STABILIZATION OF RED MELINJO PEEL (GNETUM GNEMON L.) ETHYL ACETATE EXTRACT AS ANTIBACTERIAL AGENT

Melinjo (Gnetum gnemon L.) is a typical Indonesian plant that has many benefits such as antimicrobial agent. The aim of this study was to determine the antimicrobial activity of red melinjo peel extract. In this study, extraction was conducted by maceration using ethyl acetate as solvent for 24 hours at room temperature. 4-16% red melinjo peel extract (w/v) could inhibit the growth of Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 7644 and Salmonella Typhi ATCC 14028. However, 4-16% red melinjo peel extract could not inhibit the growth of Candida albicans ATCC 10231. In stability test, the selected extract had a stable inhibition at pH 4-7, heat treatment 65 95 °C for 30 minutes, salt 1%-5%, and sugar 10%-50%. The selected extract produced the biggest inhibition diameter at low pH (pH 4) and produced the smallest inhibition diameter at neutral pH (pH 7). Heat treatment 65 °C for 30 minutes produced the biggest inhibition diameter among tested bacteria and decreased with increasing heating temperature. Addition of 1-5% NaCl and 10-50% sucrose worked synergistically with the selected extract in inhibit the growth of the tested bacteria.Abstract in English.


INTRODUCTION
Melinjo (Gnetum gnemon L.) is a typical Indonesian plant which is rich in flora diversity. Melinjo belongs to Gnetaceae family that is originated from Indo-Malaya and Melanesia and widely cultivated from Southeast Asia to Fiji (Manner and Elevitch, 2006). In Indonesia, melinjo is commonly processed into emping (chips from melinjo seeds) and vegetables in various soup (Kato et al., 2009).
According to BPS or Indonesian Central Bureau of Statistics (2016), melinjo production increased about 7.78% from 197,648 tons in 2014 becoming 213,025 tons in 2015. This increasing of melinjo production consequently will increase the melinjo peel waste as well. Unfortunately, the utilization is still limited. In fact, melinjo peel has many benefits, one of them is as an antimicrobial agent. Parhusip and Sitanggang (2011) explains that melinjo peel had antibacterial activity against Bacillus cereus, Staphylococcus aureus, Enterobacter aerogenes and Pseudomonas aeruginosa.
The use of red melinjo peel in this study was based on the study conducted by Cornelia et al. (2009) that it had the highest phenolic compounds (0.386 mg GAE/g samples) compared to yellow and green melinjo peels. The phenolic compounds can inhibit the growth of Gram positive bacteria (Septiadi et al., 2013). In this study, the melinjo peel extract will be used to test its antimicrobial activity toward Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhi, and Candida albicans which represented Gram positive bacteria, Gram negative bacteria, and yeast. The four types of microbes are pathogenic microbes that often contaminate food products and cause disease if consumed (Parhusip and Sitanggang, 2011) The melinjo peel extract was obtained through extraction and maceration using ethyl acetate as a solvent. Parhusip and Sitanggang (2011) reported that ethyl acetate extract of red melinjo peel produced a better inhibition diameter compared to ethanol extract. In addition, ethyl acetate has low toxicity, and is able to attract polar and nonpolar compounds, easily evaporated, inexpensive, and easily obtained (Putri, 2013).
In this study, ethyl acetate extract of red melinjo peel will be tested to inhibit S. aureus, Listeria monocytogenes, Salmonella Typhi, and Candida albicans, then the optimum concentration of ethyl acetate will be determined to inhibit the growth of the tested micorbes. Stability test will be tested using the selected extract which is carried out at pH of 4-5, temperature of 65-95 °C for 30 min, and addition of 1-5% NaCl solution, and 10-50% sucrose. Stability test is required to see its inhibition in certain pH, heat temperature, salt, and sugar concentration as an initial step to be applied on food products. Based on the previous study, the 1-5% salt concentration was chosen in this study because in general the addition of 1-2% salt gives a salty taste in food products. Salt with a concentration of 5-15% used to preserve food or food products (Yusmita, 2018). Meanwhile, the sugar concentration was chosen from 10% to 50%, because in general the addition of sugar concentration up to 40% gives a sweet taste to food products. If the concentration is more than 40%, the sugar will act as a preservative in food products (Utomo et al., 2015).

