CONTACT : NADHILA IDRIS nadhilaidris14@gmail.com 36 Abstract Mustard g reens Brassica juncea L. one of vegetable that is very easily damaged by microorganisms known as soft rot disease. This causes a decrease in the quality of green mustard so that it cannot last long. A study entitled “The Potential of Hexadecanoic Acid Compounds as Antimicrobials in Bacteria and Fungi that Cause Decay in Mustard Greens Brassica juncea L.". This research aims to specify effect of hexadecanoic acid compounds in inhibiting the growth of bacteria and fungi that cause decay in mustard greens. An inhibition test was carried out on Xanthomonas campestris bacteria and Fusarium oxysporum fungus using 10%, 20%, and 40% hexadecanoic acid test compounds. The results obtained showed that 10%, 20%, and 40% hexadecanoic acid extracts were able to inhibit the growth of Xanthomonas campestris bacteria and the fungus Fusarium oxysporum. Hexadecanoic acid compounds are bacteriostatic in Xanthomonas campestris and fungi Fusarium oxysporum are fungistatic. ISSN : 2580-2410 eISSN : 2580-2119 Potential of Hexadecanoic Acid as Antimicrobials in Bacteria and Fungi that Cause Decay in Mustard Greens Brassica juncea L. Nadhila Idris1*, Eva Johannes1, Zaraswati Dwyana1 1 Department of Biology, Mathematic and Natural Sciences Faculty, Hasanuddin University, Makassar, Indonesia Introduction Indonesia is an agrarian country, because of that agriculture have an crucial role within the standard the overall wheels of the country's economy. T his can be proven by the large number of people who work in agriculture as a source of livelihood which is also supported by suitable natural conditions that allow residents to plant throughout the yea r (Suratman, 2018). Green mustard Brassica juncea L. is a vegetable commodity that is in great demand by the people of Indonesia because it has commercial value and good prospects from various aspects. In addition, mustard has a high demand and is always increasing along with the increasing population in Indonesia and increasing public awareness of the importance of nutritional needs (Sarif, et al, 2015). OPEN ACCESS International Journal of Applied Biology Keyword Mustard greens Brassica juncea L; Hexadecanoic acid; Xanthomonas campestris; Fusarium oxysporum; Article History Received March 3, 2022 Accepted December 14, 2022 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. International Journal of Applied Biology, 6(2), 2022 37 But in its cultivation it often has drawbacks because it is easy for green mustard to be attacked by diseases caused by bacteria and fungi that cause decay to occur in mustard greens so that the quality of mustard greens decreases and the selling price of mustard greens decreases. Some of the causes of decay in green mustard a re Xanthomonas campestris bacteria, and there is also Fusarium oxysporum fungus which can be some of the rots in mustard greens (Semangun, 2004). Theref ore, natural preservatives are needed to inhibit the bacteria and fungi can cause decay in mustard greens . One of the natural preservatives that can be used to inhibit bacteria and fungi that cause decay in green mustard is hexadecanoic acid which is an isolate from the hydroid Aglaophenia cupressina Lamoureoux. by Johannes, 2009. Hexadecanoic acid is a deriva tive of carboxylic acid which has antibacterial and antifungal properties, so it is hoped that this study can extend the shelf life of mustard greens . Materials and Methods Materials The materials used in this study included hexadecanoic acid compounds, Xanthomonas campestris bacteria culture (InaCC B1449), Fusarium oxysporum fungus culture (InaCC F641), Potato Dextrose Agar (PDA) medium, label paper, Nutrient Agar (NA) medium, sterile distilled water, 70% alcohol, physiological NaCl 0.9%, aluminum foil, cotton, cotton swab, Ciprofloxacin, and Ketoconazole. Methods This study used the well diffusion method to test the activity of the hexadecanoic acid compound by looking at the inhibition zone f ormed a round the well. Place 5 containers on nutrient agar medium and potato dextrose agar medium in each petri dish. The media containing the suspension of bacteria and