Introduction
The digestive system function is to take in food, break it down into its nutrient molecules, absorb these molecules into the bloodstream, and eliminate indigestible remains from the body. The organs comprising the digestive system include the alimentary canal, teeth, tongue, salivary glands, esophagus, stomach, small intestine, large intestine (colon), and the rectum. The gallbladder, liver, and pancreas are accessory organs of the digestive system. (Bekdash, Lecture: Chapter 23-The Endocrine System-Part 1)
The teeth and tongue initiate digestion as chewing mechanically breaks down food into digestible pieces while saliva secreted from salivary glands chemically breaks down food into an absorbable form. The esophagus receives food from the mouth when one swallows then via muscular contractions called peristalsis delivers it to the stomach. Cells lining the stomach secrete acid and enzymes to further breakdown foods. After food is processed in the stomach, it passes to the small intestine where food is broken down using pancreatic enzymes and bile from the liver. Peristalsis also occurs in the small intestine where the duodenum is primarily responsible for the breakdown process while the jejunum and ileum are responsible for nutrient absorption into the bloodstream. Water, bile, enzymes, and mucous transform semi-solid foods arriving at the small intestine into a liquid form which passes to the large intestine. The large intestine processes waste passing it through the colon via peristalsis. The “waste” is referred to as stool composed of food debris and bacteria which move to the rectum for excretion. The rectum holds stool until it is evacuated from the body via the anus. If the brain signals that it is an appropriate time to expel the stool, rectal sphincters relax and the rectum contracts to dispose the stool. ("Digestive System | Cleveland Clinic," n.d.)
Enzymes are an important aspect of the digestive system because they are substances secreted by glands to act as a catalyst binding to a substrate to produce a chemical reaction which results in a desired product. In terms of the digestive system, the product is the continuous chemical breakdown of consumed foods by various enzymes. ("Enzymes: Function, definition, and examples," n.d.)
Salivary amylase initiates carbohydrate digestion by breaking down starch and glycogen to disaccharides. Chief cells secrete pepsin which begins protein digestion. The pancreas secretes both pancreatic amylase and pancreatic lipase which breaks down starch and glycogen into disaccharides and fats into fatty acids and glycerol. The mucosal cells from the intestines break down peptides to amino acids using peptidase and convert trypsinogen into trypsin using enterokinase.
It is important for body temperature to be at 37.5oC to ensure that each digestive enzyme is able to function and avoid denaturation. ("Chemistry for Biologists: Enzymes," n.d.) Also, the approximate optimal pH for all intestinal enzymes is 7.5 and stomach enzymes require a pH of 2 to function properly. ("Effects of pH (Introduction to Enzymes)," n.d.)
This laboratory exercise uses Physioex 9.1 with a computer simulated experiment to study the digestive system. Activity 1 studies starch digestion by salivary amylase and Activity 3 studies how pepsin secreted by chief cells aids in protein digestion. Lastly, Activity 4 shows how
lipase from the pancreas aids in fat digestion.
Material and Methods
Sign in to Physioex.com and login with username and password. Click on Exercise 8: Chemical and Physical Processes of Digestion. Do the computer simulated experiments Activity 1, 3 and 4, one at the time.
Activity 1: Assessing Starch Digestion by Salivary Amylase.
On the provided laptop computer using the Physioex 9.1 simulation software, proceed to start with Activity 1: Assessing Starch Digestion by Salivary Amylase. In the bottom right side of the site there is a forward and backward arrow that allows to view between the top bars of the activity. Review the objectives, introduction and the pre-lab quiz. Click submit to submit the answers for the pre-lab quiz. Once these steps are completed, proceed to start with the experiment. Let start by dragging the first tube into the first holder in the incubation unit, then seven more tubes will automatically be placed in the incubation unit, then click next. Now we will add the substances in the order as follow: Tube 1: amylase, starch, and pH 7.0 buffer, Tube 2: amylase, starch, pH 7.0 buffer, Tube 3: amylase, starch, pH 7.0 buffer, Tube 4: amylase, deionized water, pH 7.0 buffer, Tube 5: deionized water, starch, pH 7.0 buffer, Tube 6: deionized water, maltose, pH 7.0 buffer, Tube 7: amylase, starch, pH 2.0 buffer, and Tube 8: amylase, starch, pH 9.0 buffer, then click next. Under the first tube click the number 1, notice that the numbers will descend into the incubation unit, click next. Now click boil for tube 1, once finished boiling the tube automatically will rise, click next. Click under the second test tube, next, and now click freeze to freeze tube 2, click next. In this step observe that the incubation temperature is set at 37oC and the timer is set to 60 min. Click incubate to evenly mix the test tube rack. Notice that the simulation of 60 minutes is compressed to 10 seconds of our real time, once finished the incubation time the rack will automatically rise and the doors to the assay cabinet will open, then click next. A question will show, answer and submit the answer then click next. At this step we use reagent |K| to test for presence of starch, and Benedict’s reagent detects the presence of reducing glucose or maltose. Let start by dragging the first test tube in the incubation unit to the first small assay tube on the left side of the assay cabinet to decant. The rest of the tubes will do the decanting step automatically then click next. Dispense |K| reagent into the first assay and to the rest of the tubes and then click next. Now check the tubes for color change, the color blue-black color is a sign of positive result for starch, a negative mixture will have a pale gray color. Click record data and the results will show in the grid, then click next. A question will show, answer and submit answer. Now we will check with the Benedict’s reagent, dispense five drops into the first tube and the rest of the tubes and click next. Click boil, next, now observe for color change. A positive test for maltose or glucose will have a green to reddish color. A negative result will have no color change. Click record data, and next. Click submit to record the experiment result and proceed to answer and submit the post lab quiz and the review sheet. Once submitted and done with these steps proceed to save and review the lab report.
