01. Utama.cdr Vol.13, No.4, December 2019, p 109-117 DOI: 10.5454/mi.13.4.1 Antibacterial Activity Test of Indigenous Yeast from Sapodilla Fruit against Staphylococcus aureus and Escherichia coli * GEMILANG LARA UTAMA , MUTIARA NABILA, HENI RADIANI ARIFIN, ELAZMANAWATI LEMBONG, AND TITA RIALITA Faculty of Agro-Industrial Technology, Universitas Padjadjaran, West Java, Indonesia. The research aimed to identify indigenous yeast antibacterial activity from sapodilla fruit against Escherichia coli and Staphylococcus aureus, which conducted by experimental methods and followed by descriptive analysis. This study was done by the isolation of indigenous yeast, macroscopic and microscopic identification, yeast identification using RapID Yeast Plus System, antibacterial test by measuring the clear zone diameter, testing of pathogenic bacteria viability against indigenous yeast and identification of organic acid produced by yeast. The results of yeast isolation obtained 1 isolate (S.cereviseae 1) from fruit and 3 isolates form sapodilla skin (S.cereviseae 2, Candida famata, and Pichia anomala) which had antibacterial activity against E. coli and S. aureus except C. famata isolates. Isolates with the largest antibacterial activity against E. coli and S. aureus based on the clear zone diameter were S. cerevisiae (2) isolates. The results of organic acid analysis by HPLC found that -1 S.cerevisiae (2) isolate produced the highest organic acid namely acetic acid as much as 2.442 mg mL . Key words : antibacterial, organic acid, sapodilla fruit, yeast Penelitian ini bertujuan untuk mengidentifikasi aktivitas antibakteri khamir indigenous buah dan kulit buah sawo terhadap bakteri Escherichia coli dan Staphylococcus aureus yang dilakukan dengan metode eksperimental dan data yang diperoleh dianalisis secara deskriptif. Tahapan penelitian dilakukan dengan isolasi khamir indigenous, pengamatan khamir secara makroskopis dan mikroskopis, identifikasi khamir dengan RapID Yeast Plus System, pengujian aktivitas antibakteri khamir dengan pengukuran diameter zona hambat, pengujian viabilitas sel bakteri patogen terhadap khamir, dan pengujian metabolit asam organik yang dihasilkan oleh khamir. Hasil isolasi khamir didapatkan 1 isolat pada bagian buah dan 3 isolat pada bagian kulit buah sawo. Setelah diidentifikasi didapatkan 2 isolat Saccharomyces cerevisiae, dimana isolat S.cerevisiae (1) merupakan hasil isolasi pada bagian buah sawo, 1 isolat Candida famata, dan 1 isolat Pichia anomala yang memiliki aktivitas antibakteri terhadap bakteri E. coli dan S. aureus kecuali isolat C. famata. S. cerevisiae (2) merupakan isolat khamir indigenous yang menghasilkan aktivitas antibakteri tertinggi terhadap bakteri E. coli dan S. aureus karena menghasilkan asam-asam organik jenis laktat, asetat, sitrat, dan malat dimana asam asetat adalah jenis asam -1 organik tertinggi dengan jumlah 2,442 mg mL . Kata kunci : antibakteri, asam organik, buah sawo, khamir MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-81220272894; Email: lugemilang@gmail.com pathogenic bacteria that cause diarrhea (Gómez-García et al. 2019; Utama et al. 2015). Some bacteria that cause diarrhea like Staphylococcus aureus and Eschericia coli. These bacteria are found in food and can potentially cause infection and food intoxication so that the food consumed can cause diarrhea and even poisoning for consumers (Putri et al. 2015). The results of research by Mukhriani et al. (2017), sapodilla fruit extract was able to inhibit the growth of S. aureus with an optimum concentration of 1500 ppm -1 or 1.5 mg mL , while the results of Arsyad and Annisa (2016) the minimum inhibition concentration of sapodilla fruit extract which could inhibit total E. coli growth is 22.5% (v / v). The antibacterial properties are not only from the compounds contained in the fruit, but can also come from microorganisms such as yeast contained in the fruit. Romano et al. (2019) states that yeast can be Sapodilla (Achras zapota L.) is one type of potential fruit plant that grows in Indonesia. Sapodilla fruit consumption in Indonesia is growing rapidly along with the easy of planting sapodilla and sapodilla plants which can produce fruit throughout the year (Ying et al. 2017). Sapodilla fruit known as an herbal medicine that can cure various diseases, one of which is diarrhea. Sapodilla fruit contains flavonoids, saponins and tannins, besides sapodilla fruit also contains