Therapy of Fermented Milk Lactobacillus Casei Strain Shirota to

Level of Malondialdehyde (MDA) and Proteind Bands the Hearth the

White Rats (Rattus Norvegicus) That given High Cholesterol Dietary

D. W. E. Putri

, A. Aulanni’am

, A. Srihardyastuti

.Institut Biosains,Brawijaya University, Mayjend Pandjaitan Street, Malang, Indonesia

Department of Chemistry, Faculty Mathematics and Sciences, Brawijaya University, Malang, Indonesia

. Department of Chemistry, Faculty Mathematics and Sciences, Brawijaya University, Mayjend Pandjaitan street, Malang,

Indonesia

Keywords: Hypercholesterolemia, Fermented Milk, MDA, Protein Profile

Abstract: Cardiovascular disease is a disease that occurs due to a disturbance in the function of the heart and blood

vessels such as coronary heart disease (PJK), hypertension and stroke. One cause of PJK is a condition of

hypercholesterolemia. Hypercholesterolemia is a condition which cholesterol level more than 200 mg / dL.

High cholesterol dietary can cause an increase in free radicals that cause oxidative stress. Fermented milk

Lactobacillus casei thought to contain biopeptida as antioxsidant. This study was to determine the effect of

fermented milk Lactobacillus casei in lowering levels of MDA and repairing protein profiles of heart. Animal

that used is a rat (Rattus norvegicus, males, aged 2-3 months, weightabout 100-250 gram. Rats divided into

normal rats, hypercholesterolemia rats , hypercholesterolemia rats and therapy 1 mL and 2 mL . High

cholesterol dietary used egg yolk, cholesterol pure and cholic acid that give in forcefeeding for 4

weeks.Therapy of Lactobacillus casei fermented milk given for 2 weeks in. Level of MDA was measured

using the method of TBA and cardiac protein bands was tested using SDS-PAGE. The result of Research

obtained showed Lactobacillus casei fermented milk therapy was significantly (p <0.05) lower levels of MDA

and affect cardiac protein bands of rats. Doses of 2 mL is the best dose with decreased levels of MDA by

43.11% and is able to restore protein band profiles such as Lactobacillus casei normal. In conclusion tha

fermented milk can be used as an alternative treatment of hypercholesterolemia

1 INTRODUCTION

Cardiovascular disease is a disease that occurs due to

interference withwork functions on the heart and

blood vessels such as coronary heart disease (CHD),

hypertension and stroke. 17.3 million people are

estimated to die of cardiovascular diseasein 2008 but

this number will continue to increase to 23.3 million

deaths inyear 2030. In Indonesia alone the prevalence

of CHD more than 800,000 people in 2013

(Riskesdas, 2013).Unhealthy lifestyles such as

smoking and consuming foods high in fat can because

hypercholesterolemia as one of the causes of CHD.

One of research stated that in 2009-2010 sufferers of

coronary heart disease (CHD) caused by

hypercholesterolemia increased from 13.5% to 19.2%

(Pangestika, et.al., 2014). This matter shows that in

just one year there was an increase of 5.7%.

Induction of a high cholesterol diet causes an

accumulation of fat in the liver an increase that also

increases the amount of acetil co-A in the liver cells

to producecholesterol so that an increase in

cholesterol levels (Guyton, 1991). The build up of

cholesterol and fat in the body is called

hypercholesterolemia. The body that experiences

hypercholesterolemia willbalance cholesterol levels

with the synthesis of bile acids that also produced by-

product in the form of free radicals which is

characterized by increased levels of MDA in body

(Sugiarto, et.al., 2014). Malondialdehyde (MDA) is a

dialdehyde compound that hasmolecular formula

C3H4O2 , results from oxidation of unsaturated fatty

acids by free radicals (Winarsi, 2007). Treatment of

hypercholesterolemia so far has only used anti-

medication hypercholesterolemia which can reduce

cholesterol levels but has side effects against other

organs such as the kidneys. One study mentioned that

126

Putri, D., Aulanni’am, A. and Srihardyastuti, A.

