STARCHES WITH DIFFERENT DEGREE OF SUBSTITUTION
2. Materials and methods 99
2.1. Materials 100
Arenga starch (Arenga pinnata Merr.) 101
used for this study was obtained from Sigi 102
distric Central Sulawesi Province, Indonesia.
103
High-purity acetic anhydride 98%, sodium 104
hydroxide (NaOH) and hydrochloric acid 105
(HCl)were purchased from Merck. The 106
chemicals for analysis used in the study were 107
of analytical grade purchased at local agent.
108
2.2. Preparation of acetylated arenga starch 109
The acetylated arenga starches (AAS) 110
was prepared by a modified procedure of 111
Phillips et al. (1999) with modification. The 112
DS was determined by a titration method.
113
Starch (100 g) was dispersed in 225 mL of 114
distilled water and stirred for 60 min at 25oC.
115
The suspension was adjusted and controlled at 116
pH 8.0 ± 8.2 with 3.0% NaOH solution.
117
Acetic anhydride of (5, 10, 15 % starch base, 118
sb) was slowly added drop-wise to the stirred 119
slurry. After the complete addition of the 120
3 acetic anhydride, the reaction was continued 121
for 60 min and interrupted, setting the pH at 122
4.5 with 0.5 N HCl. The suspension was 123
precipitated for 10 min and subjected to two 124
successive washes twice with distilled water 125
and once with 95% ethanol, and then oven- 126
dried at 40oC for 48 h or up to approximately 127
12% moisture, and ground on an analytical 128
mill. Acetylation of starch with 5% (sb) acetic 129
anhydride gave the AAS with DS 0.039, with 130
10% (sb) acetic anhydride gave that with DS 131
0.078, and with 15% (sb) acetic anhydride 132
gave that with DS 0.139.
133
2.3. Determination of resistant starch 134
The RS determined as a dietary fiber 135
according to the enzymatic gravimetric 136
method (Ebihara et al., 2006). To determine 137
the content of RS, 4 g of sample was 138
suspended in 160 mL of 0.08 M phosphate 139
buffer (pH 5.5) before the addition of 40 µl of 140
heat-stable α-amylase. The solution was 141
incubated at 95°C for 75 min and then allowed 142
to cool. The pH of the solution was adjusted to 143
4.5 by adding 1 N HCl, and the sample was 144
incubated with amyloglucosidase at 60°C for 145
45 min. Aliquot (100 µl) was added with 10 146
mL of glucose oxidase reagent and the 147
mixture was incubated at 20°C for 20 min.
148
Absorbance was measured using a 149
spectrophotometer at 510 nm. The 150
concentration of RS was calculated as follow:
151
RS (g/100 g) = (1 – G × 0.9 / wt. sample) × 152
100, where wt. sample was the initial weight 153
(g), and G is the weight of glucose (g).
154
2.4. The bile acids/salts binding 155
The binding of bile acid/salts (cholic acid, 156
sodium taurocholic sodium deoxycholic) was 157
measured by in vitro analysis (Smietana et al., 158
2000). The sample (100 mg) was admixed 159
with 10 mL of solution of each bile acid. The 160
solutions were prepared in 0.1 mol phosphate 161
buffer pH 7.6for each bile acid in 2 µmol/mL 162
concentration. The samples and parallel blank 163
samples were incubated at 37°C for 30 min.
164
Centrifugation was performed at 2000 g for 165
5min. The sample (50 µl) was combined with 166
5 mL 70% sulphuric acid and 1 mL of freshly 167
prepared furfural solution (2.3 g/L) with 168
careful mixing of the whole sample.
169
Absorbance was measured at 510 nm after 80 170
min. The results were expressed as percent of 171
bile acid absorption.
172
2.5. The cholesterol binding 173
The cholesterol binding was measured by 174
in vitro analysis (Smietana et al., 2000). The 175
sample (100 mg) was combined with 2 mL 176
emulsion composed of 1% lecithin, 1.375%
177
sodium salt of deoxycholic acid and 0.225%
178
cholesterol prepared in 0.1 mol phosphate 179
buffer of pH 6.8. Incubation for 1 h at 37°C 180
was performed in a shaking water bath.
181
Cholesterol absorption by 20 µl emulsion was 182
analysed using reagent kits. The results were 183
expressed as percent of cholesterol adsorbed 184
by samples.
