Optimization of Antibacterial Metabolite Production and Antioxidant Activity of Endophytic Fungi Isolate from Kelakai (Stenochlaena palustris)

This research aims to optimize the production of antibacterial metabolites and test the antioxidant activity of endophytic isolates from kelakai ( Stenochlaena palustris ). The research results showed that the optimum growth profile of the isolate showed that exponential growth only started on day 1 and reached

This research aims to optimize the production of antibacterial metabolites and test the antioxidant activity of endophytic isolates from kelakai (Stenochlaena palustris).The research results showed that the optimum growth profile of the isolate showed that exponential growth only started on day 1 and reached a maximum on day 17.Based on pH variations, it shows that pH 7 is the optimum pH in inhibiting bacterial growth, and based on variations in carbon sources, it shows that fructose is a better carbon source than glucose.Antimicrobial activity test using ethyl acetate extract from endophytic fungal fermentate based on differences in pH and carbon source showed antimicrobial activity against S. aureus and E. coli with the inhibition zone diameters being 13.50 mm and 11.35 mm, respectively.

INTRODUCTION
Innovations based on natural ingredients that have potential as antibiotic and antioxidant compounds are being developed in line with the increasing number of types of microorganisms that are resistant to antibiotics (Rumidatul et al., 2021).Research on secondary metabolites obtained from endophytes is the focus of the search for new antibiotic compounds aimed at overcoming these problems and to ward off free radical compounds continues to increase (Aini et al., 2022;Mookherjee et al., 2020;Utami et al., 2023).Microorganisms can cause infectious diseases and various other diseases such as cancer, asthma and cardiovascular disorders (Preuttiporn & Sita, 2019).
Endophytic fungi are fungi that colonize plant tissue as hosts, without causing damage or causing disease in plants (Aini et al., 2022;Gouda et al., 2016;Jia et al., 2016).Endophytic fungi have an effective source of bioactive compounds, have low toxicity, low cost, fast growth, and use on an industrial scale provides minimal impact on the environment (Pandy et al., 2023;Preuttiporn & Sita, 2019).
Antioxidants obtained naturally are very effective in preventing destructive processes caused by free radicals.Free radicals can cause various diseases including cancer, hypertension, diabetes, neurodegenerative disorders, and others (Dewage et al., 2022;Michalak, 2022;Sharifi-Rad et al., 2020).Synthetic antioxidants have been shown to have toxic effects on the body.So, it is very important to look for new sources of antioxidants and antimicrobial compounds that are safe and easily available locally and come from natural sources (Adeleke & Babalola, 2021;Elfita et al., 2022).
Endophytic fungi isolated from Kelakai (Stenochlaena palustris) have antimicrobial activity and contain compounds that have potential as antioxidants.Three endophytic fungi isolated from Kelakai, identified as Aspergillus sp., Paecilomyces sp., and Arthrocristula sp., were tested for antimicrobial activity.Antimicrobial activity showed good inhibition results against S. aureus and E. coli (Mashar et al., 2023).In order to increase the production of bioactive compounds, optimization of the production of bioactive compounds was carried out on isolates which in previous research showed the best activity, and identified optimal conditions for production.
The production of bioactive compounds in endophytic fungi is influenced by several factors, including medium composition, nutrient availability, lighting, stirring, variations in carbon sources and pH.Optimization of bioactive compound production is carried out to obtain optimum conditions for the growth of endophytic fungi (Rumidatul et al., 2021).
The production of bioactive compounds from endophytic fungi is closely related to the condition of the medium and the length of incubation time.A suitable environment is very supportive of the production of bioactive compounds from endophytic fungi, so it is important to create optimal environmental conditions for the growth of endophytic fungi (Saleh et al., 2019;Widjajanti et al., 2022;Zhao et al., 2021).

Making Nutrient Agar Media
A total of 23 grams of NA powder was dissolved in 1000 mL of distilled water then heated.After that, measure the pH of the media and add HCl or NaOH to obtain a pH of 7. The media is then sterilized.

