Nutrition Research and Practice (2009), 3(2), 149-155
DOI: 10.4162/nrp.2009.3.2.149
?2009 The Korean Nutrition Society and the Korean Society of Community Nutrition
Effect of heat treatment of digestion-resistant fraction from soybean on retarding
of bile acid transport in vitro*
Sung-Hee Han1,2, Seog-Won Lee3 and Chul Rhee4§
1Institute of Life Science and Natural Resources, Korea University, 1 Anam-dong 5-ka, Sungbuk-gu, Seoul 136-701, Korea
2Present post; Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, 3327-204
Naka-Machi, Nara, Nara 631-8505, Japan
3Department of Food and Nutrition, Yuhan College, 185-34 Goean-dong, Sosa-gu, Bucheon, Kyeonggi 422-749, Korea
4Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, 1 Anam-dong 5-ka,
Sungbuk-gu, Seoul 136-701, Korea
Received January 29, 2009; Revised May 6, 2009; Accepted May 25, 2009
Abstract
In this study, we investigated the heat effect of digestion-resistant fraction (RF) from soybean on retarding bile acid transport in vitro. The RFs
from soybean retarded bile acid transport. A raw, unheated RF of soybean (RRF-SOY) was significantly more effective than the heated RF of
soybean (HRF-SOY). The RS1 which physically trapped in milled grains and inaccessible to digestive enzyme after 18 hrs incubation level of
content in RRF-SOY was found to be as high as 24.1% and after heating the RS1 of HRF-SOY was significantly reduced to 16.8%. The X-ray
diffraction pattern of RF from soybean was altered after heat treatment. The RFs from soybean were characterized by peak at diffraction angles
of 12.0° and 20.0° corresponding to RS content. Cellulose contents of RRF-SOY was 5% higher than that of HRF-SOY and pentosan contents
of RRF-SOY was 5% higher than that of HRF-SOY, too. Whereas the hemicellulose content of RRF-SOY was 13% lower than HRF-SOY.
Key Words: Digestion-resistant fraction, thermal processing, resistant starch, xylose
Introduction12)
properties of RF, which, in turn, affects the quality and quantity
of RS and NSP. RF from unheated source, so-called Saeng-shik
The digestion-resistant fractions (RFs) consisted of resistant
in Korea, has more RS and shows more effectiveness in lowering
starch (RS) and non-starch polysaccharides (NSP) have important
diabetes and cardiovascular disease risk compared to RF from
implications for diabetes, colon cancer and blood lipid chemistry
processed sources (Park et al., 2003). Soybeans widely ground
(Anderson et al., 2000; Hinggins et al., 2004; Leeuw et al., 2004;
and consumed in various regions of the world, are rich and
Leu et al., 2002). The potential mechanisms for this health
relatively inexpensive sources of proteins and carbohydrates. Its
include the promotion of beneficial microflora that uses NSP of
carbohydrates are composed of starch and nonstarch
RF as a substrate in colon. And the health benefits like RS is
polysaccharides as resistant starch, cellulose, hemicellulose, and
a reduced glycemic index. Particularly, RF was found to have
pentosan. It was known that they affected colon cancer, obesity
great effects on various disease risks with resistant starch 1 (RS1),
and cardiovascular disease (Han et al, 2004; Lee et al., 2006).
which is physically trapped in milled grains and inaccessible to
However, the change in the amount of RS and NSP of soybean
digestive enzyme after 18 hrs incubation (Annison & Topping,
during heating may be partly attributed to the redistribution of
1994; He et al., 2005). Also, the RF feeding had the increased
RF. Also, the governing factors for the physiological
the bile acid excretion in mice stool samples of apolipoprotein
functionality of RF have not been determined.
deficient mice after feeding RFs (Han et al., 2006)
In the present study, the effects of change of functional factors
Several studies have shown that ungelatinized starch, resistant
(RS1 and NSP) of RF from soybean on bile acid transport were
starch 2 (RS2) of RF, which is abundant in raw potato, banana,
investigated.
and high amylose maize starch, are not degraded by digestive
enzymes after 4-5 hrs incubation in vitro (Mccleary & Monaghan,
2002). Heating of plant food alters the physical and physiological
* This work was supported by a Korea University Grant.
