Chem162L Lab Report - Salivary Digestion

Experiment One: Salivary Digestion Mae Ann A. Derecho Department of Chemistry, University of San Carlos – Talamban Campus, Talamban, Cebu City, Philippines July 15, 2016 Saliva is viscous fluid with a pH around 5.6-7.9 is rich in protein such as mucins, amylase and others. In this experiment, the collected saliva sample were treated in a series of test to determine the component that were present. Mucins tested positive together with the inorganic constituents specifically chlorides and phosphates. This is determined through precipitating out their colored product formed. However, there are also components that tested negative despite being there and yet due to it being in minute amounts qualititave measures failed to detect these components. Presumptive tests were also performed and defined that ferric chloride test can be used detect the presence of thiocyanate/cyanide obtained from food or cigarettes. Factors affecting enzymatic activity were also identified using test the showed the effect of acid and base to salivary digestion.

INTRODUCTION Digestion begins in the mouth where the food is subjected to the mechanical process of grinding to break it up into smaller particles thus enabling the digestive juices to get at the food more readily. This also aids in mixing the saliva of the mouth with the food. Saliva moistens the mucous membranes of the mouth and helps to prevent tooth decay by cleansing the mouth of cariogenic carbohydrates and by neutralizing lactic acid. In addition, the salivary glands secrete an enzyme known as salivary amylase. This enzyme begins the breakdown of starches, it is responsible in the digestive function of saliva. Amylase is the responsible in breaking up immune complexes that are known to cause body system disruption. Saliva plays a very important role in the digestive process. Its effectiveness is increased with proper and careful chewing of food. Therefore, the objectives of this experiment is to identify the product formed on the salivary digestion of starch, components present in saliva, trace the salivary digestion of starch through iodine solution, the factors that affect enzymatic activity and to give the different functions a constituent play on both the digestion process and on the human body.

METHODOLOGY A. Test for Mucin In a test tube, 3 mL of saliva were added with 1-2 drops of 0.1 M acetic acid. B. Test for pH Two drops of universal indicator were added to 3 mL of saliva sample. C. Test for Proteins Two mL of the saliva sample were added with Biuret reagent/ D. Inorganic Matter Acidified 20 mL of the saliva was added with 2 drops of acetic acid, heated to boiling and filtered to remove the protein. Obtained filtrate were tested for the presence of inorganic matter following the table below for the reagents added:

Table 1. Reagents added for the test of inorganic matter in saliva samples Ions Chlorides Sulfates Phosphates Calcium

Reagents HNO3, AgNO3 HNO3, BaCl2 HNO3, (NH4)2MoO4 HNO3, K2C2O4

E. Test for Thiocyanates (Ferric chloride test) In a small porcelain dish, 1 mL of saliva was added with a few drops of ferric chloride before it was slightly acidified with dilute HCl, forming a red solution of ferric thiocyanate. Confirmatory test for the product formed was done through adding a drop of mercury (II) chloride that turned the solution colorless. Also, a control set up using distilled water instead of saliva was prepared to compare the obtained result. F. Digestion of Starch Two different set ups were prepared for this part of the experiment. The first set up consist of 1 mL saliva and 5 mL distilled water mixed together in a test tube and set aside at room temperature. The other set up was composed of 1 mL saliva and 25 mL of 1% starch solution, homogenized well in a 100 mL beaker and submerged in a water bath on a maintained temperature of 40°C. A spot plate that contained two drops of iodine in potassium iodide solution to each well were then prepared. On the first well of the spot plate, a drop of starch solution was added and observe. A drop of the saliva-starch mixture was added

to the second well of the spot plate and observed in intervals between 30 seconds for every addition of the mixture to the succeeding wells until the yellow color of the iodine remained persistent. Lastly, a 1 ml saliva-starch mixture was collected and subjected for the presence of reducing sugar using Benedict’s test. G. Effect of Acid and Base Table 2 showed the solutions prepared in two separate test tubes before it was heated at 40°C for an hour and tested for the effects of acid and base to the digestion process. Table 2. Solution preparation for the effects of acid and base in saliva digest Test tube no. 1 2

Saliva 3 3

Two drops of digested were collected each from the set up and transferred to clean spot plate. A drop of iodine solution was then added to each wells and observed for any changes that occurred.

RESULTS AND DISCUSSION Table 3 showed the experimental results gathered on the first part of the experiment. Mucin, a glycoprotein, is identified present on the saliva sample after it is precipitated out at around ph 4.5. Increase in mucin production can be consider as a symptom to any abnormalities in the body such as cancers of the pancreas, lung, breast and other tissues.

