An In vitro Approach for Evaluating Antimicrobial Activity and Production of Kojic Acid by Aspergillus flavus Isolated from Karwar Region

Biosynthesis of kojic acid by fungi has been acquired huge attention nowadays because it can be used as an alternative method to chemically synthesized compounds. The fundamental feature of the microbiological agitation practice was to obtain potential fungi to achieve greater amount of kojic acid, the fungal isolate used in this study exhibited maximum growth on PDA and was identified as A.flavus based on its microscopic and macroscopic characteristics and ITS sequence analysis. In this vignette, appropriate cultivation conditions were carried out to gain a highest quantity of kojic acid. The maximum yield of kojic acid (31g/l) was reached by formulating the medium. The massive amount of kojic acid and maximum biomass was achieved from A. flavus by utilizing yeast extract as a nitrogen source and carbon source as glucose. The optimum incubation temperature employed was at 30oC, optimal pH was 3. Antagonistic activity was analyzed against both bacterial and fungal pathogens. The ethyl acetate extract exhibit increased antibacterial activity and maximum antifungal activity comparable with methanol, and chloroform.


INTRODUCTION
Throughout evolution, an attempt was made to include marine microbes into the area of the pharmaceutical industry to discover new drugs 1 . Fungal isolates obtained from marine habitats have been identified as renowned producers of unique bioactive compounds 2 . Various Species of the genera Aspergillus have known repositories of antimicrobial substances with the highest efficiency 3 . The global health care system is facing the problem of multi-resistant microbes [4][5][6] . Most of the bacterial species exhibit antagonistic properties towards various antibiotics. In recent years, the majority of bacterial isolates such as S. aureus are exhibiting resistance mechanisms against ciprofloxacin, erythromycin, clindamycin 7-10 , Escherichia coli, Pseudomonas spp. and Klebsiella spp were involved in drugresistant bacterial septicemia in Ile-Ife, Nigeria 10 .
In the living cells, the chemical synthesis of kojic acid develops free radicals 11 . To overcome this consequence, several attempts have been made to choose a different approach to produce kojic acid. Recent investigations have concentrated on the isolation and usage of kojic acid from microorganisms as another non-toxic and secure method. In industry, biosynthesis of kojic acid from Aspergillus species is treated as one of the finest approaches 12 . More than 58 various species like Aspergillus, Penicillium, Mucor, etc, are used for Production of kojic acid 13 .
As far as the advancement of the potentiality of fungi to produce a considerable number of biologically active secondary metabolites that are involved in enhancement of ecological strength, and most of them perform the significant role as agents in defense mechanism, advancing regulators, insect attractants and as an agent for communicating with other organisms 14 . These pharmacologically active substances exhibit various properties such as antimicrobial, antitumor, antihyper-cholesterolemic, and Immunosuppressant [15][16][17][18][19] .
Additionally endophytic fungi are also renowned for the synthesis of similar bioactive metabolites from plants like Catharanthus alkaloids that are produced by endophytic fungi such as campthothecin, vinblastine, vincristine and, paclitaxel these are potent drugs for cancer 20 , In such a way endophytic fungi Aspergillus flavus influence prominent role in finding novel bioactive metabolites and they require very small quantity of substance from plants. In some tropical areas, maximum exposure of skin to sunlight can result in an elevated risk that leads to skin cancer, oxidative stress, and sunburn 21 . To prevent the hazardous cause of ultraviolet rays, kojic acid isolated from fungi has been used in many cosmetic creams, lotions, and soaps because they prevent hyperpigmentation and tyrosinase inactivity, it is also used to avoid food browning 22 .
Hence kojic acid and its derivatives have developed huge applications in medicine, Pharmaceutical industries, agricultural, chemical and food industries 22 . Along with kojic acid, its derivatives are also found to enhance solidity and solubilizing nature of kojic acid in various oily creams in the cosmetic industry [23][24][25][26] . Further other derivatives of kojic acid can have the capacity to inhibit tyrosinase activity and it has 8 times deeper potentiality than kojic acid 24 . Some earlier reports have described as flufuran (5-(hydroxymethyl)-furan-3-carboxylic acid) is an isomer of kojic acid but recent studies indicates that flufuran was misidentified 27 .
Kojic acid (5-hydroxy-2-hydroxymethylgamma-pyrone; KA) is synthesized from different microbes like fungi and some bacteria 28,29 . In the group of Aspergillus species, the oryzaeflavus-tamarii category is considered as the highest producer of kojic acid 30 . It is an essential secondary metabolite mainly obtained from carbohydrates. Kojic acid has wide applications in the medical field as an anti-inflammatory drug, it has been used as a pain killer, in the food industry it is utilized as an anti-speck, as a flavor enhancer, and in traditional foods, it gives special taste and color 31,73 . The purpose of usage of kojic acid in the agriculture field is that it acts as an anti-melanosis of agriculture products by preventing polyphenol oxidase formation 32 . Kojic acid exhibits bio-pesticide and bio-fungicidal activities in agriculture. In the agriculture field, it has been preferred for its biodegradable activity. It has various biological activities such as antibacterial, antifungal, antioxidant. It is also used as a whitening agent in the cosmetic industry 33 . Various microorganisms produce kojic acid as azidometalkojates form that exhibits antimicrobial activity. Besides, it acts as a chelating agent in insecticides production 34,35 .
Different carbon, nitrogen sources, pH and temperature can be employed for an adequate amount of kojic acid production. Until now, an ample amount of kojic acid has been obtained with the glucose that was used as a carbon source. Most of the Aspergillus and Penicillium species are potent producers of Kojic acid 36 . Among Aspergillus species, Aspergillus flavus being attentive as the maximum producer of Kojic acid. Therefore the present survey was carried out to isolate and explore the antagonistic activity of biologically active compounds obtained from Aspergillus flavus and the results achieved were used to progress the control cultural strategy to produce maximum kojic acid.