Therapy of Fermented Milk Lactobacillus Casei Strain Shirota to Level of Malondialdehyde (MDA) and Proteind Bands the Hearth the White Rats (Rattus Norvegicus) That given High

Cholesterol Dietary.

DOI: 10.5220/0009586901260132

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 126-132

ISBN: 978-989-758-483-1

fermented milk including yogurt, goat milk can

reduce cholesterol levels because it has antioxidants

which will suppress free radicals and inhibit LDL

oxidation reactions so cholesterol levels in the blood

will decrease (Pangestika, et.al., 2014).

Fermented milk products are not only limited to

yogurt. One of the bacteria that canused for

fermenting milk is Lactobacillus casei Shirota

strains. But the milk products that used bacterin has

not been much studied. Therefore, it is necessary

conducted research to determine the benefits of

Lactobacillus casei fermented milk Shirota strains

against decreased levels of Malondialdehyda (MDA)

and cardiac protein profiles.

2 MATERIALS AND METHODS

2.1 Preparation of Experimental

Animals

Rats were divided into 4 treatment groups and each

group contained 6 rat, vizgroup 1 was rat that were

not treated (normal rat), group 2 is a hypercholesterol

rat (positive control / K +), group 3 is a rat

hypercholesterolemia and given a fermented milk

dose of 1 mL (Y1). Before receiving treatment,

animal model rats ( Rattus norvegicus ) is adapted to

the laboratory environment for 7-14 days.

Feeding during the adaptation period is in the

form of standard feed as needed namely 20 grams of

feed / head / day and drinking water. Consumption of

rat feed per day rangesbetween 15-30 grams / head /

day. The feed given is in the form of standard feed.

Rats are placed in the cage according to treatment.

This is so that high cholesterol dietsgiven through

gastric sonde in each animal model can be achieved.

2.2 Preparation of Animal Model Rat

(Rattus norvegicus)

Hypercholesterolemia

Animal model of hypercholesterolemia is made by

induction of hypercholesterolemia in rat ( Rattus

norvegicus ) which is done by increasing blood

cholesterol levels in experimental animals by giving

food made from pure cholesterol as much as 2 grams,

cholic acid 0.02 grams, and boiled quail egg yolk 1

gram. Which is then dissolved in distilled wateras

much as 2 mL. The feeding of hypercholesterolemia

in rats was carried out using sondes tomach. Induction

of a high cholesterol diet is carried out for 4 weeks

(Wulandari, 2013). Level measurement of rat

cholesterol ( Rattus norvegicus ) is done once a week

before the treatment by milk containing Lactobacillus

casei bacterial fermentation of Shirota strains using

GCU tools.

2.3 Lactobacilus Casei Fermented Milk

Bacteria Shirota Strain

Lactobacillus casei fermentation of Shirota strain is

administered by sonde stomach. Treat will be done if

the cholesterol level in rats has reached 145mg / dL

in hypercholesterolemia rat. Dosage of Lactobacillus

casei fermented milk Shirota strain of 1 mL for group

Y1 and 2 mL for group Y2.Y1 group was given

Lactobacillus casei fermented milk therapeutic milk

shirota strain as much as 1 mL per day for each mouse

for 2 weeks, while the Y2 group was

givenlactobacillus casei fermented milk therapy

strains of shirota as much as 2 mL per day for each rat

for 2 weeks. Giving Lactobacillus casei fermented

milk therapy Shirota strains are carried out alternately

according to the treatment group, which begins the

group Y1 for hypercholesterolemia rat + 1 mL

therapeutic milk fermented Lactobacillus casei

bacterial Shirota strain and continued

hypercholesterolemia rat + 2 mL bacterial fermented

milk therapy Lactobacillus casei Shirota strains in a

gastric sonde.

2.4 Intake of Heart Organs

Intake of the heart organ is done after bacterial

fermented milk therapy Lactobacillus casei Shirota

strains for 2 weeks. Intake of the heart organ is carried

out after surgical dislocation of the neck. The heart

organ is removed for further storage in PBS (

Phosphate Buffer Saline ) for measurement of MDA

levels and protein profiles.

