UNIVERSIDAD DE

ZAMBOANGA
School of Allied Medicine (SAM)
Pharmacy Department
PHARM BIO SCI 3 LABORATORY
PHARMACEUTICAL BIOCHEMISTRY

PHARM BIO SCI 3 LABORATORY

PHARMACEUTICAL BIOCHEMISTRY
Activity No. 3 - MILK
GROUP WORK OUTPUT

BS Pharmacy 2-A
MWF 9-11 am

Submitted to:

Sabtula, Ben-Frazier U RPh.

Biochemistry laboratory Instructor

Submitted by:

Acabal, Jasher Dave C (Performer, Encoder and Researcher)

Tuknol, Apasra (Performer and Observer)
Latosa, Ariel (Errand and Performer)
Ramos, Ronald (Performer)
Sarahadil, Ayman (Performer and observer)
AmilHamja, Alnaliza (Performer)

PROTEIN MAZIMIZERS
Group name
 UNIVERSIDAD DE ZAMBOANGA
School of Allied Medicine
(SAM)
Pharmacy Department
PHARM BIO SCI 3 LABORATORY
PHARMACEUTICAL BIOCHEMISTRY

ACTIVITY No. 4 SALIVARY DIGESTION

A. REACTION

We placed 5 drops of resting saliva in a test tube then we tested it with red and blue litmus
paper. The red litmus paper after dipping remained red but the blue litmus turned pink at the tip
which we then concluded that the resting saliva is slightly acidic.

We then sliced a sufficient amount of paraffin and chewed it for at least 5 minutes to
produce stimulated saliva. We followed the same procedure by placing 5 drops of stimulated
saliva into the test tube and tested it with red and blue litmus paper. The results were the red
litmus paper remained red and the blue litmus paper remained blue that made us arrive to the
conclusion that the resting saliva was neutral.

Resting saliva is found in the mouth in the intervals of food taking and mastication while the
Stimulated saliva is found while chewing food that causes the muscles to compress the salivary
glands thereby producing saliva.
 The normal pH of the saliva ranges from 6.2 7.6. Measurement of the pH and buffering
capacity of the saliva gives an indication of the caries susceptibility of the patient. A high salivary
lactobacillus count reveals in most cases a high frequency of sugar intake and salivary yeast
infection in an indicator of reduced salivary flow and removable dentures.

B. PREPARATION OF MUCIN
We prepared 10mL of Saliva then we added 40mL of 95% Alcohol then we stirred the mixture.
After stirring, the solution formed a white insoluble precipitate; we then let it stood for 2 days and
the result was the precipitate settled at the bottom of the container. We then poured the
supernatant liquid then we transferred the precipitate in a filter paper then we washed it with
3mL alcohol then another 3mL of ether. We let the precipitate to dry then we divided it by 6 then
we performed the following tests:

1. Solubility
The results obtained were the following:

REAGENTS RESULTS SOLUBILITY

The precipitate spread a
3mL Water + Precipitate Insoluble
little
3mL HCl + Precipitate The precipitate swelled Insoluble
The precipitate settled at
3mL NaOH + Precipitate the bottom of the Insoluble
container.

Based on our research, mucin is a hydrophilic and has high elasticity, high
viscosity, adhesiveness and low solubility.

2. Millons Test
We prepared 3mL of Millons reagent then we added a piece of the
precipitate, there was no reaction upon and after addition. With this result, we
therefore conclude that there was no presence of tyrosine in the precipitate since
the Millons test is used to detect its presence and it didnt produce a positive
color of dark red.

3. Biuret Test
We placed 3mL Biurets reagent in a test tube then we also placed a piece
of the precipitate in the solution then upon and after addition, the precipitate
settled at the bottom. With this, we therefore conclude that there is no presence
of derived proteins such as proteases and peptones.

4. We prepared 10mL of water in a test tube then we added 2mL of dilute HCl then
we placed the remaining precipitate and we boil the solution. After boiling, there
 was no reaction or change, thats why we were not able to proceed to the next
step.

