digestion of starch experiment

Microbe Notes

Starch Digestion: Structure, Enzymes, Mechanism, Process

Dietary starches contribute as the largest energy sources by humans to meet their calorie intake. Starch is a complex macromolecule derived from plant-based foods and beverages. Because of their structural complexity, starches seem to take a longer duration to get digested and used for energy needs. The gut employs several processes, such as mechanical and chemical, to degrade the complex conformation of starch into simpler molecules.

Starch digestion is a complex process involving enzymes like salivary and pancreatic amylase that break it down into smaller molecules. These molecules, such as maltose, are further broken down by maltases and sucrases.

The resulting glucose is absorbed in the small intestine and transported to cells for energy or glycogen storage, playing a crucial role in providing a steady glucose supply for vital biological processes.

Table of Contents

Interesting Science Videos

Starch and its Structure

Glucose is the functional unit of the starch molecule. Repeating units of glucose molecules joined by glycosidic bonds compose a starch polymer. They undergo hydrolysis reactions to yield glucose molecules. They can be easily derived from the plants as starch granules in semi-crystalline form. Two components of starch include Amylose and Amylopectin.

• Makes upto 20-30% of the total starch structure.
• The polysaccharide is composed of alpha-D-glucose monomeric units.
• This component makes up the linear and unbranched polymer of starch.
• The glucose monomers are joined to each other via α-1-4 glycosidic bonds (the 1 and 4 being the carbon number of glucose molecule).
• An amylose polymer may have around 300-3000 units of glucosyl residues.
• It is observed that amylose molecules are resistant to digestion due to a lack of branching.

Amylopectin

• They contribute to about 70-80% of the total starch composition.
• They are constituted by the branching of amylose polymers via α-1-6 glycosidic bonds as the branching point of the glucose units in the amylose.
• They show branching among amylose units after every 24-30 glucose subunits.
• Amylopectin polymers are easily digested due to the presence of branching of glucose units.
• They are responsible for enhancing the gel strength and solubility in the water.

Linkage types (α-1,4 and α-1,6) between glucose molecules 

The linkages between glucose molecules in starch involve glycosidic bonds: α-1,4 and α-1,6. The α-1,4 glycosidic bonds connect the glucose units along the linear chains of amylose and Amylopectin.

These bonds are formed between the carbon atom 1 (C1) of one glucose molecule and the carbon atom number 4 (C4) of the adjacent glucose molecule. The α-1,6 glycosidic bonds, found exclusively in Amylopectin, create the branching points in the molecule. They occur between the C1 of a glucose unit and the C6 of another glucose unit, forming a branch.

Granular structure in plants

In plants, starch has a granular structure. These granules are formed inside plastids, specifically in the amyloplasts responsible for starch synthesis and storage. The size and shape of starch granules can vary depending on the plant species.

They can be round, oval, or irregular in shape. Inside the granules, both amylose and Amylopectin are present. Amylose is generally found in the inner region of the granule, forming a helical structure.

Amylopectin is more abundant and forms the outer region of the granule, creating a branched network. This granular structure allows plants to store starch as an energy reserve efficiently.

Enzymes Involved in Starch Digestion 

Salivary amylase (ptyalin), pancreatic amylase, and brush border enzymes (maltase, sucrase, and lactase) are all involved in the digestion of starch in the human body. Here’s a brief description of each enzyme:

Salivary amylase (ptyalin)

Salivary amylase is an enzyme secreted by the salivary glands in the mouth. It begins starch digestion by breaking down complex carbohydrates (starch) into smaller polysaccharides and disaccharides like maltose.

Pancreatic amylase

The pancreas generates pancreatic amylase, which is then discharged into the small intestine. It continues starch digestion by breaking down the remaining complex carbohydrates into smaller units like maltose, maltotriose, and alpha-limit dextrins.

Brush border enzymes (maltase, sucrase, and lactase)

Brush border enzymes are located on the surface of the small intestine’s absorptive cells, specifically the microvilli. They further break down the smaller units of carbohydrates into their respective monosaccharides:

• Maltase: Maltase converts maltose into two glucose molecules.
•  Sucrase: Sucrase breaks down sucrose into one glucose molecule and one fructose molecule.
•  Lactase: Lactase breaks down lactose into one glucose molecule and one galactose molecule.

Mechanism of Starch Digestion

Humans and animals consume starch granules through plant-based food sources such as wheat, potatoes, rice, leaves, and other parts of plants. Also, they are found to be consistent in the staple diet, owing to the major carbohydrate and energy source in humans and animals alike. Starch granules digestion begins in the oral cavity all the way leading to its degradation in the intestine.

Oral Cavity: Pre-Gastric Processing

As the food is ingested, mechanical and enzymatic processes act on the food. The mastication process is favored for breaking down the macroscopic structure of foods other than starch. Saliva production is commenced before the ingestion of food by sight, smell, or sound of food triggers cephalic digestion in our brain.

• As the food is masticated and turned into a saliva-infused bolus, salivary alpha-amylase enzyme acts on the starch molecule to break it down into short-chain oligopolysachharides such as maltose or isomaltose.
• Salivary amylase’s catalytic activity is enhanced by the presence of chloride ions. 
• The gene AMY1 for salivary amylase is located in chromosome 1.
• Individuals or communities that rely on a starchy diet have more number of gene repeats.
• The composition of macromolecules in the food, such as proteins, carbohydrates, and fats, is strongly associated with the release of amylase from acinar cells of salivary glands.
• Tests in rodents have shown an increased salivary amylase production by the consumption of a high-fat diet.
• Mixing of ingested food is also important for lubrication and swallowing as it reduces friction and helps in the easy movement of a food bolus from the oesophagus to the stomach for further degradation.

Stomach-Gastric Processing

Since the pH of the gastric environment is lower than the optimal amylase activity, a minimal digestion process occurs for starch granules. Further homogenization of starch via mechanical contraction of gastric chambers turns the contents into a creamy paste. This paste is allowed to pass through the pyloric sphincter to the duodenum for complete digestion in the small intestine.

Small Intestine- Final Processing

The homogenized mixture enters the duodenum with high hydrogen ion concentration, which leads to the release of cholecystokinin from the hormone enteroendocrine I cell, initiating the process of digestion in the small intestine. To diffuse the acidic influx of gastric juice along with chyme (a liquified mixture of food from the stomach), bicarbonate ions are released from the small intestinal epithelium cells, pancreatic acinar cells and hepatocytes. 

• Contractile movement of intestinal walls helps mix the food contents to bile and pancreatic juice.
• Pancreatic juice contains the active pancreatic alpha-amylase enzyme released from acinar cells in the pancreas. It breaks down starch into maltose, isomaltose, sucrose and other simple sugars.
• Pancreatic amylase gene AMY2 and AMY2B is located in chromosome 1.
• Pancreatic alpha-amylase specifically cleaves the 1,4-glycosidic bonds of the amylose component of the starch.
• Another important enzyme secreted by the intestinal epithelium is glucosidases.
• Glucosidases help in the amylolysis of starch, producing glucose, maltose, and maltotriose as the end products.
• Further digestion of disaccharides is achieved by the catalytic actions of enzymes sucrase, maltase, isomaltase, lactase, etc, to convert them into monosaccharides useful in cellular metabolism.

Feedback Mechanism and Starch Digestion

Feedback stimulus, such as food texture, taste perception, the release of enzymes, etc, is very important to upregulate or downregulate the starch digestion mechanism. 

Biological Factors controlling Starch Digestion

Biological factors that control starch digestion include

• Mastication – Responsible for the mechanical size reduction of the starch granules.
• Salivation – Breakdown of starch granules and food bolus formation.
• Gastric Peristalsis – Contraction and relaxation of gastric chambers lead to a mechanical reduction of food particle size and mixing food bolus with the gastric juices.
• Gastric Emptying – Rate of delivery and release of food chyme (food bolus mixed with the gastric juice) into the small intestine.
• Intestinal Motility – further size reduction of food particles and fusion of intestinal juices and enzymes to the food chyme.

Starch is a complex carbohydrate and a major dietary component for carbon and energy sources. They are made up of repeating units of glucose molecules as functional units. It has two main components, amylose, and amylopectin, differing in glycosidic bonds. The process of starch digestion begins in the mouth by the action of the enzyme salivary alpha-amylase secreted via salivary glands. Amylases break down the scratch into simpler sugar, such as maltose. The digestion by the intestinal juice further leads to the degradation of starch granules to sugars like glucose by the action of enzymes pancreatic amylases, maltase, sucrase, etc. These monosaccharides are absorbed by the intestinal cells and transported to the liver for cellular metabolism processes and energy requirements.

• Brownlee, Iain A., et al. “Starch digestion in the upper gastrointestinal tract of humans.” Starch‐Stärke 70.9-10 (2018): 1700111.
• Lee, Byung-Hoo, et al. “Modulation of starch digestion for slow glucose release through “toggling” of activities of mucosal α-glucosidases.” Journal of Biological Chemistry 287.38 (2012): 31929-31938.
• Nutritional qualities of starch depend on the way it is digested – https://www.sydney.edu.au/science/news-and-events/2021/12/20/nutritional-qualities-of-starch-depend-on-the-way-it-is-digested.html
• Starch – https://alevelbiology.co.uk/notes/starch/#12-degradation-in-animals-
• Li, Cheng, et al. “Biological factors controlling starch digestibility in human digestive system.” Food Science and Human Wellness 12.2 (2023): 351-358.
• https://academic.oup.com/jn/article-abstract/122/1/172/4754868
• https://academic.oup.com/jas/article-abstract/63/5/1624/4662245
• https://pubs.acs.org/doi/abs/10.1021/jf9813900
• https://pubs.acs.org/doi/abs/10.1021/jm800115x
• https://www.sciencedirect.com/science/article/pii/0016508586904361
• https://www.sciencedirect.com/science/article/pii/S0144861714009412
• https://link.springer.com/article/10.1007/BF00451611
• https://pubs.rsc.org/en/content/articlehtml/2014/fo/c4fo00115j
• https://www.sciencedirect.com/science/article/pii/S0268005X21000734
• https://www.sciencedirect.com/science/article/pii/003194227480289X

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Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

Observing earthworm locomotion

Practical Work for Learning

Published experiments

Investigating the effect of amylase on a starchy foodstuff, class practical or demonstration.

Place rice in a Visking tubing bag to model food in the gut . Investigate amylase action by adding water, amylase, or boiled amylase to the rice. Leave for 10-15 minutes in a water bath at around 37 °C then test for the presence of a reducing sugar in the water surrounding the Visking tubing bag.

Lesson organisation

This experiment could be done as a demonstration or in groups. Each group needs to set up three Visking tubing bags, so a group of 3 students is ideal.

Apparatus and Chemicals

For each group of students:.