MATERIALS AND METHODS Obtaining the fungi and its extracts
Soil samples were gathered from different regions in and around the coastal area of Karwar, Karnataka, India, during winter and summer seasons. Each sample was placed in sterilized tightly sealed polythene bags and brought to the laboratory for further investigation. Fungal isolates were separated by employing a serial dilution method on PDA medium supplemented with chloramphenicol (50ppm) to retard the growth of bacteria, incubated at 28°C at pH 6, for seven days, isolated colonies were grown separately.
Isolate that showed better growth was identified by using slide culture technique and lactophenol cotton blue staining technique and it was identified based on their morphological characteristics. Molecular characterization was carried out. Chromosomal DNA was extracted by using a spin column kit (Hi-Media, India or similar manufacturers. Fungal ITSrRNA gene (600 bp) 37 amplification was carried out by using a PCR method in a thermal cycler and were purified using Exonuclease I -Shrimp Alkaline Phosphatase (Exo-SAP) 38 . Sanger method in ABI 3500xl genetic analyzer (Life Technologies, USA) was used to sequence purified amplicons. Sequencing files (.ab1) rearranged using CHROMASLITE (version 1.5) and further evaluated by Basic Local Alignment Search Tool (BLAST) with adjacent culture sequence recovered from the National Centre for Biotechnology Information (NCBI) database that locates a range of local analogy among sequences 39 . The program correlates protein or nucleotide sequences to database sequences and determines the statistical implication of similarities 40 . The BLAST algorithms deployed to interpret practical and progressive correlation among the sequences and it also helps to relate members of gene families. (i) Fundamental examination to search probably closely linked type strain sequences using the BLASTN program22 (ii) Pairwise arrangement to enumerate the sequence analogy values within the query sequence and the sequences identified in step (i) 41 . Therefore, each isolate is reported with the first five-ten hits observed in the said database. And the strain was identified and confirmed as Aspergillus flavus. Therefore further studies were conducted by employing A.flavus for the extraction of bioactive compounds.

