APPROVAL SHEET

The complete report of Biochemistry Experiment with title is “Bile“ was

made by :
name : Wiwiana
ID : 1513441007
class : ICP Chemistry Education
group : IV (Four)
It has been checked and consulted by assistant and assistant coordinator, this
report has been accepted.

Makassar, November 2017

Assistant Coordinator Assistant

Muhammad Iqbal Hidayat Annisa Utami R, S. Pd

Known by,
Responsibility Lecturer

Dr. Halimah Husain, M. Si

ID. 19641020 199003 1 002
A. TITLE OF EXPERIMENT
Bile
B. OBJECTIVE OF EXPERIMENT
To know the physic condition of bile, musin test and anorganic compound
in bile, relationship between color substance in body, Van Den Berg test and
bile acid.
C. LITERARUR REVIEW
Bile is an important product released by the hepatocytes. It promotes the
digestion of fats from food by emulsifying them in the small intestine. The
emulsifying components of bile, apart from phospholipids, mainly consist of bile
acids and bile salts. Bile acids are steroids consisting of 24 C atoms
carrying one carboxylate group and several hydroxyl groups. Initially, the
cholesterol double bond is removed. Monooxygenases then introduce one or two
additional OH groups into the sterane framework. Finally, the side chain is
shortened by three C atoms, and the terminal C atom is oxidized to a
carboxylate group (Koolman, 2005 : 314).
Toxoplasmosis is a common parasitic disease caused by the protozoan
parasite Toxoplasma gondii. Toxoplasmosis is a zoonosis of worldwide
distribution caused by Toxoplasma gondii, an obligate intracellular coccidian
parasite that infects most warm-blooded animals including birds, humans,
domestic and wild animals. Birds can be considered important reservoirs of T.
gondii as they are often hunted by felids; they proliferate out of control as they are
not selective about food, eating food waste that may be contaminated with T.
gondii. In addition, as they fly long distances and can feed on the ground, they can
be potential hosts of this coccidian. Toxoplasmosis encompasses a large variety of
hosts including human, animals and birds ,the main sources of infecting humans,
is bird meat, so besides another indicators to detect the distribution of
T.gondiioocysts in the environment, the determination of T.gondiiprevalence in
domestic birds is of great importanc (Al-Nasrawi, 2014).
 The molecules that carry out this function are called bile salts or bile acids.
Metabolically, the liver creates them and secretes them into the gall bladder, from
where they are pumped into the duodenum. Bile salts are derived from cholesterol
and are a major end product of cholesterol metabolism. They are powerful
detergents, with a large, hydrophobic component and a carboxylic acid end-group
that is negatively charged at the pH characteristic of the small intestine. The
hydrophobic component of the bile acid will associate above a specific
concentration (termed the critical micelle concentration, or CMC) to form disc-
shaped micelles, that is, droplets (Schmidt, 2000:14).
Bile acids are produced only in the liver as the end products of cholesterol
catabolism. In addition to the classic function of bile acids in facilitating
hepatobiliary secretion of endogenous metabolites and xenobiotics and intestine
absorption of lipophilic nutrients, bile acids also play equally important roles in
controlling the metabolism of glucose and lipids in the enterohepatic system, and
energy expenditure in peripheral tissues. Because of such a close association
between bile acid signaling and metabolic homeostasis, targeting bile acid
metabolism by using bile acid receptor agonists or bile acid-binding resins have
proven to be effective in improving lipid and glucose homeostasis in obesity and
diabetes. This paper will summarize recent advances in our understanding of bile
acid signaling regulation of glucose and lipidmetabolismand the potentials of
developing novel therapeutic strategies that target bile acid metabolism for the
treatment of metabolic disorders (Li, 2012).
Probiotics are live microbial feed supplements, which beneficially affect
the host animal by improving its intestinal microbial balance defined probiotics as
a culture of specific living microorganisms, primarily Lactobacillus spp. that are
implanted in the organism and ensure the rapid and effective establishment of a