3 x 15 cm lengths of Visking tubing

Syringe barrels, sawn off, 3

Boiling tube, 3

Test tubes, 6

Test tube racks to accommodate 6 test tubes and 3 boiling tubes per group

Teat pipettes, 6

White dimple (spotting) tile

Beaker, 250 cm 3

Kettle for boiling water for Benedict’s test

Eye protection for each student

For the class – set up by technician/ teacher:

Length of Visking tubing, knotted at one end, 15 cm, 3 per group ( Note 1 )

Syringe barrel, sawn off, 3 per group ( Note 2 )

Elastic bands, 3 per group

Electric water baths set at 35-40 °C, with thermometer to show temperature accurately

Cooked rice

Iodine solution ( Note 3 )

Benedict’s reagent ( Note 4 )

Amylase solution, 5 cm 3 per group ( Notes 5 and 6 )

Boiled amylase, 5 cm 3 per group

Clinistix (as an alternative to Benedict’s reagent) ( Note 7 )

Health & Safety and Technical notes

Students should wear eye protection when handling chemicals. Electrical apparatus should be maintained and checked according to your employer’s instructions. Ensure students know how to deal with breakages of glass or thermometers

Read our standard health & safety guidance

1 Soak the Visking tubing in warm water beforehand so it is ready to use.

2 The end of an old syringe makes a convenient support for the Visking tubing, and makes it easier to take samples of the contents with a teat pipette.

3 Iodine solution (See CLEAPSS Hazcard and Recipe card): a 0.01 M solution is suitable for starch testing. Make this by 10-fold dilution of 0.1 M solution. Once made, the solution is a low hazard but may stain skin or clothing if spilled.

4 Benedict’s (qualitative) reagent. (See CLEAPSS Recipe card) No hazard warning is required on the bottle, as the concentrations of each of the constituents are low. Take care making up the reagent; sodium carbonate is an irritant to the eyes and copper(II) sulfate(VI) is harmful if swallowed. See CLEAPSS Hazcards.

5 Amylase solution: Check your amylase supply as many contain starch or reducing sugars, which would interfere with the results of this test. Alpha amylase is bacterial amylase with high activity, and does not give a positive reducing sugar test or starch test. You can use lower concentrations of this enzyme. Some bacterial amylases may survive boiling!

Using saliva: the CLEAPSS Laboratory Handbook provides guidance on precautions, including hygiene precautions, for safe use of saliva as a source of amylase. This has the advantage of being cheaper and technicians do not need to make up fresh solutions each lesson. It is directly interesting to students, and salivary amylase is reliable. It also provides an opportunity to teach good hygiene precautions, including ensuring that students use only their own saliva samples.  Provide small beakers to spit into. Students must be responsible for rinsing their own equipment. All contaminated glassware is placed in a bowl or bucket of sodium chlorate(I) for technicians wash up.

6 Working with enzymes: It is wise to test, well in advance, the activity of stored enzymes at their usual working concentrations to check that substrates are broken down at an appropriate rate. Enzymes may degrade in storage, and this allows time to adjust concentrations or to obtain fresh stocks.

7 Clinistix are quick and easy to use. Each stick can be cut into two or three pieces.

Ethical issues

There are no ethical issues associated with this protocol.

Preparation

a Prepare boiled rice, enzyme solution, boiled enzyme solution, iodine solution, and Benedict’s reagent.

b Set up a water bath at 37 °C.

c Soak Visking tubing, cut 15 cm lengths (3 per group) and set up model guts with syringe barrels, or leave for students to assemble.

Investigation

d Label 3 boiling tubes 1, 2, 3.

e Label 3 test tubes 1, 2, 3.

f Set up 3 model guts: take a wet piece of Visking tubing, tie a knot in one end, place the sawn off syringe barrel in the other end and secure with an elastic band. These may have been set up for you (see diagram).

g Use the spatula to add rice to each of the model guts until they are half full.

h Rinse the outside of each piece of Visking tubing under a running tap.

i Place the rice-filled model gut in a labelled boiling tube. Add warm water to boiling tube outside the Visking tubing until it reaches about 2 cm higher than the level of the liquid inside the Visking tubing (see diagram).

j Immediately withdraw one drop of the water you have added and test it with iodine on a dimple tile.

k Add 5 cm 3 of water to model gut 1.

l Add 5 cm 3 of amylase to model gut 2.

m Add 5 cm 3 of boiled amylase to model gut 3.

n Place all the boiling tubes containing the model guts in the water bath at approximately 37 °C.

o Leave for at least 15 minutes.

p While you are waiting:

• Place a grain of rice in a well on the white tile and add a drop of iodine.
• Put some rice in a test tube. Add 2 cm 3 of water and 2 cm 3 of Benedict’s reagent, and place into a large beaker of boiling water. Check the colour after 2-3 minutes.
• Record your results in a suitable table.

q After 15 minutes, use a teat pipette to remove 2-3 cm 3 of the water surrounding the model gut in boiling tube 1.

r Place one drop of this water in a well on the white tile and add a drop of iodine. Record the result.

s Place the rest (around 2 cm 3 ) of the water from boiling tube 1 into test tube 1. Add an equal volume of Benedict’s reagent and place test tube 1 into a large beaker of boiling water. Check the colour after 2-3 minutes. Record the result.

t Repeat steps q , r , s with water from boiling tubes 2 and 3. Record the results.

Teaching notes

The sawn off syringe barrel acts as a model mouth to the gut. It is a good idea to use cooked rice, as this is real food and can be seen in the (model) gut.

Many students will need help to understand this activity. When interpreting the results, students have to think in terms of two types of model: the model gut with Visking tubing representing the selectively permeable membranes lining the gut wall, and a simplified chemical model of large and small molecules. A further complication is that the movement of chemicals is unseen and only inferred from the results of chemical tests. An additional model could be used, with chicken wire or mesh, fruit or satsuma bags to represent the membrane, and poppet beads in chains to represent starch and singly to represent glucose.

Health and safety checked, September 2008

Related experiments

Evaluating Visking tubing as a model for a gut

Investigating the effect of pH on amylase activity

Digestion of starch by microbes

NOTIFICATIONS

Salivary amylase and starch.

• + Create new collection

In this activity, students investigate the action of salivary amylase on starch present in cooked rice. Simple tests for starch and its digestion product, maltose, are applied.

By the end of this activity, students should be able to:

• use simple chemical tests to identify soluble starch and reducing sugars like glucose and maltose
• safely use their own salivary amylase
• explain in simple terms how the enzymatic digestion of starch occurs
• recognise the need for careful control of variables such as temperature and amount of reactant in activities of this type
• describe how high temperatures can inactivate enzymes like amylase.

Download the Word file (see link below) for:

• introduction/background notes
• what you need
• student worksheets.

Related content

The article, Rate of digestion , looks at how surface area, temperature and pH all influence the rate of digestion of large food molecules. The action of salivary amylase on starch is used as an example.

The article Catalysing chemical reactions with enzymes includes an animated video outlining in detail how enzymes work.

See our Enzymes Pinterest board for more resource ideas.

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Simulation of Human Small Intestinal Digestion of Starch Using an In Vitro System Based on a Dialysis Membrane Process

Carol gonzález.

1 Department of Chemistry, Universidad Tecnológica Metropolitana, Las Palmeras 3360, Ñuñoa, Santiago 7800003, Chile; [email protected] (C.G.); moc.liamg@oipracledzelaznog (D.G.)

Daniela González

Rommy n. zúñiga.

2 Department of Biotechnology, Universidad Tecnológica Metropolitana, Las Palmeras 3360, Ñuñoa, Santiago 7800003, Chile; [email protected]

3 Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación, Universidad Tecnológica Metropolitana, Ignacio Valdivieso 2409, San Joaquín, Santiago 8940577, Chile

Humberto Estay

4 Advanced Mining Technology Center (AMTC), University of Chile, Av. Tupper 2007, AMTC Building, Santiago 8370451, Chile; [email protected]

Elizabeth Troncoso

This work deepens our understanding of starch digestion and the consequent absorption of hydrolytic products generated in the human small intestine. Gelatinized starch dispersions were digested with α-amylase in an in vitro intestinal digestion system ( i -IDS) based on a dialysis membrane process. This study innovates with respect to the existing literature, because it considers the impact of simultaneous digestion and absorption processes occurring during the intestinal digestion of starchy foods and adopts phenomenological models that deal in a more realistic manner with the behavior found in the small intestine. Operating the i -IDS at different flow/dialysate flow ratios resulted in distinct generation and transfer curves of reducing sugars mass. This indicates that the operating conditions affected the mass transfer by diffusion and convection. However, the transfer process was also affected by membrane fouling, a dynamic phenomenon that occurred in the i -IDS. The experimental results were extrapolated to the human small intestine, where the times reached to transfer the hydrolytic products ranged between 30 and 64 min, according to the flow ratio used. We consider that the i -IDS is a versatile system that can be used for assessing and/or comparing digestion and absorption behaviors of different starch-based food matrices as found in the human small intestine, but the formation and interpretation of membrane fouling requires further studies for a better understanding at physiological level. In addition, further studies with the i -IDS are required if food matrices based on fat, proteins or more complex carbohydrates are of interest for testing. Moreover, a next improvement step of the i- IDS must include the simulation of some physiological events (e.g., electrolytes addition, enzyme activities, bile, dilution and pH) occurring in the human small intestine, in order to improve the comparison with in vivo data.

1. Introduction

1.1. in vitro systems to simulate human digestion.

Evidence has shown that nutrition, and in particular postprandial glycaemia, plays a crucial role in the development of type-2 diabetes. Therefore, the study and understanding of human digestion has turned out to be of great interest to predict and control the absorption of glucose from the intake of foods with high starch content [ 1 , 2 , 3 ]. Human digestion is a complex process in which mechanical and enzymatic transformation of food macromolecules take place simultaneously and hydrolytic products are absorbed [ 4 , 5 ]. Most of the systems developed for food disintegration and nutrient release research are made as static in vitro systems. These consist of placing the food in a series of reactors or bioreactors which recreate the physicochemical and enzymatic environment of each digestive compartment (e.g., mouth, stomach and small intestine) [ 5 ]. However, these systems simplify the behavior of the human gastrointestinal tract and are unrealistic, because they cannot simulate the real physiological processes, such as mixing, peristaltic movements, injection in real time of digestive enzymes or changes in pH conditions over time [ 4 , 5 , 6 ]. Now, among the intestinal experimental absorption models, in vivo models are used to study nutrient or drug bioavailability when food or drug formulations are administered orally, or directly into the intestine, in humans or suitable animal species. These in vivo models are the most accurate way to determine intestinal permeability, fraction absorbed and bioavailability, because the impacts of different physiological parameters are considered [ 7 , 8 ]. Among the techniques used for these kind of in vivo studies, we highlight (i) intestinal perfusion to assess the effective intestinal permeability of phytochemicals, sulforaphane, quercetin-3,4′-glucoside [ 9 ], vitamin E and gallic acid [ 10 ] and drugs such as tripterine [ 11 ] and amoxicillin [ 12 ] or drugs that are incompletely absorbed in the proximal small intestine [ 13 ] and (ii) ileostomy to determine the bioavailability of folic acid [ 14 ], amoxicillin [ 15 ], sugars [ 16 ] and (poly)phenols [ 17 ]. However, in vivo models have ethical, economic and technical restrictions that limit their application. In addition, in vivo models are less applicable for phenomenological understanding of the individual impacts of physiological factors on nutrient bioavailability [ 7 ]. For this reason, a real need exists for the development of in vitro models that simulate the real physiological processes that occur in the human gastrointestinal tract during the digestion of food, which must be flexible, precise, controlled and reproducible [ 4 , 6 ]. Therefore, in recent years, there has been a development of dynamic in vitro models capable of simulating the physiology of the stomach and small intestine, which can predict in vivo behavior, such as decomposition of solids and nutrient absorption. These models can be used as a valuable research tool for the study and understanding of changes and interactions as well as the bioaccessibility and bioavailability of nutrients, drugs and non-nutrient compounds [ 5 , 6 , 18 ].