When reducing sugars are heated in the presence of an alkali they get
converted to powerful reducing species known as enediols. Enediols reduce the
cupric compounds (Cu2+) present in the Benedict's reagent to cuprous
compounds (Cu+) which get precipitated as insoluble red copper(I) oxide(Cu2O).

Unhydrolyzed mucin reduces Benedicts solution. The color of the

obtained precipitate gives an idea about the quantity of sugar present in the
solution, hence the test is semi-quantitative. A greenish precipitate indicates
about 0.5 g% concentration; yellow precipitate indicates 1 g% concentration;
orange indicates 1.5 g% and red indicates 2 g% or higher concentration.

C. INORGANIC MATTER

We placed 1mL of Saliva to each 4 test tubes and acidified them with a drop of
HNO3. We then tested all the test tubes with blue litmus paper that all resulted to red.
We then boiled them after to remove all the proteins present. After boiling, we filtered
the first test tube and tested the filtrate for chlorides using a drop of AgNO 3 that resulted
to the formation of bluish solution; for the second test tubes filtrate, we tested it for
Phosphates using a drop of Ammonium molybdate that resulted to a yellowish solution;
for the third test tube, we tested the filtrate for Sulfates using a drop BaCl 3 that resulted
to a clear and colorless solution; and for the last test tubes filtrate, we tested it for
Calcium with 3 drops of Ammonium Potassium Oxalate that also resulted to a clear and
colorless solution.
 The organic salts found abundant in saliva are Chloride and Phosphate. Chloride
is the coenzyme of ptyalin while Phosphate maintains pH level.

D. TEST FOR NITRATES

We placed 1mL of Saliva in a 5mL test tube then we added a drop of H 2SO4 then
stirred the solution using the stirring rod thoroughly. The color of the solution stayed the
same. We then added 2 drops of KI solution and 3 pinch of Starch paste that resulted
into the formation of greenish-black precipitate solution.

E. TEST FOR THIOCYANATES

We placed 1mL of Saliva in a 5mL test tube then we added a drop of FeCl 3
Solution and stirred it with the stirring rod. After stirring, we obtained a blurred white
solution. We then acidified it with 1 drop of HCl; and upon addition, we obtained a final
result of formation of peach cloudy precipitate in a blurred solution.
F. VISCOSITY TEST

We placed 2 filter papers in 2 separate funnels in each 50mL beaker. We then

poured 5mL of Starch Paste to both filter papers at the same time then added 10 drops
of saliva to one and 10 drops of water to the other at the same time too. The progress of
filtration was the starch paste with water filtered fast while the starch paste with saliva
filtered slowly. It is because the starch paste with saliva was thicker and had greater
viscosity than the starch paste with water.

G. DIGESTION OF STARCH PASTE

We placed 10mL of Starch Paste in a 50mL beaker then we added 5 drops of

Saliva then we stirred it thoroughly. After 1 minute, we added a drop of iodine. Upon
addition, the solution became dark violet. We then added another 5 drops of Saliva; the
result was still dark violet. We then stirred it for some time then we tested it again with a
drop of iodine solution and the result remained dark-violet. Since there was no blue
color produced, we proceeded by testing the solution with a drop of Benedicts reagent,
and the final result was a dark violet solution.
 If the Benedicts test is positive, it implies that the solution contains a reducing
sugar, in which case an orange-red precipitate forms when the Benedicts reagent is
added. Iodine does not react with reducing sugars and so when iodine (which is red) is
added to such a solution, no color change will be observed unless starch is present, in
which case a blue/black color change gives away the presence of starch.

Food is in the mouth for a short period of time, but this is enough to start
digestion of carbohydrates (starches). When food reaches the stomach, in spite of the
acid media, starch digestion continues in the internal part of the bolus. 30 to 40% of the
starch is digested by salivary amylase, and the remaining part is later helped along by

Pancreatic amylase. Disaccharides are later broken into monosaccharides (reducing

sugars) by intestinal epithelial membrane.

