless (10).

three

in the small

present

to digestive

food is ground in the starch

foods

digestion in the This fraction of

starch has been called resistant starch (RS) (4, 5). Starch in food may resist digestion and absorption intestine

the grinding,

dietary

by anaer-

and methods

cornflakes have

Areas include cancer

to determine

to examine

(eg, cooking,

like

in the lange intestine health benefits cur-

and

significance

is needed

RS,

human

starch oats

studies

fiber.

physiological

a method

All foods

Recent

to dietary

amylase

in the

intestine,

in the colon ( 12). Therefore, many ofthe characteristics to be beneficial ( 14), and bowel

pancreatic

naturally

for fermentation

food. In this study we describe a simple crudely the physiological conditions

Nutr

with

small

as substrate

es-

of starch

of resistant

in the

available

shown disease

ofstarch

WORDS

hydrolysis of RS occur

studied,

which

J C/in

digestion

attributed

has been vascular

and

types

in-

123-7.

cessing,

rently

Before

chewing,

water

becomes

obic bacteria it may share

chickpeas.

increased

in boiling

1 1 ). All of these

chick-

dry

boiled

persion (4,

different

by cooking

Cooling

that In this

starch

produced

g/g dry wt) was reduced

g/g dry

system

digestion.

resistant

techniques

in uncooked

(0.247

in vitro for starch

food-processing

of RS.

firm

an

conditions starch

sources

Different

0 ‘Dea

Deakin and Medical

after In was

University, Research

23. 1991. 123

124

MUIR

AND

Chewed food sample 0. 1 g carbohydrate

I 200

Incubate

Add I mL pepsin in 0.02 mol HCI/L. 31)

mm

>< g and

(RDS)].

.

(1 g/L

O’DEA

Neutralize

with

water

bufl#{232}r:these

bath

natant

NaOH

1

Add

5 mL 0.2

mol acetate

buffer/L

(pH 5.0) Readily Digested and Resistant

+

Starch free glucose

free

+

sample

Starch

Add

1 mL enzyme

incubate water

the

and

I mL enzyme

solution

and incubate overnight in shaking water bath

in shaking

To this

bath

Centrifuge

at 37 #{176}C

(10mm

measured

at 12(X) x g)

and reserve supernatant. Readily Digested Starch free glucose fraction

was

pension

boiled

1200

out up to 10 mL

Homogenize (final volume

pellet in acetate approximately

.t Total +

Resistant Readily + free

glucose

L.__

Add

Termamyl

x g) and resete

and starch

cose

pellet

in I mL

(vonex

every

DMSO.

boil

6() mm

20 mm)

.L Pool DMSO

sample

reserved

with

supernatant

.L 2 mL enzyme solution. incubate in shaking water bath

Add

for 6() mm at 51) #{176}C

collect

FIG I. Procedure 6Enzyme solution crease.

aliquots

for glucose

for measuring in vitro amounts is a mixture of amyloglucosidase

ofresistant

and

starch.

porcine

pan-

Separate

samples

cose

in each

assay.

that

samples

were

and

then

and

porcine + RS

was 98% procedure.

uals was studied (see below). Chewed samples were weighed

accurately

carbohydrate The sample

food-composition in a l-mL pepsin

Louis water, water

as calculated from was then suspended

MO) mixture pH 2.0) and bath.

This

ment

approximates

then

neutralized

exposure

time with

average. mixture. for

to an acid-proteolytic

spent

in the stomach.

sodium

hydroxide

acetate buffer/L (pH 10 mg ofa porcine

ture (Halcyon Chemicals, 28 U amyloglucosidase buffer ranges

Sandringham. (Sigma, St Louis

it is the optimal

These

samples

6 h at 37

#{176}C. This

approximates After this

0.

1 g

tables ( 16). (Sigma, St

the transit incubation,

were

solution

the

was

addition

of

time

time through samples were

was

chosen

the small centrifuged

water

taken the

ground

it

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subjected

from

this

diagram

to the and

with

glucose

assay

each

reading.

of glucose

reproducibility

exNon-

in this of this

RS amounts

free

procedure

of the

included

a source The

step

(amyloglucosidase

free glucose)

by measuring

before

between

the food

quantity

carassay

in cornflakes

individuals

was

which

chickpeas,

studied.

5 male) were prothe total number

was swallowed.

of food,

16 g roasted

number

of times

starch

Each was

subject

either

30 g boiled

oxidase

by 0.9

to convert

analysis

were

was

whole

long-grain

used

(Glucose

or rice,

with

this

free analysed

final

assay

back

by

using

All results

was

analyzer Japan). did

glucose

glucose

chewed. measured

GOD-PAP.

Tokyo,

The

was glucose

on an automatic

assay.

of variance.

food released

Mannheim.

of DMSO oxidase

the

method

Germany)

glucose Results

a particular

hydrolysis

Boehringer

tration

because

except

RDS

not

in chewing

same

(eg.

