This lab report summarizes experiments on the digestion of proteins, carbohydrates, and triglycerides. In experiment one, albumin was digested with pepsin and hydrochloric acid to test for protein breakdown into peptides. The results showed that both pepsin and hydrochloric acid are needed for protein digestion. Experiment two tested the digestion of starch into maltose using amylase, confirming starch is broken down in the small intestine. The report provides background on the multi-step digestion processes for each macronutrient and cites sources to support the results and conclusions.

1.
Lab Report for Digestive
Physiology
Leah Stone
Course 2021 Section 4
Kelly Boon
March, 23 2015

2.
Introduction:
In the experiments for the digestive physiology we focused on the digestion of
proteins, carbohydrates and triglyceride lipids. Digestion is the breakdown of food by
mechanical or chemical digestion. We have seven main organs that help our body
breakdown the food we eat, mouth, pharynx, esophagus, stomach small and large
intestine, rectum and anus. The starting process of digestion begins in our mouths.
Along with the mouth our accessory organs which include teeth, tongue, salivary
glands, liver, gallbladder, and pancreas play a vital role in helping the beginning process
in the mouth. The mechanical digestion is what beings the digestive process and the
chemical digestion is directly affected by the efficiency of the mechanical digestion. Gail
Bornhorst and R. Paul Singh explain digestion in their article Bolus Formation and
Disintegration during Digestion of Food Carbohydrates. ‘​The first step in the digestion
process is oral digestion, which begins with mastication, or chewing. During mastication,
the simultaneous processes of food comminution and lubrication occur, the end product
being the food bolus.’ ‘During the gastric digestion process, the boluses are physically
reduced in size while being chemically broken down due to the acidic and enzymatic
conditions of the gastric secretions. The rate at which foods disintegrate will control the
rate at which they are emptied from the stomach and move to the intestines where
nutrients are absorbed.’
It’s important to know the chemical digestion of proteins, carbohydrates, and
triglycerides to fully understand the digestive system as a whole process. The main

3. objective of the chemical digestion is to produce the smallest molecule obtainable for
absorption. The proteins are broken down by enzymes into peptides and then into
amino acids which are able to travel through gastrointestinal tract and be absorbed.
Main digestion of proteins happens in the stomach. Lipids are broken down by lipase
into the smallest molecules glycerol and fatty acids. Lipids are primarily digested in the
small intestine. Starch is broken down by amylase into monosaccharides and
disaccharides. Starch being a complex carbohydrate starts the digestion in the mouth
with the release of amylase and is primarily digested and absorbed the small intestine.
‘These products plus ingested disaccharides, primarily lactose and sucrose, are further
cleaved to monosaccharides by disaccharidases in the brush border membrane of the
epithelial cells. In contrast, approximately l/ to ½2 of the ingested protein is hydrolyzed
completely to free amino acids by pancreatic enzymes. What remains is peptides
averaging 2 to 6 amino acids in length which are cleaved by peptide hydrolases while
being transported through the epithelial cells. Peptide hydrolases are present not only in
the brush border membrane but also in the cytosol and it is clear that some peptide
molecules are transported intact and hydrolyzed inside the cell’, William Heizer explains
absorption in his article Normal and abnormal intestinal absorption by humans.
In experiment one we were testing for digestion of proteins by adding the enzyme
pepsin to the substrate albumin and using biuret reagent to show the presence or lack
of a protein. John Northrop explains this theory ‘The optimum hydrogen ion
concentration for the digestion of the protein must coincide with the hydrogen ion
concentration at which the concentration of protein ions and therefore the conductivity

