**9.2. Analysis for dietary fiber**

Total, soluble and insoluble dietary fiber contents of chitosan and plant fibers were analysed according to the enzymatic–gravimetric method of the Association of Official Analytical Chemists (AOAC) Official Method 991. 43 [140]. Apple, bamboo, psyllium and wheat fibers were investigated to obtain contents of main cell wall constituents (lignin, cellulose, hemicellulose). These components were determined by modifications of the method described by Robertson and van Soest [38, 141] using ANKOM200/220 Fiber Analyzer (ANKOM Technology, Macedon, NY, USA). This method measures Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF) and Lignin. Cellulose and hemicellulose contents were obtained by calculations. To determine ADF, duplicate samples were agitated under pressure with hot acid detergent solution for 60 min, rinsed in hot water and dried. To determine lignin content, duplicated samples were digested in 72% (v/v) sulfuric acid, following ADF analysis. Cellulose content of samples was calculated from ADF minus the lignin content. To determine NDF, duplicated samples were shaken with neutral detergent solution and heat-stable α-amylase for 60 min, rinsed and dried. Hemicellulose content of samples was calculated as NDF minus ADF [141].

### **9.3. Yoghurt preparation**

Yoghurt was prepared using reconstituted whole milk powder (15% w/w) and 5% sucrose. This mix was homogenized and heated to 85 °C for 30 min., cooled to ambient temperature and inoculated with 0.03% starter culture. Starter was constituted by a 1:1 mixture of Streptococcus thermophilus (CIDCA collection 321) and Lactobacillus delbrueckii subsp. bulgaricus (CIDCA collection 332). Samples were incubated at 43°C to reach a pH of 4.4–4.6 and stored at 4°C, after completion of the fermentation process 1.3% (w/w) of each dietary fiber was added to samples of yoghurt [142]. The amount of fiber was selected following US regulations for fiber-fortified products [143].

To study glucose availability 0.6 g of glucose (Sigma-Aldrich Co., St. Louis, MO, USA) was added for each sample of yoghurt with each type of dietary fiber. In calcium availability studies the digestive mimicking was done without the addition of exogenous calcium because yoghurt is a source of calcium in the diet [138]. To evaluate the interactions between the fibers and iron, 0.8% (w/w) of ferrous sulfate was added to yoghurt samples with each type of fiber [139]. This addition was in accordance with local regulations governing iron supplementation in milk products. Ferrous sulfate (FeSO4·7 H2O) of 99.9% purity was used as purchased (Sigma-Aldrich Co., St. Louis, MO, USA).

#### **9.4. Digestive chemical experimental model**

468 The Complex World of Polysaccharides

speed at 25 °C [139].

**9.2. Analysis for dietary fiber** 

samples was calculated as NDF minus ADF [141].

regulations for fiber-fortified products [143].

as purchased (Sigma-Aldrich Co., St. Louis, MO, USA).

**9.3. Yoghurt preparation** 

Deacetylation degree was obtained using FT-IR spectroscopy (Nicolet iS10 FT-IR Spectrometer, Thermo Fisher Scientific, USA) with samples in the form of KBr at a ratio of 1:2. Viscosity of 1% chitosan in 1% acetic acid solution was measured with a Brookfield model DV-IV + viscosimeter (Brookfield, USA) with spindle 21 and a 50 rpm rotational

Total, soluble and insoluble dietary fiber contents of chitosan and plant fibers were analysed according to the enzymatic–gravimetric method of the Association of Official Analytical Chemists (AOAC) Official Method 991. 43 [140]. Apple, bamboo, psyllium and wheat fibers were investigated to obtain contents of main cell wall constituents (lignin, cellulose, hemicellulose). These components were determined by modifications of the method described by Robertson and van Soest [38, 141] using ANKOM200/220 Fiber Analyzer (ANKOM Technology, Macedon, NY, USA). This method measures Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF) and Lignin. Cellulose and hemicellulose contents were obtained by calculations. To determine ADF, duplicate samples were agitated under pressure with hot acid detergent solution for 60 min, rinsed in hot water and dried. To determine lignin content, duplicated samples were digested in 72% (v/v) sulfuric acid, following ADF analysis. Cellulose content of samples was calculated from ADF minus the lignin content. To determine NDF, duplicated samples were shaken with neutral detergent solution and heat-stable α-amylase for 60 min, rinsed and dried. Hemicellulose content of

Yoghurt was prepared using reconstituted whole milk powder (15% w/w) and 5% sucrose. This mix was homogenized and heated to 85 °C for 30 min., cooled to ambient temperature and inoculated with 0.03% starter culture. Starter was constituted by a 1:1 mixture of Streptococcus thermophilus (CIDCA collection 321) and Lactobacillus delbrueckii subsp. bulgaricus (CIDCA collection 332). Samples were incubated at 43°C to reach a pH of 4.4–4.6 and stored at 4°C, after completion of the fermentation process 1.3% (w/w) of each dietary fiber was added to samples of yoghurt [142]. The amount of fiber was selected following US

To study glucose availability 0.6 g of glucose (Sigma-Aldrich Co., St. Louis, MO, USA) was added for each sample of yoghurt with each type of dietary fiber. In calcium availability studies the digestive mimicking was done without the addition of exogenous calcium because yoghurt is a source of calcium in the diet [138]. To evaluate the interactions between the fibers and iron, 0.8% (w/w) of ferrous sulfate was added to yoghurt samples with each type of fiber [139]. This addition was in accordance with local regulations governing iron supplementation in milk products. Ferrous sulfate (FeSO4·7 H2O) of 99.9% purity was used Two types of digestive simulations were performed to study the interactions between dietary fibers used and the macro and micro nutrients tested. To evaluate the interaction of glucose and calcium with the fibers, gastric and duodenal environments were simulated. To examine the interactions between the fibers and iron was used in addition, a dialysis membrane to imitate the iron passage through the intestinal wall. Digestive enzymes were not utilized in these models because they do not hydrolyze fibers. The importance of duodenal simulation in these studies is because most dietary glucose, calcium and iron are absorbed in the duodenum.