Mannheim.

intestine ( 18). for 10 mm at

above

(ie,

were

above

free glu-

and 15 g cornflakes with 70 mL water). The results were used to calculate an average and a range of RS values in relation to

705:

bath

the

and

the pepsin

being

background

in

control. variation

a glucose

ofthe intestine approximately

in a shaking

The

after

For these experiments. 10 subjects (5 female, vided with a range offoods and asked to record

After

Victoria. Australia) and MO) dissolved in acetate the length as being

are system.

monthly

with

recovery

starch)

digestion

checked

the

pH for the digestive-enzyme

incubated

incubation

The

before

no

for any

glu-

6 h digestion

to those

a schematic

polysacchanides

ofchews

environ-

1 presents

for

remaining

ofstanch

before

and

(containing

as quality

given

5.0) and 1 mL of enzyme pancreatic enzyme mix-

(pH 5.0). Although the pH along from 2 to 7 ( 1 7). 5 was selected In addition,

to contain

was

starch

(amyloglu-

after

mixture

to the for a fur-

taken

of starch

similar

average

to account

bohvdrate

(1 g/L. made up in 0.02 mol HCI/L acidified incubated for 30 mm at 37 #{176}C in a shaking

short

5 mL of0.2 mol mixture containing

whereas individ-

Figure

.

were

amounts

overnight The

total

=

solution

the enzyme

pancrease) amylase.

enzyme

or homogenized

with

back

Aliquots

were

was boiled

to dissociate

was incubated

in conjunction

Procedures

pellet and

is used

amylose)

taken

centrifuged

The

supernatant

amount

the total

ground

incubated

starch and kept constant for that food. the variation in chewing between

were

to measure

then

(0.1

the sus-

extract was then pooled transfer was carried

mixture

total

120L

(DMSO)

DMSO

reserved

retrograded

experiment

Blanks

was recorded experiments

The

(including RS.

periment chewed in other

were

and

buffer.

and

reserved.

2 mL

and

acetate

Termamyl

Samples

pancrease).

determination.

glucose

determination

porcine

mortar Homogenizer

with

sulphoxide

the

a

Denmark).

20 mm).

superbelow.

(3 X 10 s on high).

1.5 mL

amylase

combined

RDS

Xl0/20

Bagsvaerd.

at 50 #{176}C with

and

heat-stable

.L Centrifuge.

10-1

the

with

Ystral

the supernatant

adding

the pellet represented

supernatant

J. Resuspend

Germany)

every

The

60 mm

cosidase

(0 1 L/1 0 L). boil 30 mm

(10 mm at l2X)

Centrifuge

starch digested glucose

West

dimethyl

(vortexing

extract.

ther

.L

Starch

free

an

for 30 mm.

by carefully

DMSO

buffer I 1.5 mL)

and acetate

glucose.

the retrograded amylose (5). The DMSO with the reserved supernatant. Quantitative

.

Make final volume with acetate buffer

free

with

A/S.

in 1 mL

for 60 mm

starch

as described

ground

heat-stable

X g) and

resuspended Resuspend pellet in 1.5 mL buffer and centrifuge. Repeat washing step three times and pool washings with Readily Digested Starch + free glucose fraction

the

with

amylase

then

up to

Industri

was

( 10 mm,

+

added

digested

by resuspending

pooled

and

was

made

L)(Novo

then

RDS

pellet

volume

times

heat-stable

homogenized

final

L/l0 Add

were

(Ballrechten-Dottingen.

solution5

6 h at 37

or

[readily

three

1200 X g) the pellet with 1.5 mL

with

washed

pestle

glucose

.

Homogenize

mm.

treated

fraction

The

removed

was washed

washings

and

This Total

supernatant

pellet

centrifuging ( 10

pH 2 0)

at 37 #{176}C in shaking

the

The

(Hitachi The

not

by

Boehninger final

interfere

reading

was

System concenwith

the

multiplied

to polysaccharide. Student’s

are expressed

t

test

or one-way

as . ± SD ( 19).

MEASUREMENT TABLE I Effects of chewing

and grinding

on amounts

of resistant

OF

starch

RESISTANT

125

STARCH

in foods6

Total

number

Food

(11

of chews 10)

=

Resistant

starch

g/IOO g do 30 30 IS 15 16 16 6

t.

81 23 93 78 186 77

g Whole long-grain white rice g Ground long-grain white rice g Whole cornflakes gGround cornflakes g Whole. roasted chickpeas g Ground, roasted chickpeas

#{149}SD (range-from Significant Significantly

the smallest

to the largest

number

difference between smallest and largest different from whole rice. P < 0.001.

II Significantly

different

from

whole

cornflakes.

P

18 (55-109) 9 (6-50) 39 (56-190) 32 (28-137) 9 1 ( I I 7-350) 26 (25-127)

± ± ± ± ± ±

on

that

amounts

and

of RS

roasted

in boiled

long-grain

rice,

are

from

55 to 109

and rice,

in Table

I

of chews:

for 30 g boiled

whole

and

was

1). decrease

in RS amounts

and the amounts white

chewing

were .

the smallest

Table

1 ). Data

± SD compared. The of RS are significantly

rice.

= 0.80) Whole,

(ie, when

compared.

However,

between roasted

long-grain Because

there

chewed chickpeas

white these

was

no

and

largest

for chewing

number pooled

significant

(P

difference

whole and ground. roasted chickpeas. required more chewing than did whole

(P

for some of times