Activity 3: Assessing Pepsin Digestion of Protein
Click to activity 3, review the objectives, introduction, and pre-lab quiz. Submit the answer for the pre-lab quiz, click next to start the experiment. Let start by first dragging a test tube into the first holder in the incubation unit, then five more tubes will automatically be placed in the incubation unit, then click next. Add the substances as follow: Tube 1: pepsin, BAPNA, pH 2.0 buffer, Tube 2: pepsin, BAPNA, pH 2.0 buffer, Tube 3: pepsin, deionized water, pH 2.0 buffer, Tube 4: deionized water, BAPNA, pH 2.0 buffer, Tube 5: pepsin, BAPNA, pH 7.0 buffer, Tube 6: pepsin, BAPNA, pH 9.0 buffer, then click next. Click under the first tube and then next. Now click boil, once the tube automatically rise, click next. Answer and submit the answer for the question. Observe as described in the first activity the incubation temperature and timer are set to 37oC and 60 minutes (10 seconds for our real time). Click incubate to gentle agitate and evenly mix the test tubes, then click next. Answer and submit the question. Now drag the first tube into the unit holder in the spectrometer to measure how much yellow dye was liberated from BAPNA hydrolysis, then click next. Click analyze to measure the amount of light absorbed and click next. Record data to display the results in the grid and then click next. Now we will return the tube back to the incubation unit and click next. A question will show, answer and submit the answer and proceed to analyze the rest of the five tubes remaining. Once finished, click record data, next and then click submit to record the experiment results into the lab report and procced with the post lab quiz and review sheet. Once completed and submitted all answers proceed to review and save the lab report.
Activity 4: Assessing Lipase Digestion of Fat
Click to activity 4, review the objectives, introduction, and pre-lab quiz. Submit the answers for the pre-lab quiz and proceed to start the experiment. Start by setting six tubes in the incubation unit. Add the following substances as follow: Tube 1: lipase, vegetable oil, bile salts, pH 7.0 buffer, Tube 2: lipase, vegetable oil, deionized water, pH 7.0 buffer, Tube 3: lipase, deionized water, bile salts, pH 9.0 buffer, Tube 4: deionized water, bile salts, pH 7.0 buffer, Tube 5: lipase, vegetable oil, bile salts, pH 2.0 buffer, Tube 6: lipase, vegetable oil, bile salts, pH 9.0 buffer and then click next. Answer and submit question. Then click incubate to gently and evenly mix all tubes in the incubation unit and click next. Answer and submit question. The doors in the assay cabinet will open proceed to measure the final pH in the solutions with the pH meter. Move the first test tube in to the pH meter then click next. Click measure pH, record the results by clicking record data, and click next. Return the test tube back to the incubation unit and then measure the pH for the remaining tubes, one by one, record data and click next. Answer and submit question. Follow to click next and submit to record the results of the experiment and continue with the post lab quiz and review sheet. Once completed with the post lab quiz and the answer of the review sheet click view experiment results to view the lab report.
Results
I) Activity 1 Data
Table 1: Data showing the results of IKI and Benedict’s assays performed on given samples subjected to different treatments.
