Antimicrobial and Antioxidant Activity of Endophyte Bacteria Associated with Curcuma Longa Rhizome

Most cases of bacterial resistance towards antibiotics, encourage various efforts to gain new sources of antibiotics. Endophyte bacteria is a microorganism has important role as the producer of bioactive compounds. Endophyte bacteria from Curcuma longa with antimicrobial and antioxidant activities have not been studied yet. Curcuma longa has been utilized as the main ingredients of traditional herbal medicines (jamu). The objective of this research was to investigate the antimicrobial and antioxidant activity of endophyte bacteria associated with Curcuma longa rhizome. Based on morphological characteristics of bacterial colonies, eight endophyte bacteria was isolated from Curcuma longa rhizome. Screening of endophyte isolate has antimicrobial activity was done using agar well diffusion method. The culture supernatant of each endophyte isolate was dropped on agar well against pathogenic bacteria Salmonella enterica ser. Typhi, Staphylococcus aureus and yeast Candida albicans. Three endophyte isolates K3, K2 and M1b showed antimicrobial activity against pathogenic bacteria and yeast. Isolate K3 showed strong antimicrobial activity against C. albicans and S. aureus, however, isolate K2 and isolate M1b showed antimicrobial activity against Salmonella enterica ser. Typhi and S. aureus, respectively. Those endophyte bacteria also had antioxidant activity shown by scavenging ability toward DPPH radical with consecutive percentage of isolate K3 (72.3 %), K2 (51.3 %) and M1b (64.6 %). Isolate K3 showed the highest antimicrobial and antioxidant activity. Based on biochemical characteristics using Microbact 24E kit, isolate K3 was identified as Paenibacilus alvei and isolate K2 as Enterobacter agglomerans.


INTRODUCTION 
Recent main health care issues include the rise of antibiotic resistances and the rise of chronic and degenerative disease in countries throughout the world regardless of income level. The rise of antimicrobial resistance need the discovery and/or production of novel antimicrobial. Antioxidants, that have capability scavenging free radicals, are known to play important roles in preventing the degenerative, ROS-linked diseases. As the human population growth and the increase awareness on healthy life, people prefer natural compounds. Thus, the exploration of novel source of natural bioactive compound is unavoidable. One of the most promising source of natural bioactive compound is endophyte [1].
Endophytes are microorganisms, often bacteria, actinomycetes or fungi that live in healthy plant tissue intercellularly and/or intracellularly without causing any apparent symptoms of disease. Endophyte bacteria are found in roots, tubers, rhizome, nodule, stems, leaves, flowers, ovules, seeds and fruits of various plant species. In general roots have greater numbers of endophytes than above ground tissues [2]. Many evaluations of bacterial endophytes have shown that they are widespread in numerous plant kingdom. A single plant may have several different endophyte bacteria. The structure of bacterial endophyte communitas are varied, dynamic overtime, and attributed to plant source, plant age, tissue type, time of sampling, season and environment [3].
The endophyte bacteria beneficial to its host by promote plant growth and yield, suppress pathogens, help plants to tolerate biotic stress or abiotic stresses, help to remove contaminants, solubilize phosphate, or contribute in fixing nitrogen. Endophytes bacteria are also known for the production of various classes of natural products and have been reported to exhibit a broad range of biological activity. It has reported over two thousands natural products have been isolated from endophytes associated with medicinal plants, including alkaloids, flavonoids, glycosides, phenolic acid, xanthones, steroids, terpenes, tetralones, coumarins, quinones, lactone, polysaccharide, peptides. Such bioactive metabolites are found to apply as agrochemicals like insecticidal, growth-promoting, and their potential in the pharmaceutical like antibiotics, antioxidants, antitumor, antidiabetics, antiparasitics, antithrombotic, anticancer and immunosuppressants agents [4].
Medicinal plant is well known as source of precious bioactive compound. Endophytes that have long time associate with medicinal plant may participate in metaboloc pathway or gain some genetic information to produce specific bioactive compound similar to the host plant. Plant that have ethnobotanical history should be sourced of endophyte microbe. Therefore, it is needed and important to study and explore medicinal plants and endophyte microbes that live in [4]. Curcuma longa's rhizome, commonly called as turmeric has been widely used as a spice and has a long history of medicinal use in the treatment of a variety of human diseases especially in Asia regions. This study was aimed to analyze the antimicrobial and antioxidant activity of endophyte bacteria associated with turmeric rhizome.