2.5 Measurement of Malondialdehyde

Levels (MDA) and Making the

MDA Standard Curve

MDA standard curve measurements are carried out

the way the MDA standard solution is taken

onconcentrations of 1, 2, 3, 4, 5, 6, 7 and 8 µg / mL,

each taken as much as 100 µL. The solution was put

into a tube and added 550 μL of distilled water and

100 μL of distilled water TCA 10%, then

homogenized. HCl 1 N was added as much as 250 µL

and 100 µLNa-Thio 1% into the tube and

homogenized. Then, heat for 20 minutes at 100 °C,

after cold centrifugation with a speed at 5000 RPM

for 10 minutes. Obtained supplies are taken and

Therapy of Fermented Milk Lactobacillus Casei Strain Shirota to Level of Malondialdehyde (MDA) and Proteind Bands the Hearth the

White Rats (Rattus Norvegicus) That given High Cholesterol Dietary

127

measured at maximum wavelengths (λmax = 533 nm)

using a spectrophotometer to obtain the value of the

last absorbanceMDA standard curves were made.

MDA standard curves are generated based on

equations regression between absorbance (y) and

MDA concentration (x).

2.6 Measurement of MDA Levels in

Heart Organs

MDA levels were measured using the Thiobarbituric

Acid method (TBA) according to the method

performed (Aulanni’am, et.al., 2012). The testing

begins with weighing cardiac organs weighing 0.5

grams and put into the last mortar crushed until

smooth. NaCl 0.9% solution was added and

homogenized. Homogenates formed were

centrifuged at a speed of 8000 rpm for 20 minutes and

the supernatant was taken. Supernatant taken as much

as 100 µL is inserted in a microtube, added 550 µL of

aquades and homogenized. Next, 100 µL TCA 10%

was put into a microtube tube and homogenized. Then

done addition of 100 µL HCl 1 N and 100 µL Na-

Thio and homogenized with vortex.The mouth of the

micro tube is covered with aluminum foil and heated

at 100° C for 30 minutes in a water bath. After getting

cold, centrifugation is done at 5000 RPM for 10

minutes and the supernatant is taken to be transferred

to the new microtube. The absorbance samples were

measured using spectrophotometer with a maximum

wavelength (λmax = 533 nm). Measurement

complete cardiac MDA levels can be seen in

Appendix E2.

2.7 SDS PAGE Electrophoresis

2.7.1 Gel Preparation

Gels are made using a series of two glass plates. There

are two types of gels that are made namely gel as a

sample ( stacking gel ) and gel as a protein separation

medium( separatig gel ). Materials for separating gel

consists of Lower Gel Buffer (LGB), T-acrylamide,

distilled water, ammonium persulphate (APS),

Tetramethyl Ethylene Diamine (TEMED) then

dissolved into distilled water. Separating gel solution

is poured in the place gel layer and allowed to

polymerize for 10-20 minutes. Material for

manufacture Stacking gel consists of the Upper Gel

Buffer (UGB) , T-acryl, APS, and later TEMED

dissolved in distilled water. Stacking gel that has been

made is poured into a separating gel which has been

polymerized then installed a comb to form a gel and

wells.

2.7.2 Sample and Running Injection

The isolated and extracted heart organ is taken 15 µl,

then15 µl RSB was added (1: 1 ratio) and then heated

in a water bath temperature of 100° C for 5

minutes.30 µl cold extract of the heart organ was

added in gel wells that already contain protein

markers. The cathode is connected to the power

supply whereas the anode was connected to the

reservoir. The current is set at 200V for 1 hour. The

process is terminated when the blue marker reached

0.5 cm below the lower limit of the gel.