The study of in vitro hydrolysis of starch has been of great interest because it can be associated with the glycemic response, which is an indicator of the postprandial glucose response for foods with high starch content [ 19 , 20 , 21 , 22 ]. Glucose causes metabolic stress when it is present in high levels in the blood. High glucose concentration is associated with an increased risk of suffering from some diseases, such as type-2 diabetes, cancer and cardiovascular disease [ 23 ]. The realization of in vitro studies to determine the rate of starch digestion and absorption has advantages over in vivo studies, because it is much more economical and does not require ethical authorization. To simulate in vitro absorption, artificial or biological membrane systems or assays based on biological cell monolayers have been used [ 24 , 25 , 26 ]. For in vitro intestinal systems, semipermeable membranes are often used, which act as a selective barrier to keep the enzymes separated from their digestion products, avoiding the inhibition of enzymatic hydrolysis [ 18 ]. Recently, Dupont et al. [ 5 ] reviewed the validation of eight dynamic in vitro models against in vivo data, namely DGM, HGS, ARCOL, DIDGI ® , TIM, SHIME ® , ESIN and simgi ® . Of the eight models, two of them simulate the stomach (DGM and HGS), one simulates the large intestine (ARCOL) and the other five simulate the stomach and small intestine. Of the five models that simulate the small intestine, two do not take into account intestinal nutrient absorption (DIDGI ® and simgi ® ). The other three models use membranes to simulate intestinal absorption (TIM, SHIME ® and ESIN). Specifically, the TIM and ESIN models use a dialysis hollow fiber membrane to mimic the passive absorption of water and hydrolytic products from digestion in the small intestine. A description of other more established models based on membrane systems can be found in Gim et al. [ 18 ], where the advantages and limitations of existing approaches are briefly discussed. For example, in 2010, the in vitro small intestine model (SIM) [ 27 ] was fabricated to study glucose absorption using semi-permeable dialysis membranes, also mimicking intestinal peristalsis and segmentation motion; however, fluids secretion, pH and body temperature were not considered. In 2014, the SIM was improved by the dynamic duodenum (DDuo) model, which incorporated simulated intestinal secretions and pressure zones mimicking segmentations and peristalsis [ 28 ]. After two years, the human duodenal model (HDM) was proposed to estimate nutrient absorption using a membrane dialysis and to simulate peristaltic motion using pneumatic movement, which also incorporated the sigmoidal shape of the first portion of the human small intestine [ 29 ]. In spite of all the efforts to simulate intestinal digestion and absorption effectively, the complex phenomenology of intestinal mass transfer is still a challenging problem.

The use of hollow fiber membranes for the dialysis process is common practice in studies focused on hemodialysis. These kinds of membranes function as an artificial kidney, in which the membrane acts as a separation barrier for metabolic wastes [ 30 ]. During the dialysis process, the driving force is the difference in concentration of the compounds, between the lumen and the shell side of the membrane. For hemodialysis, the phenomenology of mass transfer is promoted using a diffusion process [ 30 , 31 , 32 , 33 , 34 , 35 ] and a convective effect, which is determined by a pressure difference between the lumen and shell side. This pressure difference generates a water transfer or solution, as described elsewhere in the literature [ 24 , 25 , 33 ]. Transmembrane pressure is the driving force for other membrane processes, such as micro-, ultra- and nanofiltration processes. The dialysis process is the only membrane process capable of transferring solutes with high molecular weight by diffusive mass transfer, due to a concentration difference. Considering this last point, in a previous work performed by our research team [ 18 ], we fabricated an in vitro small intestine model ( i -IDS) where a dialysis membrane system was used as an artificial small intestine, due to its ability to simulate the phenomena of diffusive mass transfer and absorption occurring in the small intestine and the arrangement of the large transfer area of the membrane module. The system allowed glucose transfer and absorption to be studied. We then proposed mathematical models to describe the diffusive and convective mechanisms involved.

1.2. Kinetics of Starch Digestion

Starch is the main source of digestible carbohydrates that contributes significantly to total energy intake, and it is present in foods such as cereals, corn, rice, wheat and potatoes [ 23 , 36 ]. Amylose and amylopectin correspond to the two main polymers that compose the starch granule [ 37 ]. These polymers contain glucose bound by α-1,4-glycosidic bonds, which can be hydrolyzed by the α-amylase enzyme to produce short-chain linear oligosaccharides. The ramifications of amylopectin are formed through α-1,6 glycosidic bonds which are resistant to the action of α-amylase [ 38 ]. The starch granule has a semi-crystalline structure which, in the presence of water and temperature above 58 °C, undergoes a structural change called gelatinization [ 36 ]. Starch gelatinization consists of the swelling of the starch granules, as a consequence of water absorption, increasing the availability of starch for enzymatic hydrolysis by digestive enzymes [ 39 , 40 , 41 , 42 ]. The physical state of the starch will determine the accessibility of the digestive enzymes. There are different states in which starch can be present in foods, namely (i) native, where digestibility is low, (ii) gelatinized, where digestibility is high or (iii) retrograded, where the digestibility is intermediate between native and gelatinized [ 3 ]. Retrogradation is the phenomenon occurring when the molecules comprising gelatinized starch begin to re-associate in an ordered structure [ 36 ]. The hydrolysis of starch consists in the breaking down of starch polymers into short chain fragments, such as glucose monomers, maltose and dextrins, where most of the hydrolysis of starch is carried out by the pancreatic α-amylase in the small intestine [ 19 , 40 ]. This process occurs during digestion in order to obtain monosaccharides that are capable of being absorbed through the walls of the small intestine and are subsequently transported by the bloodstream to the cells to be used as an energy source for different functions of the human body. In several studies, the hydrolysis of starch has been modeled by first-order reaction kinetics [ 21 , 43 , 44 , 45 ], obtaining information about the degradation rate constant and the amount of product generated. This information is very valuable for the generation of phenomenological models for enzymatic starch hydrolysis and absorption during in vitro gastrointestinal digestion.

Based on the above, the objective of this work is to deepen our previous study [ 18 ], using the same in vitro digestion system, namely the i -IDS ( Figure 1 ). In this system, a dialysis membrane was used to simulate the absorption process of glucose molecules in the small intestine. In the present study, we take on the challenge of simulating a more realistic phenomenon of the human intestinal digestion. Starting with the use of a food matrix, such as gelatinized potato starch, and using an enzyme for its degradation, we study the absorption process of the hydrolytic products generated by means of a dialysis membrane, obtaining information on the phenomenological model of generation and transfer of sugars. With these results we expect to obtain a more realistic understanding of the functioning of the digestion and absorption processes in the small intestine to achieve a simulation that resembles the operation of this organ and to reproduce and scale-up the results to human digestion.

Experimental diagram of the i -IDS to simulate starch digestion and mass transfer of reducing sugars in the human small intestine [ 18 ].

2. Materials and Methods

2.1. materials.

Commercial potato starch (Chuño Delicado, Chile) purchased from a local market ( Table 1 ) was used as a model starch-based food. α-amylase (BAN 480L, Novozymes Company, Frederiksberg, Denmark), produced by submerged fermentation of a selected strain of Bacillus amyloliquefaciens , was kindly provided by Blumos Chile (Santiago, Chile). BAN 480L is available with a standard strength of 480 KNU/g and relative activity at 37 °C of 67% and 92% at pH 7, as provided by the manufacturer. Monobasic sodium phosphate, dibasic sodium phosphate (Sigma-Aldrich, St. Louis, MO, USA) and sodium azide (Merck, Darmstadt, Germany) were used for the preparation of phosphate buffer. For the DNS method, 3,5-Dinitrosalicylic acid, crystalline phenol and sodium bisulfite (Acros Organics, Geel, Belgium), sodium potassium tartrate were used. In addition, solutions of sodium hydroxide and hydrochloric acid (Fisher Scientific, Waltham, MA, USA) were used to adjust the pH of samples by means of manual titration. Ultra-purified water (resistivity 15.0 MΩ-cm) was used for the preparation of the starch dispersions.

Composition of the potato starch (g per 100 g of dry sample weight), according to information given by the manufacturer.

2.2. Preparation of Gelatinized Starch Dispersions

The starch dispersion consisted of potato starch (1% w/v ) and phosphate buffer. The choice of this concentration ensured a Newtonian behavior of the gelatinized starch dispersion, which had a viscosity of 0.0165 ± 0.0020 Pa·s (data not shown). For the samples, it was necessary to maintain a pH equal to 7.0, simulating the conditions of the small intestine. For the preparation of 1 L of phosphate buffer the following was used: 5.421 g of monobasic sodium phosphate, 14.288 g of dibasic sodium phosphate and 0.2 g of sodium azide. For the study, 2 L of starch dispersion were prepared in a 3 L double-walled vessel, then the dispersion was stirred. Prior to the assays, water from a thermoregulated bath was circulated at 75 °C inside the walls of the vessel for 1 h to ensure the gelatinization of the starch at a temperature of 65 °C, considering the heat loss through the walls of the vessel towards the ambient temperature.

The gelatinization temperature of the starch was determined using differential scanning calorimetry equipment (DSC 1 STARe System, Mettler Toledo, Zurich, Switzerland), coupled to an immersion cooler (TC100, Huber, Offenbach). The gelatinization temperature was evaluated measuring the endothermic transition enthalpy of starch suspensions. Measurements were performed in aluminum pans (~100 μL of sample) sealed under manual pressure and reweighted after the process in order to check that no significant evaporation occurred during the measurement. An aluminum pan filled with pure water was used as reference. To assess the reversibility of the heat effects on the samples, a cooling step was performed, followed by a re-heating interval. The heating profiles were conducted from 25 to 95 °C at a heating rate of 5 °C/min followed by a cooling step from 95 to 25 °C with a 40 °C/min rate and maintained at this temperature for 5 min. The last interval was performed from 25 to 95 °C at a heat rate of 5 °C/min. These experiments were conducted in duplicate. DSC thermograms allowed the onset, peak and endset temperatures of the thermal transition of starch gelatinization to be determined, with values of 60, 65 and 71 °C, respectively (data not shown).

2.3. In Vitro Starch Digestion

2.3.1. in vitro potato starch digestion using the i -ids.