Production of kojic acid
The sterile 70ml medium in 250 ml Erlenmeyer flask employed for kojic acid production from 1ml of stock solution of A. flavus, (1 x 108 spore per ml) consisting of glucose (100 g/l); NaNO 3 (2 g/l); KH 2 PO 4 (1 g/l); MgSO 4 ·7H 2 O (0.5 g/l); KCl (0.5 g/l); and FeSO 4 ·7H 2 O (0.01 g/l). The culture was incubated at 30°C, 150rpm in incubator shaker for 30 days. Mycelium was separated by filtering through layers of sterile muslin cloth and then centrifuged at 6000rpm for 15min. Later Filtrate was transferred to the separating funnel containing ethyl acetate, chloroform, methanol (1:1 ratio) separately, aqueous and organic layers were separated followed by evaporation in a rotary evaporator. Kojic acid crystals were obtained by evaporation, these crystals were weighed and then placed in a clean vial 42 . Kojic acid concentration was determined against blank (the filtrate collected from the medium that is not inoculated) all the observations were used in triplicates.

Analytical determination of kojic acid and glucose
The analytical determination of glucose concentration was carried out by the DNS method 43 . Dinitrosalicyclic acid (DNS) reagent consisting of 3,5-dinitrosalicylic acid (10 g/l), phenol (2g/l), sodium sulfite (5 g/l) and NaOH (10g/l) was developed and 1ml of DNS reagent was added to 1ml of supernatant, boiled for 10min in boiling water bath, then it was cooled Journal of Pure and Applied Microbiology and 1ml of Rochelle salt was supplemented. The spectrophotometer was used to measure the absorbance at 575 nm.
The determination of the quantity of kojic acid was performed by a colorimetric method 44 . The experiment was conducted by mixing 1ml of supernatant with 1ml of ferric chloride (FeCl 3 ) solution, which was prepared by placing FeCl 3 .6H 2 O (1gm) in 100ml of HCl (0.1N). The reddish color was formed due to the reaction between hydroxyl and phenolic functional groups present in the samples. The absorbance of the sample was estimated at 500nm by employing a spectrophotometer and used the kojic acid standard curve to determine the kojic acid equivalent.

Screening of antimicrobial activity
The extract obtained from the isolate was bioassayed for antibacterial and antifungal activity, this was analyzed by agar-based disk diffusion method 45 , against two gram-negative and two gram-positive bacteria and one fungal isolate. 0.2 ml of an overnight grown culture of target bacteria were inoculated on Nutrient agar medium, in similar fashion 0.2 ml of cultured target fungal isolate was on sabouraud's agar medium. By using sterile spreader the two cultures were evenly spread out. A constant amount of 20µl of the extract of the isolate was applied on the sterile Whatman antibiotic assay discs of 6 mm diameter later located over the surface of the agar medium. Bacterial cultures were incubated at 37°C for 24h and fungal plates at 25°C for 72h. Diameters of inhibition zones were recorded. The analysis was achieved in duplicate.

Estimation of the dry weight of fungi
To determine the dry weight of the isolate the culture broth was drained through the sterile filter paper (Whatman 1). The mycelium was washed. The weight of the empty filter paper was measured, later weight was measured along with the fungal mycelium and then filter paper along with mycelium was placed in the oven at 95°C until the stable weight was reached, subsequently fungal dry biomass was measured 46 . Analyzing the effect of nutritional conditions on kojic acid production Different aspects were employed for the increased production of kojic acid. These aspects comprised:

The response of carbon source
Different kinds of carbon sources such as glucose, fructose, maltose, lactose, sucrose, starch, cellulose, xylose, arabinose were employed in the final concentration of carbon remained same (100g/l) in the medium, pH of the cultures were adjusted, were sterilized and incubated for 13 days in order to determine the prominence of carbon on kojic acid biosynthesis and increase of fungal biomass by measuring dry weight of mycelia. Analysis of kojic acid and the level of glucose was performed by colorimetric method 44 . A. flavus utilize glucose as the prime source to manufacture a maximum quantity of kojic acid 47 . Glucose that acquired during the agitation process converts into kojic acid by the action of various enzymes associated with cell 48,49 such as hexokinase and gluconate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose is considered as the finest precursor for the maximum kojic acid synthesis.

The response of nitrogen source
The response of prominent nitrogen on kojic acid formation as well as increased biomass was determined by using different nitrogen sources such as ammonium nitrate, ammonium dihydrogen phosphate, ammonium mono hydrogen phosphate, yeast extract, asparagine, sodium nitrate, potassium nitrate at 0.2% w/v concentration was used.