Making Potato Dextrose Yeast Media
A total of 19.8 g of GDP was dissolved in 750 ml of distilled water and then heated.Once dissolved, add 3 grams of yeast extract and heat.Measure the pH of the media and add HCl or NaOH to obtain a pH of 6, then divide it into 15 glass bottles and then sterilize.

Making Potato Dextrose Yeast Media pH 4, 7 and 8
A total of 50 mL of PDY was put into a glass bottle.Measure the pH of the media and add HCl or NaOH to obtain a pH of 4, 7 and 8 then sterilize.

Making Potato Dextrose Yeast + Glucose Media pH 4, 7 and 8
A total of 50 mL of PDY was put into a glass bottle then add 1 gram of glucose into each0020bottle.Measure the pH of the media and add HCl or NaOH to obtain a pH of 4, 7 and 8 then sterilize.

Making Potato Dextrose Yeast + Sucrose Media pH 4, 7 and 8
A total of 50 mL of PDY was put into a glass bottle then add 1 gram of fructose into each bottle.Measure the pH of the media and add HCl or NaOH to obtain a pH of 4, 7 and 8 then sterilize.

Making Starter Cultures for Endophytic Fungi Isolates
A starter culture of endophytic fungal isolates is made by inserting 1 active endophytic fungal isolate which has been incubated for 5-7 days into a glass bottle containing 50 mL of PDY media.The starter culture is then incubated for 3 days.

Making Bacterial Suspensions
Staphylococcus aureus and Escherichia coli bacterial inoculum was obtained by inoculating 1 ose pure colonies.The inoculum was diluted using 0.9% physiological NaCl to the equivalent of 0.5 McFarland standard.

Fermentation of Endophytic Fungi Isolates
To obtain secondary metabolites, the active isolate was fermented by inoculating 1 ml of starter culture into a glass bottle containing 50 ml of PDY media.Fermentation was carried out for 14 days at room temperature.

Determination Growth Profile of Active Endophytic Fungi Isolates
In the fermentation process, fermentate is taken and fungal mycelia are harvested every 24 hours to obtain the growth profile of endophytic fungi.The growth profile of endophytic fungi was created based on the increase in the dry cell weight of the fungal mycelia and testing the activity of the fermentation fluid.

Testing Fermentate Activity from Growth Profile
The fermentation liquid obtained from fermentation every day is then tested for antimicrobial activity against approximately 20 µL of Staphylococcus aureus and Escherichia coli bacteria.Then incubated at room temperature for 1 day at 37˚C.Antimicrobial activity was measured based on the inhibition zone formed around the endophytic fungus.

Optimization of Fermentation Conditions for Active Endophytic Fungi Isolates with Variations in pH and Medium Substrates in the Production of Fermentation Secondary Metabolites
A total of 1 mL of starter culture was inoculated into a glass fermentation bottle which previously contained 50 mL of PDY media, PDY+Glucose media, PDY+Sucrose media respectively with pH variations of 4, 7 and 8. Fermentation was carried out according to the optimum time for the growth of the active isolate.previously obtained.

Fermentation Activity Testing from Optimization of Fermentation Conditions
The fermentation liquid obtained from the Optimization of Fermentation Conditions was then tested for antimicrobial activity against approximately 20 µL of Staphylococcus aureus and Escherichia coli bacteria.Then incubated at room temperature for 1 day at 37˚C.Antimicrobial activity was measured based on the inhibition zone formed around the endophytic fungus.

Fermentation Production of Active Isolates on a Large Scale
As much 5 mL of starter culture was inoculated into an Erlenmeyer flask containing 250 mL of the Fermentation Condition Optimization media obtained previously.

Fermentation Media Extraction
After the fermentation process ends, the fermentation medium is separated from the fungal mycelia by filtering it using filter paper.The fermentation medium was fractionated with ethyl acetate using a ratio of 1:1.The fermentation medium and ethyl acetate were put into a separating funnel, then shaken gently for approximately 5 minutes.Next, let it sit to separate the media and ethyl acetate.The ethyl acetate fraction was separated and evaporated to obtain a thick extract.This process is carried out until a clear ethyl acetate fraction is obtained.