organic acids such as citric acid and malic acid (Murnisyazwani and Rabeta 2019). These compounds are known to have antibacterial properties. According to Jenie (1996) organic acids show antimicrobial activity against many pathogenic microorganisms, including found in a place that is rich in sugar content, for example in fruit. Sapodilla fruit has a sugar content of 14% (Jadhav 2018). The sugar content in sapodilla fruit can act as a substrate for yeast growth. The optimum sugar concentration for yeast growth is 14-18% (Ranalli 2007). It is suspected that sapodilla fruit has yeast which can inhibit the activity of S. aureus and E. coli bacteria like antibacterial activity of sapodilla fruit extract. The antibacterial properties of indigenous yeast from sapodilla fruit are derived from the results of the yeast's own metabolism. According to Raftari et al. (2009), the effect of inhibition on pathogenic bacteria by yeast is largely due to the accumulation of organic acids, where acid will cause a decrease in pH to below the pH range of bacterial growth where these acids are not dissociated and can diffuse rapidly into in pathogenic cells that cause cells to become damaged. Different types of yeast can produce organic acids with different antibacterial activity. Therefore, in this study we aimed to identify indigenous yeast antibacterial activity from sapodilla fruit against Escherichia coli and Staphylococcus aureus, which conducted by experimental methods and followed by descriptive analysis. MATERIALS AND METHODS Chemical Materials. The raw materials used in this study include the skin and sapodilla fruit which are 3 months old, S. aureus, E. coli, NA media (Nutrient Agar), NB media (Nutrient Broth), YMA media (Yeast and Mold Agar), Yeast Extract (Kraft F o o d ) , a n t i b i o t i c s ( A m o x i c i l l i n 5 0 0 m g ) , Chloramphenicol 500mg, aquades, Physiological NaCl,70% alcohol, MSA (Mannitol Salt Agar), EMB (Eosin Methylene Blue), 1% BaCl2, 1% H2SO4, citric acid, tartaric acid, maleic acid, oxalic acid, lactic acid, and acetic acid. The research method used is the experimental method using descriptive analysis. Yeast Isolation. Yeast is isolated from the skin and sapodilla fruit. 1 gram of sample is diluted using physiological NaCl solution of 0.85% until dilution to -3 10 100 µL of each dilution was inserted into the petri dish then poured Yeast and Mould Agar (YMA) media -1 with a composition consisting of 3 g L malt extract -1 agar and 3 g L yeast extract agar and incubated for 48 hours at room temperature. Characterization of yeast isolates was carried out by observing the physical characteristics macroscopically and microscopically from yeast isolates (Balia et al. 2018; Ruriani et al. 2012). Yeast Identification. Identification of yeast types is done using the RapID Yeast Plus System. The results of the color change data are inputted into the website and found yeast isolates (Utama et al. 2016). Antimcrobial Activity Test. Yeast colony swabs in 20 mL of Yeast and Mold Agar (YMA) media aseptically and incubated for 48 hours at room temperature. Swab Liquid culture Staphylococcus aureus and Escherichia coli on Nutrient Agar (NA) evenly. Plug aseptically yeast agar plate, use a sterile forceps or needle to carefully pick up the plug and place them onto each NA plates. Incubate the NA plates at 30°C for 48 h then diameter of the clear zones were measured at 24 and 48 hours (Roostita et al. 2011). Determination of Antibacterial Indigenous Yeast Activity on Viability of Test Bacteria. Fresh cultures of yeast and bacteria were inoculated as 4 -1 follows: 190 μL at a concentration of 1 × 10 cells mL of S. aureus and E. coli with 10 μL at a concentration of 6 -1 3 × 10 cells mL of each yeast, including the following controls: (i) 200 μL of bacterial culture at a 4 -1 concentration of 1 × 10 cells mL , (ii) 198 μL of 4 -1 bacterial culture at a concentration of 1 × 10 cells mL with 2 μL of amoxicilin at a concentration of 100 mg -1 mL . Incubation at 30 C for 48 hours. 