Antimicrobial activity of extracellular compounds from endophytic fungus BAR1.5 cultivated with different media

Endophytic fungi are microorganisms that live inside the plants that are known to be able to produce the same bioactive compounds as their host plants. Endophytic fungus BAR1.5 was isolated from Rhizophora stylosa leaves collected from the mangrove area of Barru, South Sulawesi. The extracellular components of fungal endophytes are known to have higher antimicrobial activity than the intracellular compounds. Furthermore, the growth of endophytic fungi is strongly influenced by nutrients in the culture media, so that it may affect the production of its secondary metabolites. The aim of this study was to compare the production of extracellular compounds and its antimicrobial activity of the fungus BAR 1.5 cultured with different media (Potato Dextrose Broth, PDB and Hagem medium). The results showed that the endophytic fungus cultured on PDB media grew faster, produced more yield of extracts and showed stronger antimicrobial activity. Its extracellular compounds inhibited the growth of the tested bacteria which were Escherichia coli, Pseudomonas aeruginosa, Candida maltosa, Bacillus subtilis and Staphylococcus aureus. The strongest antibacterial activity was against B. subtilis with the inhibition zone was 12.5±0.3 mm. The extracellular compounds of the endophytic fungus BAR 1.5 contained terpenoids, polyphenols, and flavonoids.


Introduction
Mangroves are biological resources that have been widely used as a source of traditional medicine by the community.Leaves, bark and fruit are part of the mangrove plant that is widely used.They have several biological activities such as antimicrobial, antifungal, antitumor, antiviral, insecticide and antileukemic activity [1].Rhizophora stylose is one of the many types of mangroves found in Indonesia and has potential as a source of bioactive components.Extracts from the leaves and bark of R. stylosa were expected to be effective against a range of gram positive and gram-negative bacteria.To treat a variety of bacterial infections, these extracts may serve as broad-spectrum antibiotics [2].Baharanuddin et al. [3] showed that R. stylosa fruit extract could stop the growth of many Vibro bacteria, including V. alginolyticus, V. harveyii, and V. parahaemolyticus, which induce vibriosis in mangrove crab larvae (Scylla serrata F).Moreover, Mouafi et al. [4] demonstrated that the R. stylosa leaf extract could prevent the growth of Candida albicans, E. coli, S. aureus, B. subtilis, Penicillium digitatum, and Fusarium oxysporium.However, the excessive use of parent plants will have an impact on the sustainability of 2 the plant.An effective way that can be used to efficiently source bioactive compounds from plants without disturbing the sustainability of plants is to use endophytic microbes associated with host plants.
The term "endophytic microbes" refers to microorganisms that dwell in plant tissues without causing harm to their host plants.Endophytic microbes can be fungi or bacteria obtained from the inside of the plant and are able to produce the same number of bioactive compounds as those found in the host plant without having to extract the plant parts themselves.Several studies have studied the potential of the bioactive components produced by endophytic fungi.Endophytic fungi from Colombia medicinal plant Otoba gracilipes (Myristicaceae) have antibacterial activity towards Escherichia coli and Staphylococcus aureus [5].Sibero et al. [6] reported that the endophytic fungi RS3 isolated from the anthill plant/ sarang semut (Hydnophytum formicarum) produced extracellular black pigment and has potential as a photoprotector agent.Moreover Wahjuningrum et al. [7] demonstrated Nodulisporium sp.KT29, an endophytic fungus, has the ability to act as an immunostimulant to reduce vibriosis in vannamei white shrimp raised in a marine culture system.In this study, we used endophytic fungi BAR 1.5 that isolated from leaves of R. stylose which live in Barru waters, South Sulawesi.
Endophyte fungal bioactive substances typically show up on both external (mycelia) and intracellular surfaces.Extracellular substances, however, have more biological activity than intracellular substances [8].production of secondary metabolites influenced by a variety of cultivation factors, such as nutrient availability, temperature, and pH etc.In this study we used Potato Dextrose Broth (PDB) and Hagem medium for culture of endophytic fungi BAR 1.5.These culture media have different compositions which act as carbon and nitrogen sources for the growth of fungi.Furthermore, the objective of this study was aimed to determine the antimicrobial potential of the extracellular component of the endophytic fungi of the mangrove plant R. stylose (BAR 1.5) using different culture media and to evaluate the phytochemical composition of extracellular extract of the endophytic fungal BAR 1.5.

