**10.2. Digestive chemical model and glucose, calcium and iron retention percentages**

Dietary fiber have been found to have the capacity of binding different substances like bile salts and glucose which have implications in cholesterol lipid and carbohydrate metabolism respectively, as presented in the preceding sections. However, the continuous introduction of new ingredients in the food industry requires further studies in order expand knowledge of the impact on nutrient absorption.

Figure 1 shows the behavior of samples during the digestive tract simulation to evaluate glucose and calcium retention percentages and macroscopical differences between them could be observed. Different simulated digestive contents for different fibers before dialysis in assays to determine the iron retention percentages are not shown because they are similar to those presented in Fig. 1. Simulation of gastrointestinal environment during dialysis of different yoghurts can be observed in Figure 2. Changes in pH during gastrointestinal simulation produces different behaviors depending on the type of fiber employed. The apple fiber is a fine powder with brownish color, probably due to the content of phenolics compounds [150]. When apple fiber is added to the yogurt and subjected to the gastrointestinal simulation this color persists (Figure 1). In Figure 2 it can be seen that Psyllium fiber gives a viscous dispersion [151,152]. Due to changing pH values in the Dietary Fiber and Availability of Nutrients: A Case Study on Yoghurt as a Food Model 473

(1) Wheat, (2) psyllium, (3) apple, (4) inulin, (5) chitosan and (6) bamboo fibers.

472 The Complex World of Polysaccharides

hemicellulose (%) of fibers

**percentages** 

Fibre ADF NDF Lignin Cellulose Hemicellulose

Apple 38.6 ± 0.9 44.3 ± 0.7 8.4 ± 0.8 30.2 ± 1.7 5.7 ± 1.6 Bamboo 50.2 ± 0.7 90.4 ± 0.6 5.0 ± 0.3 45.2 ± 1.0 40.2 ± 1.7 Psyllium 7.3 ± 0.4 36.8 ± 0.9 0.8 ± 0.1 6.5 ± 0.4 29.5 ± 1.3 Wheat 74.8 ± 0.3 89.7 ± 0.6 2.6 ± 0.4 72.2 ± 0.7 14.9 ± 0.9

Scientists who deal with animal nutrition usually use Van Soest's method to analyse feed. Scientists working on human nutrition use methods of the AOAC, because of their interest in soluble fiber. It is known that soluble fiber plays an important role in human health and the food industry. However, it could be useful in human nutrition to know the composition of insoluble fiber, as it is possible that insoluble fibers do not all have the same effect on human health. The NDF and insoluble fiber methods were applied to the same samples. Insoluble fiber includes hemicellulose, cellulose, lignin, cutin, suberin, chitin, chitosan, waxes and resistant starch. NDF includes hemicellulose, cellulose and lignin. Escarnot et al. [149] studied three wheat varieties and four spelt genotypes. They analysed three milling fractions from those grains for insoluble and soluble fiber contents, lignin, hemicellulose and cellulose. They found a very high correlation (r2 = 0.99) between the two methods, showing that NDF and insoluble fiber methods cover the same types of fiber. For insoluble fiber

**Table 5.** Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), lignin, cellulose and

**10.2. Digestive chemical model and glucose, calcium and iron retention** 

Dietary fiber have been found to have the capacity of binding different substances like bile salts and glucose which have implications in cholesterol lipid and carbohydrate metabolism respectively, as presented in the preceding sections. However, the continuous introduction of new ingredients in the food industry requires further studies in order expand knowledge

Figure 1 shows the behavior of samples during the digestive tract simulation to evaluate glucose and calcium retention percentages and macroscopical differences between them could be observed. Different simulated digestive contents for different fibers before dialysis in assays to determine the iron retention percentages are not shown because they are similar to those presented in Fig. 1. Simulation of gastrointestinal environment during dialysis of different yoghurts can be observed in Figure 2. Changes in pH during gastrointestinal simulation produces different behaviors depending on the type of fiber employed. The apple fiber is a fine powder with brownish color, probably due to the content of phenolics compounds [150]. When apple fiber is added to the yogurt and subjected to the gastrointestinal simulation this color persists (Figure 1). In Figure 2 it can be seen that Psyllium fiber gives a viscous dispersion [151,152]. Due to changing pH values in the

analysis, the NDF method is faster and more thorough.

of the impact on nutrient absorption.

**Figure 1.** Photograph of the macroscopic view of different fibers in the in vitro digestive tract simulation.

(1) Yoghurt without fiber, (2) chitosan, (3) psyllium, (4) wheat.