1 mL suspension of E. coli bacteria was platted in 20 mL of EMB (Eosin Methylene Blue Agar) media while the suspension of S. aureus bacteria in MSA (Mannitol Salt Agar) media at t = 0, 12, 24, 36, and 48 hours. Incubation was carried out at 37C for 24 hours and TPC calculations were carried out(Acuña-Fontecilla et al. 2017). Identification of Indigenous Yeast Organic Acid Production using HPLC. Yeast samples that have been dissolved in NB media added with yeast extract are filtered and inserted through the injector. The data is a chromatogram that displays retention times in the form of sums of peaks and surface area compared between standard organic acids and organic acids in the sample (Kim et al. 2018). RESULTS Indigenous Yeasts Identification. Based on the results of the yeast isolation of indigenous sapodilla fruit, 1 yeast isolate from sapodilla fruit and 3 yeast isolates from the sapodilla skin were observed macroscopically and microscopically. All data from macroscopic observations have characteristics such as yeast, which is white, round (circular) with prominent elevation in the middle of the colony (ambonate), 110 UTAMA ET AL. Microbiol Indones Volume 13, 2019 Microbiol Indones 111 Table 1 Microscopic and macroscopic characteristics of sapodilla indigenous yeasts smooth or glossy texture (glistening) and full (entire) edge on S.1, S.2, and S.4, while in S.3 has a flat elevation. Based on microscopic observations, the four yeast isolates have a round to oval shape with a range of 3-10 µm. Yeast isolates were then purified to form pure colonies which could be used to identify the types of yeast isolates by biochemical tests using the RapID Yeast Plus Kit. Based on the results of identification, three indigenous yeast species from four isolated isolates were obtained. There are two yeast isolates of Saccharomyces cerevisiae which isolated from fruit (S.1) and sapodilla skin (S.4), other isolates were Candida famata (S.2), and Pichia anomala (S.3) that isolated from sapodilla skin. Indigenous Yeasts Antibacterial Activities. The diameter of clear zone formed is classified into weak (d=0-3 mm), medium (d=3-6 mm), and strong (d>6 mm) (Pan et al. 2009). Based on the Fig. 1, it known the yeast that has strong antibacterial activity against E. coli is S.1 (S. cerevisiae 1) and S.4 (S. cerevisiae 2) isolates. S.3 isolates (P. anomala) have weak antibacterial activity, whereas P.2 (C. famata) isolates do not form clear zone diameters, so it can be said that these isolates do not have antibacterial activity. The antibacterial activities towards for S. aureus has shown S. cerevisiae (1) and S. cerevisiae (2) had moderate antibacterial activity (d=3-6 mm), C. famata has no antibacterial activity because there is no clear zone diameter formation, whereas P. anomala isolates have weak antibacterial activity (d = 0-3 mm) against S. aureus. Indigenous Yeasts Viability towards E.coli and S.aureus. Based on Fig. 2, it is known that almost 100% of E.coli has decreased from the 0 until 12 hour. The effectiveness of the decrease in the number of E.coli cells by S. cerevisiae (2) was 23.7% at 0 hour, while the number of viability of S. aureus decreased by 76.2% at 0 to 12 hours. Organic Acid Production. Based on the Table 3, S. cerevisiae can produce acetic, citric, malic, and lactic acid compounds as indicated by an increase in the amount of organic acid. While oxalic and tartaric acid compounds experience a decrease in the amount of organic acid. DISCUSSION Indigenous Yeasts Identification. The S. cerevisiae colonies are yellowish white, have a circular edge shape, and the surface glistening. S. 112 UTAMA ET AL. Microbiol Indones Table 2 The results of RapID Yeasts Plus System with ERIC analysis Test S.1 S.2 S.3 S.4 Glucose + + + + Maltose - - + - Sucrose + + + + Trehalose - - + - Raffinose - - + - Lipid - - - - NAGA - - - + αGlucoside + + + + bGlucoside + + + + ONPG - - - - αGalactoside - - - - bFucoside + - + - PHS - - - - PCHO - - - - Urea - - - - Prolyne - + - - Histidine + - - + Leucyl-Glycine + - - + Yeast Name S.cereviseae (1) C.famata P.anomala S.cereviseae (2) 1 Table 3 Organic acid production by potential indigenous yeast S.cereviseae (2) Blank S.cereviseae Metabolite Oxalic 2.650 2.489 -0.161 Tartaric 1.862 1.206 -0.656 Lactic 3.968 4.186 0.218 Acetic 1.677 4.119 2.442 Citric 9.542 `0.664 1.122 Malic 0.023 0.044 0.021 1 cerevisiae cells are round (spherical), sometimes ellipsoidal (oval, elongated) to cylindrical, and produce pseudomycelium. The cell size of S. cerevisiae ranges from 5-12´5-10μm (Kurtzman et al. 2011). P. anomala is white, smooth with fine serrated edges. P. anomala has round, elliptical or elongated cells. The cell size of P. anomala ranges from 2-7´2-5μm (Passoth et al. 2006). C. famata forms yellowish white colonies, smooth and shiny texture. C. famata