The experiments to study the availability of glucose and calcium were performed in the following steps: a mix of 12.5 g of yogurt with 0.3 g of each fiber was stirrer in 50 mL of 0.1 M HCl (Merck) pH 1.0–2.0, 30 rpm and 37°C to reproduce the gastric environment. After 1 hour simulations were taken from the acidic medium to pH 6.8–7.2 with 15 g/L of NaHCO3 (Sigma Chemical Co., St. Louis, MO, USA). The stirring speed was increased from 30 to 300 rpm and the temperature was maintained at 37°C to reproduce the duodenal environment. Then simulations were allowed to rest for 15 min until two phases separated. Samples to determine glucose and calcium concentration were taken from the supernatant. Glucose and calcium amounts, determined by this way, represent the bioavailability fraction of those nutrients. A control without fibers was made to consider glucose and calcium 100% availability [138].

Experiments to study the interaction of dietary fibers with iron were carried out in the following manner. Yoghurts with ferrous sulfate and each fiber were stirred in 50 mL of 0.1 M HCl (Merck) for 1 h at pH 1.0–2.0, 30 rpm and 37 °C to reproduce the gastric environment. During this first step of simulation pH was checked each 15 min with a pH Meter Hach model EC-30 (USA) and it remained constant (pH 1.0–2.0). To reproduce the chemical duodenal environment pH level was increased to pH 6.8–7.2 with 0.2 M NaHCO3 (Sigma-Aldrich Co., St. Louis, MO, USA), stirring speed was increased from 30 to 300 rpm to imitate the peristaltic movement and temperature was maintained at 37°C. Simulations were immediately transferred into a dialysis tubing cellulose membrane (D9527-100 FT, (Sigma-Aldrich Co., St. Louis, MO, USA). This cellulose membrane (molecular weight cut-off 12,400) was previously prepared, as indicated by suppliers, and it was cut into 28 cm length pieces. The loaded tubes were immersed in 100 mL of distilled water; at 37°C. Iron concentrations were determined from the dialysed medium at 30 and 60 minutes. Control yoghurt with ferrous sulfate without fibers was subjected to the digestive simulation and was considered as 0% iron retention to calculate iron retention percentages for each fiber [139].

#### **9.5. Analytical techniques**

To determine glucose concentration an enzymatic method was used. Glucose reacts with 10 kU/L glucose oxidase (GOD), and 1 kU/L peroxidase (POD) in presence of 0.5 mM 4 aminophenazone (4-AP) and 100 mM phosphate buffer (pH 7.0) containing 12 mM hydroxybenzoate (Wiener Lab Glicemia enzymatic AA Kit, Argentina). An amount of

digestive simulation solution (10 mL) was mixed with 1.0 mL of reagent, tubes were incubated for 5 min in water bath at 37°C and developed colour were read in spectrophotometer (Spectronic 20 Genesys TM, Spectronic Instrument, USA) at 505 nm. Final reaction colour is stable for 30 min. Glucose calibration curve was carried out. The amounts of glucose used in this study correspond to available carbohydrates in the human mixed diet.

To determine calcium concentration a spectrophotometric method was used. Calcium reacts with 3.7 mmol/L cresolphtalein complexone (Cpx) at pH 11 (buffer 0.2 mol/L aminomethylpropanol (AMP) solution in 35%v/v methanol) (Wiener Lab Ca-color Kit, Argentina). Assays were carried directly in spectrophotometer test tubes: 50 lL Cpx were mixed with a plastic rod and absorbance was read in spectrophotometer (Spectronic 20 Genesys TM, Spectronic Instrument, USA) at 570 nm (internal blank), then 20 mL of each digestive mimicking sample were added, immediately mixed and read after 10 min. A standard curve was developed [138].

To determine iron concentration in the dialysates a spectrophotometric method was used, 500 μL of dialyzates was reduced with 2 mL of mercaptoacetic acid (succinic acid buffer, pH 3.7). Then, iron reacted with one drop of pyridyl bis-phenil triazine sulfonate (PBTS) producing a pink color due to the complex formed (Wiener Lab Fe-colour Kit, Rosario, Argentina). Absorbance was read on a spectrophotometer (Spectronic 20 Genesys Thermo Electron Scientific Instruments Corp., Madison, WI, USA) at 560 nm (internal blank). All glassware used in sample preparation and analysis was rinsed with 10% (v/v) concentrated HCl (37%) and deionised water before using, to avoid mineral contamination. A regression equation (y = 2.5333x + 0.0042, R2 = 0.995) derived from data generated from standards of FeSO4 was used to calculate iron concentrations in the samples. Iron retention percentages for each studied fibers were calculated as a percentage of the amount of iron measured in the dialysed medium obtained with the control yoghurt without fibers [139].

### **9.6. Statistical analysis**

Experiments were performed at least five times for each dietary fiber using freshly prepared yogurt. For total iron concentration in dialyzates, each individual sample was run in duplicate. Averages and standard deviations were calculated and expressed in each case as the mean ± SD for n replicates. Normality of the data was checked with the Lilliefors test. The influence of different dietary fibers on the retention percentages of glucose, calcium and iron were statistically analyzed by a one-way analysis of variance (ANOVA) (p < 0.05) to find significant differences and Tukey's test to compare means.
