Antituberculosis agents of Lactobacillus plantarum and Pediococcus acidilactici Lactic Acid Bacteria in Breast milk isolates against Mycobacterium tuberculosis

: Tuberculosis (TB) is one of the infectious diseases that have become a major problem in Indonesia. This disease is caused by the Mycobacterium tuberculosis bacteria. The bacteria are commonly treated with antibiotics. However, the use of irrelevant antibiotics is the most influential factor of antibiotic resistance. Therefore, natural ingredients are required as novel antibiotic agents, some of which are sourced from lactic acid bacteria. This study aims at investigating the antituberculosis activities of Pediococcus acidilactici and Lactobacillus plantarum isolated from breast milk against M. tuberculosis . Pediococcus acidilactici and Lactobacillus plantarum breast milk isolates were rejuvenated with MRS broth media, and the supernatant was then neutralized to pH 7.0 using a pH meter by adding 1N NaOH solution. Antituberculosis activity test was performed with Lowensten Jensen media to investigate the growth of M. tuberculosis . The results of this study showed that Pediococcus acidilactici had antituberculosis activity at 48 hours at concentrations of 80% and 90%, while the secondary metabolites of L. plantarum had antituberculosis activity at 24 hours and 48 hours at concentrations of 60%, 70%, 80%, 90%. Therefore, this study concludes that P. acidilactici and L. plantarum bacteria have the potential to be developed as antituberculosis agents.


INTRODUCTION
Indonesia is one of the most populous countries in the world, ranking fourth with a population of 273,523,615 people 1 . This large number requires the government to prosper its people because numerous problems are likely to arise, including political, social, economic, and health issues.
One of the prevalent health issues in Indonesia is tuberculosis (TB), an infection caused by Mycobacterium tuberculosis bacteria 2 , which can trigger symptoms in the respiratory tract. A person suffering from tuberculosis will experience symptoms of continuous cough, fever, and chest pain 3 . This disease can be transmitted through close contact with patients. Droplets resulted from coughing and sneezing are spread by the wind and then stick to other people. Rajni and Laxman 4 reported that this disease is one of the death-causing illnesses that are most difficult to control properly and effectively.
There have been many studies researching tuberculosis and Mycobacterium tuberculosis. Palomino and Anandi 5 reviewed the molecular basis and mechanism of drug resistance in Mycobacterium tuberculosis. Igarashi 6 also investigated the development of antituberculosis drugs derived from natural products. Furthermore, several related studies were conducted to obtain more effective drugs or methods for treating this disease, one of which is the potential of a secondary metabolite as an antimicrobial agent that can inhibit the growth of microbes 7 .
Secondary metabolites are antimicrobial peptides produced by bacteria that are effective to treat tuberculosis. These compounds serve as chemical defenses against microorganisms that are resistant to heat. In the pharmaceutical field, these have been widely developed in the food industry and have great potential as antimicrobials that can kill pathogens. Sharma et al 8 reported that secondary metabolites of Bacillus subtilis GAS101 can inhibit the growth of Staphylococcus epidermidis and Escherichia coli in broad ranges of temperature [30-121°C] and pH [2][3][4][5][6][7][8][9][10][11][12]. Aguilar-Perez et al 9 researched the ability of secondary metabolites that have antibacterial activity against M. tuberculosis.
Bacteriocins are produced by lactic acid bacteria (LAB). These bacteria are gram-positive, catalase-negative, and rod-shaped, and can perform carbohydrate fermentation 10 . LAB can be found or isolated from a variety of sources, including breast milk. Breast milk is known to contain various bacteria beneficial for babies, particularly those that aid in immune system development. Staphylococcus, Lactobacillus, Pseudomonas, Enterococcus, and other bacteria are frequently found in breast milk 11 , 12 . Pediococcus pentosaceus OZF is isolated from breast milk 13 .
Studies on the potential of L. plantarum and P. acidilactici bacteria as antituberculosis agents isolated from breast milk in inhibiting the growth of M. tuberculosis have not been carried out. Therefore, a study that investigates the potential of those bacteria as antimicrobials and their further development into antituberculosis agents that can inhibit the growth of M. tuberculosis bacteria is required.

Rejuvenation of Lactobacillus plantarum and Pediococcus acidilactici bacteria
L. plantarum and P. Acicilactici bacteria isolates were obtained from breast milk 14 . The isolates were then rejuvenated in the medium of MRS agar with 1% CaCO 3 and incubated at 35±2ºC for 2-3 days under anaerobic conditions. The clear zone formed around the bacterial colonies was suspected to be LAB.

Isolation of secondary metabolites from P. acidilactici and L. plantarum
P. acidilactici and L. plantarum were cultured in MRS broth. After being cultured on the medium, the bacteria were incubated for 24 hours and then centrifuged to obtain the content of secondary metabolites in LAB. The supernatant produced was saturated with ammonium sulfate to 80% under cold conditions, and the precipitate was filtered and dried. It was then dissolved with potassium phosphate buffer at pH 7 and secondary metabolites with neutral pH were obtained.