**Figure 2.** Different fiber behaviors in the dialysis step of digestive simulation.

digestive tract, Chitosan precipitates while passing through the first portion of the small intestine, forming flocculus. Chitosan, that is a positively charged polysaccharide, is insoluble in neutral and alkaline pH. It is only soluble in acidic pH because below pH 6.5 (pKa = 6.5), the amine groups of chitosan are positively charged. When it is solubilised in dilute acid, chitosan has a linear structure [153]. At pH > 6.5, the polymer loses its charges from the amine groups and therefore becomes insoluble in water and precipitate forming flocculus.

Using the model that reproduces *in vitro* gastrointestinal conditions we determined glucose availability reduction and the results are shown in Figure 3. Significant differences (p < 0.05) are observed in glucose availability reduction percentage for the different fiber samples. In the gastrointestinal conditions chitosan formed a flocculus that entrapped glucose so its availability reduction is the highest. Psyllium increases viscosity medium and glucose availability reduction is 15.3 ± 1.8%; wheat has 9.5 ± 2.1% of glucose retention and inulin 5.7 ± 1.8%, apple and bamboo showed no availability reduction. This *in vitro* study supports the view that certain types of dietary fiber reduce the rate of glucose absorption but chitosan has the most pronounced effect. The behavior in delaying absorption could be likely to alter the gut endocrine response both by carrying material further down the small intestine prior to absorption as well as by producing a flatter blood glucose profile.

**Figure 3.** Glucose availability reduction

On the other hand, dietary fiber may influence the availability of minerals, such as calcium, magnesium [154] and iron [155]. Animal studies have found that dietary chitosan possibly arrests the absorption of calcium [156,157].

To study calcium availability the same model for glucose was used but without the addition of exogenous calcium because yogurt is an important source of this mineral in the human diet. Data are shown in Figure 4. Statistical analysis confirmed significant differences (p < 0.05) among the behavior of the different fibers with calcium. It is observed 16.5 ± 1.6% of calcium availability reduction for apple fiber that have significant differences with the others fibers. However availability reduction responses, between insoluble fibers (wheat and bamboo) and soluble ones (inulin and psyllium plantago), have no significant differences (p < 0.05) by Tukey's test. Again, like results obtained with glucose, this study demonstrated that the chitosan effect is more pronounced and higher than for the other studies [138].

**Figure 4.** Calcium availability reduction.

474 The Complex World of Polysaccharides

flocculus.

digestive tract, Chitosan precipitates while passing through the first portion of the small intestine, forming flocculus. Chitosan, that is a positively charged polysaccharide, is insoluble in neutral and alkaline pH. It is only soluble in acidic pH because below pH 6.5 (pKa = 6.5), the amine groups of chitosan are positively charged. When it is solubilised in dilute acid, chitosan has a linear structure [153]. At pH > 6.5, the polymer loses its charges from the amine groups and therefore becomes insoluble in water and precipitate forming

Using the model that reproduces *in vitro* gastrointestinal conditions we determined glucose availability reduction and the results are shown in Figure 3. Significant differences (p < 0.05) are observed in glucose availability reduction percentage for the different fiber samples. In the gastrointestinal conditions chitosan formed a flocculus that entrapped glucose so its availability reduction is the highest. Psyllium increases viscosity medium and glucose availability reduction is 15.3 ± 1.8%; wheat has 9.5 ± 2.1% of glucose retention and inulin 5.7 ± 1.8%, apple and bamboo showed no availability reduction. This *in vitro* study supports the view that certain types of dietary fiber reduce the rate of glucose absorption but chitosan has the most pronounced effect. The behavior in delaying absorption could be likely to alter the gut endocrine response both by carrying material further down the small intestine prior to

On the other hand, dietary fiber may influence the availability of minerals, such as calcium, magnesium [154] and iron [155]. Animal studies have found that dietary chitosan possibly

absorption as well as by producing a flatter blood glucose profile.

**Figure 3.** Glucose availability reduction

arrests the absorption of calcium [156,157].

To study iron retention percentages by the fibers tested in the present study, the introduction of cellulose dialysis tubes in the digestive chemical experimental model is utilised. The use of a membrane dialysis tube reproduces, in the laboratory, the duodenum wall and its utilisation is presumably a significant factor that determines iron absorption according to Miret et al. [158].