Cultivation of endophyte fungus on different medium.
Endophytic fungus of BAR 1.5 was cultured on PDB and Hagem medium for 18 days with shaking condition (120 rpm).The pH of the culture media was measured throughout the cultivation process.In addition, fungal mycelia were harvested every 3 days which was done by filtration using filter paper to obtain mycelia and culture media.The fungal mycelia were then dried using an oven at 40°C for 24 hours.The weight of the dry mycelia was used to determine the mold growth curve, while the culture media was then extracted to obtain extracellular bioactive components from the endophytic fungi.BAR 1.5.Extracellular compound was extracted from broth of medium culture.Liquid-liquid extraction method was applied with ethyl acetate as a solvent extraction for 3 days (shaking condition) [8].A total of 70 mL of culture media was extracted using ethyl acetate solvent in a ratio of 1:1.The separation of the culture media from the ethyl acetate extract was carried out using a separating funnel.To transform the extract into a paste, it was condensed using a rotary vacuum evaporator at a temperature of 40 o C.

Antibacterial assay.
Antibacterial assay of extracellular compound extract was obtained by agar well diffusion method which refers to previous references [9].Microorganisms used in the antimicrobial activity test were B. subtilis, P. aeruginosa, E. coli, S. aureus and Candida maltosa.The test microbes were rejuvenated by inoculating one ose of microbes into the NA medium, incubated at 37 o C for 24 hours.After that, the test microbes were inoculated on NB media, incubated at 37 o C for 18-24 hours.The turbidity of the culture growth was measured using a UV-Vis spectrometer at a wavelength of 600 nm until it reached an OD of 0.5-0.8.Furthermore, the test medium was made by adding 20 L of culture into sterile MHA media, homogenized with a vortex, and poured into a petri dish.The solid media was perforated to make a 6 mm diameter well.Endophytic mold extract was inserted into the hole in the amount of 0.5 mg; 1 mg; 2 mg along with positive control and negative control.Positive control used chloramphenicol antibiotics while negative control used ethyl acetate as solvent.The plates were incubated at 37 o C for 24 hours and the inhibition zone formed was measured using a ruler (in millimeters).Each extract treatment was repeated twice.The inhibition of antimicrobial substances was measured by reducing the diameter of the inhibition zone formed by the diameter of the well.According to the level of the inhibitory effect (diameter zone), the following values were assigned: high (≥17 mm), middle (14 ≤ diameter zone <16 mm), and low (diameter zone ≤13 mm) [10].

Phytochemical analysis.
The active components from the extract of fifteenth days cultivated fungi were separated using thin layer chromatography.The aluminum plate with silica gel (G60 F254 Merck) was dried in an oven at 105 o C for 10 minutes.The extract was spottened using a capillary tube to create a small circle, and the plate was then placed into a container that have contained eluent (a mixture of dichloromethane, ethyl acetate, and n-hexane in a ratio of 2:3:1) [11].The plate is propped up and left until the eluent reaches the boundary line, then the plate is removed and allowed to dry.Separation was carried out with two replications.One of the plates was used for the observation of the stains produced under a UV lamp at wavelengths of 254 nm and 366 nm.
The phytochemical test was carried out by spraying the reagent on a dry TLC plate.The reagents used include ammonia vapor, FeCl3, and sulfuric acid anisaldehyde.Ammonia vapor was used to detect flavonoid compounds, positive the presence of yellow or yellow brown spots showed positive.FeCl3 reagent was used to detect polyphenol compounds, the presence of black spots showed positive.Sulfuric acid anisaldehyde reagent was used for testing terpenoid/steroid compounds, the presence of purple-redor purple-colored spots showed positive [12].

Bioautographic assay using B. subtilis.
The bioautographic test was carried out by placing the eluted plate in a petri dish and then adding MHA media containing B. subtilis bacteria.Then, incubated at 37 oC for 24 hours.The inhibition zone formed was observed.

Growth curve and pH condition of Endophytic fungi BAR 1.5
Endophytic fungi BAR 1.5 was isolated from leaves of R. stylose which live in Barru waters, South Sulawesi.This fungus has a characteristic white color, spreading hyphae with fibrous edges resembling roots, the surface of the hyphae is slightly rough, and the underside is white-brown.The appearance of the endophytic fungi BAR 1.5 shown in Figure 1.The dried biomass of mycelia cultivated on PDB medium was higher than that cultivated on Hagem medium (Figure 2).The highest biomass weight on PDB media was 1.727±0.53g produced on the 15th day of harvesting, while on Hagem media it was 0.728±0.06g produced on day 9 of harvesting.Moreover, the endophytic fungus BAR 1.5 cultivated on PDB medium passed the log phase up to day 9 th which indicated an increase in the number and activity of cells.After that, it entered the stationary phase on the 12 th day and entered the death phase on the 15 th day of cultivation.Meanwhile, endophytic fungi BAR 1.5 cultivated on Hagem media had entered the death phase on the 12 th day of cultivation.These results suggested that the composition of the cultivation media was influenced by the growth of fungi, especially carbon and nitrogen sources.The endophytic fungus BAR 1.5 has grown better on PDB than Hagem medium with more yield of dried mycelia.The fungal nutrient sufficiency in their media is closely linked to their growth phase [13].We used two different kinds of culture media with different amounts of carbon, nitrogen, and trace elements.PDB medium just had potato infusion solids and dextrose, whereas Hagem medium had been enhanced with malt extract, yeast extract, D-glucose, NH4Cl, and some trace elements.It has been demonstrated that the type of carbon or nitrogen sources employed, as well as their complexity, trace elements, and their dose, affect the growth of fungi [14][15][16].
The pH condition of the substrate or growth medium is one of the determining factors for fungal growth.Fungi will grow well over the pH range 3-7 [17].The pH of the BAR 1.5 fungal endophytic culture medium did not change during cultivation, except for the PDB medium that decreased to 4 on day of 15th of cultivation.This decrease in pH may be due to the formation of organic acids as a result of glucose metabolism [18].