beneficial intestinal population. Modes of action of probiotics in poultry include
1) maintaining a beneficial microbial population by “competitive exclusion” and
“antagonism”. The pre-condition for probiotic microbe to favour animal’s
performance is colonization in the gut which is best attained if the organism being
administered originates from the gut of same species. It is inevitable to identify
the predominant probiotic bacteria to evolve the species specific probiotic
organisms (Gunasekaran, 2012).
Application of concentrated solutions of neutral salts (e.g. KCl or NaCl) is
often effective in removing precipitated/aggregated proteins, or other material
retained in the column via ionic interaction with the media. The use of buffers
containing EDTA or other chelating agents helps remove any metal ions
associated with the gel. Use of (dilute) detergent solutions is effective in removing
lipid and a whole range of other contaminants. Solvent-containing (e.g. ethanol,
butanol, isopropanol) buffers may also be used in this regard Increasing the
column temperature to 50–608C may sometimes be considered, particularly if
lipid appears to be a major column contaminant (most lipids liquefy at such
temperatures). Chromatographic systems are also designed to facilitate effective
CIP. Internal surfaces of the column, its valves and piping, are smooth,
impervious and devoid of recesses which could harbour microorganisms or other
contaminants (Walsh, 2003 : 104).
Chicken anemia virus (CAV), a negative single-stranded DNA virus is the
only member of genus Gyrovirus of the Circoviridae. Isolated chicken infectious
anemia virus and described this disease for the first time. In maternal antibody-
free chicks (less than 2 weeks old), CAV causes subcutaneous hemorrhage,
thymic, bursal and bone marrow atrophy and severe anemia which result in
lymphocyte depletion of both cortex and medulla and develop a profound
immunosuppression with enhanced susceptibility to a wide range of viral and
bacterial pathogens. Maternal antibody-positive chicks become resistant to the
disease by one month of age and thus the disease appear subclinically. Because of
high transmission rate of CAV and possibility of transmitting the infection to
susceptible commercial chickens, finding its prevalence rate in native chicks and
controlling CAV seem to be necessary. In this paper we describe molecular
detection of CAV in native Larry-breed chickens in Chaharmahal-va-Bakhtiyari
province in Iran for the first time (Eskandarzade, 2015).
Although products of fat digestion, including cholesterol, are absorbed in
the first 100 cm of small intestine, the primary and secondary bile acids are
absorbed almost exclusively in the ileum, and 98–99% are returnedto the liver via
the portal circulation. However, lithocholic acid, because of its insolubility, is not
reabsorbed to any significant extent. Only a small fraction of the bile salts escapes
absorption and is therefore eliminated in the feces. Nonetheless, this represents a
major pathway for the elimination of cholesterol. Each day the small pool of bile
acids (about 3–5 g) is cycled through the intestine six to ten times and an amount
of bile acid equivalent to that lost in the feces is synthesized from cholesterol, so
that a pool of bile acids of constant size is maintained (Murray, 2003: 227).
Many investigations have focused on peptides with angiotensin converting
enzyme (ACE) inhibitory and antioxidant effects. Bile acid binding by dietary
fiber is well reported. Thus, various studies have been carried out on bile acid
binding from different materials such as corn bran dietary fiber, extruded potato
peels, chitosan, soy bean, black eye bean, garbanzo and lima bean; kidney bean,
black gram, bengal gram and moth bean. However, few reports are available on
peptides with bile acid binding capacity. Some researchers reported the
involvement of post-digestion hydrophobic peptides in plasma cholesterol-
lowering effect of dietary plant proteins, bile acid binding activity of buckwheat
protein and the binding of bile acids by lupin protein isolates and their
hydrolysates (Zhang, 2011).
In bile there is compoundtahat important, its beteen is bile salt, bile
pigment, lecithin, cholesterol, and anorganic salt. Bile salt have a role is fat