§ Corresponding Author: Chul Rhee, Tel. 82-2-3290-3023, Fax. 82-2-928-1351, Email. rhee2@korea.ac.kr
150
Effect of heating on digestion-resistant fraction
Material and Methods
though 2? range from 5° to 40°.
Preparation of RFs
Content of cellulose and hemicellulose
Soybeans [Glycine max(L.) Merr.] was purchased from Sunhan
Cellulose was analyzed according to Kim et al. (1986).
Nonghyup in Korea. Fat of soybean were defatted before
Digestion-resistant fractions (RFs) were hydrolyzed by a cellulase
preparation of digestion-resistant fractions (RFs) by hexane. RFs
mixture [Trichoderma viride cellulase: Aspergillus niger
were prepared using a modification of Goni et al. (1996).
cellulase, 10:1 (W/W)]. One hundred twenty mg of the cellulase
Soybeans were finely ground before heat treatment and
mixture were added to 500 mg of RFs and 15 mL of sodium
autoclaved under standard conditions (121?, 15 min, 1 atm).
acetate buffer (0.49 g/15 mL, pH 4.8). The mixtures were allowed
Heat-treated and raw, unheated samples were then processed
to react in a 50? shaking water bath (120 rpm) for 1 hr. Then
further. First, proteins in 10 g of samples were digested by
the mixtures were centrifuged (3,500 rpm, 20 min) and analyzed
incubation with pepsin in 1000 mL of KCl-HCl buffer (pH 1.5)
for reducing sugars.
for 60 min at 40?. After protein digestion, carbohydrates were
Cellulose content of RFs was determined by calculation of
digested by adding 900 mL of ?-amylase solution (4 mg/100
reducing sugar content using a DNS method (Lee et al., 2005).
mL) in tris-maleate buffer (25.5 g/ 550 mL, pH 6.9) for 18 hrs
In brief, 0.5 mL of supernatant and 1.5 mL of DNS reagent (1,000
at 40?. After two digestion reactions, the samples were
mL distilled water + 7.5 g dinitrosalicylic acid + 14 g NaOH
centrifuged (3000 × g, 15 min), the supernatant was discarded,
+ 216 g potassium sodium tartrate + 5.4 mL phenol + 5.9 g
and the pellet was washed three times with distilled water. The
Na2S2O5) were mixed well. After mixing, the samples were
pellet was then incubated in a series of alkaline and acidic
allowed to stand for 5 min in boiling water, and then cooled
solutions for 30 min at room temperature (KOH [67.3 g/ 300
to room temperature in ice water. The absorbance of the samples
mL], followed by HCl [14.0 mL/ 550 mL]). The solution was
was measured at 550 nm and the cellulose of samples was
further incubated with sodium acetate buffer (9.8 g/ 300 mL,
calculated according to the following equation:
pH 4.75). Finally, the residuals were digested with
amyloglucosidase (Sigma A-7255) for 1 hr at 60?. Each
B
Cellulose (%) =
× 100
digestion reactions were performed with appropriate shaking. The
A
final sample was washed, centrifuged (3000 × g, 15 min), and
A: the absorbance of reducing sugar produced from reagent-
the lyophilized pellet was used as an RF in the experiments.
grade cellulose.
Two types of RFs were prepared: a raw, unheated RF (RRF)
B: the absorbance of reducing sugar produced from samples
and a heated RF (HRF) from soybean (SOY). The RFs were
designated as RRF-SOY, and HRF-SOY, respectively.
Hemicelluloses in RFs were measured according to others (Lee
et al., 2004; Nagata et al., 2001). First, to remove the amount
Retarding effect of digestion-resistant fraction in vitro on bile
of protein in RFs from soybean, One hundred mL of sodium
acid transport
chloride (5 g/ 95 mL) were added to 1 g of sample and
homogenized for 20 min. The samples were then centrifuged at
Retarding effects of digestion-resistant fractions (RFs) on in
10,000 rpm for 10 min. 100 mL of alcohol (80 mL/ 100 mL)
vitro bile acid transport were investigated by dialysis (Sigma
were added in the precipitate, extracted, and filtrated.
D7884: M.W. cut-off<1,200) using a glucose transport model.