A previously designed system in our laboratory ( i -IDS, in vitro intestinal digestion system) ( Figure 1 ) [ 18 ] was used for the simulation of the human small intestine. The system consists of two reservoirs. The first one is a jacketed glass vessel (capacity 3 L) used as a feed tank, which is maintained at 37 °C by circulating warm water through the jacket. This tank contained the starch dispersion which was pumped from the feed tank to the mass transfer stage using a peristaltic pump. The second reservoir is a graduated vessel (capacity 25 L) made of high density polyethylene, which was used as a dialysate tank where ultra-purified water was stored at 37 °C and pumped from the dialysate tank to the mass transfer stage using a peristaltic pump. The mass transfer stage consists of a hemodialysis hollow fiber membrane module (Nipro, ELISIO™17H-PP, USA) with a molecular weight cut-off of less than 20 kDa. The feed solution was circulated through the membrane lumen side, while ultra-pure water (dialysate liquid), coming from the dialysate tank, was fed into the shell of the membrane module. The feed flows were defined according to the operating conditions recommended by the supplier.

Three assays were carried out according to three ratios of feed flow (mL/min)/dialysate flow (mL/min): 225/386, 389/386 and 389/248 (nominally named as 250/400, 400/400 and 400/250, respectively), since it is expected that the flow rate influences the mass transfer of reducing sugars obtained from starch digestion. These feed/dialysate flow ratios were set at the same values used in our previous work [ 18 ] in order to compare results between studies. Furthermore, the selection of these flow ratios was based on the minimization of the transmembrane pressure. For each experiment, the feed tank was filled with 2 L of feed solution (i.e., gelatinized potato starch dispersion), while the dialysate tank was filled with 19 L of ultra-pure water and 1 L of phosphate buffer solution with continuous stirring at 37 °C, adjusting the pH to 7.0. Prior to the start of the simulation, 6.6 µL of α-amylase was added to the feed vessel. The enzyme was left to act for 1 min while continuing to homogenize, after which the system was put into operation. The enzyme:starch (dry weight basis) ratio was 0.26:1 v / w , following the procedure performed by Dartois and co-workers [ 46 ]. The feed flow circulated through the membrane lumen side, dialysate solution was fed into the shell side of the membrane module, and both tanks were maintained at 37 ± 1 °C during the assays. Each test was carried out for 1 h. In order to determine the reducing sugars generated by the enzymatic digestion of starch and the reducing sugars transferred in the system, simulating the digestive and absorptive functions of the human small intestine, aliquots (2 mL) were taken simultaneously from both the feed and the dialysate tanks during the process time (1 h). The aliquots were taken at intervals of 2 min during the first 20 min of the experiment and then at intervals of 5 min until completing 1 h of assay. In addition, the volume of both tanks was registered during the assays.

2.3.2. In Vitro Potato Starch Digestion Under Batch Conditions

In order to determine the maximum mass of reducing sugars generated by the enzymatic digestion, assays of in vitro starch digestion under batch conditions were carried out. For these studies, a reservoir with identical characteristics to the feed tank (3 L double-walled vessel) was used. Two liters of gelatinized potato starch dispersion (1% w/v ) at pH equal to 7.0 were prepared in the vessel, following the procedure described above. The vessel was maintained at 37 °C by circulating warm water through the jacket. Prior to the assay, 6.6 µL of α-amylase was added to the vessel, leaving it to act for 1 h min while continuing to homogenize the sample. Two mL aliquots were taken every 2 min during the first 20 min of the experiment, and then at intervals of 5 min until completing 1 h of assay, which were collected to be subjected to measurements of reducing sugars concentration.

All experiments were done in triplicate and results are presented as mean values with standard deviations.

2.3.3. Measurements of Reducing Sugars Concentration

The aliquots obtained from the assays of starch digestion using the i -IDS (from the feed and the dialysate tanks) and those obtained from the assays of in vitro starch digestion under batch conditions were collected into 3 mL Eppendorf tubes (Mundolab, Santiago, Chile), which contained 20 µL of 1 N hydrochloric acid (Merck, Darmstadt, Germany). These samples were kept for 30 min. At the end of 30 min, the concentration of reducing sugars was measured using Millier’s spectrophotometric method with 3,5-Dinitrosalicylic acid (DNS) [ 47 , 48 ]. For the analysis of the samples, aliquots of 0.5 mL were taken from the Eppendorf tubes and placed in test tubes and mixed with 1.5 mL of DNS solution to be heated for 5 min using a bath of boiling water and then were placed into a cold bath for 3 min. After this process, the samples were diluted with 10 mL of ultra-purified water and the absorbance of the samples was then measured using a spectrophotometer (Shimadzu UV Visible, UVmini-1240, Kyoto, Japan) at 600 nm. To determine the reducing sugars, a calibration curve made with glucose (Sigma Aldrich, St. Louis, MO, USA) was previously performed, allowing the interpolation of the measured absorbance and the calculation of the reducing sugar concentration, expressed as mg glucose equivalent per mL sample.

2.4. Dialysis Membrane Process and Phenomenological Approach

In the simulation system, the generation of hydrolytic products from potato starch corresponding to reducing sugars, such as glucose monomers, maltose and dextrins occurs [ 19 , 40 ]. To predict the amount of hydrolytic product formed over time, the reaction kinetics of starch degradation were modeled using the first order equation (Equation (1)) [ 21 ]:

where rs t is the reducing sugars mass (g) generated over time, rs∞ is the maximum mass of reducing sugars (g) produced at each operational condition in the i -IDS, k is the degradation rate constant (1/min) and t is the time (min). It is relevant to mention that Equation (1) represents a kinetic model of reducing sugar generation.

The amount of reducing sugars generated by starch digestion in relation to the initial potato starch mass can be expressed as a percentage according to Equation (2):

where M 0 is the initial mass of potato starch (g).

The membrane efficiency is defined as the ability to allow the passage of the hydrolytic products generated during digestion in the i -IDS from the feed to the dialysate, which can be quantified in Equation (3) as:

where M rsD is the dialysate reducing sugars mass (g) and M rsF is the feed reducing sugars mass (g).

The expected overall efficiency of the mass transfer stage in the i -IDS with respect to the maximum mass of reducing sugars generated from the batch digestion of starch was determined according to Equation (4):

where M T is the maximum mass of reducing sugars generated (g) from batch experiments.

From the experimental results, a phenomenological model is proposed to simulate the reducing sugars transfer process in the in vitro system, based in our previous work [ 18 ]. This model is a useful tool for the operation and scaling-up of the i -IDS to the human small intestine, including the digestion process of starch. Through the experimental data it became possible to propose a model that allows the concentration of reducing sugars in the feed vessel over time to be known. For the development of the model, the use of the transmembrane pressure values estimated in our previous study [ 18 ] was considered.

2.5. Statistical Analysis of Data

All experiments were carried out in triplicate using freshly prepared samples, and results are presented as mean values with standard deviations. Analysis of variance was carried out when required using Statgraphic Centurion XVI (version 16.1, Statistical Graphics Corporation, Rockville, MD, USA), including multiple range tests ( p > 0.05) for separation of least square means.

3. Results and Discussion

In this study, time-concentration curves of reducing sugars in the feed for different operating conditions of feed flow/dialysate flow ratio in the i -IDS were obtained ( Figure 2 ). The shape of these time-concentration curves describes the continuous generation of hydrolytic products from starch digestion, together with the corresponding transfer of these products to the dialysate. The increase in concentration of reducing sugars (by the enzymatic digestion of starch) in the feed tank with time occurred until a maximum value was reached, after which a decrease in reducing sugars concentrations as the transfer occurs was noted. The latter was promoted by the operation of the membrane, where the sugars transfer from the feed side to the dialysate side will be determined by the minimum size reached by these molecules, which are capable of penetrating the membrane pores (molecular weight cut-off < 20 kDa). In Figure 2 , the experimental results were compared with those previously obtained by our research team [ 18 ] in a study of glucose transfer. In this study, the same system was used, where a feed consisting of glucose solution (15 mg/mL) was employed in the i -IDS to simulate and extrapolate results of the process of glucose transfer in the human small intestine. The previous study did not consider the effects of other phenomena interacting simultaneously with the sugar transfer process, such as digestion and consequent generation of hydrolytic products. For this reason, the development of the current study was based on a more complex phenomenology, simulating the starch digestion (main source of digestible carbohydrates) in a more realistic manner, which will allow a more detailed understanding of some critical aspects that must be considered when operating new or pre-existing models of human small intestine simulation.

Experimental curves of glucose concentration in the feed tank obtained by Gim-Krumm et al. [ 7 ] (primary axis) and concentration of reducing sugars in the feed generated from the enzymatic starch digestion (secondary axis) operating the i -IDS at different feed flow/dialysate flow ratios.

3.1. Concentration of Reducing Sugars in the i-IDS under Different Operational Conditions

During starch digestion in the i -IDS, the reducing sugars concentration over time in the feed and dialysate tanks for different operational conditions of feed flow/dialysate flow ratio was determined ( Figure 3 ). From the experimental curves, it can be observed that the feed flow/dialysate flow ratio affected the behavior of mass transfer of reducing sugars. Unlike the results previously obtained [ 18 ], where the glucose concentration in the feed tank only decreased, the results in Figure 3 show an increase in the concentration of reducing sugars from time 0 until a maximum was reached at a certain time, in function of the operating conditions. From this maximum value, a decrease in concentration begins. The rise in concentration is explained by the generation of reducing sugars, such as glucose, oligosaccharides or dextrins, due to the enzymatic digestion of starch induced by α-amylase. Since the dialysis module is already operating, the transfer of these sugars will be determined by the minimum size that is reached by these molecules. At this point, it should be considered that the analytical measurement included reducing sugars and not directly glucose, as previously described. For the concentration curves, the time at which the inflection point occurs depends on the feed flow/dialysate flow ratio tested (250/400, 400/400 and 400/250). As can be seen from Figure 3 , all concentration curves show a delay in the detection of reducing sugars in the dialysate, although there is an evident concentration gradient that promotes the mass transfer between both zones (feed and dialysate). It is known that the most common final products of starch hydrolysis are maltodextrins, glucose, fructose or maltose [ 36 ]. However, in the early stages of starch hydrolysis, maltodextrins are generated. These are a mixture of poly- and oligosaccharides with a broad molecular weight distribution. In fact, the molecular weight of commercial maltodextrin with different dextrose equivalent values (2–19) has been found in the range from 9 to 155 kDa [ 49 ]. Moreover, results reported by Bednarska (2015) [ 50 ] demonstrated that the majority of the products generated after the first 15 min of enzymatic starch hydrolysis had a molecular weight of 70 kDa, as a result of the attack of the enzyme on the accessible linear fragments of high molecular weight amylopectin. For this reason, hydrolysis products with a molecular weight higher than the cut-off of the membrane cannot be transferred to the dialyzed zone, but they can be detected using the DNS method in the feed zone through the reaction with the reducing-end groups of these high molecular weight fractions. In this regard, the membrane behaves as a selective barrier to small molecular weight sugars, just as the human small intestine does.

Concentration of reducing sugars in the feed and dialysate tanks over time for different operational conditions of feed flow/dialysate flow ratio and transmembrane pressures.