Yield of extracellular compound extract
Extraction was carried out on PDB and Hagem culture media to obtain extracellular components produced by endophytic fungi BAR 1.5.Tarman et al. [8] showed that culture media extracts had higher extract yields and higher antimicrobial activity than fungal biomass extracts.Extraction of culture media from endophytic fungi of BAR 1.5 was carried out with ethyl acetate solvent using maceration method.Ethyl acetate is used because it has medium polarity (semipolar) so that it is able to dissolve non-polar to polar compounds.The appearance of the endophytic fungal culture media BAR 1.5 before extracting the extracellular components can be seen in Figure 3  The appearance of the culture media of endophytic fungi BAR 1.5 shows the change in the color of the culture media during cultivation time (Figure 3).The PDB culture media (EX-PDB) changed color from clear yellow to reddish brown with the length of time of cultivation, while the yellowish white Hagem media (EX-HAG) did not experience a significant color change.The color change in both culture media was related to the yield of the extract produced by fungi BAR 1.5 during its growth phase whereas as the yield of the extract, the color of the resulting culture media also became more concentrated.Yield extracellular compounds of endophytic fungi BAR 1.5 on different media cultures are presented in Table 1.The yield extract from PDB media had a higher percentage than the Hagem media.The highest percentage yield on PDB media was found on the 15 th day of cultivation (0.139%) where the cultivation time indicated the end of the stationary phase.While the highest yield on Hagem media was found on the 9 th day of cultivation (0.1% ) which is the beginning of the stationary phase.These results indicate that the production of extracellular components is maximally produced at the stationary phase (Figure 2), where the weight of fungal mycelia is also the highest in that phase.This is in line with the results of research from Melliawati and Harni [20] showed that the yield of the extract produced was higher because the generation of secondary metabolites peaked when the number of fungi reached its highest level.

Antimicrobial activity of extracellular compound extract
The agar well diffusion method was used to evaluate the antibacterial activity.Chloramphenicol was employed as a positive control because of its broad spectrum of activity against both Gram-positive and Gram-negative bacteria [21].The negative control used was ethyl acetate as a solvent on dilution.Tests were carried out on 5 microbes representing Gram-positive bacteria (S. aureus and B. subtilis), Gramnegative (E. coli and P. aeruginosa), and yeast (C.maltosa).The extract was tested for microbial inhibition, and to see the effect of cultivation time on antimicrobial activity.The antimicrobial activity of the PDB and Hagem culture media extracts is shown in Figure 4.
Extracellular extract of endophytic fungi BAR 1.5 can inhibit five types of bacteria tests.Antibacterial activity of extracellular compound of fungi BAR 1.5 was significantly increased in the stationary phase (Figure 4).In addition, the higher antibacterial activity showed on the highest of yield of extract.This result suggested that The extracellular component of the extract which plays an important role as an antibacterial is thought to be a secondary metabolite.The production of secondary metabolites is produced by fungi at the end of the exponential phase or the beginning of the stationary phase until the end of the death phase at which time the nutritional sources are limited [22].Srikandace et al. [23] stated that the secondary metabolites are molecules produced as a result of the catabolic process and are released when primary nutritional sources, such as carbon and nitrogen, are depleted.Figure 4 shows that extracellular extract from PDB has higher antibacterial activity than Hagem which B. subtilis could be inhibited well by the extracellular fungal extract.The diameter of the inhibition zone on B. subtilis bacteria was relatively larger than the other tested microbes, which was 12.47 mm for the extract from PDB media and 10.17 mm for the extract from Hagem media.It is categorized as a strong antibacterial agent (>17 mm) based on the categorization by CLSI [9].The availability of nutrients (carbon and nitrogen sources), together with other factors, affects the ability of micro-fungi to produce any metabolites [16].According to Sánchez et al. [24], carbon sources have a significant role in the development of antibiotics.