Alcohol-insoluble RFs were extracted by KOH (22.4 g/ 100 ml)
0.2 g of RF in 6 mL of phosphate buffer (pH 7.0) with sodium
and centrifuged. The pH of the supernatant was then adjusted
azide (0.65 g) and taurocholic acid (0.05 g) were added to a
to pH 4 with hydrochloric acid. The residue of hemicelluloses
dialysis tube. The dialysis bag was then placed in a 150 mL
was lyophilized and the yield was calculated.
capped cylindrical container containing 100 mL of sodium azide
and shaken at 75 rpm in a 37? water bath for 24 hrs. Aliquots
of 1 mL of the dialysate were removed at regular intervals, and
Pentosans
assayed for taurocholic acid content according to others (Ju et
The total pentosans were analyzed according to others
al., 2003; Lee et al., 1996; Lee et al., 2003).
(Hashimoto et al., 1987; Delcour et al., 1999) with some
modification. The samples were weighed (10 mg) and 2 mL of
X-ray diffraction
HCl (14.0 mL/ 550 ml) were added. The mixtures were then
hydrolyzed at 100? for 3 hr. After cooling, the samples were
X-ray diffraction analysis for digestion-resistant fractions (RFs)
neutralized by adding 2 mL of sodium carbonate (0.21 g/ml).
was performed with X-ray diffraction image processor (Bruker
Saccharomyces cerevisiae yeast (25 mg/mL of sodium phosphate
D8 discover, Germany). Analysis parameters were 40 kV and
buffer [0.16 g/ mL], pH 7.0) was added and the samples were
40 mA; Cuk radiation ? = 0.154 nm. The samples were scanned
incubated in a shaking water bath at 37? for 1 hr. The mixture
Sung-Hee Han et al.
151
was then centrifuged at 3000 × g for 10 min. The supernatant
35
)
was analyzed by the Orcinol-HCl method (Albaum & Umbreit,
1947).
is (% 30
i
alys 25
h
d
Analysis of monosaccharides in digestion-resistant fractions
ug
ro 20
(RFs)
th 15
Monosaccharide composition of digestion-resistant fractions
(RFs) from soybean was determined by using of a high
s
p
o
r
ted
10
performance anion exchange chromatography (HPAEC) (Bio-LC;
tran
Dionex Co., Sunnyvale, California, U.S.A). Ten mg of RFs were
5
hydrolyzed in 1 mL of trifluoroacetic acid (TFA, [57.2 mg/mL])
Bile acid 0
for 2.5 hr at 100?. An analytical column for carbohydrate
0
2 3 4
18
detection (CarboPac PA1; Dionex Co., Sunnyvale, California,
Dialysis time (hours)
USA) and an electrochemical detector (ED50A; Dionex Co.,
Control
RRF-SOY
HRF-SOY
Sunnyvale, California, USA) were used. Separation of the various
monosaccharides was achieved using eluent A (NaOH [0.72 g
Fig. 1. Retarding effects of digestion-resistant fraction on bile acid transport in
vitro. RRF-SOY: RF from raw soybean; HRF-SOY: RF from heated soybean.
/ mL]) followed by application of a linear gradient for 30 min
using eluent B (NaOH [8 g/ mL]) (Seo et al., 2004)
contents of RFs were nearly zero. The RF from raw, unheated
Statistical analysis
soybean had relatively high level of resistant starch (RS)
compared to that of heated sample. After heating, the content
The results are expressed as mean ± standard division (n=3).
of RS reduced, but the fiber was vice versa.
An analysis of variance (ANOVA) was performed, and the
differences among the samples were determined by Duncan’s
Multiple Range Test using the Statistical Analysis System
Retarding effect of digestion-resistant fraction in vitro on bile
(Version 9.13, SAS Institute, GA, USA). P values<0.05 were
acid
considered significant.