When the i -IDS was operated at a flow ratio of 250/400 ( Figure 3 ), the whole experimental time period (60 min) could not be completed, since when working at a higher flow rate of dialysate compared to the feed flow rate, there is a higher transmembrane pressure on the dialysate side (−7.25 mm Hg). This fact promoted the transfer of water and reducing sugars to the lumen side, causing the overflow of the feed tank in a short period (25 min), and as a consequence, it was necessary to finish the process earlier than anticipated. Furthermore, the convective flow under this condition competes with the diffusive flow, because there is a transfer of reducing sugars across both sides of the membrane, a result that is consistent with those previously observed [ 18 ]. However, when the i -IDS was operated at a flow ratio of 400/400 ( Figure 3 ), the whole experimental time period was completed. Under this condition, the system tends to equalize the concentrations of reducing sugars in both tanks, tending to an equilibrium state. In turn, the mass transfer rate of hydrolytic products decreases, since the convective and diffusive transfers depend on the concentration gradient between the dialysate and the feed. It was also found under this condition ( Figure 3 ) that the concentration of reducing sugars in the feed tank reaches a maximum, which favors the diffusive mass transfer induced by a higher concentration gradient between the lumen and the shell side of the membrane module, after which this gradient decreases. For this case, the transmembrane pressure is low (19.66 mm Hg), a condition which favors the diffusive mass transfer of reducing sugars in the system.

For the case in which the i -IDS was operated at a feed flow/dialysate flow ratio of 400/250 ( Figure 3 ), the reducing sugars transfer was influenced by a diffusive and convective process from the feed side to the dialysate side. Here, the amount of reducing sugars generated from the starch digestion during the testing time was higher in comparison with the other cases studied (see next section). This generated a higher concentration gradient between the lumen and the shell side, and favored the transfer of reducing sugars to the dialysate which is reduced gradually in time, and decreased the rate of reducing sugars transfer to the dialysate to the extent that the concentrations in both tanks are equal. The higher feed flow compared to the dialysate flow generated a pressure difference (transmembrane pressure of 59.73 mm Hg) that favored the mass transfer by convective effect. In this case, the simulated absorption process of reducing sugars turns out to be faster compared to the previous tests, and an equilibrium state for the concentrations between both tanks was reached at ~60 min of testing.

3.2. Mass of Reducing Sugars Obtained under Different Operating Conditions of the i-IDS and the In Vitro Batch Digestion of Starch

The amounts of hydrolytic products generated during each test, promoted by the in vitro starch hydrolysis and expressed as mass of reducing sugars, for the different operating conditions of feed flow/dialysate flow in the i -IDS, and the generation of reducing sugars obtained from the batch digestion of starch are shown in Figure 4 .

Reducing sugar mass in the feed, dialysate and total over time for different operational conditions of the i -IDS and mass of reducing sugars generated under batch condition.

From Figure 4 , it can be observed that at the end of the assay (25 min at a ratio of 250/400) ~3 g of reducing sugars were generated. This value is similar to that obtained from the test at a ratio of 400/400 (2.86 g) after 60 min of assay and similar to the sugars mass obtained at 14 min (2.83 g) when the assay was carried out at ratio of 400/250. As previously discussed for the results of concentration, the behavior found in Figure 4 at a ratio of 250/400 can be associated with the differences in pressure between the lumen side and the shell side of the membrane, promoting the transfer of water and reducing sugars to the lumen. The low amount of reducing sugars measured in the dialysate suggests that the transfer of water from the dialysate into the feed could be a factor that affects the passage of hydrolytic products through the membrane. When analyzing the results at an operating ratio of 400/400 ( Figure 4 ), it was observed that the transport of reducing sugars from the feed into the dialysate was initiated at ~25 min, the longest time period for which these sugars were transferred. In any case, and as previously commented, under this operating condition the process is controlled only by a diffusive transport, as found in the human small intestine [ 27 ]. Unlike the results mentioned above, the operation of the i -IDS at a feed flow/dialysate flow ratio of 400/250 favored the rapid transfer of hydrolytic products into the dialysate, as soon as the generation of reducing sugars begins. Under this condition, a higher amount of reducing sugars generated was obtained (4.9 g), in comparison with the other flow ratios analyzed (2.92 g at 250/400 and 2.86 g at 400/400). This fact can be explained by the higher rate of starch hydrolysis attributed to the prompt transfer of hydrolytic products with molecular weight lower than 20 kDa, since it is known that compounds produced during the starch hydrolysis (e.g., glucose and maltose) can induce uncompetitive inhibition of the catalytic action of α-amylase [ 1 , 51 ]. Alternatively, the rapid starch hydrolysis in the feed tank and subsequent removal of the reducing sugars generated could be related to the higher mass transfer as the feed viscosity reduces, since at low viscosity starch and α-amylase can move freely to contact each other. Furthermore, when the system is operated at a flow ratio of 400/250, it results in a higher loss of water in the feed tank ( Figure 5 ). In consequence, the concentration of substrate and α-amylase in this container is increased, promoting the increase in the rate of starch degradation. This agrees with Michaelis-Menten’s kinetics [ 1 ], which describes the rate of generation of hydrolytic products during starch digestion as a function of initial starch concentration. Therefore, given the results in Figure 5 , it would be expected that the increase in the concentration of starch and α-amylase in the feed tank would be more significant when the feed flow was higher in comparison with the other operating conditions assayed.

Volume variation of the feed tank, for different operational conditions of the i -IDS. Positive values mean accumulation and negative values mean water transfer.

From the in vitro assays of batch starch digestion, the maximum amount of reducing sugars that can be obtained during one hour of hydrolytic reaction with α-amylase under controlled conditions of temperature was determined, with a value equal to 6.7 g ( Figure 4 ). In this study, the degradation rate constant of starch (k) was obtained from the mass generation curve under batch condition and was estimated in 0.037 1/min. This value is in the range of those reported by Goñi and co-workers [ 21 ] (k = 0.03 − 0.16 1/min), who applied an in vitro procedure to measure the rate of starch digestion in starchy common foodstuffs, and by Butterworth and co-workers [ 43 ] (k = 0.025 − 0.038 1/min), who analyzed the starch amylolysis using plots for first-order kinetics. Now, considering that initial mass of gelatinized starch for all the experiments carried out was of 20 g, it was estimated that the starch hydrolysis obtained at the end of the batch digestion (60 min) was of ~33.5%, based on the 6.7 g of reducing sugars generated and which could be effectively available for absorption in the i -IDS. From this value, the expected overall efficiency (%, Equation (4)) of the i -IDS was defined, with the purpose of comparing the results obtained from this system with those obtained from the static models commonly used to simulate the in vitro food digestion. When operating the i -IDS at feed flow/dialysate flow ratios of 250/400, 400/400 and 400/250, the expected overall efficiencies were 10.2%, 31.5% and 66.4%, respectively ( Table 2 ). These percentages are in accordance with the argumentation previously presented in relation to the impact of the operating conditions on starch hydrolysis and mass transfer of hydrolytic products. However, it highlights the fact that both systems analyzed (i.e., dynamic and static) have marked differences in the results obtained in evaluating the in vitro starch digestion.

Percentages of efficiency of the in vitro intestinal digestion system ( i -IDS).

The final conversions of starch to reducing sugars in the i -IDS were 14.6%, 14.3% and 24.5%, at flow ratios of 250/400, 400/400 and 400/250, respectively ( Table 2 ). The operation of the i -IDS at a ratio of 400/250 presented the higher membrane efficiency (91.5%), in line with the explanation given above. On the contrary, when operating at ratios of 250/400 and 400/400, lower membrane efficiencies were obtained due to the lower generation of reducing sugars. With all this in mind, it is important to highlight the impact of the operating conditions of the i -IDS, or other similar dialysis membrane-based systems, when studying in vitro digestion of different food matrices, since these conditions determine the diffusive and convective transfers that control the process.

3.3. Fouling Analysis and Its Effect on Dialysis Performance

In this study, differences found in the dialysis performance at different operating conditions of the i -IDS could also be explained by the potential effect of membrane fouling on the system behavior. Different contents of reducing sugars in the dialysate tank were achieved by absorption and dialysis of hydrolytic products through hollow fiber membranes in the i -IDS. From the experimental results, it was established that different phenomena determine the behavior of the system. Therefore, in order to understand in a better way the response of the variation of reducing sugars concentration in the feed with time, a mathematical modeling was performed adopting the realization of a mass balance based on our previous work [ 18 ].

The transfer phenomena of reducing sugars by convective and diffusive effects from the feed tank to the dialysate tank can be represented by the following expression (Equation (5)):

where J rs is the total flux of reducing sugars in the dialysis membrane process (g/m 2 min), J D is the flux of reducing sugars transferred by diffusion (g/m 2 min), and J C is the flux of reducing sugars transferred by convection (g/m 2 min).

The overall mass transfer of reducing sugars through the membrane (N rs , g/ min) can be described as the difference of concentration (diffusive or osmotic effect) and transmembrane pressure (convective or hydrostatic effect) through the Equation (6) [ 18 ]:

where K is the overall mass transfer coefficient (m/min), A T is the total area of mass transfer (m 2 ), K UF is the ultrafiltration coefficient reported by the supplier of the dialysis membrane modules (m 3 /Pa min), ΔP is the transmembrane pressure (Pa), C F rs is the concentration of reducing sugars in the bulk of the feed (g/m 3 ), and C D rs is the concentration of reducing sugars in the bulk of the dialysate (g/m 3 ).

The mathematical model proposed previously [ 18 ], which represents the process of reducing sugars transfer in the membrane system, was described in Equation (7) as:

where C rs 0 is the initial reducing sugars concentration (g/m 3 ) and V F is the feed volume (m 3 ). This last equation has been taken as a good starting point for modeling the results obtained in this study, considering the incorporation of the kinetics of starch digestion (Equation (1)) and eliminating the term C rs 0 , since in this case this value was equal to 0, which increases gradually over time.

Following the approach mentioned above, the hydrolytic products in the system will depend on the kinetics of reducing sugars generation, together with the diffusive and convective effect. This means that the mass transferred through the membrane (N rs ) is equal to the mass of reducing sugars generated over time, which can be obtained by deriving the kinetic equation (Equation (1)). Therefore, this mass balance can be expressed by the following Equation (8):

By discretizing Equation (8), it is possible to determine the concentration of reducing sugars in the feed ( C F rs ) in function of time (Equation (9)). The mass of reducing sugars generated over time (rs t and rs t−1 ) by the in vitro starch digestion can be determined from the generation of hydrolytic products (Equation (1)).

where rs t−1 corresponds to the moles of reducing sugars generated in the previous time period (g), and Δt is the time step (min).