Solutions to be 0.5% (w/w) 0 3

Table 3. Qualitative test for the components of saliva Test Mucin pH Proteins (Biuret Test) Inorganic Matter  Chlorides  Phosphate s  Sulfates  Calcium Thiocyanates (Ferric Chloride Test)

Result cloudy soln with white ppt yellow green soln with yellow green ppt clear, colorless soln with indigo ppts

   

turbid soln with cloudy white ppt clear, colorless soln with pale yellow ppt slightly turbid soln clear, colorless soln

The soln turned pale orange soln with pale orange curds/ ppt formation. The soln turned colorless after the addition of mercury (II) chloride. In comparison to the control set up, the soln turned milky white with white ppt formation after the confirmatory test using the mercury (II) chloride solution. With the saliva solution turning to yellow green ammonium phosphomolybdate precipitate (refer with precipitate formation after the addition of to equation 2). The function of inorganic the universal indicator, the normal pH range for phosphate includes: contributes to solubility saliva is considered to be 5.6 to 7.9. This product of calcium phosphate, which is crucial explains why the pH balance in the body is close in maintaining tooth structure, important as a to neutral. However, several factors influence buffer and an essential nutrient for oral the pH nature of the saliva and varies depending microflora for metabolic pathways. to the food and drinks intake of an individual. 12(NH4)2MoO4 + H3PO4 + 21HNO3 → Qualitative determination of salivary proteins (NH4)3PMo13O40(s) + 21NH4NO3 + 12H2O can be performed through the use of Biuret reagent. Biuret test is a specific to test the (eqn. 2) presence of peptide bones which is also present The presence of calcium in saliva can be in proteins. The bluish complex formation of investigated through reacting it with potassium copper hydroxide confirms the presence of oxalate soln in slightly acidic medium. The proteins in saliva. There are number of proteins presence of the ion based from the obtained present in saliva such as amylases, defensins, result was not determined and may be cystatins, histatins, immunoglobulins, statherin, influenced by several factors. The content of lactoperoxidase, lysozyme and lactoferin. calcium can be related to the tooth enamel of an The inorganic constituents of saliva includes individual. A study conducted by Hubbell and Na+, K+, Ca2+, Cl-, HCO3− and HPO4−. Chloride Bunting (1932) showed that as the number of was tested to be present in the saliva through carious teeth increases, the calcium of the saliva precipitating out as AgCl (refer to equation 1) in decreases; supporting the absence of calcium on white form after silver nitrate is added. The the experimental result. The last ion, sulfates, chloride ions help in maintaining the osmotic investigated showed a negative result indicating balance in the mouth preventing any excess that the ion is in minute amount on the saliva inflow or outflow of water from water tissues. sample. Sources of sulfates can be from dietary sulfates and is essential to the humans as sulfates AgNO3 + Cl− → AgCl(s) + NO3 (eqn. 1) contribute to the protein structure of the saliva. Inorganic phosphates were also identified to be present in saliva through the formation of the

Thiocyante is an endproduct of detoxification of hydrogen cyanide present in cigarette smoke. Thiocyanate can also be present in several foods such as cabbage and broccoli, hence, its presence in the saliva. Test for thiocyante are now widely used to identify individuals who are cigarette smoker through salivary test to which will give a white ppt in a soln as a positive result (refer to equation 3). 3CNS- + Fe3+ → Fe(CNS)3

indicating the presence of maltose (reducing sugar) on the salivary digest. The degree of visible result for the Benedict’s test subjected before achromatic point can be possible however differs with the ones obtained after. This is seen through the color ranging from green to orange.

(eqn. 3)

Table 4. Variables tested in saliva Test Digestion of starch

Result  

 Effect of acid and base

 

At 40°C, the salivary amylase is able to digest the starch molecule. The formation of the deep blue coloration after the addition of I2 (see Table 4) denotes that starch reacts the iodine molecule an indicating that it is still a coiling polysaccharide compound made up of several glucose units. On the other hand, the presence of the yellow solution after achromatic point showed that starch is already digested and broken down to its individual glucose. Achromatic point is the moment wherein the solution is ‘without color’. It is the point where starch does not five a colored solution during I2 test. Benedict’s test can be used to evaluate the presence of reducing sugar; this is observed through the brick red precipitate that will form

Hydrochloric acid is naturally produced in the stomach and one of its function is to change the pH to acidic. Salivary amylase (enzyme) can work in optimum condition when it is in alkaline medium, resulting for the enzymatic activity to slow down until it comes into halt. This explains the negative result obtained to the digest treated with 0.01 M HCl when I2 was added.

CONCLUSION Saliva contains 98% water and essential substances such as electrolytes, mucus and various enzymes. The digestive functions of saliva include moistening food and helping the task of swallowing a lot easier. It contains several components that play essential role in human’s body system, particularly the inorganic matter inherent to man. These ions maintain the osmotic balance in our mouth and the structure of the tooth enamel, however, qualitative measure for the presence of these ions varies to each individual. As a result, some test gave negative result. Saliva can also be used as presumptive test to determine any symptoms of serious illness such as cancer and the identification of a cigarette smoker. Factors that affect the enzymatic activity in salivary digestion were also defined; the normal pH range for saliva was identified to be close to neutral, hydrochloric acid was known to be naturally produced in our stomach that also alters the pH to the acidic side disallowing the enzymatic activity to run at optimum conditions.

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