absorption and vitamins A, D, E and K that soluble in fat. Bile salt lower of stains
surface and enlarge emulsifiying fat capacity. Thus will facilitate the work of
lipase. To the next bile salt react with fatty acid producing complex
compound that easily to soluble and easily absorbed as result of lipolysis
process (Tim Dosen Biokimia, 2015: 10).
These include variation in the level of acidic conditions and bile secretion
at different incubation time simulating the physiological aspects of human
digestive system. Viability of these bacteria upon ingestion and sufficient survival
through the transit to GI tract is crucial to confer any health benefits to the host
although some discrepancy between in vitro and in vivo observations does exist
due to the complex nature of the human system. no significant differences were
observed between in vitro and in vivo data, indicating the predictive value of the
model for the survival of the bacteria. Thus, in this study, an in vitro methodology
was employed to assess the transit tolerance of the probiotic samples. The effects
of bile on probiotic strains are more difficult to assess by in vitro due to variation
of bile concentration in the system at any given moment (Sahadeva, 2011).
In vertebrates, before ingested triacylglycerols can be absorbed through
the intestinal wall they must be converted from insoluble macroscopic fat particles
to finely dispersed microscopic micelles. This solubilization is carried out by bile
salts, such as taurocholic acid (p. 355), which are synthesized from cholesterol in
the liver, stored in the gallbladder, and released into the small intestine after
ingestion of a fatty meal. Bile salts are amphipathic compounds that act as
biological detergents, converting dietary fats into mixed micelles of bile salts and
triacylglycerols. Micelle formation enormously increases the fraction of lipid
molecules accessible to the action of water-soluble lipases in the intestine, and
lipase action converts triacylglycerols to monoacylglycerols (monoglycerides) and
diacylglycerols (diglycerides), free fatty acids, and glycerol (step 2 ). These
products of lipase action diffuse into the epithelial cells lining the intestinal
surface (the intestinal mucosa) (step 3 ), where they are reconverted to
triacylglycerols and packaged with dietary cholesterol and specific proteins into
lipoprotein aggregates called (Nelson, 2004 :632).
Cholic acid and chenodeoxycholic acid, known as the primary bile acids,
are quantitatively the most important metabolites of cholesterol. After being
biosynthesized, they are mostly activated with coenzyme A and then conjugated
with glycine or the non-proteinogenic amino acid taurine. The acid amides formed
in this way are known as conjugated bile acids or bile salts. They are even more
amphipathic than the primary products. Deoxycholic acid and lithocholic acid are
only formed in the intestine by enzymatic cleavage of the OH group. They are
therefore referred to as secondary bile acids (Koolman, 2005: 314).
 According to (Li, 2012: 6), that Extensive investigations conducted in the
past decade have shown that bile acids are important regulators of glucose and
lipid metabolism. The identification of bile acid-activated nuclear receptor FXR
and cell surface G protein coupled receptor TGR5 has significantly advanced our
understanding on how bile acid signaling regulates cellular metabolism in
physiological and diseased conditions. The identification of these regulatory
mechanisms also provided molecular basis for developing bile acid receptor
agonists and receptor antagonists for treating human metabolic diseases. On the
other hand, conflicting studies in the field are present, which not only reflects the
complex nature of the bile acid signaling in regulation of whole body metabolism,
but also implies the difference between physiological role and pharmacological
role of bile acid signaling in metabolic control. Furthermore, studies that focus on
the regulation of bile acid metabolism in diseased conditions, especially obesity
and diabetes, are still insufficient. Future advances in the field are needed to
improve our understanding in the bile acid control of metabolism, which is also
critical in developing better drug therapies for the treatment of metabolic
disorders.