fungi pour into each petri dish and are allowed to solidify. The reservoir is removed to form a well for the test solution (Pangemanan, et al, 2016). Hexadecanoate extract with 3 different concentrations (10%, 20%, 40%), positive control (Ciprofloxacin) for antibacterial test, positive control (Ketoconazole) for antifungal test, and negative control (aquades) was dripped as much as 50 l on each Each different well was carried out in duplicate and then incubated in an incubator at 37 oC for 24 hours for bacteria and 72 hours for fungi. Diameter of the inhibition zone formed was observed and measured using a caliper (Pangemanan, et al, 2016). Results and Discussion Hexadecanoic Acid Activity Test Against Xanthomonas campestris Bacteria The results of the observation of the inhibition zones formed at extract concentrations of 10%, 20%, and 40% on Xanthomonas campestris bacteria after incubation 24 hours and 48 hours can be seen in Figure 1 below. International Journal of Applied Biology, 6(2), 2022 38 Figure 1. The results of the inhibition test of 10%, 20% and 40% hexadecanoic acid extract and control against Xanthomonas campestris after incubation for 24 and 48 hours. Table 1. Average diameter of inhibition zone of hexadecanoic acid extract with concentrations of 10%, 20%, 40%, and control against Xanthomonas campestris bacteria after 24 hours and 48 hours incubation periods. Concentrations Average diameter of inhibition zone (mm) Xanthomonas campestris 24 hours 48 hours 40% extract 48,22 47,87 20% extract 43,35 43,15 10% extract 32,37 32,04 + Control 37,4 34,3 - Control 0 0 BaseduonuTable 1,uthe measurement resultsuofuthe hexadecanoic acid extract with concentrations of 10%, 20%, and 40% showed a clear zone around the well in the Xanthomonas campestris bacterial culture. T he admi nistration of 10%, 20% and 40% hexadecanoic acid extract and positive control (Ciprofloxacin) in each well showed a clear zone which was the inhibition zone of each treatment. The measurement results above show a decreaseuin the diameteruof the inhibition zone after observations were made at incubation ti mes of 24 hours and 48 hours. This indicated that the hexadecanoic acid extract was bacteriostatic against Xanthomonas campestris bacteria. According to Sinurat, et al, (2019), Bacteriostatic means a substance can inhibit theugrowth ofubacteria which is characterized decrease in theuarea of the inhibition zone which is directly proportional to the increase in the incubation period. When the administration of antimicrobial compounds is stopped, microbial growth will increase again because the compounds given cannot kill but only inhibit microbial growth. It could be seen that the inhibitionuzone formed will have a larger diameter when given hexadecanoic acid extract with a higher concentration. According to Cappucino (1978), the difference in the size of the large or small diameter of the inhibition formed is influenced by the growth rate of the microbe, the sensitivity of the microbe to the active substance, the ability of the active ingredient to diffuse in the medium, and the viscosity of the medium used. The cause of the inhibition by antimicrobial substances is due to interference with cell membranes from microbes, inhibition of enzyme work, disruption of protein and nucleic acid synthesis, or inhibition of cell wall synthesis (Pelczar dan Chan, 1998). There is a reaction between the hydroxyl group of lipopolysaccharide which is a constituent of the cell wall with hexadecanoic acid, causing changes in the structure of the lipopolysaccharide membrane from the cell wall to being asymmetrical. This causes the cell 24 hours 48 hours International Journal of Applied Biology, 6(2), 2022 39 to become lysed or damaged due to disruption of the balance of the lipid membrane structure so that it will disrupt the integrity of the bacterial cell membrane (Sjafaraenan, et al, 2021). Based on the observations, it can beuseen that the 40%uconcentration has the largest inhibition area among the other concentrations used, this indicates that the hexadecanoic acid extract has antibacterial activity. Of the three concentrations, 10%, 20%, and 40% were