4. due to the protein is at a maximum.’ in his article The mechanism of an enzyme reaction
exemplified by pepsin digestion. My hypothesis was for test tube 2 to test positive for
proteins test tubes 3 and 4 to test positive for peptides after the digestion of albumin
with pepsin and HCL. Test tube 1 was negative for peptides, it was our basic and our
control for the experiment. Test tubes 3 and 4 contained hydrochloric acid which is
released in your stomach during the digestion of food. Test tube 3 was in 37 degree
celsius water and tested negative for peptides. Test tube 4 was put in an ice chest and
tested negative for both peptides and proteins. These results showed that pepsin alone
cannot digest proteins. Based on the article, Enhanced efficiency of biological excess
sludge hydrolysis under anaerobic digestion by additional enzymes, does a study on
enzymatic efficiency and temperatures. ‘​Based on the results of single enzymes
hydrolysis, the parabolic effects of mixed­enzyme on hydrolysis should be further
explored. Since particulate organic matters were changed into soluble organic matters
after enzymatic hydrolysis, VSS concentration of sludge samples was lower than control
test (​Fig. 3​). The observations confirmed that there was virtually no significant
relationship between temperature and VSS reduction for the control test, while it had
dramatically effect on enzyme­treated samples within the temperature range of 40–50
°C. Apparently, the VSS concentration of control test only decreased from 5650 mg/L
(40 °C) to 5350 mg/L (50 °C). However, the obvious decrease of VSS concentration to
2420 mg/L was observed at the mass ratio, approximately 1:3 for protease and amylase
at the temperature of 50 °C, which seemed to improve VSS reduction effectively’

5. According to the lab information provided and information provided in text by
Barbara E. Goodman in the article ‘Insights into digestion and absorption of major
nutrients in humans’ stated ‘​Consumed proteins or polypeptides begin to be broken
down in the stomach under the action of the protease pepsin (​4​). Pepsin is secreted by
chief cells in the gastric mucosa as pepsinogen, a larger inactive form of the enzyme,
also known as a zymogen. Gastric acid (HCl, secreted by the parietal cells) alters the
conformation of pepsinogen so that it can cleave itself and become active pepsin in the
stomach. Gastric acid also denatures the proteins, which partially unfolds them so that
proteases have better access to their peptide bonds. Pepsin (an endopeptidase) in the
stomach begins to hydrolyze proteins at various cleavage points to smaller
polypeptides’ confirming that the presence of both HCL and pepsin should have allowed
the breakdown of protein.​This experiment showed the process and results of the
enzyme pepsin reacting with hydrochloric acid in the breakdown of proteins into
peptides.
Experiment two was the digestion of carbohydrates and had 2 parts. It was
testing for broken down carbohydrates polysaccaride into oligosaccharides and
disaccarides using the enzyme amylose. Levin explains the breakdown of
carbohydrates in his article Bolus Formation and Disintegration during Digestion of Food
Carbohydrates ‘Hydrolysis of maltose, sucrose, and lactose by the brush border
disaccharidase produces glucose, galactose, and fructose. Although lactose absorption
may be rate limited by its slow hydrolysis, the rate of absorption of monosaccharides
produced by hydrolysis of other disaccharides at the brush border membrane should be

6. the same as from other monosaccharide solutions of equimolar concentration.’ Lugol’s
iodide was used to show the substrate amylose and benedicts reagent was used to
show the product maltose. Due to the amylose in the saliva beginning the process of
digestion for carbohydrates, the second and third part of this experiment needed to be
set up and timed out precisely.
The amylase only partially breaks down oligosaccharides and polysaccharides in
the mouth, moving through the stomach the oligosaccarides and polysaccarides break
further down but it is not until the small intestine that the starch is broken down and
absorbed. Experiment 2 my hypothesis was test tube 1 testing positive for starch, test
tube 2 testing negative for maltose, test tube 3 testing negative for starch showing
digestion of starch and leaving maltose and test tube 4 testing positive for maltose with
digestion of starch. Test tube 1 had 2 ml of 1% starch with 2­4 drops of lugol’s IKI. Test
tube 2 had 2 ml of 1% glucose and 2­4 drops of lugol’s IKI. Test tube 3 had 2 ml of 1%
starch and 1 ml of benedict’s and sad in boiling water for 2 minutes. Test tube 4 had 2
ml of 1 % glucose and 1 ml of benedict's and was put in boiling water for 2 minutes.
Test tube 1 tested positive for amylose, test tube 2 tested negative for maltose, test
tube 3 tested negative for starch and test tube 4 tested negative for maltose. These
were our control tubes.
Part two of this experiment was testing for the product which was the maltose.
Ros E explains ‘W​hile in health the intestinal absorption of triglycerides is very efficient,
the same does not apply to cholesterol absorption. Besides being generally inefficient,
cholesterol absorption is highly variable, with a between­subject variability that depends