D. APPARATUS AND CHEMICALS

1. Apparatus
a. Picnometer 50 mL 1 unit
b. Measuring glass 10 mL 2 units
c. Measuring glass 25 mL 1unit
d. Tube react 7 units
e. Rack tube 1 unit
f. Beaker 50 mL 2 units
g. Beaker 100 mL 2units
h. Drop pipette 10 units
i. Funnel 1 unit
j. Rough cloth 1 unit
k. Smoth cloth 1unit
l. Analytical balance 1 unit
m. Pinset 1 unit
n. Spray bottle 1 unit
o. Oven 1unit
p. Eksicator 1 unit
q. Clamp 1 unit
2. Chemicals
a. Chicken bile
b. Acetic Acid 10 % (CH3COOH)
c. Ammonium molibdate ((NH4)2Mo7O24)
d. Iodide 5% in alcohol (I2)
e. Silver nitrate (AgNO3)
f. Barium cloride (BaCI2)
g. Nitric Acid consentrate (HNO3)
h. Sulphate acid concentrate (H2SO4)
i. Aquadest (H2O)
j. Molisch reagent
k. Tissue
l. Universal Indicator
E. WORK PROCEDURE
1. Test phisycal condition
a. Colour, smell, shape, acidity degree and density of bile was interested and
checked.
b. Density of bile was determined by :
1) Picnometer empty was cleaned.
2) Picnometer was heated in oven and then dried in exicator and weighed.
3) Picnometer was added bile and then weighed.

4) Density of bile was measured according to obtained of data.

2. Musin test
a. Bile was diluted with acetic acid 10 %
b. Bile diluted was tested content of Cl-, by added AgNO3.
 c. Bile diluted was tested content of SO42- by added BaCl2.
d. Bile diluted was tested content of PO43-, by added ammonium molibdate
3. Gmelin test and Smith test
a. Gmelin test
3 mL of HNO3 concentrate was added with 3 mL of bile dilute drop by drop
b. Smith test
3 mL of bile was added 3 mL of I2 drop by drop until I2 in top layer. See the
solution
4. Acidic test of bile
a. 1 mL of bile was diluted with 5 mL of water.
b. 3 mL of bile dilute was added with sucrose solution
c. Solution was shaked
d. Solution was added H2SO4 concentrate.
F. OBSERVATION RESULT
1. Test physical condition of bile
No Physical Condition Result
.
1 Color Green
Smell Stink
Acidity (pH) 6
Shape Liquid
Density m1 = 17.3017 gr
m2 = 43.2335 gr
m1+m 2
M average = =25.9333 gr
2
m 25.9333 gr
ρ= = =1.0373 gr /ml
v 25 ml

2. Musin test
No Activity Result
.
 1 Bile diluted
CH3COOH 10% Drop by drop AgNO3 5% White precipitate
2 Bile diluted
CH3COOH 10% Drop by drop BaCl2 5% Precipitate, green
solution.
3 Bile diluted
CH3COOH 10% Drop by drop ammonium Formed 2 layers
molibdate and top is green ,
bottom is dark
green

3. Gmelin test and Smith test

a. Gmeiln test
HNO3(l) + Bile Top layer + Bottom layer + there is ring
(dark green) (Colorless) (brown)
b. Smith test
Bile + I2 in alcohol Top layer (I2) + Bottom layer + there is ring
(orange) (dark green) (green)
4. Acidic test of bile
1mL of Bile diluted
+ H2O 5 mL + sucrose Green colour + bubble
added
H2SO4 (l) Formed 2 layer,
top layer is green and
bottom layer is dark green
G. DATA ANALYSIS
Known : m1 = 17.3017 gr
m2 = 43.2335 gr
v of bile = 25 mL
Asked: density of bile ?
m1+m 2
Answer : M average = =25.9333 gr
2
m 25.9333 gr
ρ= = =1.0373 gr /ml
v 25 ml