The digestion-resistant fractions (RFs) from soybean were
retarded bile acid transport (Fig. 1). In only bile acid solution
(control) was dialyzed and its transport continually increased up
Results
to 18 hrs. But samples of RFs including were significantly
retarded the bile acid transport. Also, digestion-resistant fraction
Chemical composition of digestion-resistant fractions (RFs)
from raw, unheated soybean (RRF) induced low level of bile
acid transport than that of digestion-resistant fraction from heated
Raw, unheated (R) and heat-treated (H) digestion-resistant
soybean (HRF) up to the limit time, although level of transport
fractions (RFs) were prepared from soybean (SOY). The yield
increased rapidly in early stage of dialysis (<4 hrs). The decrease
of RF was approximately 5 g per 100 g unheated materials.
in transport level brought by adding RRF-SOY and HRF-SOY
However, the yield was markedly reduced after the heat treatment
were 26.7 and 48.2% for bile acid. This retarding effect is in
(2.4 g per 100 g of soybean). The composition of two types
accordance with results of previous in vivo experiments (Lee et
of RFs was shown in Table 1. Carbohydrate, protein, and lipid
al., 2003). As shown the Table 1, heat treatment changed
compositions of RF. So it seems to be that the composition of
Table 1. Composition of digestion-resistant fractions from soybean(%, w/w)
RFs from soybean affected the functionality of RF.
Digestion-resistant fraction
Components
(resistant starch and fibers)
Raw
Heat- treated
X-ray diffraction patterns
Moisture
11.10 ± 0.01
9.00 ± 0.02
The X-ray diffraction patterns of digestion-resistant fractions
Carbohydrate
0.00 ± 0.01
0.00 ± 0.00
(RFs) were showed in Fig. 2. The RFs from soybean were
Protein
0.99 ± 0.02
0.66 ± 0.07
characterized by peak at different angles (2?) corresponding to
Ash
2.10 ± 0.03
2.03 ± 0.02
Lipid
0.22 ± 0.02
0.28 ± 0.05
resistant starch (RS) content. The structures of RFs were
Resistant fraction
85.59
88.03
influenced by heating. The peak of raw soybean starch as a
- Non-starch soluble fiber
30.17 ± 0.15
31.91 ± 0.08
control of X-ray diffraction had the strong intensity at diffraction
- Non-starch insoluble fiber
31.32 ± 0.05
39.32 ± 0.05
angles of 12.0 and 20.0°. The RF from unheated soybean
- Resistant starch (RS1)
24.10 ± 0.50
16.80 ± 0.36
(RRF-SOY) showed similar the peak intensity to raw soybean
152
Effect of heating on digestion-resistant fraction
35
Control
180
A
30
150
25
120
20
90
oses of RFs(%) 15
60
e
l
l
ol
C 10
180
RRF-SOY
5
150
0
120
RRF-SOY
HRF-SOY
it
y
ns
(A)
Inte
90
50
B
60
40
HRF-SOY
%)
180
s(
f
RF 30
150
o
ses
lo
120
20
i
c
e
llu
90
Hem
10
60
10
15
20
25
30
0
RRF-SOY
HRF-SOY
2? (Angle)
(B)
Fig. 2. Digestion-resistant fractions X-ray pattern of raw and heated soybean.
Control: soybean starch; RRF-SOY: RF from raw soybean; HRF-SOY: RF from
Fig. 3. Celluloses (A) and hemicelluloses (B) content of digestion-resistant
heated soybean.
fraction from raw and heated soybean. RRF-SOY: RF from raw soybean;
HRF-SOY: RF from heated soybean.
starch, control. However, the RF from heated soybean
35
(HRF-SOY) was characterized by a peak at a diffraction angle
(2?) of 19.8°. The X-ray pattern of HRF-SOY were reduced
30
peaks at diffraction angles of 12 and 20° compare to RRF- SOY.
) 25
%
Cellulose and hemicellulose
RFs ( 20
Cellulose and Hemicellulose content of digestion-resistant
of
n
s
fractions (RFs) from unheated and heated soybean was shown
15
in Fig. 3. The RFs of two samples had cellulose over 20%. The
n
t
osa
Pe
RF from unheated soybean (RRF-SOY) had significantly high
10
level of cellulose content than RF from heated soybean
(HRF-SOY). However, in case of hemicellulose, the
5
hemicellulose content of RRF-SOY and HRF-SOY showed
different trend compared to the results of cellulose of RFs.
0
RRF-SOY
HRF-SOY
Hemicellulose of RFs from soybean had over 30%.
Fig. 4. Pentosans of digestion-resistant fractions from raw and heated soybean.
Hemicellulose of RRF-SOY was low level than HRF-SOY.