categorized as having very strong inhibito ry power because they showed an inhibitory power of more than 20 mm, which means they are classified as very strong (Davis dan Stout,1971 in Palupi dan Nugraha, 2014). ,In this study, Ciprofloxacin was used as a positive control in the inhibition test of Xanthomonas campestris bacteria. Ciprofloxacin was used as a comparison of the effects of drugs, antimicrobials, standard with hexadecanoic acid extract test solution. Ciprofloxacin is a fluoroquinolone antibiotic that has the ability to inhibit bacterial D NA synthesis so that it becomes anti-microbial. Ciprofloxacin is also an antibacterial that is can againstuGram- positiveuanduGram- negativeubacteria, theref ore Ciprofloxacin is often used as a treatment for several infections caused by bacteria (Castro, et al, 2013). Ciprofloxacin was used as a positive control because Ciprofloxacin has an antibacterial against Xanthomonas campestris. The data obtained above shows a reduction in the area of the inhibition area which indicates that Ciprofloxacin is bacteriostatic against Xanthomonas campestris bacteria. Similar results were reported by Rojas, et al. (2019) which stated that Ciprofloxacin was bacteriostatic against Xanthomonas campestris which was characterized by a reduction in the area of the inhibition area. Hexadecanoic Acid Activity Test Against Fusarium oxysporum Fungus Figure 2. Resultsuof the inhibition test of hexadecanoic aciduextract at concentrationsuof 10%, 20% and 40% and control of the fungus Fusarium oxysporum after incubation for 48 and 72 hours. 48 hours 72 hours International Journal of Applied Biology, 6(2), 2022 40 Table 2. Average diameter of inhibition zone of hexadecanoic acid extract with concentrations of 10%, 20%, 40%, and control against Fusarium oxysporum fungus after 48 hours and 72 hours incubation periods. Concentrations Average diameter of inhibition zone (mm) Fusarium oxysporum 48 hours 72 hours 40% extract 22,6 21,45 20% extract 19,25 18,4 10% extract 11,7 10,34 + Control 35,4 34,65 - Control 0 0 BaseduonuTable 2, it can beuseen that there were clear zones formed in the 10%, 20%, 40% hexadecanoic acid extract and in the positive control (Ketoconazole). The hexadecanoic acid extract could inhibit the fungus Fusarium oxysporum which was seen from the inhibition zone that appeared around the well. , it can beuseen that the inhibition area formed atua concentra tion of 40% had the largest area of the inhibition zone compa red to other extract treatments. After observing the incubation time of 48 hours and 72 hours, there was a decrease in the area of inhibition because the hexadecanoic acid extract was fungistatic. According to Jana, et al (2020) in Sjafaraenan (2021), an antimicrobial substance is fungistatic if there is no increase in the a rea of the inhibition zone after incubation and the second observation is because the antimicrobial antimicrobial substances are not able to kill microbial growth. Based this results above, the concentrati onuof 40% has the largest inhibition among other concentrations. The area of the inhibition zone at a concentration of 40% is included in the very strong group because it has an inhibitory zone area of more than 20 mm. Meanwhile, at concentrati ons of 20% and 10%, it is included in the strong group because it has an area between 10-20 mm according to Davis and Stout (1971) in Palupi and Nugraha (2014). Hexadecanoic acid has the capability to inhibit fungal accretionby forming complex compounds when it will bind to the active groups of fungal cell walls. Fungal cells have chitin compounds in their cell walls. Although there is a reaction between hexadecanoic acid and cell wall active groups, this reaction will only reac t with the outer ring structure (CO2-OH) and cannot da mage the main structure of chitin in fungal cell walls. This causes the lack of this reaction affects the integrity of the fungal cell wall so that the hexadecanoic acid compound only inhibits or does not kill fungal cells. (Johannes, 2013). In wells with positive control, Ketoc onazole ha d an antifungal effect. The data obtained above showed a reduction in the inhibiti on