Material Preparation and Red Melinjo Peel Extraction (Parhusip and Sitanggang, 2011)
Melinjo seed was sorted, washed, and drained, then, the peel was separated from its seed. Melinjo peel was dried in a cabinet dryer until its moisture content was around 10%. The dried peel was grinded using a blender and sifted to obtain red melinjo peel powder.
The dry powder of melinjo peel was mixed with ethyl acetate as an extraction solvent with a ratio of (1:4 w/v). The mixture was stirred using a shaker at 150 rpm for 24 h at room temperature, then filtered using a Whatman filter paper No. 1 and vacuum pump. The filtrate was concentrated using a rotary evaporator at temperature of 55 °C and blown with Nitrogen to obtain a crude extract. The crude extract was diluted into 4-16% concentration by weighting 0.4, 0.8, 1.2, and 1.6 mg in 10 mL ethyl acetate.

Antimicrobial Activity Test of Melinjo Extract with Well Diffusion (Parhusip and Sitanggang, 2011)
A 40 µL culture of each test microbe was distributed into the media on a petri dish and contacted with the extract, so the concentration of each test microbe is about 10 4 -10 5 CFU/40 Μl. Let the media solidify for 30 minutes and make 5 holes with 6 mm diameter each. A 0.5 mL of each concentration of red melinjo peel extract was aseptically inserted into wells 1-4 (4%, 8%, 12%, and 16% (w/v) respectively). Well 5 was a control which ethyl acetate solution was used as a control. Then, the dish was incubated for 24 h at 37 °C and observed their inhibition diameter on the next day by measured to the nearest mm. The most effectiveness extract concentration obtained in this test will be further used for stability test, it is done by statistical analysis.

Stability Test of the Selected Extract at pH, Temperature, Salt and Sugar Concentration (Romson et al., 2011, Winarti et al., 2008, and Adriansyah et al., 2003)
The selected extract was put into a test tube containing a different pH solution on each tube. The four pH levels were pH 4, 5, 6 and 7. The temperatures for stability were 65, 75, 85, and 95 °C for 30 minutes.
The sugar stability test was done by inserting the selected extract into a tube containing 10%, 20%, 30%, 40% and 50% sucrose, respectively. The stability test for salt was done by inserting the selected extract into 6 tubes containing NaCl solution with different concentrations (1%, 2%, 3%, 4% and 5%). The inhibition diameter was observed using well diffusion method with the selected extract (no sucrose or NaCl added) as a control.