RRF-SOY: RF from raw soybean; HRF-SOY: RF from heated soybean.
Sung-Hee Han et al.
153
250
200
8 - 12.923
7 - 12.045
150
6 - 9.100
)
n
C
100
1 - 1.557
o
u
l
omb (
C
11 - 16.333
50
n
a
no
4 - 4.967
10 - 14.318
2 - 2.487
5 - 7.705
12 - 18.348
0
14 - 25.220
16 - 29.250
3 - 4.657
9 - 13.595
13 - 22.430
15 - 26.512
-500.0
5.0
10.0
15.0
20.0
25.0
30.0
Retention time (min)
(A)
250
6 - 8.842
200
1 - 1.453
7 - 11.632
150
n
C)
100
o
u
l
omb (
C
50
10 - 13.802
n
a
no
3 - 4.553
5 - 7.498
9 - 13.130
12 - 17.728
13 - 25.065
14 - 28.992
0
2 - 3.468 4 - 6.775
8 - 12.458
11 - 15.868
-500.0
5.0
10.0
15.0
20.0
25.0
30.0
Retention time (min)
(B)
Fig. 5. HPAEC analysis of the monosaccharide composition of RRF-SOY (A) and HRF-SOY (B). The peaks correspond to rhamnose (5), arabinose (6), galactose (7),
glucose (8), xylose (9), mannose (10), and fructose (11).
Pentosans of digestion-resistant fractions from soybean
effect of RF on bile acid in vitro.
Pentosan content of digestion-resistant fraction (RF) from
unheated and heated soybean was shown in Fig. 4. Pentosan
Monosaccharides in digestion-resistant fractions
content of RF from unheated soybean (RRF-SOY; 29.6%) was
Peaks for rhamnose, arabinose, galactose, glucose, xylose,
significantly higher value than RF from heated soybean
mannose, and fructose in digestion-resistant fraction (RF) from
(HRF-SOY; 25.1%). This was similar trend like those of RS and
unheated soybean (RRF-SOY) and RF from heated soybean
cellulose in RRF-SOY. Pentosan is a polymer of various
(HRF-SOY) were assigned by comparison with standard (Fig.
pentoses, arabinose, xylose, and ribose and that sort of things.
5 and Table 2). In RRF-SOY, glucose, galactose, and arabinose
Particularly, arabinose and xylose were well known to have the
were found to have the highest levels at 32.5, 25.2, and 15.0%,
physiological functional components (Marlett & Fischer, 2003).
respectively. Whereas in HRF-SOY sample had glucose,
So, this result could be explains a correlation with the retarding
galactose, and arabinose levels at 1.9, 30.1, and 31.3%. On the
154
Effect of heating on digestion-resistant fraction
Table 2. HPAEC analysis of the monosaccharide composition of RRF-SOY and
functionality on retarding bile acid. After heating, RS in RFs
HRF-SOY
content were markedly reduced. And the levels of cellulose and
Retention
Peak
Height
Area
Relative area
RFs
Number
pentosan in RFs from unheated soybean were higher, whereas
time (min)
name
nC
(nC·min)
(%)
the levels of RS in RFs from heated soybean were reduced. The
1
1.56
UK.3)
88.96
18.94
7.85
2
2.49
UK.
5.35
0.74
0.31
retarding effect showed difference with different RFs content.
3
4.66
UK.
12.40
1.98
0.82
We conformed reduction of RS in RFs after heating by X-ray
4
4.97
UK.
20.12
3.60
1.49
diffraction. The peak of RS was reduced in RF from soybean
5
7.71
rhamnose
4.44
1.05
0.44
so that NSP peak was distinguished after heating, relatively.