In yoghurt, caseins are modified as a consequence of its production process. Bioactive peptides are formed from caseins during the elaboration of milk products (cheese, yoghurt) under the action of endogenous enzymes of milk (plasmin, cathepsin, among others) or of microorganisms [159]. These peptidic fragments that are already present in yoghurt, could fix iron and calcium according to Bouhallab and Bouglé [159]. Then, these complex matrices

(yoghurts with each type of fiber and iron or calcium) are subjected to the gastrointestinal simulation. Control yoghurt with ferrous sulfate without fiber was also subjected to the digestive simulation and considered to be 0% iron retention (100% iron dialyzated) to calculate iron retention percentages for each fiber. Similarly, control yoghurt without fibers was subjected to the digestive simulation to estimate calcium 100% availability. With these control yoghurts, we could consider the interaction of iron or calcium with casein peptidic fragments.

Iron retention percentages of different fibers are presented in Figure 5. Bamboo and wheat fibers, both insoluble, have low iron retention percentages between 2–5% at 30 min with a maximum of 10% at 60 min. There are no significant differences (p < 0.05) between them by Tukey's test. Bamboo and wheat are high in cellulose content. Cellulose could retain iron by physical adsorption according to results reported by Torre et al. [15]. They worked with high dietary fiber food materials studying the physicochemical interactions with Fe(II), Fe(III) and Ca(II) without an *in vitro* digestive model. They found that the interaction between Fe(II) and cellulose could be explained better by physical adsorption than complex formation. Inulin, a soluble fiber, has no iron retention at 30 or 60min of simulation. This result is in accordance with studies that confirm that inulin does not interfere with iron absorption [17,20,160,161].

Although psyllium and apple fiber contain both soluble and insoluble fractions, they have significantly different responses (p < 0.05). The apple fiber incorporated in yoghurt has no

**Figure 5.** Iron retention percentages of yoghurts with different studied fibers

influence on iron retention. Psyllium shows, on average, 44.6 ± 3.8% iron retention at 60min, which may be mainly attributed to the formation of high viscous dispersion that could be interfering with iron absorption (Figure 2). In addition, the differing behaviors between apple fiber and psyllium could be explained by the different chemical composition of these fibers. Psyllium has high hemicellulose content and apple has the highest lignin content and cellulose. However, bamboo has a low iron retention percentage. The bamboo behavior could be explained due to the composition with equal proportions of soluble and insoluble fractions. More research is needed of this type of fiber.

476 The Complex World of Polysaccharides

absorption [17,20,160,161].

fragments.

(yoghurts with each type of fiber and iron or calcium) are subjected to the gastrointestinal simulation. Control yoghurt with ferrous sulfate without fiber was also subjected to the digestive simulation and considered to be 0% iron retention (100% iron dialyzated) to calculate iron retention percentages for each fiber. Similarly, control yoghurt without fibers was subjected to the digestive simulation to estimate calcium 100% availability. With these control yoghurts, we could consider the interaction of iron or calcium with casein peptidic

Iron retention percentages of different fibers are presented in Figure 5. Bamboo and wheat fibers, both insoluble, have low iron retention percentages between 2–5% at 30 min with a maximum of 10% at 60 min. There are no significant differences (p < 0.05) between them by Tukey's test. Bamboo and wheat are high in cellulose content. Cellulose could retain iron by physical adsorption according to results reported by Torre et al. [15]. They worked with high dietary fiber food materials studying the physicochemical interactions with Fe(II), Fe(III) and Ca(II) without an *in vitro* digestive model. They found that the interaction between Fe(II) and cellulose could be explained better by physical adsorption than complex formation. Inulin, a soluble fiber, has no iron retention at 30 or 60min of simulation. This result is in accordance with studies that confirm that inulin does not interfere with iron

Although psyllium and apple fiber contain both soluble and insoluble fractions, they have significantly different responses (p < 0.05). The apple fiber incorporated in yoghurt has no

**Figure 5.** Iron retention percentages of yoghurts with different studied fibers

Chitosan presents the highest iron retention percentages at 30 min (53.2 ± 3.7%) and 60 min (56.8 ± 4.5%), which shows significant differences (p < 0.05) with other fibers. This biopolymer, which has an animal origin, contains 98% insoluble fiber, and flocculates in the first portion of the small intestine. This flocculus (Figure 2), which could entrap iron, clearly decrease iron dialysis. However, certain amount of iron could go through the cellulose membrane and could be measured to calculate the iron retention percentage. Certain amount of casein-peptide-fragments interacting with iron could remain in solution. Nevertheless, their presence does not interfere with the calculation of iron retention percentages as proven by the digestive simulations performed with control yoghurts.