MATERIALS AND METHODS Study Area
Sample of turmeric rhizomes were collected from Mondo Village, Mojo District, Kediri Regency. The soil type in the research site is aluvial, along the area of Brantas Watershed. Soil acidity in the plant site is 6.34. Optimum pH of soil for most plants range 5.5 -7.0 and nutrient will adsorbed well in the range pH 5.5 -6.5 [5]. Refer to the soil pH at the sample site, it has qualified for the plant to grow well. Healthy ten months old plants were selected as source of rhizome for endophyte bacteria isolation.

Surface Sterilization of Turmeric rhizome
Rhizomes of turmeric were washed with running tap water. The procedure includes sequential immersion of rhizomes parts in 70% ethanol for 3 minutes, sodium hypochlorite 2% for 5 minutes and 70% ethanol for 30 seconds, then rhizomes was washed using sterilized distilled water for five times [6]. The last twice washing solutions were plated on Nutrient Agar (NA) to confirm the effectiveness of sterilization treatments. The surface of turmeric rhizomes were pilled out using aseptic technique and the inner tissues of rhizomes were macerated using a sterile mortar and pestle [7].

Isolation of Endophyte Bacteria from Curcuma longa Rhizome [8,9]
Total of 10 g turmeric rhizomes were extracted then performed a serial dilution in saline solution (0.85% NaCl) and plated out in Nutrient Agar (NA) to recover endophyte bacteria present in the rhizome. All the plates were incubated at 28-30°C (room temperature) for 48 hours. The isolated bacteria were preliminary characterized according to their morphological characteristics. The distinct colony types were picked up from Nutrient Agar (NA) plates and were purified through three rounds of streaking and single colony was selected an refresh in the same medium.

Test Microorganisms
Pathogenic strain yeast of Candida albicans, Gram-positive bacteria Staphylococcus aureus and Gram-negative Salmonella enterica ser. Typhi clinical isolates were used as test microorganism in this study. All pathogenic strains were obtained from Department of Microbiology, Medical Faculty, University of Brawijaya. After 18-24 hours of incubation at 37⁰C (for bacterial strains) in NA and 30⁰C (for yeast strain) in PDA, a loopful of each test strains was suspended in sterile distilled water water until obtained 1 x 10 6 cfu.mL -1 for bacteria and 10 5 cfu.mL -1 for yeast.

Assays of Antimicrobial Activity
Isolated endophyte bacteria from turmeric rhizomes were cultured in 5 mL Nutrient Broth (NB) medium at room temperature (28-30⁰C) for five days. After five days, culture medium was centrifuged at 4000 rpm for 15 minutes and supernatant was screened for antimicrobial activity by agar-well diffusion technique on NA media that was previously seeded with test pathogens. Supernatant (50μL) was added into wells (7 mm) formed by cork borer on the NA medium [10]. Sterile NB was set as control. As a positive control for antimicrobial activity towards test microorganism, we used amoxicillin antibiotic dose 10 µg.mL -1 [11] for S. enterica ser. Typhi and dose 25 µg.mL -1 [12] for S. aureus. While for the antimicrobial activity on yeast, we used anti fungal nystatin 12 µg.mL -1 as positive control [13]. The plates were incubated in suitable temperatures for 24-48 hours; the zone of inhibition was measured and recorded.

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Antimicrobial and Antioxidant Activity of Endophyte Bacteria with C. longa Rhizome (Sulistiyani et al.)