2.7.3 Staining

The running gel is colored by soaking the gel in a

staining solution for 30-60 minutes by matching using

a magnetic stirrer. Elimination of the color is done by

soaking the gel in destaining solution by shaking

using a magnetic stirrer until the gel turns clear

2.7.4 Determination of Molecular Weight

Determination of molecular weight can be done by

comparing the results of electrophoresis and protein

markers so that the types of protein in the extract can

be identified. The mathematical determination used

the formula:

Rf =

Distance of protein movement from the starting place (cm)

Color movement distance from starting place (cm)

Rf is the Retardation factor , then a standard curve

is made with the price of Rf asthe X axis and the

logarithmic price of molecular weight as the Y axis

and plotted so it willthe molecular weight can be

known.

2.7.5. Data Analysis

Data analysis was performed using the ANOVA test

( One Way Analysis of Variance ) and BNJ test

(Honestly real difference) to determine whether there

is a difference for the MDA level test and for the

analysis of protein profiles using SDS PAGE.

3 RESULTS

3.1 Effect of Induction of High

Cholesterol Diet and Therapy of

Fermented Milk on Levels Rat

Cholesterol (Rattus norvegicus)

Induction of a high cholesterol diet is done by

providing feed containing quail egg yolk, pure

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cholesterol and cholic acid for 4 weeks caused by an

increase in cholesterol levels. Rats can be catagorized

as hypercholesterol rats if the cholesterol level have

exceeded 145 mg / dL. Hypercholesterolemia rats

were treated with fermented milk at a dose of 1 mL

and 2 mL within 2 weeks.Cholesterol levels reduction

showed on Table 1.

Table 1 : Rats (Rattus norvegicus) Cholesterol Levels

Treatment

After

Acclimation

After

Induction

After

Therapy

Normal 30,4±4,16 31,6±3,57 30±2,91

Hyper

cholesterol

34±6,82

147,8±20,

194,4±23,

Hyper

cholesterol

+ 1 mL

fermented

milk

31,6±2,3

191,6±17,

104,8±3,5

Hyper

cholesterol

+ 2 mL

fermented

milk

31,8±4,54 182±16,76 87±3,16

Hypercholesterolemia rat experienced a significant

increase in cholesterol levels after induction by high

cholesterol diet. Induction of a high cholesterol diet

contains saturated fats resulting in increased levels of

triglycerides in the blood whereas triglycerides

themselvesis a cholesterol precursor. Saturated fats

will increase LDL levels and decrease levels of HDL.

Rat with hypercholesterolemia were given fermented

milk therapy with a dose of 1 mL and 2 mL.

Fermented milk therapy could reduce the cholesterol

levels of rats.

3.2 Effect of Induction of High Cholesterol

Diet on MDA (Malondialdehyde)

Levels of Rats (Rattus norvegicus)

Hypercholesterolemia

MDA levels were obtained after measurement using

the TBA methodhypercholesterolemia MDA levels in

rat hearts will experience whereas in rat treated MDA

levels of rats heart will decrease.

Table 2: MDA levels of the rat heart induced by a high

cholesterol diet

Treatment

Average of

MDA Levels

(µg/mL)

MDA Levels

Increase

Decrease

Normal 1.47±0,06

- -

Hyper

cholesterol

7,81±0,23

81,17 -

Hyper

cholesterol

+ 1 mL

fermented

milk

5,97±0,78

23,56

Hyper

cholesterol

+ 2 mL

fermented

milk

4,44±1,42

- 43,11

From table 2 showed that the induction of a high

cholesterol diet increased MDA levels of rat heart

(7.93 ± 0.259) µg / mL or experiencing 85.88% of

MDA levels of normal rat heart organ that is equal to

(1.12 ± 0.213) µg / mL. MDA levels of rats

hypercholesterolemia which has been treated with 1

mL of fermented milk is (5.11 ± 0.701) mg / dL or

decreased when compared with hypercholesterol rat

by 35.51%whereas in hypercholesterol rat treated

with mL fermented milk, MDA levels were obtained

by (3.88 ± 0.721) mg / dL which decreased by

50.99%.

