Antimicrobial Activities of Fungus Comb Extracts Isolated From Indo-Malayan Termite Macrotermes Gilvus Mound

The embodiment of antimicrobial components into the food packaging material has the ability to prevent microbial contamination. Fungus comb could be an alternative source of natural antimicrobial agents. In this study, n-hexane, ethyl acetate, methanol, and water extracts from fungus comb isolated from Indo-malayan termite Macrotermes gilvus Hagen mound were analysed for its antibacterial and antifungal activities against food spoilage microorganisms, including Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Aspergillus avus, and Aspergillus niger. The antimicrobial activity of the fungus comb extracts was evaluated using Kirby-Bauer disc diffusion and microdilution method. The result showed that ethyl acetate extract exhibited the biggest diameter inhibition zone for all bacteria and fungi tested. Ethyl acetate extract showed antibacterial activity in all bacteria with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.39 mg/mL and 0.78 mg/mL, respectively. This extract also inhibited A. avus and A. niger with MIC value of 0.78 mg/ml. Ethyl acetate extract contained guaiacol and syringol, which were predicted as the main antimicrobial components in fungus comb. Whereas n-hexane extract only inhibited Gram-positive bacteria. S. aureus ATCC 25923 was the most sensitive bacteria tested using all extracts. In addition, A. avus was more sensitive compared to A. niger. Overall, fungus comb extract exhibited antimicrobial activity against E.coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, A. avus, and A. niger. This study revealed that the fungus comb extract, especially ethyl acetate, could be considered as a new antimicrobial agent. A. niger FNCC 6114. Ethyl acetate extract of fungus comb contained phenolics compound of guaiacol and syringol, predicted as important antimicrobial substances. S. aureus ATCC 25923 was the most sensitive bacteria tested using n-hexane, ethyl acetate, methanol, and water extracts from fungus comb the instrument. Each well microplate was lled Mueller Hinton Broth (HiMEDIA, India) suspension of E.coli ATCC 25922, P. aeruginosa ATCC 2785, S. aureus ATCC 25923, and fungus comb extract. The number of test microorganisms used in the liquid culture microdilution method has been standardized with a standard turbidity solution McFarland 0.5. Each well has a different concentration of fungus comb extract, starting from a concentration of 100 mg/ml to 0.1 mg/ml. The positive control used a medium with the addition of bacterial suspension. The microplate was then incubated at 37°C for 18 hours, and then observed the MIC values. Observations were made visually based on turbidity. Turbid indicates the presence of bacterial growth and indicates the media is not overgrown with bacteria. To determine the MBC value, it was done by inoculating bacterial growth results in a microplate on Mueller Hinton Agar (OXOID, United Kingdom) media by adopting the concentration indicated as the MIC value. After incubation for 18 hours at 37°C, the MBC value can be seen from the lowest concentration that did not occur in bacterial growth on Mueller Hinton Agar (OXOID, United Kingdom) media. constituents in fungus comb. Application of ethyl acetate extract of fungus comb as antimicrobial agent in packaging material need to be further investigated.


Introduction
Fruits and vegetables are well-known as essential components of a healthy diet. Recently, fresh fruits and vegetables have been associated with diverse foodborne illness outbreaks in many countries. Fruits and vegetables are usually quite acidic and hence quite resistant to invasion by fungi and bacteria. The fruit and vegetable commodities commonly affected by fungi, especially Aspergillus avus and Aspergillus niger are corn, tomatoes, grape, strawberries, gs, peaches, pears, apples, citrus, melons, and mangoes (Bui-Klimke and Wu, 2016). A. avus is a well-recognized producer of a atoxin, and A. niger is known as ochratoxin producer (P iegler et al. 2020). In addition, fresh herbs like basil, parsley, and leafy greens, especially spinach and lettuce, are noteworthy potential sources of bacterial contamination (FAO/WHO, 2008). Denis et al. (2016) reported bacterial infections in fresh vegetables and fruits at retail in Canada were identi ed as Escherichia coli, Salmonella, and L. monocytogenes. Other pathogenic bacteria that often contaminate food are Staphylococcus aureus, and Pseudomonas aeruginosa.
The microbial attack on food products can occur during pre-harvest, transportation, storage, and food processing (Bbosa et al. 2013). In tropical countries, the risk of contamination in postharvest is much higher than in the eld due to warm and humid environments. Therefore, it is essential to maintain the temperature remain low between 2-3 °C and relative humidity around 90% and 95% to avoid decay-causing fungi (Boer et al. 2009).
Although much effort has been made to adjust the environment, some fruits and vegetables have a high susceptibility to mechanical damage during transportation and chance of atmospheric damages due to oxygen levels decreasing and carbon dioxide increasing. According to Feliziani and Romanazzi (2016), the consumer request for new technologies of fruit and vegetable packaging which are safe and environmentally friendly has increased signi cantly, such as the application of edible or biodegradable coatings. In addition to a barrier to mechanical injury, the coating can be injected by antimicrobial agents that can inhibit the growth of any food spoilage fungi and bacteria. Therefore the use of antimicrobial agents in food packaging has become a potential solution for preventing more postharvest losses.
Natural products are the most abundant source of antimicrobials. Fungus combs as a natural product are able to minimize potentially pathogenic fungi and are competitive in the environment to maintain the health of their colonies (Arango, 2015). Fungus comb is a special structure created by termite colonies from the sub-family Macrotermitinae (Isoptera: Termitidae) in their nests as a substrate for the growth of the only one fungus species, Termitomyces sp. (Arshad andSchnitzer 1987, Arinana et al. 2016). It is proposed that chemical compounds in the fungus comb might suppress the growth of another species. Therefore, fungus comb is a potential candidate as a source of active compounds for an organic antimicrobial agent. In a previous study, Nandika et al. (2021) extracted and determined the chemical components of fungus comb from Indo-Malayan termite Macrotermes gilvus Hagen (Isoptera:Termitidae). It is composed phenol, hydroquinone, steroids, terpenoids, and saponin compounds. The study also showed that ethyl acetate extract inhibited the growth of Aspergillus foetidus, a fungus that attacks wooden raw materials, including rubberwood (Hevea brasiliensis Muell. Arg.). However, the bioactivity of fungus comb extract from Indo-malayan termite Macrotermes gilvus mounds as an antifungal and antibacterial agent has not been reported. Therefore, in this research, fungus comb extracts' antifungal activity has been tested against A. avus and A. niger. Antibacterial activity has been tested against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923.