Assay of Antioxidant Activity by Scavenging DPPH Free Radical
Endophyte bacteria culture (in NB medium) were centrifuged at 4000 rpm for 15 minutes, 4⁰C and then the supernatants were assayed their antioxidant activity by scavenging DPPH free radical methode [14] described with any modification. The supernatant (0.5 mL) was added to 3 mL of 0.1 mM DPPH in methanol solution. Methanol 1.5 mL was then added thus the final volume of solution was 5 mL. For control, supernatant of each sample was replaced by steril Nutrient Broth (NB). Methanol was used as blank. Discoloration of DPPH radical solution was measured at 517 nm in triplicate after incubation in the dark for 5 hours. Ascorbic acid was used as the positive control. Percentage of scavenged DPPH radical was calculated using following formula A 0 is the absorbance of control and A 1 is the absorbance of sample (supernatant of endophyte bacteria culture) or standard. Ascorbic acid was taken at various concentrations as a known antioxidant for comparative analysis. Then the percentage of scavenging were plotted against respective concentrations used, and from the graph, EC 50 was calculated.

Statistical analysis
The experimental results of biological activity tests were expressed as mean ± standard deviation (SD) of three replicates. The results were processed using Microsoft Excel 2007 and SPSS software. The data of antimicrobial activity assay results was analyzed using Kruskal-Wallis test followed by t-test and Tukey test whereas antioxidant activity using Anova following Tukey test.

RESULTS AND DISCUSSION
Based on the morphology characteristics of colony, we obtained eight isolates K 1 , K 2 , K 3 , K 4 , M 1a , M 1b , M 5 and M 6 of endhopytes bacteria. Each pured isolate was tested further for the antimicrobial and antioxidants activities.

Antimicrobial Activity
Three of eight isolates of turmeric endophyte bacteria has inhibition activity towards pathogenic test microorganism. Isolate K 3 inhibit the pathogenic yeast C. albicans (Fig. 1a) and pathogenic bacteria S. aureus (Fig. 1b). Otherwise, isolate K 2 inhibit the pathogenic bacteria Salmonella enterica ser. Typhi (Fig. 1c) and isolate M 1b inhibit S. aureus (Fig. 1d). The results showed that the inhibition zone of isolate K 3 towards C. albicans was greater than antifungal nystatin (12 µg.mL -1 ). It also showed similar results for inhibition zone of isolate K 3 to S. aureus, which is greater than amoxicilin (25 µg.mL -1 ) and isolate K2 to Salmonella enterica ser. Typhi than amoxicilin (10 µg.mL -1 ). Otherwise, the inhibition zone of isolate M 1b to S. aureus was relatively similar to the inhibition zone of amoxicillin (25 µg.mL -1 ) (Fig 2).
Microbes produce any substance for defense systems or survival mechanism. These include antibiotics, bacteriocins, metabolic by-products, lytic agents, numerous types of protein exotoxins, and short chain fatty acid [15]. This study found that Isolate K 3 and M 1b (Gram positive bacteria ) showed inhibition of growth towards the pathogenic bacteria S. aureus as Gram-positive bacteria and showed no inhibition towards Gram-negative bacteria S. enterica ser. Typhi. In contrast, isolate K 2 showed inhibition to Gram-negative bacteria S. enterica ser. Typhi and showed no inhibition to the Gram-positive bacteria S. aureus and yeast C. albicans. These properties are similar or corresponding to the nature of bacteriocins that they have a relatively narrow killing spectrum and are toxic only to bacteria closely related to the producing strain. But further test is needed to confirm that the

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Antimicrobial and Antioxidant Activity of Endophyte Bacteria with C. longa Rhizome (Sulistiyani et al.) substance is bacteriocin. In addition more than 99% of bacteria can produce at least one bacteriocin and within a species tens or even hundreds of different kinds of bacteriocins are present [15].

