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Effect of Probiotic Milk with Lactic Acid Bacteria against

Mycobacterium tuberculosis growth in Agar Diffusion Bioassay

1. Introduction.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis which that

primarily attacks the lungs still a public health problem in world. There are 8.80 million

new cases of pulmonary tuberculosis occur each year which 3.90 million is for lung

tuberculosis with Acid Fast-Bacilli (AFB) positive and 80% occur in developing

country and a third in Southeast Asia. WHO estimated that 140 thousand people die

caused by TB every year in Indonesia (WHO, 2004).

The further studies mention that the use of probiotic for preventiontuberculosis

infection develops from every time. From the research, found that the Lactococcus lactis

which is Lactic Acid Bacteria can produce a BacteriocinNisin and Lacticin that inhibit

growth of Mycobacterium tuberculosis(James Carroll, LorraineA.Draper,

PaulaM.O'Connor, AidanCoffey, ColinHill, R.PaulRoss,PaulD.Cotter, JimO'Mahony.

2010.

ComparisonoftheactivitiesoftheLantibioticsNisinandLacticin3147againstclinicallysignifi

cantMycobacteria.InternationalJournalofAntimicrobialAgents, Volume36, Issue2,

August2010, Pages132-136).In other studies, Lactic Acid Bacteria such as

Lactobacillus Rhamnosus and Bifidobacterium bifidum can improve immune system

due to intracellular Mycobacterium tuberculosisinfection (Darab Ghadimi, Michael de

Vrese, Knut J. Heller, Juergen Schrezenmeir. Lactic acid bacteria enhance autophagic

ability of mononuclear phagocytes by increasing Th1 autophagy-promoting cytokine

(IFN-γ) and nitric oxide (NO) levels and reducing Th2 autophagy-restraining cytokines

(IL-4 and IL-13) in response to Mycobacterium tuberculosisantigen.International

Immunopharmacology, Volume 10, Issue 6, June 2010, Pages 694-706).

2. Material and Method.

2.1 Mycobacterium Tuberculosis culture conditions

1. Sputum added NaOH 4% (which has been given the phenol red indicator)

approximately in thesame amount.

2. Shake for 2-3 minutes until homogeneous.

3. Put it in an incubator at 370C for 30 minutes in order to make the sputum

become thin.

4. Suspense of sputum is neutralized withHCI 2N until it changes color.

5. Centrifuge at 3000 rpm for 30 minutes.

6. Supernatant discarded into a place that contains a disinfectant, with the residue:

a. Grown on Lowenstein Jensen medium. Incubate the medium which had been

planted in the Incubator at 37 C for 6-8 weeks.

b. Acid-fast staining was given to look for Acid-Fast Bacilli.

2.1.2 Lowenstein Jensen Media1. Formula / Liter

L-Asparagine .......................................................................... 3.6 g

Monopotassium Phosphate..................................................... 2.5 g

Magnesium Sulfate................................................................. 0.24 g

Sodium Citrate ........................................................................ 0.6 g

Malachite Green ...................................................................... 0.4 g

Potato Flour............................................................................. 30 g

Supplements

Glycerol, 12 mL

Egg Suspension, 1000 mL

2. Lowenstein Jensen with selective Mycobacterium

Similar to the procedure no 1 was added by Cycloheximide 0.64 g, Lincomicin 3.2

mg, and nalidixic acid 56 mg.

3. Directions:

a. Dissolve 37.3 g of the medium in 600 mL of purified water containing 12 mL

of glycerol.

b. Heat with frequent agitation to completely dissolve the medium.

c. Autoclave at 121°C for 15 minutes.

d. Prepare 1000 mL of a uniform suspension of fresh eggs under aseptic

conditions. Avoid whipping air into suspension during the collection and

mixing.

e. Aseptically mix the 1000 mL of egg suspension with 600 mL of the sterile

Lowenstein-Jensen Medium cooled to 50 - 60°C, avoiding air bubbles.

f. Dispense the finished medium into sterile screw-cap test tubes. Place the tubes

in a slanted position and heat at 85°C for 45 minutes.

g. Test the finished product performance using stable culture control.

2.2 Making the Probiotic milk

The process of making probiotic milk involves several steps. The milk is

retrieved from cows, then sterilized and cooled. After the curing process, the milk

receives the 3 strain of Lactic Acid Bacteria (Lactococcus Lactis, Lactobacillus

Rhamnosus, and Bifidobactrium bifidum) which the specific manufacturer has decided

to use. After being treated with the bacteria, put the milk into the containers and

incubatedat 370C for 24 hours, then cooled again until the analysis begin.

2.3 Experimental Procedure

2.3.1 Sterilization of Tools and Materials

Petri dishes, Reaction Tubes, Erlenmeyer, Tweezers, Spatula, Lowenstein Jensen

Media, and all tools and materials (except dairy Probiotics) which will be used are

sterilized in the autoclave for 30 minutes by adjusting the pressure of 15 dyne/cm3

(1 atm) and at the temperatures of 1210 C dried then wrapped in paper.

2.3.2 Sample Preparation.

Createda variouslevelsofprobioticmilkconcentration ranging from0% (as negative

control), 20%, 40%, 60%, 80% and100% fromoriginallevels and Kanamisin

(Positive control).

2.3.3 Preparation ofinoculum

1osepure cultures ofMycobacteriumtuberculosisfrom thestockculturemedium

swiped inintoLowensteinJensen slant media. Thenkept the tubeinan incubator

withtemperature at370Cfor24hours. 5ml ofphysiological solution(saline) was

added into thetube, shakenuntilallculturesreleased

untilobtained3x108colonyformingunits (cfu). Measure the

Bacterialinoculumsolutionusingspectrophotometerat580nm wavelength.

2.3.4 Analytical method

Prepare2 pieces oftest tubes, each of them containing sterileLowenstein

Jensenmedia,7.5mlfor seed layerand12.5mlfor Base Layer. Pour

thebaselayermediaintosterilepetri dish, wait until thickness. Put

10μlMycobacteriatuberculosisinoculumintothe seedlayerat 40-450C, shake

itwithvortex. Thenpour itonthe surface ofthe baselayerin apetri dishuntil

homogeneous and become thickness. Makea holeinthemediawith

thepositionlikeFigure 1. Filleachholewith a50μlsample ofprobioticmilk, and

thenwrapit withpaper. Petri disheswere incubatedwithtemperature370Cfor24hours.

Observeandmeasure thediameter ofthe lightzoneis formedbyusinga millimeters

ruler.

Figure 1.

2.3.5 Statistical Analysis

Toanalyze thedata, used a statistic method likeCompletely Randomized

Design(CRD) and analyzed byAnalysis ofVariance(ANOVA) tofind

outwhetherthere isa differenceoreffectoneachsample.

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