Phytochemical and bioautography profile of extracellular compound extract
According to the results of the antimicrobial activity test (Figure 4), the extracellular extract from the endhopyc fungus of BAR 1.5 that was chosen was the extract from harvesting days 15 on PDB media and 9 on Hagem media.The extract was then eluted on a TLC plate for phytochemical and bioautography tests.The results of the elution on the TLC plate are shown in Figure 5.The extracellular extract from PDB medium had eight spots that could be seen on the TLC chromatogram as opposed to six spots for the Hagem media treatment (Figure 5).It was believed that fungus BAR 1.5 produced more active chemicals in PDB medium than in Hagem media.The chromatogram's Rf value, which was discovered, shows that the extract contains bioactive substances.The results of phytochemical screening using the reagent spraying method on the TLC plate showed that both extracts of the extracellular component contained terpenoids/steroids, flavonoids, and polyphenols, which can be seen in Table 2.After the compounds had been separated using TLC, bioautography testing was done to evaluate which active fraction has the ability to halt the growth of the B. subtilis bacteria.
Agar overlay is the technique used for bioautography.According to Wang et al. [25], applying this method has the benefit that it only requires a small amount of extract and may be used to bioassays that lead in the isolation of active compounds.The bioautographic profile of the BAR 1.5 fungal extract is presented in Figure 6 and Table 3.
Figure 6 shows that the extract of the extracellular components of the fungus BAR 1.5 has antimicrobial activity against B. subtilis bacteria which is indicated by the presence of inhibition zones on the agar medium.The extract of extracellular components from the media showed 3 clear zone spots at Rf values of 0.75, 0.5 and 0.1.Meanwhile, the extract from Hagem's medium only had one clear zone spot at an Rf value of 0.56.This shows that the active fraction in the extracellular extract from PDB medium is more than the extract from Hagem media.This result is in line with the previous test (Figure 4) which showed that the antimicrobial activity of extracts of extracellular components from PDB medium was the highest as indicated by the larger diameter of the clear zone.Based on the phytochemical profile (Table 3) it is known that the extracellular components of the BAR 1.5 fungus that play a role in inhibiting bacteria there are 3 types of active compounds, such as terpenoids/sterols and flavonoids, but there is one spot that has not been identified.while in the extract from Hagem media there were only steroid/terpenoid compounds that acted as antimicrobials.The combination of the three active fractions contained in the PDB culture media extract was thought to have contributed greatly to inhibiting bacterial growth compared to the Hagem culture media extract which only had one active fraction.
Each bioactive compound has a unique mechanism for acting as an antimicrobial.Cowan [26] asserts that bacterial cell membrane damage is assumed to be the mechanism through which steroid compounds inhibit microorganisms.Flavonoids are secondary metabolites that function as protective elements in nature, such as preventing bacterial invasion [27].Cells' proteins may get denatured as a result of the antibacterial effects of flavonoid compounds.This is caused by the OH group's capacity to bind to internal proteins in cell membranes in flavonoid compounds [28].Pelczar and Chan [29] suggest that phenolic compounds and their derivatives can change cell permeability, which can prevent cell development or cause cell death by damaging cell membranes.According to Jawetz et al. [30], the phenol compounds' ability to attach to the sulfhydryl group of proteins is the mechanism of inhibition.The target cell membrane protein has a confirmatory change as a result.The control of protein structure, active transport, selective permeability, and bacterial cell lysis are all affected by protein structure instability, which results in the loss of shape and lysis of the cells.

Conclusion
Compared to Hagem Media, the endophytic fungus cultured on PDB media grew faster, produced more yield of extracts and showed stronger antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Candida maltosa, Bacillus subtilis and Staphylococcus aureus.The strongest antibacterial activity was against B. subtilis.The extracellular compounds of the endophytic fungus BAR 1.5 contained terpenoids, polyphenols, and flavonoids.

Figure 2 .
Figure 2. Growth rate ( Hagem media, PDB media) and pH condition of culture media ( Hagem media, PDB media) for endhopic fungi BAR 1.5 during 18 th days cultivation.

Figure 4 .
Figure 4. Antimicrobial activity of crude extract from endophytic fungi BAR 1.5 in Hagem and PDB media against S. aureus, P. Aeruginosa, B. subtilis, E. coli and Candida maltosa.

Table 1 .
Yield extract of extracellular compound of endophytic fungi BAR 1.5 during cultivation with different medium.

Table 2 .
Phytochemical profile of extracellular compound of endophytic fungi BAR 1.5.

Table 3 .
Diameter zone inhibition of active fraction from extracellular compound extract of endhophytic fungi BAR 1.5.
Figure 6.Bioautography profile of of the BAR 1.5 fungal extract cultured oo PDB media (EX-PDB) and Hagem media (EX-HAG).