is elongated round (ovoid) with cell size 2.0-3.5´3.5-5.0μm (Dmytruk and Sibirny 2012). Based on observations, it is known that all isolates can hydrolyze substituted glycosides, namely αGLU and β-GLU compounds enzymatically. All isolates are known to hydrolyze glucose and sucrose types of carbohydrates, but only S.3 isolates can hydrolyze maltose and trehalose carbohydrate compounds. HIST and LGY compounds which are amino acid groups can only be hydrolyzed by isolates S.1 and S.4. S. cerevisiae can hydrolyze simple sugars such as glucose and fructose, and can hydrolyze disaccharides like sucrose because it produces the enzyme sukrase (invertase) which converts sugar to be easily fermented (Sainz-Polo et al. 2013). P. anomala can utilize all types of sugar such as glucose, maltose, sucrose, galactose, Volume 13, 2019 Microbiol Indones 113 Fig 2 Indigenous yeasts viabity towards (a) E. coli; (b) S. aureus. (a) (b) Fig 1 Sapodilla indigenous yeasts antibacterial activities towards (a) E. coli; (b) S. aureus. (a) (b) 114 UTAMA ET AL. Microbiol Indones and raffinose, except lactose (Tao et al. 2011). C. famata can ferment glucose, sucrose, and trehalose (Gientka et al. 2016). Indigenous Yeasts Antibacterial Activities. S.cerevisiae isolates can produce antimicrobial metabolites such as organic acids, phenolic compounds, besides S. cerivisiae is known to produce several proteins that have antimicrobial properties (Roostita et al. 2011). S. cerevisiae produces high concentrations of ethanol which are toxic to many microbial species and are capable of producing volatile compounds such as a r o m a t i c a l c o h o l s i n v o l v e d i n i n h i b i t i n g microorganisms (Jouhten et al. 2016). The antibacterial activity of S. cerevisiae with an incubation time of two days caused by the primary metabolites produced by yeast, namely organic acid compounds. Organic acids produced by S. cerevisiae such as acetic acid, malic acid, succinic acid, and lactic acid have strong antimicrobial activity '(Fakruddin et al. 2017). Based on research by Younis et al. (2017), S. cerevisiae showed moderate antibacterial activity against E. coli bacteria and showed weak antibacterial activity against S. aureus. E. coli population decreased after exposed acetic acid, lactic acid, propionic acid, and formic acid, where the reduction in the E. coli population increased with an increase in the concentration of organic acids (Raftari et al. 2009). P. anomala produces ethanol under limited oxygen conditions and produces acetic acid in aerobic conditions. P. anomala produces volatile compounds such as ethyl acetate, ethyl propanoate, phenyl ethanol, and 2-phenylethyl acetate (Passoth et al. 2006). P. anomala can reduce fungal growth in several ways: production of killer poisons, secretion of β-1-3- glucanase, ethyl acetate production or by competition for nutrition (Muccilli and Restuccia 2015). P. anomala is known to produce acetic acid where acetic acid has an inhibitory effect on bacteria. Raftari et al. (2009) stated that E. coli and S. typhimurium had a high susceptibility to lactic acid and acetic acid. P. anomala had the most influential antimicrobial activity on E. coli growth (Walker 2011). The absence of a clear zone indicates no antibacterial activity in C. famata isolates. This can be caused because most of the inhibition of bacteria by C. famata is with produce the optimal killer toxin at pH 4.5 with a temperature of 20 °C. Above pH 4.5 and 20 °C killer toxin activity decreases (Muccilli and Restuccia 2015). Tests are carried out above 20 °C, so the production of killer toxin is inhibited and antibacterial activity cannot take place. The antibacterial activity of indigenous yeast to sapodilla fruit against S. aureus bacteria is lower when compared to E. coli, this can occur because test bacteria have different resistance to different types of organic acids. S. aureus test bacteria have high acid resistance when compared to E. coli, this is caused by differences in the cell wall structure of the two types of bacteria (Lopez-Romero et al. 2015). E. coli bacteria grow at pH 7.0-7.5 while S. aureus bacteria grows at pH 4.0-9.8 (Padan et al. 2005). Indigenous Yeasts Viability towards E. coli and S. aureus. The optimum temperature for the growth of E. coli and S. aureus is 35-37 °C, so that antibacterial testing of yeast carried out to determine the effectiveness antibacterial origin of yeast that