Antioxidant Activity
The test of antioxidant activity on the eight isolates of endophyte bacteria showed that all isolates has the ability of scavenging to DPPH radical (Fig. 3). Three isolates with the highest antioxidant activity are isolate K 3, K 2 , and M 1b . Bacterial growth curve were made for the three isolates to obtain optimum time for sampling to test the antioxidant activity.
Antioxidant compound produced by the endophyte bacteria, as reported by scholar, is consisted of various substances. Antioxidant substances produced by endophyte bacteria are EPS [16], surfactin [17], L-asparaginase [18], carotenoid pigment [19], and several enzymes [20]. Most of the compounds were produced maximally at the end of exponential phase. Thus the sample for antioxidant activity was collected at the 14 th hour (the end of exponential phase).
Before the test of antioxidant activity, OD of liquid culture of endophyte bacteria was equated. The test of antioxidant activity showed that isolate K 3 has the highest ability of antioxidant activity compared to the other two isolates (Table 1) and then the EC 50 of K 3 isolate was determined.
Efficient Concentration or EC 50 value is defined as the concentration of substrate that causes 50% loss of the DPPH activity (colour) [21]. Isolate K 3 supernatant had EC 50 value 70.26 µL.mL -1 and vitamin C (as standard) had EC 50 value 3.71 µg.mL -1 . In this study isolate K 3 supernatant was still in original liquid and had not evaporated yet or extracted in to concentrate, so it was intelligible that the EC 50 value was too lower than vitamin C. For next study may be required further processing of the supernatant.

Species Identification
Isolates K 3 and K 2 had both antimicrobial and antioxidant activity significantly , therefore need to characterize these isolates furthermore. Isolate K 3 and K 2 was characterized based on the biochemical characteristic using Kit Microbact System 24E. Biochemical characteristics of isolate K 2 were analyzed by the software Microbact System 24E and isolate K 3 were analyzed refer to Identification flow chart from Microbiology Laboratorium, The University of Ottawa Canada base on Bergey's Manual of Determinative Bacteriology [22]. The results of characterization showed that isolate K 2 was assumed as Enterobacter agglomerans with 99.9% similarity, whereas isolate K 3 is Bacillus alvei or Paenibacillus alvei with 91.2% similarity.
Paenibacillus alvei are rod-shaped, Grampositive, motile, spore-forming , catalase-positive bacteria and grow on simple media (NA/NB). Paenibacillus alvei are common found in honeybee colonies , soil , milk, mosquito larvae, the wax mot, humans and very rarely pathogenic for vertebrates. It has reported that Paenibacillus alvei produce antimicrobial substance: paenibacillin P and paenibacillin N [23], peptide AN5-1 [24], cyclic lipopeptides [25], depsipeptide [26]. Some of the antimicrobial substance show active againts pathogen S. aures and C. albicans [23,24,26,27] and consistent with this findings, this study showed endophyte bacteria from Curcuma longa's rhizome, isolate K 3 that assumed as Paenibacillus alvei show antimicrobial activity to S. aureus and C. albicans. Isolate K 3 also show antioxidant activity, it promote previous research that Paenibacillus alvei's metabolite have antioxidant activity Exopolysaccarides (EPS) [16,28,29]. Enterobacter agglomerans are rod shape, Gram negative, motile, non-sporforming bacteria. These bacteria first were isolated from plants, vegetable, fruits, seeds, and they are also commonly found in the ecological niches such as water, soil, sewage, feculent material, foodstuffs, clinical specimens [30]. It has reported that Enterobacter agglomerans produce antimicrobial substance that have inhibition growth to any pathogen bacteria and fungi. Some of them have inhibition growth to Salmonella sp. like herbicolin O [31], phenazine [32], and consistent with these finding, isolate K 2 that assumed as Enterobacter agglomerans has antimicrobial activity to Salmonella enterica ser.Typhi. Isolate K 2 also has antioxidant activity, it promote previous research that Enterobacter agglomerans has free radicalsscavenging ability [33].

CONCLUSION
The study obtained eigth isolates of endophyte bacteria from the Curcuma longa rhizomes. Three isolates of endophyte bacteria have antimicrobial activity, i.e. isolate K 3 to C. albicans yeast and S. aureus bacteria; isolate K 2 to S. enterica ser. Typhi, and isolate M 1b to S. aureus. All isolates of endophyte bacteria from Curcuma longa rhizomes has the antioxidant activity. The highest antioxidant and strong antimicrobial to Gram positive pathogenic bacteria activity was showed by isolate K 3 which identified as Paenibacillus alvei. The strong antimicrobial activity to Gram negative pathogenic bacteria and had high relative antioxidant activity was showed by Isolate K 2 which identified as Enterobacter aglomerans by biochemical characterization.