Test Activity of Ethyl Acetate Extract Active Isolate of Endophytic Fungi
The antimicrobial activity test of ethyl acetate extract from endophytic fungal samples was carried out on Staphylococcus aureus and Escherichia coli using the disk diffusion method.The paper disc is soaked in the sample with a concentration of 100% (extract in DMSO solvent).The paper disc was then placed in NA media containing bacterial culture, then incubated at room temperature for 1 day.The diameter of the inhibition zone formed was observed and measured.In this test, a control is also used to compare the test results against the sample.The controls used were 3 µl DMSO (negative control), and 3 µl Tetracycline 1.2 mg/ml (positive control).Each test uses three replications, and the mean value ± standard deviation is also described.

Antioxidant Activity Test
Antioxidant activity was determined by the DPPH method using methanol as a blank.The extract was diluted with methanol and divided into several concentration variations, namely 10, 20, 30, 40, and 50 µg/mL.To each solution, 28 µg/mL DPPH solution was added with a ratio of 1:5.Incubated ondark room for 30 minutes.After that, the absorbance was measured at a wavelength of 517 nm using UV-vis spectrophotometry (Gurgel et al., 2023;Rammali et al., 2022).

Determination Growth Profile of Active Endophytic Fungi Isolates
The growth of endophytic fungal mycelia that has been fermented for 21 days is shown in Figure 1.These results show that endophytic fungi from kelakai can grow well on PDY media, so it is hoped that they can produce good bioactive compounds too.The curve graph shows that in the initial stage of growth it is still in the adaptation phase so that exponential growth only begins on day 1 and reaches a maximum on day 17.Static conditions were seen on days 3 to 16, and began to decline on day 18.The growth curve was measured by measuring the weight of the mycelia during the fermentation process.Vol.2, No.12, 2023: 3175-3190 3181 mm and against E. coli was shown on the 12 th day with a bland zone diameter of 9.20 mm.

Optimization of Fermentation Conditions and Antimicrobial Activity for Active Endophytic Fungi Isolates with Variations in pH and Medium Substrates in the Production of Fermentation Secondary Metabolites
The optimization of fermentation conditions for active endophytic fungal isolates was carried out based on variations in pH (4, 7, and 8).The test results show that pH 7 is the optimum pH in inhibiting bacterial growth S. aureus and E. coli with inhibition zone diameters of 13.15 mm and 11.29 mm, respectively (Figures 2 and 3).
The optimization of fermentation media conditions was carried out based on variations in carbon sources, using fructose and glucose.The test results show that fructose is a better carbon source than glucose (Table 2).The results of antimicrobial activity test using ethyl acetate extract from the fermentate of endophytic fungal show that the ethyl acetate extract from the fermentate of endophytic fungi resulting from optimization with a concentration of 60% has a weak inhibitory response to S. aureus and E. coli (Table 3).

Optimized fermentation media
Diameter of the inhibition zone (mm)

Antioxidant Activity Test
Testing the antioxidant activity of the extract shows IC50 value ethyl acetate extract and vitamin C respectively is 24.29 and 14.99 µg/ml (Table 4).This value shows that the extract has very strong antioxidant activity.