Certain operational variables can impact membrane separation of poly- and oligosaccharides, affecting in turn the overall efficiency of the process [ 52 ]. The membrane fouling often causes dramatic decreases of the flux. This explains the fact by which the membrane fouling should be considered when studying its implications for the separation efficiency of the process. With this in mind, in this study a parameter of mass transfer resistance (R MT , dimensionless) was proposed, which represents the formation and degradation of a dynamic fouling layer that decreases the mass flux over time through the membrane. In this context, Equation (8) can be modified by incorporating the term of mass transfer resistance, according to Equation (10):

In the current study, the membrane fouling could be due to (i) solid impurities from the commercial starch, deposited on the membrane, (ii) formation of a biopolymer layer (i.e., composed by undigested starch and reducing sugars generated by enzymatic hydrolysis, whose sizes are larger than ~20 kDa) at the membrane surface and/or (iii) the gelatinized starch itself filling the pores of the membrane [ 53 ]. This fact is in line with reported in vivo observations of gastrointestinal mucoadhesion. For example, some studies, which focused on drug bioavailability, indicate that, by adhering to the mucus layer of the gastrointestinal tract, mucoadhesives induce rapid drug absorption. However, mucoadhesion can be significantly limited by the constant passage of food or by colloidal particulate systems, which generates an unavoidable interaction leading to fouling on the mucous gel layer [ 54 , 55 ]. Due to this, the study of intestinal absorption using simplified models based on membranes that act as an epithelial barrier has promoted new research lines focused on improving nutrient bioavailability. In fact, different chemical modifications of synthetic membranes (e.g., cell cultures-based membrane) have been studied, since it has been recognized that several constituents lead to polymer membrane fouling, such as dissolved organic/inorganic compounds, colloids, cells and suspended solids [ 56 ].

Thus, the fouling for the dialysis process in the i -IDS can be considered as a dynamic phenomenon, since the hydrolytic products from the starch reaction vary with time, and also material adhering on the membrane surface can be removed during the process time. In consequence, the value of R MT for each operating condition can be determined by rewriting Equation (10) as follows (Equations (11) and (12)):

In Equation (12), the mass transfer coefficient (K) and ultrafiltration coefficient (K UF ) were estimated using the same mathematical methodology described by Gim-Krumm and co-workers [ 18 ]. As it can be expected, and in agreement with the results of concentration and mass of reducing sugars generated from the in vitro starch digestion in the i -IDS discussed in the Section 3.1 and Section 3.2 , respectively, the value of R MT was a function of time and of the operating conditions of the i -IDS ( Figure 6 ). First of all, this highlights the fact that for the three feed flow/dialysate flow ratios analyzed, a mass transfer resistance was observed, which occurred immediately after starting the i -IDS operation. It is known that fouling can occur in a variety of membrane systems after a few minutes of operation [ 57 ]. However, this did not occur in our previous study [ 18 ], since we only worked with a glucose solution. Therefore, the glucose (size ~180 Da) was completely available for the transfer process, since there was a high concentration gradient, which promoted mass transfer in the system without being altered by some interference phenomenon. Nevertheless, in the current study, a more complex matrix based on starch was digested in the i- IDS, where the process becomes dependent on the generation rate of reducing sugars for their subsequent absorption. In turn, the operating conditions influenced the loss or gain of water in both sides of the system, which can impact the formation of large hydrolytic products that favor membrane fouling for the dialysis process [ 58 , 59 , 60 ].

Variation of the mass transfer resistance (R MT ) with time, for different operational conditions of the i -IDS.

When the i -IDS was operated at a flow ratio of 250/400 ( Figure 6 ), an increase of the values of R MT with time was obtained, and the lowest values after 25 min of assay, when comparing with the other operating conditions, were reached. This last behavior can be associated with the continuous inflow of water from the dialysate to the feed, which generates a cleaning effect of the fouling layer on the membrane surface. However, at a ratio of 400/250, it was possible to observe an increase in the mass transfer resistance over time up to 20 min of operation reaching a maximum. Then, this resistance decreases gradually over time, which reduces the fouling effect and favors the mass transfer to the dialysate. These increases in mass transfer are also favored by the flow of water from the feed to the dialysate, facilitating the transport of hydrolytic products with sizes lower than 20 kDa. Moreover, for the assay performed at a ratio of 400/400, the resistance values were similar to those obtained at a ratio of 400/250 up to 18 min, and then, R MT continued to rise up to ~30 min of assay, after which it was held constant throughout the experiment. For this condition, the evolution curve of R MT shows an inflection point close to 25 min, a time that coincides with the beginning of the mass transfer of reducing sugars to the dialysate. In addition, at an operating ratio of 400/400, not only does fouling formation occur, but also, in comparison with the other conditions, there is no significant flow of water between the feed and the dialysate in the system ( Figure 5 ). This minimizes the interference to the mass transfer towards the dialysate, so there is an additional effect on the mass transfer resistance associated with the transmembrane pressure that turned out to be similar.

3.4. Scaling-Up to the Human Small Intestine

The experimental results of reducing sugars absorption of this study were extrapolated to those found in the human small intestine. For this purpose, the phenomenological model described by Equation (11) and the overall mass balance of the system were used as follows: (i) the mass of reducing sugars transferred in the human small intestine was used to estimate an expected efficiency in the i -IDS, (ii) the overall mass balance and the expected efficiency allowed a final concentration of reducing sugars in the feed solution to be determined, and (iii) by using the above concentration of reducing sugars and Equation (11), the operation time needed to reach the same mass of reducing sugars transferred in the i -IDS was computed for each operating condition. This approach implies that the absorption process of any nutrient towards the human small intestine can be represented using the i -IDS, according to fixed experimental parameters. For this analysis, glucose transfer in the small intestine was used as a reference, since predicting and controlling the glucose absorption due to the ingestion of starchy food is of great interest worldwide. Furthermore, the most widely used method for estimating the kinetics of in vitro starch digestion consists of simulating gastrointestinal conditions in order to measure the glucose release at different times [ 1 , 61 ].

The results of scaling-up the i -IDS to the human small intestine are shown in Table 3 . The operating time in the i -IDS required to reach the mass absorbed of reducing sugars in the human small intestine (1.06 g) ranged between 30 and 64 min, according to the feed flow/dialysate flow ratio operated in the system. These time values are lower than those found in the human small intestine (180 min), according to literature data [ 27 , 53 ]. These differences are mainly associated with the higher feed flows used in the i -IDS (250–400 mL/min) with respect to intestinal flow (3 mL/min [ 62 , 63 , 64 ]) and also with the lower area of mass transfer (1.7 m 2 vs. 30 m 2 [ 65 ]). In our previous work using the i -IDS [ 18 ], glucose absorption, understood as glucose transfer, was reached at lower times (2.1–2.7 min at similar feed flows), which was mainly explained by the fact that the system had a high concentration of glucose from the beginning of assay and no hydrolytic reaction was involved (i.e., system without digestion). For this reason, there was no time period that would generate a product and therefore increase the total process time. Neither was there a high concentration gradient from 0 min, by which the mass transfer immediately began. On the contrary, and for this case study, a longer time of mass transfer was obtained because of the time required by the enzyme to hydrolyze the gelatinized starch in the feed.

Scaling-up results of the i -IDS to the human small intestine.

1 Based on the value of glucose transfer reported in literature [ 66 ]. 2 Mean value obtained for concentrations of reducing sugars in the dialysate for all the experimental runs. 3 The stomach volume was defined as a mean value from literature data [ 6 , 67 , 68 ]. 4 Value of the small intestine transfer area reported in literature [ 65 ]. 5 Based on the value reported by Gim-Krumm and co-workers (2018) [ 18 ]. 6 Value of the flow fed into the human small intestine reported in literature [ 62 , 63 , 64 ]. 7 Intestinal digestion time based on literature [ 27 , 53 ].

By operating the i -IDS, it is possible to study the generation and absorption of hydrolytic products obtained from diverse starchy food matrices and to scale-up the results to the human small intestine using Equation (11). The extrapolation to the human small intestine will require the use of an initial concentration of starch ( C F   starch 0 ), such that the amount of products generated (rs t ) be greater compared to the flow of products transferred towards the intestine (N rs ), considering that the human small intestine is capable of transferring ~96% [ 66 ] of the hydrolytic products generated from the digestion of starch. However, the scaling-up results obtained here are limited to the operating conditions used. Hence, the testing time (maximum 25 min for the assay at 250/400 and 60 min at ratios of 400/400 and 400/250), the maximum generation of products for each feed flow/dialysate flow ratio and the mass transfer resistance (R MT ) can effectively describe the experimental behavior under these considerations. However, it should be noted that the mathematical modeling can also be used for longer processing times than those used in this approach.

The mathematical model proposed includes real phenomena occurring in the human small intestine, such as osmotic and hydrostatic transport. Therefore, this simple model could be used to simulate and understand, when testing in vitro systems, the complex behavior of the human small intestine.

In summary, the i -IDS can be used to study food digestion and absorption, using similar feed flows and flows ratios as tested here, if the dialysis membrane module is the same. When using a different dialysis membrane module and different flows, the evaluation of results can be performed using the phenomenological model proposed. Thus, the times resulting from a different food matrix in a different laboratory prototype can be related to the real operation of the human small intestine by using the mathematical modeling and scaling-up methodology here proposed.

Finally, it is proper to point out that the i -IDS is a versatile system, which makes it suitable for hypothesis building and hypothesis testing when studying digestion and absorption processes in the human small intestine of different starch-based food matrices. The main advantage of the i -IDS is the possibility that hydrolytic products generated from digestion can be selectively removed during the digestion process (i.e., absorption). Nevertheless, the formation of membrane fouling during the separation processes needs further studies to propose a phenomenological interpretation with respect to the physiological behavior. In fact, it has been recognized that effective human intestinal permeability of rapidly absorbed compounds in vivo (e.g., glucose) is mainly determined by the membrane permeability, which it implies that the membrane of the intestinal mucosa is the main diffusion barrier for both passively and actively absorbed solutes [ 69 ]. On this basis, an additional effort must be made for a next improvement step of the i -IDS where membrane fouling can be controlled and interpreted. In addition, this model has the challenge of incorporating physiological events that mimic the functioning of the human small intestine. For the latter, a good starting point will be the application of the new standardized protocol developed by the COST INFOGEST network, where physiological intestinal conditions such as electrolytes incorporation, enzyme activities, bile, dilution, pH and time of digestion will be simulated [ 70 ]. In addition, the i -IDS could also be used as an exploratory system for studying and understanding digestion and absorption processes using other food matrices. However, further studies using this model are required if food matrices based on fat, proteins or more complex carbohydrates are of interest for testing.

4. Conclusions

This work deepens our understanding of starch digestion and the consequent absorption process of the hydrolytic products generated, as found in the human small intestine. By carrying out assays of in vitro digestion with α-amylase, gelatinized starch dispersions were digested in an in vitro intestinal digestion system ( i -IDS) based on a hollow fiber dialysis membrane process. This study is innovative with respect to previous research conducted by our research team, because it considers the impact of simultaneous digestion and absorption processes occurring during intestinal digestion of starchy foods and adopts phenomenological models that deal in a more realistic manner with the behavior found in the human small intestine.