This study shows that the effect of chitosan on iron absorption is more pronounced and higher than those measured for the other studied plant fibers, as dietary fiber is a significant factor that influences iron absorption. The iron retention percentages of different fibers used in this work could be explained mainly as a result of physicochemical phenomena, like adsorption, formation of viscous dispersion and flocculus.

Yoghurt contains peptidic fragment s from caseins. The caseins are amphiphilic phosphoproteins and their isoelectric point (p*I*) value is 4.6. At pH above the *pI*, caseins are negatively charged and soluble in water. The caseins have an electronegative domain preferentially located in small peptidic fragments known as αs1-Casein, β-casein and κcasein. These structural features of the caseins may render these molecules adept at forming complexes with multivalent cationic macromolecules, such as chitosan [153]. In yoghurt (pH = 4.4–4.6) aggregation of the casein-peptide-fragments occur because of a reduction in the electrostatic repulsion at around their *pI* value. Anal et al. [153] studied the interactions between sodium caseinate and chitosan, under a range of conditions. This study showed that soluble or insoluble chitosan–caseinate complexes can be formed depending on the pH. The characteristics of the complexes are determined by the biopolymer types and their concentration, as well as by environmental conditions. In a certain pH range (5.0–6.0), nanocomplexes of chitosan and sodium caseinate with diameter between 250 and 350 nm were formed. The chitosan and sodium caseinate complexes associated to form larger particles, which resulted in phase separation appear when the pH was either in the range 4.0–4.5 or >6.5. At pH 3.0–3.8, where chitosan and sodium caseinate have similar charges, they may dissociate from each other and become solubilized in solution. According to these authors, yoghurts with chitosan could contain chitosan-casein-peptidic complexes apart from free chitosan molecules in solution.

Besides, calcium existing in yoghurt or the added iron could interact with free chitosan molecules and those complexes. In our work, yoghurts with chitosan are subjected to gastrointestinal simulations. In the first step, our food passes through the simulated stomach (pH = 1.0–2.0) and it could be expected that casein peptidic fragments, chitosan, iron or calcium, all remain in solution. While the food passes through the first portion of the simulated small intestine, changes in pH can lead to formation of chitosan-casein peptidic complexes and iron or calcium could be interacting with them. At pH 6.8–7.0, free chitosan molecules and chitosan-casein-peptidic complexes precipitate forming flocculus. The force of the coagulum formed is high and can be seen in Figures 1 and 2. The results reported by Ausar et al. [162] indicate that hydrophobicity of the casein-chitosan complex is the main mechanism by which the casein-chitosan flocculation is produced.

Chitosan is essentially a positively charged polysaccharide. Iron and calcium are cations. Anal et al. [153] measured zeta potential of chitosan solutions, sodium caseinate solutions and chitosan-caseinate mixtures in a range of pH (3.0–6.5). They found that the pure chitosan solutions were strongly positively charged between pH 3.0 and 6.0. The zeta potential values of chitosan solutions decreased with increasing pH and were slightly negative (approximately − 2.5 mV) at pH 6.5. In our study, in the range of pH 3.0–6.0, isolated molecules of chitosan were probably interacting with iron or calcium by adsorption rather than by electrostatic forces. Besides, Anal et al. [153] observed that the zeta potentials of the chitosan–caseinate solutions were negative at pH > 5.5. In this range of pH, in our work, electrostatic interaction could exist between chitosan-caseinate complexes and iron or calcium. However, when chitosan precipitates, it captures the iron or calcium either by electrostatic forces or by adsorption [138,139].

The behavior of chitosan with calcium and iron in the digestive simulations were similar and can be explained in the same manner. However, the behavior between the other fibers used and the same micronutrients in the digestive simulations were significantly different. The flocculus formation by chitosan is a very strong kind of behavior which is independent of the use of the dialysis membrane. Evidently other types of interactions are brought into play for the other fibers that need further studies to determine them.

## **11. Conclusion**

Results showed that the different plant fibers decreased glucose, calcium and iron availabilities whereas the effect of chitosan (fiber from animal source) was more pronounced. These findings could be positive or negative depending on the nutrient and the nutritional stage or health of the population who would receive the food under study. However, the *in vitro* digestive chemical experimental model may be used to increase the understanding of the interactions between animal and plant fibers with nutrients and micronutrients. This knowledge is very important from the point of view of health and for food industry and technologists.