Fungus comb material preparation
Fungus comb extract was extracted from Indo-Malayan Termite Macrotermes gilvus Hagen (Isoptera: Termitidae) using four different solvents (nhexane, ethyl acetate, methanol, and water), according to Nandika et al. (2021). A sample of 0.5 mg extract was dissolved in 500 mL of its solvent.
Culturing the bacteria Culture of Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 were grown on Nutrient Agar (OXOID, Basingstoke, England) media, and then incubated at 37°C for 18 hours. Culture suspensions of E. coli ATCC 25922, S. aureus ATCC 25923, and P. aeruginosa ATCC 27853 were prepared using a physiological solution containing 0.85% NaCl. The bacterial suspension was then compared with standard McFarland 0.5 solution. This aimed to visually determine the density of bacteria in the suspension with an estimate of having a wavelength of 625 nm measured using a GENESYS™ 150 Vis/UV-Vis Spectrophotometer.

Antibacterial Susceptibility Assay
Antibacterial susceptibility assay was detected using the disc diffusion method, according to the standards set by The Clinical Laboratory Standard Institute (CLSI). A hundred microliter of an overnight culture was diluted in saline solution to about 1.5 × 10 8 CFU/mL (0.5 McFarland turbidity standard). This suspension was ooded into Mueller-Hinton agar (Oxoid Ltd., Basingstoke, UK). The sample's paper disc was dispensed into the inoculated plate and then incubated at 37°C for 24 h. The diameters of the clear zones around each paper disc were measured after incubation. Each

Antifungal Susceptibility Assay
The antifungal activity of fungus comb extracts against A. avus FNCC 6181 and A. niger FNCC 6114 cultures was performed by disc diffusion method of Kirby-Bauer using 6 mm diameter-lter paper discs (OXOID, United Kingdom). First, the cultures were diluted in 1000 µl tween solution 0.05% to prepare a homogenous single-celled suspension. Then, inoculated using streak/spread method on Potato Dextrose Agar (MERCK, United States) medium by sterile cotton bud. The test was carried out with three extract concentrations: 2.5 mg/ml, 25 mg/mL, and 50 mg/ml. The susceptibility test was determined by measuring the zone of inhibition (mm) after 48 hours of incubation at 30°C. The samples were compared to an antifungal agent,

Antibacterial activity
Antibacterial susceptibility assay of fungus comb extracts against Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC 2785) and Grampositive (S. aureus ATCC 25923) bacteria were performed using the Kirby-Bauer agar diffusion method. The diameter inhibition zone (DIZ) resulting from the exposure of n-hexane, ethyl acetate, methanol, and water extracts of fungus comb were shown in Table 1. In addition, the clear zone on bacteria tested due to the fungus comb extracts was presented in Figure 1.
A higher dosage of extracts resulted in bigger DIZ in all bacteria tested. Ethyl acetate extract showed the biggest DIZ against all bacteria tested. S. aureus ATCC 25923 was the most sensitive to all extracts. Unfortunately, due to the limitation of n-hexane extract, DIZ data of high doses are unavailable for E. coli ATCC 25922 and P. aeruginosa ATCC 2785. Nevertheless, this extract in the lowest dose inhibited S. aureus ATCC 25923 but not others ( Figure 1C). Ampicillin and Cipro oxacin were used as the positive control. Cipro oxacin in a lower dosage than the fungus comb extracts showed strong inhibition against all bacteria tested. While ampicillin inhibited only E. coli ATCC 25922 and S. aureus ( Table 1) Figure S1, Figure S2).