Figure 1 : MDA content of rat heart

Statistical analysis using One-Way ANOVA showed

that the treatment of fermented milk significantly (p

<0.05) can reduce MDA levels rat ( Rattus norvegicus

) hypercholesterol induced high cholesterol diet as

shown in table 2. Test results using Tukey or

MDAcontent

(μg/mL)

Treatmentgroup

Therapy of Fermented Milk Lactobacillus Casei Strain Shirota to Level of Malondialdehyde (MDA) and Proteind Bands the Hearth the

White Rats (Rattus Norvegicus) That given High Cholesterol Dietary

129

Honestly Significant Difference (BNJ) showed that

normal rat and hypercholesterol rat were significantly

different so the notation were different.In

hypercholesterol rat and rat that have been given

therapy show a difference

significant so that the notation is different whereas

in rat given ratshypercholesterolemia + 1 mL therapy

and rat hypercholesterolemia + 2 mL therapy showed

significant difference so the notation was different.

Feeding hypercholesterolemia in Rattus norvegicus

rat will increase the amount of free radicals in the

body is indicated by an increase in MDA levels

(Wulandari et.al., 2013). MDA levels are an indicator

of the presence of free radicals caused by lipid

peroxidation process. Normally, free radicals are

produced by the body in small amounts as a result of

various metabolic processes in the body. Free radicals

produced by some of the constituent components of

cells, such as mitochondria, plasma membranes,

lysosomes,endoplasmic reticulum and nucleus. Free

radicals produced are the resultthe side effects of

oxidation or cell metabolism that take place during

respiration cells and digestion. Fermented milk

therapy reduced MDA levels allegedly because of the

presence of active biopeptides. Active biopeptides in

fermented milk have the potential to play a role

antioxidants by capturing reactive oxygen species

(ROS) compounds so that the effect of free radicals

will decrease.

3.3 Effect of Induction of a High

Cholesterol Diet on the Protein Profile

of Rats ( Rattus norvegicus )

Hypercholesterolemia

White rat heart protein profiles were measured using

the SDS-PAGE method. Band results protein can be

seen in figure 2.

Figure 2: SDS-PAGE results

The results in figure 2 and table 3 showed normal rat,

and hypercholesterol rat + 2 mL fermented milk

therapy there is no severe protein molecules 70 kDa

and 23 kDa whereas in hypercholesterol rat and

hypercholesterol rat+ 1 mL therapy, there are proteins

with molecular weights of 23 kDa and 70 kDa

Table 3 : Molecular Weight (MW) Loss

H+1 mL

Fermented

Milk

H+2 mL

Fermented

Milk

100 kDa

√ √ √ √

85 kDa

√ √ √ √

70 kDa - √ √ -

66 kDa √ √ √ √

48 kDa √ √ √ √

35 kDa √ √ √ √

25 kDa - √ √ -

17 kDa √ √ √ √

(MW: Molecular Weight; N: Normal; H: Hypercholesterol)

A protein with a molecular weight of 23 kDa can be

said to be CRP (C- Reactive Protein ) which is a class

of pentraxin protein that binds calcium with defense

M : Marker

N : Normal

H : Hypercholesterol

T1: Hypercholesterol + 1 mL fermented milk

T2: Hypercholesterol + 2 mL fermented mil

N HM T1 T2

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properties immunological.The formation of CRP due

to an inflammatory response in the heart whichproves

that induction of a high cholesterol diet increased

ROS production. Protein with a molecular weight of

70 kDa is the HSP protein (Heat Shock Protein). The

HSP is a protein which results from genetic based

responses to induce genes encoding molecular

chaperons, proteases and other proteins that are

important in recovery and defense and cell response

from various physiological disorders and

environment. The appearance of protein with BM 70

kDa in hypercholesterolemia rat were an

inflammatory response from free radicals due to the

induction of a high cholesterol diet.In therapy 1

protein bands with molecular weights of 23 kDa and

70 kDa are still formed, however,the band formed is

thinner than the protein band formed in rat

hypercholesterolemia. This showed that

hypercholesterolemia rat + 1 mL fermented milk

therapy. Fermentation has the effect of reducing

cholesterol levels so that protein bands were formed

not as thick as a hypercholesterol rat. Therapy 2 did

not show the formation of protein bands at a

molecular weight of 23 kDa and 70 kDa. This

happened due to the active biopeptides that played a

role as an antioxidant suppresses inflammation that

occured so that the rat proteins band profiles

hypercholesterolemia treated with 2 mL of fermented

milk approached normal rat protein band profiles.