DISCUSSION
In previous research, 3 endophytic fungi were isolated from Kelakai.Based on identification results macroscopic and microscopic, the three isolates were identified as the genus Aspergillus sp., Paecilomyces sp., and Arthrocristula sp.The various endophytic fungi that can be isolated from kelakai show that every plant around us is a breeding ground for several endophytic fungi (Strobel, 2003).The number of types of endophytic functions that can be obtained from plants is greatly influenced by factors such as temperature, humidity, chemical variations, anatomical structure and age of the colonized host organs (García-Sánchez et al., 2020).
The isolate used was an isolate with the code Sp1Htm, identified as Aspergillus sp., which was the result of isolation in previous research (Mashar et al., 2023).This isolate code was chosen because it is the isolate that has the largest barrier zone compared to the other 3 isolates.The initial stage carried out was optimizing the production time of secondary metabolites.Optimization of production time aims to determine the right time to produce secondary metabolites optimally (Syarifuddin et al., 2021).Optimization was carried out by graphing the relationship between fermentation time and the diameter of the inhibition zone.
Production of secondary metabolites from endophytic fungi with the code Sp1Htm using PDY media.Fermentation was carried out using a closed system and the fungus used came from Potato Dextrose Agar (PDA) media which was 5 days old.PDY media was chosen because it can shorten the lag phase in fungal growth.PDY media has a similar composition to PDA media, the only difference is the consistency (Ginovart et al., 2011).The Sp1Htm fungus is expected to be able to adapt quickly to PDY media because it has previously adapted to PDA media.The purpose of shortening the lag phase is because a short lag phase will allow the fungus to enter the logarithmic phase more quickly, so it is hoped that the formation of secondary metabolites will occur more quickly in the stationary phase (Rolfe et al., 2012).Apart from that, the same content in PDY and PDA media will guarantee that the production results from the fermentation process are the same as those produced in PDA media.The fermentation process was carried out for 21 days, this was done to determine the growth of the mycelium.Apart from that, during these 21 days it is estimated that the stationary phase has been reached (Rumidatul et al., 2021).The achievement of the stationary phase is marked by a color change in the media, an increase in the viscosity of the media, and the volume of mycelia in the fermenter does not increase.PDY media is used in the fermentation process because it is more effective in increasing fungal cell biomass and secondary metabolites compared to fermentation on agar media.With liquid media, an agitation process can be carried out which plays a role in maintaining the homogeneity of nutrients in the media so that the process of absorbing nutrients by fungi becomes more optimal (Zhou et al., 2018).After reaching the 21st day, the mycelia and media were separated using a filtering technique (Singgih et al., 2017).
As a step to carry out initial identification of its ability as an antimicrobial, an antimicrobial activity test was carried out on the fermentate.The best activity against S. aureus was shown on day 15 with an inhibition zone diameter of 12.15 mm and against E. coli was shown on the 12th day with a bland zone diameter of 9.20 mm.The diameter of the inhibition zone of a sample in the range of 7-11 mm is included in the weak activity category, while >16 mm is included in the strong activity category (Monks et al., 2002).
Next, optimization of fermentation conditions for active endophytic fungal isolates was carried out based on variations in pH (4, 7, and 8) and carbon sources, using fructose and glucose, in the production of fermentation secondary metabolites.This stage was carried out in accordance with the optimum time for growth of active isolates obtained previously.Optimization based on pH variations aims to determine the pH range isolates can grow and produce bioactive compounds optimally.pH plays an important role in enzymatic reactions and influences the growth of fermentation microorganisms and causes changes in the structure of bioactive compounds which can have an impact on biological activity, including antioxidants.At optimum pH, growth also stimulates the production of bioactive compounds (Bishop et al., 2022;Hur et al., 2014).
Different pH environments affect the type and quantity of metabolites produced by microbes.Neutral pH encourages the formation of organic acids by Aspergillus sp (Qayyum et al., 2019).In general, the optimal pH for the production of antimicrobial compounds ranges between 7 and 8. Changes in the pH of the growth medium for microorganisms have a bad impact on the production of antimicrobial compound metabolites.(Vijayakumar et al., 2012).
Next, optimization of fermentation media conditions was carried out based on variations in carbon sources, using fructose and glucose.Media is a source of nutrition for the growth of microorganisms, able to provide a source of carbon and other minerals needed for the growth of microorganisms (Andualem & Gessesse, 2013).Carbon sources are the main nutrients needed by fungi, playing a role in the life and metabolism of fungi.Different carbon sources were chosen in order to produce more organic acids (Qayyum et al., 2019).
Sucrose is the most popular carbon source in tissue culture.Sucrose has efficient absorption capabilities across the plasma membrane.Various carbon sources can be used in growth media.Media given monosaccharides such as glucose or sucrose can produce consistently high cultures of microorganisms with higher growth rates compared to fructose.The use of sucrose is a good carbon source for culturing microorganisms (Swamy et al., 2010).Based on these results, fungal isolates from the genus Aspergillus sp can grow optimally and show good antimicrobial activity in media conditions with pH 7 and fructose as a carbon source.The Aspergillus genus has nutritional potential in various environmental conditions, including host tissue.Gene expression of enzymes involved in metabolic processes allows fungi to be highly effective in upregulating the tricarboxylic acid cycle and increasingly able to metabolize other secondary carbon sources (Nji et al., 2023;Qayyum et al., 2019).
Environmental factors greatly influence the production of bioactive compounds in Aspergillus fungi, including pH and carbon sources.Suitable environmental conditions are needed to support the sporulation process and secretion of bioactive compounds (Calvo et al., 2002;Oliviero et al., 2022).
After the fermentation process ends, the optimized fermentation media is separated from the fungal myceliafractionatedwith ethyl acetate using a ratio of 1:1.The ethyl acetate fraction was separated and evaporated to obtain a thick extract.Ethyl acetate solvent is used because it has semipolar properties so it can dissolve polar and nonpolar bioactive compounds (Rumidatul et al., 2021).
The antimicrobial activity test using ethyl acetate extract from the fermentate of endophytic fungal samples was carried out on S. aureus and E. coli using the disk diffusion method.The extract concentration used is 60%.This concentration was chosen because previous research stated that the antimicrobial activity test results showed that Sp1Htm and Sp2HjK ethyl acetate extracts at concentrations of 15, 30 and 45% had a weak inhibitory response to S. aureus and E. coli that the concentration of the extract is increased.
Testing the antioxidant activity of the extract was compared with standard vitamin C using concentrations of 10, 15, 20, 25, and 30 µg/ml.The antioxidant capacity of the samples was identifiedusing the IC50 value parameter (Inhibitory Concentration 50%).IC50 value is a parameter that is widely used to measure the antioxidant activity of a sample.IC50 is the concentration of antioxidant compounds that can reduce DPPH activity by 50%.A lower IC50 value indicates high antioxidant activity (Akullo et al., 2023).
The IC50 value is obtained by making a linear equation between the test concentration and percent inhibition.If the IC50 value obtained is <50 µg/ml, it indicates that the antioxidant activity of the sample is in the very strong category (Bahriul et al., 2014).This shows that the kelakai endophytic fungus has great potential as an antioxidant that can be used for the development of raw materials for traditional medicine.