Starch Digestion by Ptyalin is when ptyalin is mixed with food in the mouth,
where it acts upon starches. Although the food remains in the mouth for only a short
time, the action of ptyalin continues for up to several hours in the stomachuntil the
food is mixed with the stomach secretions, the high acidity of which inactivates ptyalin.
Whereas Starch acid digestion is when after starches leave the mouth and enter the
stomach, the ptyalin is destroyed by stomach acids. The remaining starches are broken
down by the stomach acids to an extent, but they dont remain in the stomach long
enough to be completely broken down. Gastric juices start protein digestion.

H. DIGESTION OF RAW STARCH

We placed a pinch of raw Starch in a 5mL test tube and added 1mL of water then
we shook the container. After shaking, we got a white blurry solution. We then added 5
drops of Saliva into the solution and after addition, the solution was still blurry. We then
mixed it well and let it stood for 20 minutes. After 20 minutes, there was a formation of
white residue in a blurry solution. We then filtered it in a beaker then we tested the
filtrate with 1mL of Benedicts reagent, resulting into a neon blue solution. We then
added a drop of Toluene. Upon addition, there was a translucent spot formation in the
neon blue solution. We then set it aside for 2 days, and we got a result of a clear neon
blue solution again so we tested it with a drop of Toluene again; then we obtained a less
translucent spot formation in the solution that disappeared after a few minutes.

Our conclusion based on our findings is that since Benedicts test is used to
detect the presence of monosaccharides and generally all the reducing sugars, it will
give a positive color of green, yellow, orange, red-brick or red ppt. But since the filtrate
remained blue, we therefore conclude that there was no presence of reducing sugar in
the filtrate. The purpose then of adding toluene (a neutral hydrophobic) to the solution is
for neutralization so as the reduction of the copper (II) ions will not take place in acidic
conditions- if excess acid is present.
CONCLUSION:

Upon doing this experiment, we were able to dig deeper and learn the nature and
properties of human saliva by preforming the different tests and researches. For the
Reaction of saliva, the normal range of resting saliva is 5.6 to 7.5 according to the
International Journal of Drug Testing and the normal range of the stimulated saliva is 5.6 up
to 8. Besides flow rate, the pH also depends on the concentration of salivary proteins,
bicarbonate and phosphate ions that have considerable buffering capacity.

For preparation of Mucin, Mucin is large polymeric glycoproteins that are largely
responsible for the gel-like properties of the saliva. It has high elasticity, high viscosity,
adhesiveness and low solubility. It is tested with Millons and Biuret test to detect the
presence of tyrosine and peptide bonds. And when heated in the presence of alkali, gets
converted into a powerful reducing species known as Enediols and Unhydrolyzed mucin
reduces Benedicts solution by giving the positive color of greenish, yellow, orange, and red.
In Inorganic Matter, the organic salts found abundant in saliva are Chloride and
Phosphate. Chloride is the coenzyme of ptyalin while Phosphate maintains pH level. And
the test for nitrates is to detect the presence of nitrates obtained from food. Nitrates levels in
biological fluids have been used as a diagnostic biomarker in many diseases. Test for
thiocyanates are indicator of cigarette smoking in adolescents. The thiocyanates found in
body fluids result, in part, from detoxification of hydrogen cyanide in cigarette smoke. These
observations have led to utilization of serum thiocyanate levels to document adult smoking
cessation.

For viscosity test, Starch paste with water filtered faster than with that of saliva
because it has less viscosity and thickness. Saliva has high viscosity making it thicker and
difficult to pass the filter paper. For digestion of starch paste, if Benedicts test is positive, it
implies that the solution contains a reducing sugar, in which case an orange-red precipitate
forms when the Benedicts reagent is added. Iodine does not react with reducing sugars
and so when iodine (which is red) is added to such a solution, no color change will be
observed unless starch is present, in which case a blue/black color change gives away the
presence of starch.
 And for Digestion of Raw starch, Benedicts test is positive, it will give a positive color
of green, yellow, orange, red-brick or red ppt. But if it is negative, the purpose then of
adding toluene (a neutral hydrophobic) to the solution is for neutralization so as the
reduction of the copper (II) ions will not take place in acidic conditions- if excess acid is
present.