The anti-mycobacterium activity of P. acidilactici and L. plantarum
Antituberculosis activity test of secondary metabolites of P. acidilactici and L. plantarum isolates from breast milk against M. tuberculosis was performed using Lowenstein Jensen (LJ) media. Secondary metabolites of P. Acidilactici and L. Plantarum with concentrations of 90%, 80%, 70%, and 60% were incubated and then contacted with M. tuberculosis for 1 hour, 24 hours, and 48 hours.

RESULTS AND DISCUSSION
Based on the results of the study, isolated microbes of Lactobacillus plantarum and Pediococcus acidilactici were obtained. Lactobacillus plantarum are gram-positive, rod-shaped, convex, and entire, while Pediococcus acidilactici are gram-positive, catalase-negative, round-shaped, and non-motile. From the two isolates, secondary metabolites which had been cultured on MRS media were isolated. Further, the secondary metabolites were tested for antimicrobial activity against M. tuberculosis as pathogenic bacteria that cause tuberculosis (TB).
The antimicrobial activity test of secondary metabolites of L. plantarum and P. acidilactici showed different results based on the variations in the contact periods, including 1 hour, 24 hours and 48 hours, as presented in Table 1. These results indicate that the secondary metabolites of each isolate have different abilities in inhibiting the growth of M. tuberculosis.   Table 1 demonstrates that secondary metabolite of L. plantarum with contact period of 1 hour and concentrations of 60%, 70%, 80%, and 90% show positive results against M. tuberculosis. The results exemplify that secondary metabolites that are administered to M. tuberculosis are not effective to inhibit the growth of M. tuberculosis. With a one-hour initial contact period, the antimicrobial activity of secondary metabolites may not work optimally, allowing M. tuberculosis to continue actively growing and reproducing. Figure 1 shows that M. tuberculosis is developing rapidly and a large number of yellow bacterial colonies grow on the media.
Positive results were also shown by the secondary metabolite activity of P. acidilactici with one hour period and the same concentration variations of 60%, 70%, 80%, and 90% without any inhibitory activity against the growth of M. tuberculosis bacteria. Even at a concentration of 90%, the bacteria that cause tuberculosis could continue to grow and reproduce. Further, within the 24 hour incubation period, secondary metabolites of L. plantarum were effective in hindering the growth of M. tuberculosis even at a concentration of 60%. The study by Lin 15 also reported that secondary metabolites produced by L. plantarum showed effective antimicrobial activity against Vibrio parahaemolyticus. Moreover, Hasan et al 16 found the activity of secondary metabolites as pathogenic antimicrobials in the isolates from yogurt. However, different results were shown by secondary metabolites of P. acidilactici. With various concentrations (60%, 70%, 80% and 90%) and the same incubation period of 24 hours, the secondary metabolites did not demonstrate any inhibitory activity against M. tuberculosis, as summarized in Figure 1. The bacteria causing TB kept growing and reproducing well.
Furthermore, with an incubation period of 48 hours, the secondary metabolite of L. plantarum appeared to show better inhibitory (antimicrobial) activity, as proven by no more growth of M. tuberculosis on MRS media. In other words, the medium was free from M. tuberculosis. Secondary metabolites of P. acidilactici with an incubation period of 48 hours also demonstrated the ability to inhibit the growth of M. tuberculosis. However, the inhibitory activity only occurred at high concentrations (80% and 90%). Meanwhile, at the concentrations of 60% and 70%, P. acidilactici did not show any growth inhibition activity. Gaspar et al. (2018) reported that secondary metabolites can block the growth of urogenital pathogenic bacteria, such as Gardnerella vaginalis, Streptococcus agalactiae and Pseudomonas aeruginosa.

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Therefore, these results signify that secondary metabolites are effective in inhibiting the growth of M. tuberculosis pathogenic bacteria. In particular, secondary metabolites of L. plantarum isolated from breast milk have a better inhibiting ability than P. acidilactici. Thus, further research is required to investigate their potential as antituberculosis agents and identify the benefits.

CONCLUSION
Secondary metabolites of Lactobacillus plantarum and Pediococcus acidilactici can inhibit M. tuberculosis bacteria. The secondary metabolites of L. plantarum can inhibit the growth of M. tuberculosis during the 24 hour incubation period with concentrations of 60%-90%. Meanwhile, the secondary metabolites of P. acidilactici demonstrate inhibitory activity at 48 hours of incubation with concentrations of 80% and 90%, respectively. Thus, the secondary metabolites of L. plantarum have better antimicrobial ability than P. acidilactici. However, both secondary metabolites have the potential as pathogenic antimicrobials to kill M. tuberculosis.

AUTHORS' CONTRIBUTIONS
All authors contributed equally to this work.