Activity 1: Assessing Starch Digestion by Salivary Amylase Results
In this activity, a given tube comprised of a specific combination of reagents was subjected to one of three treatments (boiled, frozen, or none). Incubation time and temperature were held constant at 60 minutes and 37 degrees Celsius. Both IKI and Benedict’s assays were conducted, indicating the presence or absence of a substrate. As shown in Table 1, Tube 1 was the only tube that was boiled. It yielded a positive in it’s IKI assay and a negative in Benedict’s assay. Tube 2, which was frozen, contained reagents identical to that of Tube 1, resulting in a negative in the IKI assay and a double positive in Benedict’s assay. Tubes 4, 5, and 6 all consisted of a substrate, deionized water, and pH 7.0 buffer. Their assay results are listed in this table. Tubes 7 and 8 contained identical reagents, except Tube 7 had a pH 2.0 buffer while Tube 8 had a pH 9.0 buffer. When assayed, these tubes both displayed identical results, testing positive in both IKI and Benedict’s assays.
II) Activity 3 Data
Table 2: Data obtained from spectrophotometer indicating optical density of a given sample.
Activity 3: Assessing Pepsin Digestion of Protein Results
In this activity, a given tube with a combination of reagents was analyzed for its optical density. Incubation time and temperature were held constant at 60 minutes and 37 degrees Celsius. As shown above, Tube 1, which was the only tube that was boiled, had an optical density of 0.00. Tubes 3 and 6 also had optical densities of 0.0. Tube 2, which contained pepsin, BAPNA, and a pH 2.0 buffer, had the highest optical density of 0.40.
III) Activity 4 Data
Table 3: Data showing the measured pH for a given sample consisting of several reagents.
Activity 4: Assessing Lipase Digestion of Fat Results
In this activity, a given sample consisting of several reagents underwent incubation. Incubation time and temperature were held constant at 60 minutes and 37 degrees Celsius. As shown in Table 3, the sample pH was measured for each sample after incubation. Tube 5, consisting of lipase, vegetable oil, bile salts, and a pH 2.0 buffer produced the lowest pH of 2.00. Tube 3, which consisted of lipase, deionized water, bile salts, and a pH 9.0 buffer produced the highest pH of 9.00. This pH was also recorded for Tube 6 which consisted of lipase, vegetable oil, bile salts, and a pH 9.0 buffer.
Discussion and Conclusion
Activity 1: Assessing Starch Digestion by Salivary Amylase.
Test tubes with different reagents were treated by either boiling, freezing or nothing. The time and temperature of the treatments were constant, and the temperature used was 37 degrees Celsius because that is the same as the normal body temperature. The first three test tubes used the same reagents (amylase, starch and a buffer of pH 7.0), but were treated differently. Amylase is an enzyme that digests starch with glucose or maltose as products. Test tube 1 was treated by boiling and it resulted in a positive IKI assay and a negative Benedict’s assay. A positive IKI demonstrates that starch is present, and a negative for Benedict’s assay means that the digested products of starch such as glucose or maltose, are absent. The results for test tube 1 indicate that starch was not digested when boiled because boiling denatured the enzyme and decreased amylase activity for digesting starch as predicted. Therefore, starch was not digested even with the presence of amylase. Test tubes 2 and 3 both had the same results of a negative for IKI assay and a double positive for Benedict’s assay. Both test tubes were treated differently but had the same results because the freezing treatment done of test tube 2 did not have an effect on the enzyme, and so did test tube 3 that wasn’t treated with anything. Tubes 4, 5, and 6 were not treated with anything and they all used deionized water, a substrate, and a buffer of pH 7.0. Tube 4 did not have starch as one of its reagents, so the results of IKI and Benedict’s assays were both negative. Tube 5 with starch and deionized water resulted in a positive for IKI assay and negative for Benedict’s assay because amylase was not present to digest the starch, and starch could not be digested with deionized water. Maltose and deionized water in tube 6 showed negative for IKI because Maltose is a digested product of starch, and therefore its presence appeared in the results of Benedict’s assay. No treatment was used in tubes 7 and 8, and both consisted of starch and amylase, but both had different pH levels for buffers used. Tube 7 tested with a pH 2.0 buffer, and tube 8 tested with a pH 9.0 buffer. Both tubes resulted with a positive in IKI and Benedict’s assays. This is because amylase is most active at pH 7.0 (same as the mouth’s pH) where starch is digested completely, and a pH other than 7.0 results in the presence of starch or a positive IKI assay. The controls are necessary in this experiment to make comparisons, and they could be positive or negative. Tube 4 is an example of a negative control because the results were negative, showing that amylase was not contaminated with maltose. Tube 5 is another example of control used in the experiment because the enzyme that tests for contamination of glucose in starch was absent. Based on the results of this activity, amylase is a necessary enzyme in the digestion of starch.