Antifungal activity
In this study, the antifungal activity of fungus comb extracts against A. avus FNCC 6181 and A. niger FNCC 6114 was carried out using disc diffusion method Kirby-Bauer and determined by its clear zone diameter (mm) ( Table 3 and Figure 2). Ethyl acetate extract exhibited the biggest DIZ against A.
avus FNCC 6181 and A. niger FNCC 6114 (Table 3). Methanol and water extracts also inhibited both fungi at high dosage (25 mg and 50 mg). Unfortunately, both fungi were resistant to n-hexane extract. Nystatin at 100 U dosage was used as the positive control.
MIC and MFC investigation supported the antifungal susceptibility assay. The lowest MIC and MFC values were observed for ethyl acetate extract against A. avus FNCC 6181 and A. niger FNCC 6114. Whereas MIC and MFC value of n-hexane extract was not detected for both fungi (Table 4, Figure  S3, Figure S4).

Discussion
In this study, we reported the bioactivity of fungus comb extract from Indo-malayan termite Macrotermes gilvus H. mounds as an antifungal and antibacterial agent against food spoilage microorganisms. Antibacterial activity of fungus comb extracts has been tested against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923. In addition, antifungal activity has been tested against A. avus FNCC 6181 and A. niger FNCC 6114.
The bacterial inhibitory action of fungus comb extracts has been shown to diverge considerably within species. Ethyl acetate extract displayed the largest diameter inhibition zone (DIZ) against both Gram-positive and Gram-negative bacteria tested, followed by methanol extract and then water extract (Table 1 and Figure 1). Water extract inhibited all bacteria tested at a high dosage of extract but not for the lowest dosage. S. aureus ATCC 25923 was the most sensitive bacteria to all extracts. Unfortunately, n-hexane extract inhibited only gram positive bacteria but not gram negative bacteria. Gram-positive bacteria are generally more sensitive than Gram-negative bacteria due to the lack of an outer membrane (lipopolysaccharide and lipoprotein). In addition, the cell wall architecture of the Gram-positive bacteria tolerates hydrophobic molecules to easily permeate into the cells (Nazzaro et al. 2013).
The microdilution method is used to determine the smallest concentration of antimicrobials to inhibit the growth of microorganisms known as the Minimum inhibitory concentration (MIC). In addition, we also determined the minimum bactericidal concentration (MBC). MBC is the smallest concentration that can inhibit the growth of microorganisms and kill microbes characterized by the absence of colonies on agar media after scraping from each microplate well after incubation (Migliato et al. 2010 Figure S1, Figure S2).
Comparable to the antifungal agent such as nystatin, fungus comb extracts showed positive activity on inhibition (Table 3 and Figure 2). The highest antifungal activity could be observed from ethyl acetate extract and followed by methanol. Meanwhile, water extract showed the lowest antifungal activity. Unfortunately, n-hexane extract did not inhibit A. avus FNCC 6181 nor A. niger FNCC 6114. The largest DIZ was achieved by fungus comb extracted with ethyl acetate at 50 mg dosage against A. avus FNCC 6181. The result showed that A. avus FNCC 6181 was more sensitive than A. niger FNCC 6114 ( Table 3). The lowest MIC and MFC values were observed for ethyl acetate extract against A. avus FNCC 6181 and A. niger FNCC 6114 ( Table 4).
Among these compounds, bis(2-ethylhexyl) phthalate (DEHP) is an ester of phthalic acid and naturally synthesized by plants or microorganisms such as fungi or bacteria, which demonstrate different biological activities (Ortiz and Sansinenea 2018). DEHP isolated from Calotropis gigantea plant presented antimicrobial activity against E. coli, S. aureus, Bacillus subtilis, Shigella dysenteriae Shigella shiga, Shigella sonnei, Sarcina lutea, and A.
avus (Habib and Karim, 2009  ). DEHP is the major component of n-hexane extract of fungus comb. We proposed that termite Macrotermes gilvus H. might synthesize DEHP to suppress the growth of another species. Unfortunately, n-hexane extract only inhibited Gram-positive bacteria with MIC of 1.56 mg/ml and MBC of 3.13 mg/ml but not fungi Aspergillus. Therefore, there is another compound in the fungus comb that is responsible as an antifungal agent.
Phenolic compound are extensively available in the tissues of plants. This compound also has a critical role to play in highly effective bioactivity.

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