could be use as disinfectant for E. coli and S. aureus in the environment. Temperature greatly influences the growth and physiological activities of microorganisms. Temperature differences can affect the speed of bacterial enzyme synthesis, enzyme inactivation, changes in metabolic processes and cell shape, and bacterial growth rate (Suriani et al. 2013). The antibacterial activity of S. cerevisiae with an incubation time of two days was caused by the primary metabolites produced by yeast, namely in the form of organic acid compounds. Organic acids produced by S. cerevisiae such as acetic acid, malic acid, succinic acid, and lactic acid have strong antimicrobial activity (Fakruddin et al. 2017). Organic acids have a bactericidal effect whose effects increase with increasing concentration (Abbott et al. 2009). Differences bacterial viability against organic acids can caused by the ability of strains to adapt in different acidic environments. Gram-positive bacteria such as S. aureus have murein compounds that cause cell wall resistance in gram negative bacteria to be lower than gram-positive bacteria(Nazzaro et al. 2013). The ability of S. aureus to survive in acidic environment is with the phase of adaptation to the acidic environment , namely by pumping protons out of the cell to maintain normal pH and also by increasing the concentration of alkaline compounds in cells to prevent cytoplasmic acidification, repair and degradation mechanisms damaged protein (Bore et al. 2007). Organic Acid Production. Organic acids produced by yeast result in the accumulation of acidic end products and a decrease in pH which will inhibit the growth of both gram-positive and gram-negative bacteria (Kim et al. 2018). The decrease in organic acid is caused by the use of organic acids by yeast for the fermentation process (Walker and Stewart 2016). Volume 13, 2019 Microbiol Indones 115 Changes in the concentration of organic acids illustrate that yeast can freely use organic acids as a source of energy and provide organic acids as intermediate compounds in cell metabolism. During fermentation S. cerevisiae can utilize acid because of the hydrolysis process by enzymes derived from yeast cells (Azhar et al. 2017). S. cerevisiae cannot metabolize tartaric acid, where tartaric acid is the most commonly used acid for pH adjustment in the wine industry (Jolly et al. 2014). Oxalic acid hydrolysis is carried out with enzymes produced by microorganisms (Pal et al. 2016). The highest type of organic acid produced by S. cerevisiae isolates is acetic acid followed by citric acid. S. cerevisiae produced acetic acid where acetic acid production increased with increasing fermentation temperature, where the optimum temperature of acetic acid production was at 30 °C (Shang et al. 2016). The fermentation temperature corresponding to the optimum temperature of acetic acid production allows the highest production of acetic acid by S. cerevisiae. Acetic acid formed from glucose fermentation under aerobic conditions (Gomes et al. 2018). The synthesis of citric acid by S. cerevisiae is higher with dissolved oxygen in the media that is getting higher (Walker and Stewart 2016). Yeast can produce small amounts of lactic acid. Lactic acid produced at 28−30 °C. Lactic acid formed by the reduction of pyruvic acid and the transformation of malic acid (Vilela 2019). The best temperature for producing malic acid is 18−25 °C with optimum fermentation time for 2 to 3 days. Based on observations of S. cerevisiae yeast produce small amounts of malic acid, this can be caused because most of the malic acid produced has been transformed into lactic acid or used as an energy source. Several studies have reported the inhibitory effects of various organic acids on pathogenic or destructive microbes, including Blom et al. (1997) which states that the use of organic acid in the form of 2.5% lactic acid and 0.25% acetic acid can extend the shelf life of roast pork for up to 5 weeks (Silano et al. 2018). Other studies have also been reported by Castilo et al. (2001) who used lactic acid solution at a concentration of 4% (v / v) by spraying on beef carcass turned out to be effective in reducing pathogenic microbes such as E. coli (Castillo et al. 2001). Giving of citric acid 5.5%, 0.75% lactic acid and 0.5% malic acid can inhibit the growth of S. aureus and E. coli (Al-Rousan et al. 2018). 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