6
9.10
arabinose 138.79
36.08
14.96
These results seem to be that the RS in RFs was melted by
7
12.05
galactose 168.26
60.73
25.18
heating. The X-ray diffraction depended on the chain lengths
8
12.92
glucose
202.02
78.46
32.53
making up the amylopectin lattice, amylose and the density of
RRF-SOY1)
9
13.60
xylose
6.23
2.12
0.88
packing within the granules. Therefore, X-ray diffraction had
10
14.32
mannose 16.83
7.35
3.05
affect heat treatment (Sajilata et al., 2006; Xie et al., 2006). And
11
16.33
fructose 58.39
25.89
10.73
Sievert and et al reported that effect of autoclaving and
12
18.35
UK
3.46
2.28
0.95
autoclaving-cooling cycles of RS. After autoclaving, the peak
13
22.43
UK
1.60
1.14
0.47
of X-ray diffraction was lower than those native starches and
14
25.22
UK
0.47
0.35
0.15
control. Also, autoclave cycle was increased as the peak of X-ray
15
26.51
UK
0.30
0.22
0.09
diffraction was decreased (Siever et al., 1991). Therefore
16
29.25
UK
0.36
0.28
0.11
HRF-SOY may have also to be more affected by NSP than
1
1.45
UK
196.51
39.52
21.56
RRF-SOY because RS was melted by heat treatment (Ruan et
2
3.47
UK
2.20
1.45
0.79
al., 2004). These were supported by result of cellulose,
3
4.55
UK
29.56
5.66
3.09
hemicallulose and pentosan. In case of hemicellulose, that of
4
6.78
UK
0.74
0.20
0.11
RRF-SOY was lower level than HRF-SOY. Whereas cellulose
5
7.50
rhamnose
7.84
1.98
1.08
content of RRF-SOY was higher level than HRF-SOY. So the
6
8.84
arabinose 203.95
57.36
31.28
RRF-SOY of low hemicellulose content or high cellulose content
7
11.63
galactose 159.62
56.47
30.08
HRF-SOY2)
will affect on the physiological function (Takahashi et al). The
8
12.46
glucose
10.13
3.49
1.90
chemical structure of cellulose is different. Cellulose is linear
9
13.13
xylose
3.76
1.05
0.57
polymer of ?-1,4-linked glucose units. Hydrogen bonding
10
13.80
mannose
35.27
13.22
7.21
11
15.87
UK
1.46
1.13
0.61
between sugar residues in adjacent chains imparts a crystalline
12
17.73
UK
1.46
0.78
0.42
microfibril structure. Hemicelluloses are a cell wall polysaccharides
13
25.07
UK
0.76
0.80
0.44
containing backbones of ?-1,4-linked pyranosides sugars, but
14
28.99
UK
0.36
0.26
0.14
differ from cellulose in that they are smaller in size, contain a
1) RRF-SOY: RF from raw soybean
variety of sugars, and are usually branched. Moreover, the
2) HRF-SOY: RF from heated soybean
researcher reported that these structure characteristic of
3) Unknown peak
hemicellulose from soybean influenced on serum antibody levels
other hand, arabinose is a component of hemicellulose. But the
and activation of microphage in rats (Nagata et al., 2001). And
hemicellulose of HRF-SOY was higher than did it RRF-SOY.
cellulose seems to play a role in bile acid-binding when it
These results indicate that the functional factors of RF from
stabilizes the cell wall architecture after the digestion process
soybean on retarding effect on bile acid might be not much
(Dongowski 2007). Also, different sugar content in RFs
influenced by arabinose. And the retarding effect of RF on bile
supported that effect of thermal processing of soybean on the
acid transport might be big influenced by glucose as a component
sugar composition of RFs (Periago et al., 1997). In sugar
part of RS. Also, xylose content of RRF-SOY was higher 35.2%
composition, the glucose and xylose content of RF from unheated
than that of HRF-SOY.
soybean was higher than the RF from heated soybean. The
xylooligosaccarides is well known to have functional monosaccharide
on retarding effect of bile acid. Moreover, the retarding effect
Discussion
of RF on bile acid also had been affected by the difference of
sugar content (Vazquez et al., 2000).
RFs are known to have an affect on various diseases.
The level of RS, pentosan, glucose, and xylose were inversely
Particularly, effect of RFs from soybean on cholesterol metabolism
correlated to that of the in vitro retarding effect on bile acid.
was reported (Han et al., 2004). In this study, we examined the
Therefore, this study suggests that heating soybean has affected
retarding effect of heat treatment of digestion-resistant fraction
to the RF of functional components. Also, we think that the RS
from soybean on retarding of bile acid transport in vitro. In
and the monosaccharide composition like xylose are important
consequence, heat treatment had affected to RFs content and it’s
factors for retarding effect on bile acid transport.
Sung-Hee Han et al.
155
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