185
2.6. Preparation and operation of batch 186
fermenters 187
Fresh faecal samples from a healthy 188
human volunteer who had no history of 189
antibiotics treatment in the previous 3 months 190
were first diluted (1:10) in anaerobic buffer 191
containing 0.1 M phosphate-buffered saline 192
(PBS) solution, pH 7.0, and then were 193
homogenized using a magnetic stirrer at 194
normal speed for 2 min. A 10 mL portion of 195
the filtered faecal slurry was added to 90 mL 196
growth medium in 250 mL batch fermentation 197
vessels. The growth medium contained the 198
following ingredients: peptone water (2 g/L), 199
yeast extract (2 g/L), NaCl (0.1 g/L), K2HPO4
200
(0.04 g/L), KH2PO4 (0.04 g/L), MgSO4.7H2O 201
(0.01 g/L), CaCl2.6H2O (0.01 g/L), NaHCO3
202
(2 g/L), Tween 80 (2 mL/L), Hemin (0.05 203
g/L), Vitamin K (10 µL/L), L-cysteine (0.5 204
g/L) and bile salts (0.5 g/L). The NAS and 205
BAS (1% w/v) were added to each vessel just 206
before addition of fecal slurry (10% w/v). The 207
vessels were maintained under anaerobic 208
4 conditions by continuous sparging with 209
oxygen-free nitrogen. Temperature was 210
automatically controlled at 37 °C. The batch 211
experiments were performed in duplicate with 212
two different fecal donors for each substrate.
213
At each experiment a 5 mL sample was taken 214
from each vessel at 0, 12, 24, and 48 h for 215
analysis (Vardakou et al., 2008; Ramnani et 216
al., 2012).
217
2.7. Bacterial enumeration 218
The samples from each vessel were 219
immediately transferred to an anaerobic 220
cabinet containing an atmosphere of 10% H2, 221
10% CO2, and 80% N2, and were serially 222
diluted with pre-reduced half strength peptone 223
water, pH 7, supplemented with 0.5 g 224
cysteine–HCl L-1. Portions of 1 mL from each 225
dilution were plated, in duplicate, onto agar 226
plates (incubation anaerobic condition at 37oC 227
for 48 h). The selective growth media used 228
were total plate count, for total bacterials;
229
trypticase soy agar, supplemented per litre 230
with 75 mg kanamycin, 5 mg haemin, 75 mg 231
vancomycin and 50 mL laked horse blood, for 232
Bacteroides spp.; reinforced clostridial agar, 233
supplemented per litre with 8 mg novobiocin 234
and 8 mg colistin, for Clostridium spp.; rogosa 235
agar, supplemented with 1.32 mL glacial 236
acetic acid L-1, for Lactobacillus spp.;
237
Columbia agar containing per litre 5 g 238
glucose, 0.5 g cysteine HCl, and 0.5 mL 239
propionic acid, pH 5.0, for Bifidobacterium 240
spp. All agars were purchased from Oxoid 241
(Basingstoke, Hants, UK) and prepared 242
according to the supplier's instructions. All the 243
antibiotics used in the preparation of media 244
were purchased from Sigma.
245
2.8. Calculation of prebiotic index 246
The equation used to estimate the 247
prebiotic index (PI) values (Palframan et al., 248
2003) was:
249
PI = (Bif /Total) – (Bac / Total) + (Lac / 250
Total) – (Clos /Total) 251
where Bif, Bac, Lac, Clos, and Total are the 252
numbers of bifidobacteria, bacteroides, 253
lactobacilli, clostridia, and total numbers of 254
bacteria, respectively, at the time of sampling 255
relative to their respective numbers at the time 256
of inoculation. The equation embodies the 257
assumption that an increase in the populations 258
of bifidobacteria and/or lactobacilli is a 259
positive effect while an increase in bacteroides 260
and/or clostridia is a negative effect.
261
2.9. Determination of pH 262
The pH of the samples was determined 263
using a digital pH metre (Eutech instruments, 264
Cyber scan) calibrated with buffers at pH 4.0 265
and 7.0 (Fisher Scientific, UK).
266
2.10. Determination of organic acid 267
Culture medium (1 mL) was centrifuged 268
at 4500 x g for 30 min. The supernatant was 269
filtered through a 0.22 mm filter into a 1.5 mL 270
eppendorf tube for storage at 4oC until use.
271
Samples of 1 µL were injected into a high- 272
resolution gas chromatography (GC Shimadzu 273
8A) equipped with a flame ionization detector 274
and an HP Innowax 19091 - 136 column (GP 275
10%-SP 1200/1% H3PO4 on 80/100 mesh 276
chromosorb WAW, 60m x 0.250 mm). The 277
carrier gas was nitrogen with a flow rate of 1.8 278
mL/min, and the split ratio was 40:1. The 279
oven temperature was maintained at 90oC for 280
0.5 min, and then increased to 110oC at a rate 281
of 10oC/min, increased to 170oC at a rate of 282
5oC/min and finally increased to 210oC at a 283
rate of 20oC. Injector and detector 284
temperatures were 230oC. SCFA mixture 285
containing acetate, propionate and butyrate at 286
specific concentration were used as standard.
287
2.11. Statistical analysis 288
The data experiments were analyzed 289
using SPSS (version 17.0) software and 290
subjected to one-way analysis variance. The 291
5 difference of means between groups was also 292
analyzed using Duncan’s multiple comparison 293
test. The level of P < 0.05 was considered as 294
significant. All experiments were repeated 295
once or twice at later dates.
296
3. Results and discussions