7. in part on genetic factors and an intra­individual variability, which may be modulated by
physiological and dietary conditions.’ The digestion of carbohydrates is a complex task
that is not encountered with the digestion of other nutrients. ​Using the test tubes from
part 1 of experiment 2 we labeled the test tubes 2 min, 4 min, 6 min, 8 min, 10 min, and
20 min. My hypothesis for this experiment was the longer the starch­amylase mixture
were left in the boiling water the substrate would break down into the product maltose
(red). Test tube 1 tested positive for starch, test tube 2 tested negative for glucose, test
tube 3 tested negative for starch and benedict’s solution and test tube 4 tested negative
for glucose and benedict’s solution.
The third part of this experiment was testing for the substate amylose was
broken down into maltose. It was time sensitive and required a spot plate. The results of
the reaction between amylase and starch mixed with lugol’s IKI were recorded every
minute for 15 minutes, 5 minute lapse period then last recorded reaction at 20 minutes.
My hypothesis for this experiment was that at 1 minute it would be indigo (testing
positive for amylase) and each minute that passed the first depression on the spot plate
would grow increasingly more amber, breaking down into maltose. All of the
depressions tested positive for amylase at each minute recorded (with the exception of
the 9 minute mark where it tested slight positive for maltose) and at the 20 minute mark.
Experiment 3 was the digestion of triglyceride lipids into fatty acids by
lipases focusing on emulsification.’An important step in the intestinal digestion of these
lipids is their emulsification with bile salts [1–4]. Emulsification makes lipids better
substrates for hydrolysis by various lipases found in the lumen of the intestine (Fig. 1).

8. Triglyceride hydrolysis releases FFAs and monoacylglycerols (MAGs).’ In the third
experiment we tested for digestion of triglyceride lipids by recording the color of the
products. Test tube 1 had the beginning color of being a light, cloudy purple, at 2 and 4
minutes there was no change, at 6 minutes the color changed to pink. Test tube 2
started out as a lilac color with no change at 2 and 4 minutes and ending with a pink
color. Test tube 3 began with a milky purple with no change at 2 and 4 minutes and
ended in dark purple. Test tube 4 started as light blue/purple color with no change at 2
and 4 minutes ending in light blue.
Methods and materials:
(Texas Woman’s University 2015)
Results:
Digestion of Protein
Observations after Biuret
test (color change)
Interpretations (negative,
protein, or peptide)
Test Tube #1
(Water & Albumin)
­At Room Temp
Light Blue Negative
Test Tube #2
(Water, Albumin, and
Pepsin)
­At 37 degree celsius in
water bath
Light Purple Protein
Test Tube #3
(Albumin, Pepsin and HCL)
Clear Clear (negative for
peptides)

9. ­At 37 degrees celsius in
water bath
Test Tube #4
(Albumin, Pepsin, and
HCL)
­Ice chest
Clear Negative for both
As shown in the tableabove test tube 1 was negative for albumin. Test tube 2
tested positive for albumin and negative for pepsin. Test tube 3 came out negative for
peptides. The result of test tube 4 was negative for both albumin and pepsin.
Digestion of Carbohydrates
Color of solution Observations
Test Tube 1
(1% starch)
Dark Indigo Positive
Test Tube 2
(1% glucose)
Yellow Negative
Test Tube 3
(1% starch & Benedict’s
solution)
Cloudy Blue Negative
Test Tube 4
(1% glucose & Benedict’s
solution)
Light Blue Negative
The table above shows the results of the attempted or successful digestion of a
polysaccharide into oligosaccharides and disaccharides. Test tube 1 tested positive for

10. starch. Test tube 2 tested negative for glucose. Test tube 3 tested negative for starch
and benedict’s solution. Test tube 4 tested negative for glucose and benedict’s solution.
Digestion of Carbohydrates
Exp 2
The amylose and starch mixture combined with lugol’s iodide was tested for 15
minutes total and data was recorded of digestion every minute. Each minute tested
negative for maltose except at minute 9 where the product tested slightly positive.