CONCLUSIONS AND RECOMMENDATIONS
The growth curve graph shows that exponential growth only begins on day 1 and reaches a maximum on day 17.Based on pH variations, it shows that pH 7 is the optimum pH, while based on variations in carbon sources, it shows that fructose is a better carbon source than glucose in inhibiting bacterial growth.Antimicrobial activity test using ethyl acetate extract showed antimicrobial activity against S. aureus and E. coli with the respective inhibition zone diameters being 13.50 mm and 11.35 mm.IC50 value Ethyl acetate extract is 24.29 µg/ml which shows that the extract has very strong antioxidant activity.However, other potential benefits remain to be explored in future research.

Formosa
Journal of Science and Technology (FJST)

Testing Fermentate Activity from Growth ProfileTable 1 .
Fermentate Antimicrobial Activity Test Results Staphylococcus aureus ATCC 25923) and gram-negative bacteria (Escherichia coli ATCC 25922) for 21 days.The results of this screening show that this fermentate has inhibitory activity against S. aureus and E. coli (Table1).The best activity against S. aureus was shown on day 15 with an inhibition zone diameter of 12.15

Table 2 .
Source optimization profile against S. aureus and E. coli

Table 3 .
Fermentate activity test results from optimization of fermentation conditions based on differences in pH and carbon sources

Table 4 .
IC50 Value of Ethyl Acetate and Vitamin C Extract