From the operation of the i -IDS at different flow/dialysate flow ratios, different curves were obtained for the generation and transfer of reducing sugars. This demonstrates that the operating conditions affected the behavior of mass transfer of reducing sugars because of diffusive and convective effects. However, the mass transfer behavior was also greatly affected by membrane fouling, a dynamic phenomenon occurring in the i -IDS, which was observed for all the experimental conditions tested. Finally, the experimental results obtained were extrapolated to those found in the human small intestine, where the times reached to transfer the hydrolytic products ranged between 30 and 64 min, according to the flow ratio used.

The i -IDS is a versatile system that can be used to predict digestion and absorption behaviors in the human small intestine of different starch-based food matrices, but could also be used as an exploratory system for studying and understanding these processes using other food matrices based on fat, proteins or more complex carbohydrates. However, further studies using the i -IDS are required to control and interpret membrane fouling, and also to simulate relevant physiological conditions as found in the human small intestine.

Acknowledgments

The authors gratefully acknowledge the financial support of CONICYT through FONDECYT project 11140543, FONDECYT project 1191858, and project fund CONICYT-PIA Project AFB180004.

Nomenclature

Author Contributions

Conceptualization, E.T., H.E. and R.N.Z.; methodology, E.T., R.N.Z., C.G., D.G. and H.E.; formal analysis, E.T., H.E. and R.N.Z.; investigation, C.G. and D.G.; resources, E.T.; data curation, C.G., E.T., H.E. and R.N.Z.; writing—original draft preparation, C.G., E.T., H.E. and R.N.Z.; writing—review and editing, E.T., H.E. and R.N.Z.; supervision, E.T., H.E. and R.N.Z.; project administration, E.T.; funding acquisition, E.T. and R.N.Z. All authors have read and agreed to the published version of the manuscript.

This research was funded by the National Commission for Scientific and Technological Research (CONICYT Chile) through FONDECYT project 11140543, FONDECYT project 1191858, and project fund CONICYT-PIA Project AFB180004.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Starch Digestion

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23.7 Chemical Digestion and Absorption: A Closer Look

Learning objectives.

By the end of this section, you will be able to:

• Identify the locations and primary secretions involved in the chemical digestion of carbohydrates, proteins, lipids, and nucleic acids
• Compare and contrast absorption of the hydrophilic and hydrophobic nutrients

As you have learned, the process of mechanical digestion is relatively simple. It involves the physical breakdown of food but does not alter its chemical makeup. Chemical digestion, on the other hand, is a complex process that reduces food into its chemical building blocks, which are then absorbed to nourish the cells of the body ( Figure 23.28 ). In this section, you will look more closely at the processes of chemical digestion and absorption.

Chemical Digestion

Large food molecules (for example, proteins, lipids, nucleic acids, and starches) must be broken down into subunits that are small enough to be absorbed by the lining of the alimentary canal. This is accomplished by enzymes through hydrolysis. The many enzymes involved in chemical digestion are summarized in Table 23.8 .

Carbohydrate Digestion

The average American diet is about 50 percent carbohydrates, which may be classified according to the number of monomers they contain of simple sugars (monosaccharides and disaccharides) and/or complex sugars (polysaccharides). Glucose, galactose, and fructose are the three monosaccharides that are commonly consumed and are readily absorbed. Your digestive system is also able to break down the disaccharide sucrose (regular table sugar: glucose + fructose), lactose (milk sugar: glucose + galactose), and maltose (grain sugar: glucose + glucose), and the polysaccharides glycogen and starch (chains of monosaccharides). Your bodies do not produce enzymes that can break down most fibrous polysaccharides, such as cellulose. While indigestible polysaccharides do not provide any nutritional value, they do provide dietary fiber, which helps propel food through the alimentary canal.

The chemical digestion of starches begins in the mouth and has been reviewed above.

In the small intestine, pancreatic amylase does the ‘heavy lifting’ for starch and carbohydrate digestion ( Figure 23.29 ). After amylases break down starch into smaller fragments, the brush border enzyme α-dextrinase starts working on α-dextrin , breaking off one glucose unit at a time. Three brush border enzymes hydrolyze sucrose, lactose, and maltose into monosaccharides. Sucrase splits sucrose into one molecule of fructose and one molecule of glucose; maltase breaks down maltose and maltotriose into two and three glucose molecules, respectively; and lactase breaks down lactose into one molecule of glucose and one molecule of galactose. Insufficient lactase can lead to lactose intolerance.

Protein Digestion

Proteins are polymers composed of amino acids linked by peptide bonds to form long chains. Digestion reduces them to their constituent amino acids. You usually consume about 15 to 20 percent of your total calorie intake as protein.

The digestion of protein starts in the stomach, where HCl and pepsin break proteins into smaller polypeptides, which then travel to the small intestine ( Figure 23.30 ). Chemical digestion in the small intestine is continued by pancreatic enzymes, including chymotrypsin and trypsin, each of which act on specific bonds in amino acid sequences. At the same time, the cells of the brush border secrete enzymes such as aminopeptidase and dipeptidase , which further break down peptide chains. This results in molecules small enough to enter the bloodstream ( Figure 23.31 ).

Lipid Digestion

A healthy diet limits lipid intake to 35 percent of total calorie intake. The most common dietary lipids are triglycerides, which are made up of a glycerol molecule bound to three fatty acid chains. Small amounts of dietary cholesterol and phospholipids are also consumed.

The three lipases responsible for lipid digestion are lingual lipase, gastric lipase, and pancreatic lipase . However, because the pancreas is the only consequential source of lipase, virtually all lipid digestion occurs in the small intestine. Pancreatic lipase breaks down each triglyceride into two free fatty acids and a monoglyceride. The fatty acids include both short-chain (less than 10 to 12 carbons) and long-chain fatty acids.

Nucleic Acid Digestion

The nucleic acids DNA and RNA are found in most of the foods you eat. Two types of pancreatic nuclease are responsible for their digestion: deoxyribonuclease , which digests DNA, and ribonuclease , which digests RNA. The nucleotides produced by this digestion are further broken down by two intestinal brush border enzymes ( nucleosidase and phosphatase ) into pentoses, phosphates, and nitrogenous bases, which can be absorbed through the alimentary canal wall. The large food molecules that must be broken down into subunits are summarized Table 23.9

The mechanical and digestive processes have one goal: to convert food into molecules small enough to be absorbed by the epithelial cells of the intestinal villi. The absorptive capacity of the alimentary canal is almost endless. Each day, the alimentary canal processes up to 10 liters of food, liquids, and GI secretions, yet less than one liter enters the large intestine. Almost all ingested food, 80 percent of electrolytes, and 90 percent of water are absorbed in the small intestine. Although the entire small intestine is involved in the absorption of water and lipids, most absorption of carbohydrates and proteins occurs in the jejunum. Notably, bile salts and vitamin B 12 are absorbed in the terminal ileum. By the time chyme passes from the ileum into the large intestine, it is essentially indigestible food residue (mainly plant fibers like cellulose), some water, and millions of bacteria ( Figure 23.32 ).

Absorption can occur through five mechanisms: (1) active transport, (2) passive diffusion, (3) facilitated diffusion, (4) co-transport (or secondary active transport), and (5) endocytosis. As you will recall from Chapter 3, active transport refers to the movement of a substance across a cell membrane going from an area of lower concentration to an area of higher concentration (up the concentration gradient). In this type of transport, proteins within the cell membrane act as “pumps,” using cellular energy (ATP) to move the substance. Passive diffusion refers to the movement of substances from an area of higher concentration to an area of lower concentration, while facilitated diffusion refers to the movement of substances from an area of higher to an area of lower concentration using a carrier protein in the cell membrane. Co-transport uses the movement of one molecule through the membrane from higher to lower concentration to power the movement of another from lower to higher. Finally, endocytosis is a transportation process in which the cell membrane engulfs material. It requires energy, generally in the form of ATP.

Because the cell’s plasma membrane is made up of hydrophobic phospholipids, water-soluble nutrients must use transport molecules embedded in the membrane to enter cells. Moreover, substances cannot pass between the epithelial cells of the intestinal mucosa because these cells are bound together by tight junctions. Thus, substances can only enter blood capillaries by passing through the apical surfaces of epithelial cells and into the interstitial fluid. Water-soluble nutrients enter the capillary blood in the villi and travel to the liver via the hepatic portal vein.

In contrast to the water-soluble nutrients, lipid-soluble nutrients can diffuse through the plasma membrane. Once inside the cell, they are packaged for transport via the base of the cell and then enter the lacteals of the villi to be transported by lymphatic vessels to the systemic circulation via the thoracic duct. The absorption of most nutrients through the mucosa of the intestinal villi requires active transport fueled by ATP. The routes of absorption for each food category are summarized in Table 23.10 .

Carbohydrate Absorption

All carbohydrates are absorbed in the form of monosaccharides. The small intestine is highly efficient at this, absorbing monosaccharides at an estimated rate of 120 grams per hour. All normally digested dietary carbohydrates are absorbed; indigestible fibers are eliminated in the feces. The monosaccharides glucose and galactose are transported into the epithelial cells by common protein carriers via secondary active transport (that is, co-transport with sodium ions). The monosaccharides leave these cells via facilitated diffusion and enter the capillaries through intercellular clefts. The monosaccharide fructose (which is in fruit) is absorbed and transported by facilitated diffusion alone. The monosaccharides combine with the transport proteins immediately after the disaccharides are broken down.

Protein Absorption

Active transport mechanisms, primarily in the duodenum and jejunum, absorb most proteins as their breakdown products, amino acids. Almost all (95 to 98 percent) protein is digested and absorbed in the small intestine. The type of carrier that transports an amino acid varies. Most carriers are linked to the active transport of sodium. Short chains of two amino acids (dipeptides) or three amino acids (tripeptides) are also transported actively. However, after they enter the absorptive epithelial cells, they are broken down into their amino acids before leaving the cell and entering the capillary blood via diffusion.

Lipid Absorption

About 95 percent of lipids are absorbed in the small intestine. Bile salts not only speed up lipid digestion, they are also essential to the absorption of the end products of lipid digestion. Short-chain fatty acids are relatively water soluble and can enter the absorptive cells (enterocytes) directly. The small size of short-chain fatty acids enables them to be absorbed by enterocytes via simple diffusion, and then take the same path as monosaccharides and amino acids into the blood capillary of a villus.

The large and hydrophobic long-chain fatty acids and monoacylglycerides are not so easily suspended in the watery intestinal chyme. However, bile salts and lecithin resolve this issue by enclosing them in a micelle , which is a tiny sphere with polar (hydrophilic) ends facing the watery environment and hydrophobic tails turned to the interior, creating a receptive environment for the long-chain fatty acids. The core also includes cholesterol and fat-soluble vitamins. Without micelles, lipids would sit on the surface of chyme and never come in contact with the absorptive surfaces of the epithelial cells. Micelles can easily squeeze between microvilli and get very near the luminal cell surface. At this point, lipid substances exit the micelle and are absorbed via simple diffusion.