This showed that 2 mL of fermented milk therapy has

a decreased effect which is more effective so as not to

form protein bands with molecular weights of 23 kDa

and 70 kDa.

4 CONCLUSIONS

Based on the results of this research, it can be

concluded that the higher the Lactobacillus casei

fermented milk therapy, the greater MDA levels

decrease. The best dose of therapy is 2 mL of

fermented milk. Lactobacillus casei fermented milk

therapy can suppress the expression of CRP and HSP

so that the therapeutic protein profile resembles the

normal rat protein profile.

REFERENCES

Data Riskesdes 2013 Republik Indonesia

Guyton AC, 1991, Basic neuroscience: anatomy and

Physiology, 2nd ed, WB Saunders, Philadelphia, PA

Sugiarto M, Padaga, M.C. dan Wurangil, D.K., 2014, Efek

Terapi Yogurt Susu kambing Terhadap Ekspresi

Inducible Nitric Oxyde Synthase (iNOS) dan Kadar

malondialdehida (MDA) Pada Aorta Hewan Model

Tikus (Rattus norvegicus) Hiperkolesterolemia

,Pedidikan Dokter Hewan, Universitas Brawijaya.

Winarsi H., 2007, Antioksidan Alami dan Radikal

Bebas, Penerbit Kanisius, Yogyakarta.

Pangestika A.R, Mahdi, C. dan Padaga, M. C., 2014 ,Efek

Terapi yogurt Susu Kambing terhadap Ekspresi Tumor

necrosis Factor Alpha (TNF-α)dan Picturean

Histopatologi Aorta pada Tikus (Rattus norvegicus)

Model Hiperkolesterolemia, Program studi Kedokteran

Hewan, Universitas Brawijaya.

Wulandari D.Y, Padaga M.C, dan Herawati., 2013 ,Kadar

Malondialdehida (MDA) dan Picturean histopatologi

Organ hati Pada Hewan Model Tikus (Rattus

norvegicus) Hiperkolesterolemia setelah Terapi

Ekstrak Air Benalu Mangga (dendrophthoe pentandra

L.Mig), Program Studi Kedokteran Hewan, Universitas

Brawijaya.

Risfianti D.K., 2016, Terapi Serat Kedelai hitam(Glycine

max(L.)merr) pada Tikus Hiperkolesterolemia

Terhadap Kadar Malondialdehida, Aktivitas

Superoksida Dismutase dan Histopatologi Hepar,

Thesis, Kimia, Universitas Brawijaya, Malang.

Muray, R.K, D.K. Granner, P.A. Mayos, dan V.W.

Rodwell.,2003, Harper’s Illustrated

Biochemistry.Twenty Six Edition, Lange Medical

Book/McGraw-Hill Medical Publishing Division.

Tomkin, G.H and D, Owens. 2012. LDL as a Cause of

Atherosclerosis. The Open Atherosclerosis&

Thrombosis Journal 13-21.

Lichtenstein A.H., 2006, Diet and Lifestyle

\Recommendations Revisition : A Scientific Statement

From the American Hearth Association Nutrition

Comminte, Circulation 114:82-96.

Wulandari D.Y, Padaga M.C, dan Herawati., 2013 ,Kadar

Malondialdehida (MDA) dan Picturean histopatologi

Organ hati Pada Hewan Model Tikus (Rattus

norvegicus) Hiperkolesterolemia setelah Terapi

Ekstrak Air Benalu Mangga (dendrophthoe pentandra

L.Mig), Program Studi Kedokteran Hewan, Universitas

Brawijaya.

Mohammadi M, M Alipour, M.R, Alipour and A.M,

Vatankhah., 2006, Effect of High Cholesterol Diet

and Parallel Chronic Exercise on Erythrocyte Primary

Antioxidant Enzyme and Plasma total Antioxidant

Capacity in Dutch Rabbits, International Journal

Endocrinol Metabolism 4:30-40.