area which indicated that Ketoconazole was fungistatic against the fungus Fusarium oxysporum. Ketoconazole was used as a positive control because Ketoc onazole is a synthetic broad-spectrum antifungal drug that bel ongs to the imidazole group. Imidazoles and triazoles are from the azole group which are synthetic compounds. Ketoconazole was the first oral azole to be used clinically from several other drugs (Lely, et al, 2017). In addition to the negative control, in this study aquadest was used as negative control which was also used as a solvent for the hexadecanoic acid extract. The purpose of using distilled water as a negative control is to prove that the solvent used does not affect International Journal of Applied Biology, 6(2), 2022 41 the antimicrobial test results of the compounds to be tested. Table 1 and table 2 state that distilled water did not show antibacterial and antifungal activityiwhich was indicatedibyithe absence of aniinhibition area formed around the well. Thus it can be believed that the use of distilled water as a solvent does not affect the antimicrobial test results of the extract . Conclusions Extracts of hexadecanoic acid compounds have an effect on inhibiting bacteria and fungi that cause decay in mustard Brassica juncea L. based on inhibitory and bacteri ostatic tests on Xanthomonas campestris and fungistatic on Fusarium oxysporum. International Journal of Applied Biology, 6(2), 2022 42 References Cappucino, G. J., dan Welsh, C., 2018, Microbiology A Laboratory Manual Eleventh Edition, Pearson Education Limited, England. Castro, W., Navarro, M., dan Biot, C., 2013, Medicinal Potential Of Ciprofloxacin and Its Derivatives, Journal Future Medicinal Chemistry, Vol. 5(1). Johannes, E., 2013. Pemanfaatan Senyawa Bioaktif Hasil Isolat Hydroid Aglaophenia cupressina Lamoureoux sebagai Bahan Sanitizer pada Buah dan Sayuran Segar, Disertasi. Universitas Hasanuddin, Makassar. Lely, N., Pratiwi, R. I., dan Imanda, Y. L., 2017, Efektivitas Antijamur Kombinasi Ketokonazol dengan Minyak Atsiri Sereh Wangi (Cymbopogon nardus (L.) Rendle), Indonesian Journal of Information Systems, Vol. 7(2). Pangalinan, F. R., Kojong, N., dan Yamlean, P. V. Y., Uji Aktivitas Antijamur Ekstrak Etanol Kulit Batang Rambutan (Nephelium lappaceum L.) Terhadap Jamur Candida albicans Secara In Vitro, PHARMACON. Pangemanan A., Fati mawali., dan Budiarso F., 2016, Uji daya hambat ekstrak rimpang kunyit (Curcuma longa) terhadap pertumbuhan bakteri Staphylococcus aureus dan Pseudomonas sp., Jurnal e-Biomedik, Vol. 4(1); 81-85. Palupi, N. W., dan Nugraha, A. S., 2014, Pemanfaatan Benalu Kapas Sebagai Salah Satu Sumber Bahan Antimikroba Alami : Kajian Aktifitas Antimikroba, Jurnal Ilmiah INOVASI, Vol. 14 (1). Pelczar M.J., Chan ECS., 1988 . Dasar-dasar mikrobiologi 2. Diterjemahkan oleh Hadioetomo RS, Imas T, Tjitrosomo SS, Angka SL. Jakarta: Penerbit Universitas Indonesia. Rojas, M., Pena, M., Vera, M. J. P., Sulbaran, M., Perez, E., Velasquez, C. L., 2019, Characterization and determi nation of antimicrobial and metal resistant profiles of Xanthomonas strains isolated from natural environments, Journal of Analytical & Pharmaceutical Research, Vol. 2. Sarif, P., Hadid, A., dan Wahyudi, I., 2015, Pertumbuhan dan Hasil Taanaman Sawi (Brassica juncea L.) Akibat Pemberian Berbagai Dosis Pupuk Urea, e-Journal Agrotekbis, Vol. 3 (5). Semangun, H., 2004, Penyakit-penyakit Tanaman Hortikultura Di Indonesia, Gadjah Mada University Press,Yogyakarta. Sinurat, A. A. P., Renta, P. P., Herliany, N. E., Negara, B. F. S. P., Purnama, D., 2019, Uji Aktivitas Antibakteri Ekstrak Metanol Rumput Laut Gracilaria edulis Terhadap Bakteri Aeromonas hydrophila, Jurnal Enggano, Vol. 4(1). Sjafaraenan, Johannes, E., dan Tuwo, M., 2021, Efektivitas Senyawa Asam Heksadekanoat dan β-Sitosterol Isolat Dari Hydroid Aglaophenia cupressina Lamoureoux Sebagai Bahan Antimikroba Pada Bakteri Salmonella thypi dan Jamur Aspergillus flavus, BIOMA : Jurnal Biologi Makassar, Vol. 6(1). Suratman, Y. Y. A., 2018, Analisis Pendapatan Usahatani Sawi (Brassica juncea L.) Di Kelurahan Landasan Ulin Utara Kecamatan Liang Anggang Kota Banjarbaru, Jurnal Ziraa’ah, Vol. 43 (2).