The response of incubation temperature
Temperature plays a vitalizing role in the greatest recovery of kojic acid and the maximum yield of biomass. Different ranges of temperature 15, 20, 25, 30, 35, 40, 45°C were studied to understand the effect of temperature.

The Response of pH
The fermentation medium was adjusted to various pH ranging from 2, 3, 4, 5, 6, 7, 8 to examine the consequence of an action of pH on the improvement of kojic acid biosynthesis and dry mass.

RESULTS AND DISCUSSION Microscopic observation and molecular characterization
Macroscopic, microscopic study ( Fig.  1 and 2) and molecular identification revealed that the fungal isolate was belonging to the genus Aspergillus and it is confirmed that isolate was A. flavus. The Genbank Accession number is MK521427.

Recovery of kojic acid and the estimation of biomass
The A.flavus was isolated for its potentiality to manufacture the highest kojic acid first on the PDA medium and then it was cultured on the production medium [glucose (100 g/l); NaNO 3 (2 g/l); KH 2 PO 4 (1 g/l); MgSO 4 ·7H 2 O (0.5 g/l); KCl (0.5 g/l); and FeSO 4 ·7H 2 O (0.01 g/l).] at 28±2°C. Kojic acid was extracted with ethyl acetate, chloroform and methanol (1:1 ratio), aqueous and organic layers were separated, evaporated in a rotary evaporator to acquire maximum 24.1g/l of kojic acid and increased dry weight 8.2 g/l with ethyl acetate extract, while kojic acid and dry mass from methanol extract was 20.4g/l and 7g/l, and from chloroform extract 14.3g/l and 6.5g/l was achieved. Hence ample amount of kojic acid and dry mass were obtained with ethyl acetate extract as the fermentation process progressed up to 13days. Such type of performance of kojic acid biosynthesis and dry mass by A.flavus was reported [50][51][52][53][54][55][56] .
Previous studies described that several species and strains of Aspergillus genus such as A. flavus, A. oryzae and A. parasiticus [57][58][59][60][61][62][63][64][65][66] are potent producers of kojic acid. Some earlier results showed that A. flavus strain 44-1 acquire 32.50 g/l of kojic acid by employing rotary shaker 66 . Our results were comparable with reporter 67 who demonstrated that an increased amount of kojic acid 39.9g/l was obtained by A. flavus strain 44-1 by applying SMF. Contrarily, a minimum level of kojic acid production 20 g/l by A. oryzae NRRL 484 in shake flask culture was reported 68 . However, the kojic acid biosynthesis by A. oryzae NRRL 484 was recorded 28 as 24 g/l. After the recovery of kojic acid, it was estimated quantitatively by using the standard method 44 . The highest amount of kojic acid 24.1 g/l was attained by ethyl acetate extract   (Table 1). Antifungal activity by the isolate was conducted against C. albicans that was comparable with fluconazole as a standard antibiotic ( Table 2). The diameter of the zone of inhibition was measured and was taken as a sensitivity test of bacteria and fungi.