The free fatty acids and monoacylglycerides that enter the epithelial cells are reincorporated into triglycerides. The triglycerides are mixed with phospholipids and cholesterol, and surrounded with a protein coat. This new complex, called a chylomicron , is a water-soluble lipoprotein. After being processed by the Golgi apparatus, chylomicrons are released from the cell ( Figure 23.33 ). Too big to pass through the basement membranes of blood capillaries, chylomicrons instead enter the large pores of lacteals. The lacteals come together to form the lymphatic vessels. The chylomicrons are transported in the lymphatic vessels and empty through the thoracic duct into the subclavian vein of the circulatory system. Once in the bloodstream, the enzyme lipoprotein lipase breaks down the triglycerides of the chylomicrons into free fatty acids and glycerol. These breakdown products then pass through capillary walls to be used for energy by cells or stored in adipose tissue as fat. Liver cells combine the remaining chylomicron remnants with proteins, forming lipoproteins that transport cholesterol in the blood.

Nucleic Acid Absorption

The products of nucleic acid digestion—pentose sugars, nitrogenous bases, and phosphate ions—are transported by carriers across the villus epithelium via active transport. These products then enter the bloodstream.

Mineral Absorption

The electrolytes absorbed by the small intestine are from both GI secretions and ingested foods. Since electrolytes dissociate into ions in water, most are absorbed via active transport throughout the entire small intestine. During absorption, co-transport mechanisms result in the accumulation of sodium ions inside the cells, whereas anti-port mechanisms reduce the potassium ion concentration inside the cells. To restore the sodium-potassium gradient across the cell membrane, a sodium-potassium pump requiring ATP pumps sodium out and potassium in.

In general, all minerals that enter the intestine are absorbed, whether you need them or not. Iron and calcium are exceptions; they are absorbed in the duodenum in amounts that meet the body’s current requirements, as follows:

Iron —The ionic iron needed for the production of hemoglobin is absorbed into mucosal cells via active transport. Once inside mucosal cells, ionic iron binds to the protein ferritin, creating iron-ferritin complexes that store iron until needed. When the body has enough iron, most of the stored iron is lost when worn-out epithelial cells slough off. When the body needs iron because, for example, it is lost during acute or chronic bleeding, there is increased uptake of iron from the intestine and accelerated release of iron into the bloodstream. Since females experience significant iron loss during menstruation, they have around four times as many iron transport proteins in their intestinal epithelial cells as do males.

Calcium —Blood levels of ionic calcium determine the absorption of dietary calcium. When blood levels of ionic calcium drop, parathyroid hormone (PTH) secreted by the parathyroid glands stimulates the release of calcium ions from bone matrices and increases the reabsorption of calcium by the kidneys. PTH also upregulates the activation of vitamin D in the kidney, which then facilitates intestinal calcium ion absorption.

Vitamin Absorption

The small intestine absorbs the vitamins that occur naturally in food and supplements. Fat-soluble vitamins (A, D, E, and K) are absorbed along with dietary lipids in micelles via simple diffusion. This is why you are advised to eat some fatty foods when you take fat-soluble vitamin supplements. Most water-soluble vitamins (including most B vitamins and vitamin C) also are absorbed by simple diffusion. An exception is vitamin B 12 , which is a very large molecule. Intrinsic factor secreted in the stomach binds to vitamin B 12 , preventing its digestion and creating a complex that binds to mucosal receptors in the terminal ileum, where it is taken up by endocytosis.

Water Absorption

Each day, about nine liters of fluid enter the small intestine. About 2.3 liters are ingested in foods and beverages, and the rest is from GI secretions. About 90 percent of this water is absorbed in the small intestine. Water absorption is driven by the concentration gradient of the water: The concentration of water is higher in chyme than it is in epithelial cells. Thus, water moves down its concentration gradient from the chyme into cells. As noted earlier, much of the remaining water is then absorbed in the colon.

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VanCleave's Science Fun

Your Guide to Science Projects, Fun Experiments, and Science Research

Starch Digestion in Your Mouth

By Janice VanCleave

Digestion is the process by which your body changes the food you eat to forms that your body can use for repair, building, and energy. Digestion starts in your mouth where special digestive juices change starch into sugar. Starch is a large chemical generally stored in plants and sugar is a type of chemical that your body used to produce energy for everything your body does, from movement to all the chemical reactions that occur.

When you chew a cracker, the bits of cracker mixes with saliva. My mom told me to chew each bite 32 times. Not sure it takes that much chewing for every bite of food, but it is important that the food is thoroughly crushed and mixed with saliva.

An easy experiment for a great science fair project is to chew crackers for different amount of time, spit the chewed cracker into containers, and then test the material for the presence of starch.

CAUTION: Since spit, like all body fluids can contain contagious diseases, properly discard your testing materials, such as flushing them in the toilet.

Test for Starch

Tincture of iodine is used to treat wounds. It can be purchased where first aid supplies are sold. Iodine turns starch a dark blue-black color.

Problem: What affect does chewing time have on the amount of starch digested in the mouth?

Independent Variable: chewing time

Dependent Variable : amount of starch digested

Controlled Variables: size of cracker, type of cracker, testing procedure

Hypothesis: If the size and type of cracker remain the same, as the chewing time increases the amount of starch digested increases.

[In other words, the longer you chew the cracker , more of the starch in the cracker is digested.]

Experiment:

7 small crackers are 7 equal-sized cracker pieces

7 transparent plastic cups

1 tablespoon

1/2 cup of tap water

eye dropper

tincture of iodine

7 craft sticks

1. Place a cracker in your mouth.

2 . Chew the cracker for 60 seconds.

3. Spit the chewed cracker into a cup.

4. Add one tablespoon of water, and then add two drops of tincture of iodine.

5. Use a clean craft stick to thoroughly stir the contents of the cup. Place the craft stick in the trash.

6. Take a picture of the contents for a record of the color of the results.

7. Repeat steps 1 through 6 five times, each time reduce the chewing time 10 seconds.

1. Place a cracker in the remaining cup.

2. Add 1 tablespoon of water, and then add two drops of iodine.

3. Using the remaining clean craft stick, thoroughly stir the contents of the cup.

Food and Nutrition for Every Kid

(Paid Link)

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Action of Salivary Amylase on Starch

To study the action of salivary amylase on starch solution.

The interaction between salivary amylase and starch constitutes a fundamental aspect of our digestive process, providing a glimpse into the complex biochemical mechanisms that enable our bodies to extract energy from the food we consume. Salivary amylase, an enzyme secreted by salivary glands, initiates the breakdown of complex starch molecules present in our diet. As food enters the mouth, the enzyme catalyses the hydrolysis of starch into simpler sugars like maltose. This enzymatic action marks the first step in carbohydrate digestion.

Experiment Procedure

To demonstrate the experiment on the action of salivary amylase on starch, we need to follow the given procedure:

• First of all, rinse your mouth with fresh water and collect saliva using a spatula/spoon.
• Then, filter saliva through a cotton swab.
• Now, take 1 mL of filtered saliva in a test tube and add 10 mL of distilled water to the test tube. Label it as “saliva solution.”
• Next, take 2 mL of 1% starch solution in 2 labelled test tubes (A and B).
• Add 1 mL diluted saliva to test tube B and shake well.
• We will not add anything to test tube A and keep it in control.
• After 5 minutes, take 5 drops from test tube A on a tile or a glass slide.
• Add 2 drops of 1% iodine solution in it, mix and observe colour.
• Place 5 drops from test tube B away from A’s mixture.
• Add 2 drops of 1% iodine solution to B’s drops, mix and observe.
• Repeat the iodine test after 5, 10, 15, and 20 minutes.

In conclusion, the experiment about “Action of Salivary Amylase on Starch” shows how enzymes in our bodies help break down food. By collecting saliva, diluting it, and mixing it with starch, it demonstrates how starch changes into simpler sugars. This change is important for getting energy from our food. Iodine solution is used to see this change, which makes the colour of the mixture different. These findings remind us how our bodies work with chemicals to stay alive. Understanding how salivary amylase acts on starch helps us see how our bodies make use of the food we eat.

FAQs on the Action of Salivary Amylase on Starch

Q.1 what is salivary amylase and its role in digestion.

Ans. Salivary amylase is an enzyme produced by the salivary glands, primarily in the mouth. Its main role is to initiate the digestion of complex carbohydrates, specifically starches, into simpler sugars.

Q.2 How does salivary amylase work on starch?

Ans. Salivary amylase breaks down the starch molecules into smaller fragments by catalysing the hydrolysis of the glycosidic bonds that link the glucose units in the starch molecule. This results in the production of maltose and other shorter carbohydrate chains.

Q.3 What factors can affect the activity of salivary amylase on starch?

Ans. The activity of salivary amylase can be affected by factors like pH, temperature, and the presence of inhibitors. An optimal pH level is necessary for its activity, and extreme temperatures or certain inhibitors might denature or inhibit the enzyme’s function.

Q.4 Why do we rinse the mouth with fresh water at the beginning of the experiment?

Ans. Rinsing the mouth with fresh water helps remove any residual food particles or substances that might interfere with the experiment. It ensures that only the collected saliva is being used in the experiment.

Q.5 What happens to the pH during salivary amylase action on starch?

Ans. Salivary amylase works optimally in a slightly acidic to neutral pH range, typically around pH 6.7. It starts the starch digestion process in the mouth, where the slightly acidic environment due to the presence of acids from foods and beverages helps activate the enzyme. 

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Effects of feeding rates on growth performance and liver glucose metabolism in juvenile largemouth bronze gudgeon ( coreius guichenoti ).

Simple Summary

1. introduction, 2. materials and methods, 2.1. experimental design and breeding management, 2.2. sample collection and growth index calculation, 2.3. liver physiological parameters detection, 2.4. liver tissue section examination, 2.5. rna isolation, reverse transcription, and quantitative real-time pcr analysis, 2.6. data statistics and analysis, 3.1. effects of feeding rates on the growth performance, 3.2. effects of feeding rates on liver tissue sections and glycogen synthesis, 3.3. effects of feeding rate on plasma glucose, liver glycolysis, and gluconeogenesis metabolism-related enzyme activities and gene expression, 4. discussion, 4.1. effects of feeding rate on the growth performance, 4.2. effects of feeding rate on glucose metabolism, 5. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

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Chen, P.; Qu, H.; Yang, J.; Zhao, Y.; Cheng, X.; Jiang, W. Effects of Feeding Rates on Growth Performance and Liver Glucose Metabolism in Juvenile Largemouth Bronze Gudgeon ( Coreius guichenoti ). Animals 2024 , 14 , 2466. https://doi.org/10.3390/ani14172466

Chen P, Qu H, Yang J, Zhao Y, Cheng X, Jiang W. Effects of Feeding Rates on Growth Performance and Liver Glucose Metabolism in Juvenile Largemouth Bronze Gudgeon ( Coreius guichenoti ). Animals . 2024; 14(17):2466. https://doi.org/10.3390/ani14172466

Chen, Pei, Huantao Qu, Jing Yang, Yu Zhao, Xu Cheng, and Wei Jiang. 2024. "Effects of Feeding Rates on Growth Performance and Liver Glucose Metabolism in Juvenile Largemouth Bronze Gudgeon ( Coreius guichenoti )" Animals 14, no. 17: 2466. https://doi.org/10.3390/ani14172466

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