11.
Digestion of Carbohydrates
Exp 3
The figure above shows the results of experiment 3, the digestion of
carbohydrates. Testing for level of digestion using the scale as shown on graph. Test
Tube 1 at 2 minutes tested mildly positive. At 4 and 6 minutes it tested mildly positive.
At 8 and 10 minutes it tested slightly more positive. At 20 minutes the products tested
most positive.

12. Digestion of Triglyceride Lipids
Observations Test Tube 1 Test Tube 2 Test Tube 3 Test Tube 4
Beginning color Light Cloudy
Purple
Lilac Milky Purple Light
Blue/Purple
2 minutes No change No Change No Change No Change
4 minutes No Change No Change No Change No Change
6 minutes Pink Pink Dark Purple Light Blue
This graph displays the results with the experiment for digestion of triglyceride
lipids. Test tube 1 had the beginning color of being a light, cloudy purple, at 2 and 4
minutes there was no change, at 6 minutes the color changed to pink. Test tube 2
started out as a lilac color with no change at 2 and 4 minutes and ending with a pink
color. Test tube 3 began with a milky purple with no change at 2 and 4 minutes and
ended in dark purple. Test tube 4 started as light blue/purple color with no change at 2
and 4 minutes ending in light blue.
Discussion and Conclusion:
In experiment one the results were test tube 1 was light blue in color and tested
negative for albumin. Test tube 2 was a light purple indicating the presence of proteins
and tested positive for albumin and negative for pepsin. The protein was not digested in
test tube 2 because without the aid of the hydrochloric acid the pepsin was unable to
breakdown the protein. Test tube 3 came out negative for peptides with a clear color.
The ​Test tube 4 was negative for both albumin and pepsin and produced no color

13. change as well the end result should have been positive for peptides confirming the
digestion of protein. ​The reagent used was out of date and could have been a possible
hindrance as to why the protein did not breakdown. Possibly if there were more time to
allow the reaction to take place the results could have been different.
In experiment two part one ​test tube 1 tested positive for starch and produced a
dark indigo color indicating presence of starch no breakdown into oligosaccharides or
disaccharides.. Test tube 2 tested negative for glucose with a yellow color indicating a
level of digestion breakdown. Test tube 3 tested negative for starch and benedict’s
solution with cloudy blue color and test tube 4 tested negative for glucose and benedicts
solution with light blue color. The color blue could indicate levels of sugars present.
In experiment two part two each minute tested they were all negative for maltose
except at minute 9 when there was an unintended excess of the amylase/ starch
solution. The product of minute 9 was noticeably more amber colored than the rest of
the substrates. Indicating that it’s possible with an increase of added amylase there
would be more of a reaction producing in maltose. Part three the results were recorded
for 1 test tube 2 minutes tested mildly positive at 4 and 6 minutes it tested mildly
positive at 8 and 10 minutes it tested slightly more positive and at 20 minutes the
products tested most positive. The results were comprehensive for 1 test tube but the
lab indicated the results were expected to be at two minute intervals for 6 test tubes.
Although the results were unconfirmed hypothetically each tube at two minute intervals
would have indicated different stages of starch breakdown determined by a 0­5 scale of
colors ranging from light green to red.

14. Experiment 3 the breakdown of triglyceride lipids for digestion of triglyceride
lipids by recording the color of the products. Test tube 1 had the beginning color of
being a light, cloudy purple, at 2 and 4 minutes there was no change, at 6 minutes the
color changed to pink. Test tube 2 started out as a lilac color with no change at 2 and 4
minutes and ending with a pink color. Test tube 3 began with a milky purple with no
change at 2 and 4 minutes and ended in dark purple. Test tube 4 started as light
blue/purple color with no change at 2 and 4 minutes ending in light blue.
References:
1. Digestive Physiology: Spring Semester 2015 Protocol – Department of Biology, Zoology
Labs. 2015. Available from: Texas Woman’s University, Denton, Texas.
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carbohydrates, IFT, [Internet]. 2012, Vol 11, 101
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4. Levin R. J., Digestion and absorption of carbohydrates­­from molecules and membranes
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