FUNCTIONAL AND PREBIOTIC PROPERTIES OF ACETYLATED ARENGA STARCHES WITH DIFFERENT DEGREE OF SUBSTITUTION
2. Materials and methods 1. Materials
Arenga starch (Arenga pinnata Merr.) used for this study was obtained from Sigi distric Central Sulawesi Province, Indonesia. High- purity acetic anhydride 98%, sodium hydroxide (NaOH) and hydrochloric acid (HCl)were purchased from Merck. The chemicals for analysis used in the study were of analytical grade purchased at local agent.
2.2. Preparation of acetylated arenga starch The acetylated arenga starches (AAS) was prepared by a modified procedure of Phillips et al. (1999) with modification. The DS was determined by a titration method. Starch (100 g) was dispersed in 225 mL of distilled water and stirred for 60 min at 25oC. The suspension
Rahim et al. , Carpathian Journal of Food Science and Technology 2018, 10(2), 25-36
27 was adjusted and controlled at pH 8.0 ± 8.2 with 3.0% NaOH solution. Acetic anhydride of (5, 10, 15 % starch base, sb) was slowly added drop-wise to the stirred slurry. After the complete addition of the acetic anhydride, the reaction was continued for 60 min and interrupted, setting the pH at 4.5 with 0.5 N HCl. The suspension was precipitated for 10 min and subjected to two successive washes twice with distilled water and once with 95%
ethanol, and then oven-dried at 40oC for 48 h or up to approximately 12% moisture, and ground on an analytical mill. Acetylation of starch with 5% (sb) acetic anhydride gave the AAS with DS 0.039, with 10% (sb) acetic anhydride gave that with DS 0.078, and with 15% (sb) acetic anhydride gave that with DS 0.139.
2.3. Determination of resistant starch
The RS determined as a dietary fiber according to the enzymatic gravimetric method (Ebihara et al., 2006). To determine the content of RS, 4 g of sample was suspended in 160 mL of 0.08 M phosphate buffer (pH 5.5) before the addition of 40 µl of heat-stable α-amylase. The solution was incubated at 95°C for 75 min and then allowed to cool. The pH of the solution was adjusted to 4.5 by adding 1 N HCl, and the sample was incubated with amyloglucosidase at 60°C for 45 min. Aliquot (100 µl) was added with 10 mL of glucose oxidase reagent and the mixture was incubated at 20°C for 20 min.
Absorbance was measured using a spectrophotometer at 510 nm. The concentration of RS was calculated as follow:
RS (g/100 g) = (1 – G × 0.9 / wt. sample) × 100, where wt. sample was the initial weight (g), and G is the weight of glucose (g).
2.4. The bile acids/salts binding
The binding of bile acid/salts (cholic acid, sodium taurocholic sodium deoxycholic) was measured by in vitro analysis (Smietana et al., 2000). The sample (100 mg) was admixed with 10 mL of solution of each bile acid. The
solutions were prepared in 0.1 mol phosphate buffer pH 7.6for each bile acid in 2 µmol/mL concentration. The samples and parallel blank samples were incubated at 37°C for 30 min.
Centrifugation was performed at 2000 g for 5min. The sample (50 µl) was combined with 5 mL 70% sulphuric acid and 1 mL of freshly prepared furfural solution (2.3 g/L) with careful mixing of the whole sample.
Absorbance was measured at 510 nm after 80 min. The results were expressed as percent of bile acid absorption.
2.5. The cholesterol binding
The cholesterol binding was measured by in vitro analysis (Smietana et al., 2000). The sample (100 mg) was combined with 2 mL emulsion composed of 1% lecithin, 1.375%
sodium salt of deoxycholic acid and 0.225%
cholesterol prepared in 0.1 mol phosphate buffer of pH 6.8. Incubation for 1 h at 37°C was performed in a shaking water bath. Cholesterol absorption by 20 µl emulsion was analysed using reagent kits. The results were expressed as percent of cholesterol adsorbed by samples.