H. DISCUSSION
1. Tes Keadaan Fisik Empedu
 Percobaan ini bertujuan untuk mengetahui keadaan fisik empedu dengan
memeriksa warna, bau, keadaan wujudnya, derajat keasaman (pH) serta berat jenis
empedu. Untuk mengetahui warna, bau, dan wujud empedu terlebih dahulu cairan
empedu fi keluarkan dri kantong empedu. Dari pengamatan di peroleh bahwa
empedu berwarna hijau, berbau amis, dan agak kental. Hal ini telah sesuai dengan
teori dari Poedjiadi dan Panil bahwa empedu merupakan cairan berwrna kehijauan
dan agak kental dan berbau amis. Kemudian melakukan pengukuran pada empedu
yang telah di encerkan dan pHnya di ukur dengan menggunakan indicator
universal, hasil yang diperoleh yaitu pH empedu adalah 6. Hal ini tidak sesuai
dengan teori Poedjiadi (2009) Yang menyatakan bahwa cairan empedu
mempunyai pH antara 6,9 sampai 7,7. Empedu di encerkan terlebih dahulu
sebelum pengukuran pH karena untuk mempermudah pengamatan pada indicator
universal. Adapun berat jenis empedu yang di peroleh 1.0373 gr /ml, secara teori
yaitu 0,89 gram/ml.

2. Tes Musin Dan Senyawa Anorganik Pada Empedu

Percobaan ini bertujuan untuk mengetahui kandungan protein dan
senyawa-senyawa anorganik dalam empedu seperti Cl-, SO42-, dan PO42-. Dimana
pada percobaan ini empedu tersebut terlebih dahulu di encerkan untuk
mempermudah pengamatan. empedu encer kemudian di tambahkan dengan asam
asetat. Penambahan asam aetat berfungsi sebagai pengasam dan untuk
mengendapka musin yang terdapat dalam empedu setelah penambahan asam,
terbentuk endpan berwarna putih yang menandakan bahwa pada empedu postitf
mengandung protein. Larutan kemudian disaring untuk meisahkan endapan
dengan filtratnya. karena endapan tersebut dapat menggangu proses identifikasi.
Kemudian filtratnya dibagi 3 untuk identifikasi senyawa anorganik pada empedu.
a. Uji klorida (Cl-)
Pada pengujian ini, filtrat ditambahkan perak nitrat yang berfungsi untuk
mengetahui adanya ion klorida dengan terbentuknya endapan putih AgCl. Dan
dari percobaan yang dilakukan diperoleh endapan putih. Ini berarti dalam empedu
mengandung ion Cl. Hal ini sesuai dengan teori yang menyatakan bahwa jika ada
ion Cl maka terbentuk endapan putih. Adapun reaksi yang terjadi yaitu :
Cl- + AgNO3 → AgCl (endapan putih) + NO3
b. Uji ion SO42-
Pengujian ini dilakukan dengan penambahan larutan BaCl2. Penambahan
larutan BaCl2 berfungsi untuk mengetahui adanya ion sulfat dengan terbentuknya
endapan BaSO4 . Dari hasil percobaan yang telah dilakukan, tidak terbentuk
endapan putih. Hal ini tidak sesuai dengan teori yang menyatakan bahwa jika ada
ion SO42- akan terbentuk endapan. Hal ini juga berarti empedu negative
mengandung senyawa anorganik. Adapun Persamaan reaksinya adalah :
SO42- + BaCl2 → BaSO4 (endapan) + 2Cl-
c. Uji in phospat
Pengujian ini dilakuka dengan penambahan larutan ammonium molibdat.
Penambahan ammonium molibdat berfungdi untuk mengetahui adanya ion pospat
(PO4). Dari hasil percobaan yang dilakukan tidak di peroleh endapan kuning,
artinya pengujian negative, tidak terdapat senyawa anorganik. Hal ini tidak sesuai
dengan teori yang menyatakan bahwa adanya ion fospat ditandai dengan
terbentuknya endapan. Reaksi yang terjadi adalah :
PO42- + (NH4)2Mo7O24 (NH4)2PO42- + Mo7O242-
(kuning)
Ketiga hasil ini dapat disimpulkan bahwa pada sampel empedu ayam
negatif/ tidak mengandung senyawa-senyawa anorganik (SO42-, dan PO42-), dan
mengandung senyawa anorganik (Cl-). Hal ini disebabkan karena pengaruh
makanan yang dikonsumsi setiap hari tidak bergizi.
3. Tes Zat Warna Empedu
a. Tes Gmelin
Percobaan ini bertujuan untuk mengetahui zat warna yang terdapat dalam
empedu. Pada percobaan ini, cairan empedu ditambahkan dengan larutan aam
nitrat. Asam nitrat berdungsi untuk mengoksidasi zat warna empedu. Penambahan
asam nitrat dilewatkan melalui dinding tabung agar terlihat cincin coklat yang
terbentuk. Dari hasil pengamatan diperoleh cincin ocoklat, yang berarti terdapat
zat warna bilirubin. Rekasinya yaitu :
C2H5-SH C2H5-SH