Antimicrobial Activity of Red Melinjo Peel
The inhibition diameter observed on the NA media indicated the antimicrobe activity of the melinjo peel extract. Ethyl acetate extract of red melinjo peel showed the ability to inhibit the growth of test bacteria from concentrations of 4% to 16%. However, the extract was not able to inhibit the growth of C. albicans at such Concentrations (Table 1 and Fig. 1).
The inability melinjo peel extract to inhibit C. albicans caused by the presence of chlamydospore in yeast, an asexual spore at the end of hyphae which forms a thick wall thus cannot penetrate by the antimicrobial compounds (Jawetz et al., 2005). Besides, yeast is a eukaryotic organism which has more stable membrane than prokaryotic organisms due to sterol component in its cytoplasm. This caused the red melinjo extract hardly interfere the yeast cell permeability (Madigan et al., 2009  The inability melinjo peel extract to inhibit C. albicans caused by the presence of chlamydospore in yeast which is difficult to penetrate with antimicrobial compounds. Chlamydospore is an asexual spore at the end of hyphae which forms a thick wall so that it is difficult to penetrate antimicrobial compounds (Jawetz et al., 2005). Besides that, yeast is eukaryotic organism which has more stable membrane than prokaryotic organisms due it has a sterol component in its cytoplasm. This caused the red melinjo ethyl acetate extract at concentrations of 4%, 8%, 12%, and 16% couldn't interfere yeast cell permeability (Madigan et al., 2009).
The 16% extract produced the largest diameter inhibition compared to other concentrations. S. aureus and L. monocytogenes had a larger inhibitory diameter than S. Typhi. This result was match with theory that Gram negative bacteria have more complex cell walls composed of lipopolysaccharide, proteins and have peptidoglycan (Madigan et al., 2009). From the results of visual observations and statistical analysis showed that each concentration could be used as the chosen concentration. The selection of selected extract will be based on extract efficiency and suitability with the increase in inhibition diameter produced. The selection of extracts concentrations referred to Widyasanti et al. (2016) theory, that the inhibition diameter of 20 mm or more classified as very strong, the inhibition diameter is 10-20 mm classified as strong category, the inhibition diameter is 5-10 mm classified as medium category, and the inhibition diameter is 5 mm or less classified as weak category. Based on the theory, red melinjo peel ethyl acetate extract was classified as strong category. Table 1 showed that at 12% concentration, all test bacteria produced inhibition diameters greater than 10 mm. Therefore, the extract selected in this study was 12% concentration extract.

Extract Stability Test on pH
Testing the extract at the pH aims to see the stability of the selected extract in various acidic to neutral conditions (pH 4-7). The selected extract itself has a pH of 4.93 before it was tested into various pH solutions. Most foods are produced at that pH range, thus was chosen in this study (FDA, 2008). Fig. 2 shows that the average inhibition diameter for each pH level was greater than 10 mm, thus can be classified as a strong inhibitory category (Widyasanti et al., 2016). This means that red melinjo ethyl acetate extract had a good stability and strong inhibition capacity, even though the pH varied from 4 (acid) to 7 (neutral). pH 4 gave the highest value, while other pH levels were not significant. According to Silhavy et al. (2010), bacteria would be more difficult to grow at acidic condition. This stability test reflects that red melinjo extract is potential to be applied for food products that have pH ranging from 4 to 7, such as meat, fish, milk, corn, spinach, asparagus, beets, and yellow walnut (FDA, 2008).

Figure 2. Test results of pH level affect the average of inhibition diameter on pH stability test
There was no interaction between the type of test bacteria and the pH level on the inhibitory diameter., However, significant effect was seen on the inhibitory response of the test bacteria (Fig. 3). S. aureus had the highest inhibition diameter (14.81 mm), followed by L. monocytogenes and S.
Typhi (both were not significant) with inhibition diameter of 13.57 mm (Fig. 4). These results are in accordance with the study of of Silhavy et al. (2010) that the growth of Gram-positive bacteria was easier to be inhibited than Gram negative due to differences in the composition of membrane cell.

Extract Stability Test on Temperature
Heating temperature of the selected extract was chosen between 65 and 95 °C to see extract ability for heat treatment, especially for pasteurization and blanching.    (Kyung et al., 1997). From the statistical analysis showed that there is an interaction between the type of bacteria and the diameter of inhibition produced. S. Typhi produced the smallest diameter average (resistance) to the heating temperature and S. aureus produced the largest (vulnerable) average diameter of the heating temperature. This results are in accordance with the study of Silhavy et al. (2010) that Gram positive bacteria are easier to inhibit growth than Gram negative Elevated heating above 65°C showed a decrease in the inhibition diameter of each test bacteria. The heating treatment at 95 °C produced the smallest inhibition diameter compared to the control or other heating temperatures. This was caused by bioactive component damage when exposed to high temperatures (Ewald et al., 1999). From the stability test of extracts to heating temperature, red melinjo ethyl acetate extract had to be applied for pasteurized food products, such as milk and fruit juice drinks, as well as leaf products, such as fruit and vegetables on blanching process (Singh and Lovedeep, 2009).