Optimization conditions on kojic acid production by A. flavus
The kojic acid production by A. flavus was achieved by optimizing cultural conditions like carbon source, nitrogen source, incubation temperature and pH of the medium. To test the effect of carbon sources on the formulation of kojic acid and mycelia, different carbon sources were supplemented separately to the medium in constant concentration (100g/l). The outcome of the present work showed in (Table 5) indicates that the perfect carbon source for synthesis of kojic acid with dry mass was glucose (30.1g/l) and (8.2g/l) subsequently sucrose, fructose, lactose (28.3, 25.2, 18.4 g/l respectively) (8, 7.6, 7.5g/l respectively). According to some workers 49,70,59 , the structure of glucose is almost identical to kojic acid, during the  The zone of inhibition of each extracts were expressed as the mean ± standard deviation. Ca-Candida albicans, Fc-Fluconazole fermentation process, direct conversion of glucose to kojic acid without the formation of smaller fragments will occur. Among the groups of carbon sources glucose, fructose, sucrose, and lactose exhibit the maximum amount of kojic acid and dry mass by a mold A.flavus when compared to maltose, starch, cellulose, xylose, and arabinose.
In agreement with this report various kojic acid synthesizing strains utilize glucose, sucrose, and fructose as a carbon source to obtain 0.5 to 0.6 g of kojic acid per g of glucose 70 . Hence glucose is considered as both precursors for kojic acid biosynthesis and mycelial growth 69,70 .
Among the various factors regulating the biosynthesis of kojic acid, the optimal source of nitrogen is a major component. The effect of nitrogen displayed in (Table 6) indicates that yeast extract, ammonium nitrate, and ammonium dihydrogen phosphate were the prominent nitrogen sources for manufacturing kojic acid (30.4, 24.3, 15.6 g/l respectively) with the consumption of glucose (81,73, 68 g/l respectively) and the dry biomass were (8.5,5.1,4.7 g/l respectively). The increased amount was obtained when yeast extract was used as a source of nitrogen with consumed glucose (81g/l). Our results were parallel with reporters who explained that the immobilized cell cultures exhibit consumption of glucose and the progression of kojic acid synthesis was maximum and are proportional with the rise in nitrogen concentration but at increased concentration, production of kojic acid was reduced and consumption of glucose was highest 71 .
Temperature plays a leading role in mycelial growth and kojic acid synthesis by potential A.flavus was appeared at 30°C (30.1g/l) and dry biomass weight reached (8.2g/l). Some investigators showed that 25-30°C was the optimal temperature for the manufacturing of kojic acid by various fungi. 71 Various ranges of pH were employed to achieve the greatest kojic acid and the highest level of mycelial growth by A.flavus. The potential fungi A.flavus showed better growth at pH 3 to 5 (7.5, 8.4, 7.1 g/l) on the contrary at pH 6 to 8 the growth was completely reduced (5.2, 2, 1 g/l) while, biosynthesis of increased quantity of kojic acid (31.5 g/l) was obtained with maximum consumption of glucose (86 g/l ) at pH 3 however the amount of kojic acid was gradually decreased at pH s 4, 5, 6 and pH 7 and 8 production was entirely nil as displayed in (Table 8). Our results are coinciding with the results 72,28 reported that the strains, A. flavus, A. parasiticus, and A. oryzae exhibit optimum pH values 4.5, 6.2, 6.5 for the secretion of kojic acid. Comparably, A. oryzae produces kojic acid at pH 4 73 , in contrast, the kojic acid values appeared in our investigation were not consistent with other reporters, in which the largest amount of kojic acid was obtained from glucose at very acidic pH (1.9 to 3.0) 74 . During growth progress at pH 4.5 to 6, the fungi use enzymes to convert glucose into kojic acid 36 . However, the growth of the fungi was continuous at pH between 2 and 3 the production of kojic acid appeared 75 . The change in the outcome of our vignette with the values of earlier results can  Apart from Kojic acid various fungal isolates produce some other organic acids, according to some investigators, organic acids such as Malic acid (50.5M) from (Aspergillus flavus) 77 , Itaconic acid (87.32g/l )from (Aspergillus terreus) 78 , Cyclopiazonic acid from (Aspergillus tamarii) 79 and (Aspergillus flavus) 80 are obtained from several species of Aspergillus.
Besides, most of the plants are also notable for the production of various organic acids, for example, Tartaric, Citric, Malic, Lactic,  sources. Kojic acid can be obtained by employing other carbon sources like sucrose, fructose, and lactose. The optimum incubation temperature was 30°C and pH 3 (31.5 g/l ). Hence, in the present work, authors attribute kojic acid production from marine Aspergillus flavus, and compared to other acids, Kojic acid achieved from Aspergillus species has wide applications in various fields. Additionally, further investigation is needed to boost human power to fight with various microbial diseases. Succinic, Acetic, Oxalic acids 81 , some Malus species generously incorporated malic acid and citric acid 82 , there are no shreds of evidence for kojic acid production from plants.

CONCLUSION
The present investigation was focused to optimize cultural conditions to synthesize maximum kojic acid from A.flavus. Glucose and yeast extract were the optimal carbon and nitrogen g/l g/l Nitrogen source