2.6. Preparation and operation of batch fermenters
Fresh faecal samples from a healthy human volunteer who had no history of antibiotics treatment in the previous 3 months were first diluted (1:10) in anaerobic buffer containing 0.1 M phosphate-buffered saline (PBS) solution, pH 7.0, and then were homogenized using a magnetic stirrer at normal speed for 2 min. A 10 mL portion of the filtered faecal slurry was added to 90 mL growth medium in 250 mL batch fermentation vessels. The growth medium contained the following ingredients: peptone water (2 g/L), yeast extract (2 g/L), NaCl (0.1 g/L), K2HPO4 (0.04 g/L), KH2PO4 (0.04 g/L), MgSO4.7H2O (0.01 g/L), CaCl2.6H2O (0.01 g/L), NaHCO3 (2 g/L), Tween 80 (2 mL/L), Hemin (0.05 g/L), Vitamin K (10 µL/L), L-cysteine (0.5 g/L) and bile salts (0.5 g/L). The NAS and BAS (1%
Rahim et al. , Carpathian Journal of Food Science and Technology 2018, 10(2), 25-36
28 w/v) were added to each vessel just before addition of fecal slurry (10% w/v). The vessels were maintained under anaerobic conditions by continuous sparging with oxygen-free nitrogen. Temperature was automatically controlled at 37 °C. The batch experiments were performed in duplicate with two different fecal donors for each substrate. At each experiment a 5 mL sample was taken from each vessel at 0, 12, 24, and 48 h for analysis (Vardakou et al., 2008; Ramnani et al., 2012).
2.7. Bacterial enumeration
The samples from each vessel were immediately transferred to an anaerobic cabinet containing an atmosphere of 10% H2, 10% CO2, and 80% N2, and were serially diluted with pre-reduced half strength peptone water, pH 7, supplemented with 0.5 g cysteine–
HCl L-1. Portions of 1 mL from each dilution were plated, in duplicate, onto agar plates (incubation anaerobic condition at 37oC for 48 h). The selective growth media used were total plate count, for total bacterials; trypticase soy agar, supplemented per litre with 75 mg kanamycin, 5 mg haemin, 75 mg vancomycin and 50 mL laked horse blood, for Bacteroides spp.; reinforced clostridial agar, supplemented per litre with 8 mg novobiocin and 8 mg colistin, for Clostridium spp.; rogosa agar, supplemented with 1.32 mL glacial acetic acid L-1, for Lactobacillus spp.; Columbia agar containing per litre 5 g glucose, 0.5 g cysteine HCl, and 0.5 mL propionic acid, pH 5.0, for Bifidobacterium spp. All agars were purchased from Oxoid (Basingstoke, Hants, UK) and prepared according to the supplier's instructions. All the antibiotics used in the preparation of media were purchased from Sigma.
2.8. Calculation of prebiotic index
The equation used to estimate the prebiotic index (PI) values (Palframan et al., 2003) was:
PI = (Bif /Total) – (Bac / Total) + (Lac / Total) – (Clos /Total) where Bif, Bac, Lac, Clos, and Total are the numbers of bifidobacteria, bacteroides, lactobacilli, clostridia, and total numbers of bacteria, respectively, at the time of sampling relative to their respective numbers at the time of inoculation. The equation embodies the assumption that an increase in the populations of bifidobacteria and/or lactobacilli is a positive effect while an increase in bacteroides and/or clostridia is a negative effect.
2.9. Determination of pH
The pH of the samples was determined using a digital pH metre (Eutech instruments, Cyber scan) calibrated with buffers at pH 4.0 and 7.0 (Fisher Scientific, UK).
2.10. Determination of organic acid
Culture medium (1 mL) was centrifuged at 4500 x g for 30 min. The supernatant was filtered through a 0.22 mm filter into a 1.5 mL eppendorf tube for storage at 4oC until use.
Samples of 1 µL were injected into a high- resolution gas chromatography (GC Shimadzu 8A) equipped with a flame ionization detector and an HP Innowax 19091 - 136 column (GP 10%-SP 1200/1% H3PO4 on 80/100 mesh chromosorb WAW, 60m x 0.250 mm). The carrier gas was nitrogen with a flow rate of 1.8 mL/min, and the split ratio was 40:1. The oven temperature was maintained at 90oC for 0.5 min, and then increased to 110oC at a rate of 10oC/min, increased to 170oC at a rate of 5oC/min and finally increased to 210oC at a rate of 20oC. Injector and detector temperatures were 230oC. SCFA mixture containing acetate, propionate and butyrate at specific concentration were used as standard.
2.11. Statistical analysis
The data experiments were analyzed using SPSS (version 17.0) software and subjected to one-way analysis variance. The difference of
Rahim et al. , Carpathian Journal of Food Science and Technology 2018, 10(2), 25-36
29 means between groups was also analyzed using Duncan’s multiple comparison test. The level of P < 0.05 was considered as significant. All experiments were repeated once or twice at later dates.
3. Results and discussions