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 prevention tuberculosis infection develops from every time. From the research, found that the Lactococcus lactis which is Lactic Acid Bacteria can produce a Bacteriocin Nisin and Lacticin that inhibit growth of Mycobacterium tuberculosis (James Carroll, Lorraine A. Draper, Paula M. O'Connor, Aidan Coffey, Colin Hill, R. Paul Ross , Paul D. Cotter, Jim O'Mahony. 2010. Comparison of the activities of the Lantibiotics Nisin and Lacticin 3147 against clinically significant Mycobacteria. International Journal of Antimicrobial Agents, Volume 36, Issue 2, August 2010, Pages 132-136). In other studies, Lactic Acid Bacteria such as Lactobacillus Rhamnosus and Bifidobacterium bifidum can improve immune system due to intracellular Mycobacterium tuberculosis infection (Darab

Ghadimi, Michael de Vrese, Knut J. Heller, Juergen Schrezenmeir. Lactic acid bacteria enhance autophagic ability of mononuclear phagocytes by increasing Th1 autophagypromoting cytokine (IFN-) and nitric oxide (NO) levels and reducing Th2 autophagyrestraining cytokines (IL-4 and IL-13) in response to Mycobacterium tuberculosis antigen. 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 the same 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 with HCI 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 Media 1. 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 121C 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 - 60C, avoiding air bubbles. f. Dispense the finished medium into sterile screw-cap test tubes. Place the tubes in a slanted position and heat at 85C 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 incubated at 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. Created a various levels of probiotic milk concentration ranging from 0% (as negative control), 20%, 40%, 60%, 80% and 100% from original levels and Kanamisin (Positive control). 2.3.3 Preparation of inoculum 1 ose pure cultures of Mycobacterium tuberculosis from the stock culture medium swiped in into Lowenstein Jensen slant media. Then kept the tube in an incubator with temperature at 370C for 24 hours. 5 ml of physiological solution (saline) was added into the tube, shaken until all cultures released until obtained 3x108 colony

forming

units

(cfu).

Measure

the

Bacterial

inoculum

solution

using

spectrophotometer at 580nm wavelength. 2.3.4 Analytical method Prepare 2 pieces of test tubes, each of them containing sterile Lowenstein Jensen media, 7.5 ml for seed layer and 12.5 ml for Base Layer. Pour the base layer media into sterile petri dish, wait until thickness. Put 10l Mycobacteria tuberculosis inoculum into the seed layer at 40-450C, shake it with vortex. Then pour it on the surface of the base layer in a petri dish until homogeneous and become thickness. Make a hole in the media with the position like Figure 1. Fill each hole with a 50l sample of probiotic milk, and then wrap it with paper. Petri dishes were incubated with temperature 370C for 24 hours. Observe and measure the diameter of the light zone is formed by using a millimeters ruler.

Figure 1. 2.3.5 Statistical Analysis To analyze the data, used a statistic method like Completely Randomized Design (CRD) and analyzed by Analysis of Variance (ANOVA) to find out whether there is a difference or effect on each sample.