Extract Stability Test on Salt
The addition of 1-5% salt concentration was chosen in this study because in general the use of 1-2% salt gives an acceptable salty taste in food products. Meanwhile, salt with a concentration of 5-15% is used to preserve food or food products (Yusmita, 2018). Table 3 and Fig. 6 showed the greater salt concentration added to the selected extract, the greater inhibition diameter produced in each test bacteria. There is an interaction between salt concentration and the response of each bacteria. Statistical analysis showed that the inhibition diameters produced by the control differed significantly from the inhibition diameter at 1-3% salt concentration. Meanwhile there was no significant difference in inhibition diameter produced at concentrations of 1-3%, but differed significantly from the concentration of 4%. The diameter of inhibition produced at a concentration of 4% was significantly different from the concentration of 5% (p≤0.05). These results reaffirm that the ethyl acetate extract of red melinjo peel had a good stability and works synergistically with salt concentration.  Figure 6. Inhibition diameter of L. monocytogenes (A), S. Typhi (B), and S. aureus (C) at 1-5% NaCl solution Salt works synergistically with the extract in inhibiting the growth of test bacteria as salt has a high osmotic pressure that causes plasmolysis in bacterial cells (Ahillah et al., 2017). In addition, NaCl is hydroscopic thus it can bind water molecules, resulting in lower Aw (Madigan et al., 2009).
Based on this stability test, red melinjo extract had a potential to be applied for food products that contain 1-5% salt, such as biscuits, extruded foods, cakes, noodles, porridge, chocolate drinks, canned tuna, and selected seafoods (NHRI, 2018).

Extract Stability Test on Sugar Addition
The sugar concentration was chosen from 10% to 50% in this study, because the addition of sugar concentration up to 40% to food products generally gives an acceptable sweet taste. If the concentration is more than 40%, the sugar will act as a preservative in food products (Utomo et al., 2015).
Based on the stability test (Table 4 and Fig. 7), the greater the concentration of sugar added to the extract, the greater diameter of inhibition produced in each test bacteria. The presence of sugar works synergistically with the extract in inhibiting the growth of test bacteria as it can reduce the water content of bacterial cells, thus limits the microbial living activities. This result in disruption of cell metabolism which leads to cell death. When sugar with high concentrations added to food, it can block the microbial growth and decrease the water activity (aw) (Buckle et al., 2009).
There is an interaction between the sucrose concentration and inhibition diameter of test bacteria (Fig. 10). S. aureus produced the largest (sensitive) average inhibition diameter when added with sugar concentration, while S. Typhi produced the smallest inhibition diameter (resistant). Statistical analysis showed that both controls and 10-50% concentrations did not differ significantly.   This confirmed that the red melinjo peel ethyl acetate extract had a good stable at 10-50% sugar concentration and synergistic with extract. Silhavy et al. (2010) revealed that Gram positive bacteria are more sensitive to the antimicrobial agent than Gram negative because the differences in their cell membrane structures. From this stability test, red melinjo ethyl acetate extract had a potential to be applied as an antimicrobial agent to food products that have 10-50% sugar content, such as carbonated drinks, fruit juices (Walker, 2014), formula milk, yogurt, wafers, brownies, crackers, donuts, and some baby foods (Walker and Goran, 2015).

CONCLUSION
A 12% red melinjo peel ethyl acetate extract was able to inhibit the growth of S. aureus, L. monocytogenes, and S. Typhi with categorized strong inhibitory strength (average inhibition diameter was 13.08 mm). The selected extract had a good stability at 4-7 pH, 65-95 o C for 30 minutes heating temperature, 1-5% NaCl concentration, and 10-50% sucrose concentration. So, red melinjo peel ethyl acetate extract had a potential to be applied to food products widely. Further research is needed regarding quantitative phytochemical testing to see the percentage of bioactive components contained in the extracts. In addition, an in vivo toxicity test is needed to be carried out as an initial step for the extract application as an antibacterial agent in food products.