Activity 3: Assessing Pepsin Digestion of Protein
BAPNA is a colorless solution that turns yellowish when digested by pepsin (Chemical & Physical Processes of Digestion), showing an optical density that is higher than zero because the light rays have a harder time passing through the hydrolyzed BAPNA containing tube. BAPNA has covalent bonds that are similar to the bonds found on proteins, so it is used in this case to detect the activation or inhibition of Pepsin by binding to the Pepsin. Pepsin will have the highest activity in an environment similar to that of the stomach resulting in the digestion of proteins. In this case tubes 1 and 2 contained the same exact components (BAPNA, pH 2.0 and Pepsin), the one thing that was different was the boiling of tube 1. The boiling of tube 1 inhibited the activation of Pepsin and the BAPNA digestion, resulting in the optical density of zero for tube 1, since tube 2 wasn’t boiled the pepsin was activated and helped with the digestion of BAPNA resulting in an optical density of 0.40. Tube 3 contained similar components as tubes 1 & 2 but the BAPNA was replaced with deionized water, the absence of BAPNA gave an optical density of zero because there was no digestion happening, therefore there was no activation of pepsin, and finally resulting in an optical density of zero. Tube 3 was also a negative control because the optical density was zero and no activation happened. Tubes 4 & 5 contained the same exact components (Pepsin, BAPNA, pH 7.0 buffer), resulting in the activation of pepsin to digest the BAPNA and having an optical density of 0.03. The reason behind the low optical density is that pepsin is activated and digests proteins best in an environment that is similar to that of the stomach with a pH between 1 and 3, but since pH 7.0 buffer was added, that lowered the activation of pepsin therefore lowering the optical density (Encyclopaedia Britannica). Tube 4 is a negative control because of the very low optical density of 0.03. Tube 6 had the same components as tubes 4 & 5 (pepsin and BAPNA) except it contained a more basic buffer of pH 9.0, this resulted in an optical density of zero because the environment in that tube was too basic for the pepsin to activate and help in the digestion. Finally, after comparing all 6 tubes, the results from table 2 indicate that the more active pepsin was, that resulted in an increase in the digestion of BAPNA and having a higher optical density (greater than zero).
Activity 4: Assessing Lipase Digestion of Fat
The main component in this lab was lipase, since lipase helps in the digestion or breakdown of fat (vegetable oil in this case). Lipase is an enzyme that is located in the pancreas and digestive tract and it works in an environment that has a similar 7.0 pH to that of the pancreas (SFGate). The bile, which is usually produced by the liver, that is added in almost all of the tubes also helps with the breakdown or digestion of the vegetable oil (SFGate).
In this activity, the pH was determined for tubes 1 through 6, this helps in the understanding of how lipase works in different levels of pH. Tube 1 contained lipase, vegetable oil, bile salts and pH 7.0 buffer, this mix resulted in a pH of 6.21, since the 7.0 pH is similar to that of the pancreas, it made it more effective for the lipase and bile to break down the vegetable oil.
Tube 2 had similar components to that of tube 1(lipase, vegetable oil, pH 7.0 buffer) but the bile salts was replaced with deionized water, this decreased the effectiveness of lipase because bile salts help lipase with the digestion of fat (vegetable oil), resulting in a basic 7.0 pH.
The components in Tube 3 were lipase, deionized water, bile salts and pH 9.0 buffer, the absence of fat (vegetable oil) in this tube didn’t activate lipase and the bile salts as much as it would when the fat was present in the other tubes, resulting in a basic 9.0 pH.
Tube 4 contained deionized water, vegetable oil, bile salts, and a pH 7.0 buffer. Lipase was replaced with deionized water. This makes it difficult for the fat or vegetable oil to be digested because it needs lipase to be digested, resulting in a neutral pH of 7.0 in the absence of lipase activity.
Tube 5 contained lipase, vegetable oil, bile salts, but had a buffer with a pH of 2.0. Even though lipase and bile salts were present to digest the fat, this tube contained a very low pH for lipase activity to be at its highest. A pH of 7.0 is ideal for fat digestion, therefore, a pH of 2.0 resulted in the lowest pH of 2.0 in Table 3.
Tube 6 also contained lipase, vegetable oil, and bile salts, but had a buffer with a pH of 9.0. A pH of 9.0 is not ideal for activating lipase. This matched the result of a basic pH of 9.0 because lipase was not active to digest the fat.
In this activity, a combination of lipase, bile salts and a pH of 7.0 buffer results in the highest lipase activity in digesting fat or vegetable oil in the case of this activity. Our predictions were proved in the results listed in Table 3.
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