H-C-COOH + HNO3 H-C-COOH + NO3-

NH2 NH3+
b. Test Smith
Pengujian ini digunaka larutan I2 dalam alcohol yang direaksikn dengan
empedu encer. Dari percobaan diperoleh larutan berwarna hijau yang berarti
terdapat zat warna empedu yaitu biliverdin yang berwarna hijau. Hasil ini telah
sesuai dengan teori yang menyatakan bahwa pada test Smith akan diketahui zat
warna empedu berupa biliverdin yang berwarna hijau. Reaksinya yaitu:
C2H5-SH C2H5-SH

H-C-COOH + I2 H-C-COOH + I-

NH2 NH3+
4. Tes Asam Empedu
Percobaan ini bertujuan untuk mengetahui adanya asam empedu. Empedu
diencerkan terlebih dahulu dan ditambahkan Kristal sukrosa yang berfungsi untuk
meningkatkan tegangan permukaan, setelah itu ditambahkan dengan asam sulfat
pekat dan terbentuk 2 lapisan, lapisan atas berwarna hijau dan lapisan bawah
berwarna hijau muda.
I. CONCUSION AND SUGGESTION
1. Conclusion
Base on the experiment, we can conclude that bile positively contained
Chloride but negatively contained phospate and sulphate. Characteristic of bile are
have dark green color, stink smell, and pH= 6. To Gmelin and Smith test produce
brown ring and dark green ring.
2. Suggestion
 To next apprentice to carry bile with big volume, because my group
deplete bile at experiment.

BIBLIOGRAPHY

Al-Naswari, et all. 2014. Molecular Detection Of Toxoplasma gondii In Human

and Chicken By Real-Time PCR Technique. International Journal Of
Advanced Research. Vol.2. Issue 3.

Ezkandarzade Neda, et all. 2015. Molecular Detection Of Ckicken Anemia Virus

from Native Larry-breed Chicken in Chaharmahal-va-Bakhtiyari
Province, Iran. International Journal Of Medical Laboratory. Vol.2,
Issue 2.

Gunasekaran, et all. 2012. Identivication and In Vitro Evaluation of Species

Specific Probiotic for Feeding Broiler Chicken Using Probiotik
Scores. International Journal Of Veterinary Science. Vol 1, Issue 2

Koulman, J and K. H. Roehm. 2005. Color Atlas of Biochemistry. New York :

Thieme

Li Tiangang and John Chiang. 2012. Bile Acid Signaling in LiverMetabolism and
Diseases. Journal of Lipida. Vol 2012, Issue 9

Murray, Robert K et all. 2000. Harper’s Illustrated Biochemistry. America : Mc

Graw Hill

Nelson, David L and Michael M. Cox. Lehninger Principles of Bochemistry

Fourth Edition

Sahadeva et all. 2011. Survival of Commercial Probiotic Strains to pH and Bile.

International Food Research Journal. Vol 18, Issue 4

Schamidt, Frank. 2000. Biochemistry II. Amerika : IDG Books

Walsh, Gary. 2003. Biopharmaceuticals Biochemistry and Biotechnology Second

Eddition. England : Wiley
Zhang, Hui et all. 2011. In Vitro Binding Capacity of Bile Acids by Defatted Corn
Protein Hydrolysate. International Journal of Molecular Science. Vol
2011, Issue 2

DOCUMENTATION

Mucin Test Gmelin Test Smith Test

Bile Acid Test