Silalahi J., 2001, Free radicals and antioxidant vitamints in

degenerative disease, JIMA II(5):1-3.

Burcham PC,1998. Genotoxic lipid peroxidation products :

their DNA damaging properties and role in formation

of endogenous DNA adducts. Mutagenesis 1998, 13 :

287 – 305.

Robinson R.K., 1981, Dairy Microbiology: The

Microbiology of Milk Products. Volume 11, London :

Applied Science Publishing.

Rose., 1982, Economic Microbiology Fermented Foods.

Volume V11, London Academic Press.

Therapy of Fermented Milk Lactobacillus Casei Strain Shirota to Level of Malondialdehyde (MDA) and Proteind Bands the Hearth the

White Rats (Rattus Norvegicus) That given High Cholesterol Dietary

131

Sunaryanto R., Martius E. dan Marwoto B., 2014, Uji

kemampuan Lactobacillus casei Sebagai Agensia

Probiotik, Jurnal Bioteknologi dan Biosains Indonesia

Vol 01 No 01.Issn : 2442-2606.

Wenno, M.,R, Suprayitno, Aulanni;am, Hardoko, 2016,

The Physichochemical Characteristics and Angiotensin

Converting Enzyme (ACE) Inhibitory Activivty of

Skipjack Tuna (Katsuwonus pelamis)”Bekasang”,

Jurnal Teknologi.78:4-2(2016)119-124

www.jurnalteknologi.utm.my . eISSN 2180-3722.

Padaga, M.,C, Aulann’iam, Sujuti.,H, Widodo, 2015,

Blood Pressure Lowering effect and Activity of Casein

DerivedFrom Goat Milk Yogurt in DOCA-salt

Hypertensive Rats.International Journal of PharmTech

Research.ISSN:0974-4304.Vol.8, No,6 pp 322-330.

Ebringer, L., M. Ferencik and J.krajovic, 2008,Beneficial

Health Effect of Milk and Fermented Dairy Product-

Review. Comenius University, Bratislava,

Slovakia.Folia Microbiol 53(5) 378-394. 21

Marie P, Edward R.F, dan Peter J.H., 2000, Consumption

of Fermented and Nonfermented Products: Effect on

Cholesterol Concentrations and Metabolism, Am J

Clin Nutr. 71: 674-81.

Baroutkoub A, Roushan Z.M, Razmik, B, Julayi, H

Sohrabi, Z, Mazloomi, S. M, dan Eskandari, M. H.,

2010, Effects of Probiotic Yoghurt Consumption on

the Serum Cholesterol Levels in Hypercholestromic

Cases in Shiraz, Southern Iran, Scientific Research

and Essays 5 (16): 2206-2209.

Muttaqin, A, 2008, Buku Ajar Asuhan Keperawatan Klien

dengan Gangguan Sistem Persarafan, Jakarta:Penerbit

salemba

Nursyah D.A., 2012, Picturean Siklus Estrus Tikus Putih

Rattus norvegicus Ovariektomi yang Diberi Tepung

Daging Terpang holothuria Scabra, Fakultas

Kedokteran Hewan, Institute Pertanian Bogor.

Dewi D.I., 2010, Tikus Riul (Rattus norvegicus

Berkenhout), BALABA Vol.6, No.02, Des 2010 : 22-

23.

Harini M, Astirin O. P., 2009, Blood Cholesterol Levels of

Hypercholesterolemia Rat (Rattus norvegicus) after

VCO Treatment, Nusantara Bioscience 1: 53-58.

Krinke G. J., 2000, The Hand Book of Laboratory Animal,

The Laboratory Rat, Midas Printing Ltd,Scotland 349-

353.

Aulanni’am, Roosdiana, A and Rahmah, N.R, 2012. The

Potency Of Sargassum duplicatum Bory Extract on

Inflammantory Bowel Disease Therapy in Rattus

norvegicus, Journal of life Sciences 6 (2012) 144-154.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

132