Antibacterial (Staphylococcus aureus and Escherichia coli) and Antifungal (Saccharomyces cerevisiae) Activity Assay on Nanoemulsion Formulation of Ethanol Extract of Mangosteen Leaves (Garcinia mangostana L.) as Fruit Preservative

Fruits after harvesting will decay faster if not handled properly. Fruits can be demaged by bacterial and fungal. Mangosteen leaves contain xanthons which are antibacterial and antifungal. 50% ethanol extract of mangosteen leaves is formulated in Nanoemulsion preparations using the SNEDDS (Self-Nanoemulsifying Drug Delivery System) method. The mangosteen leaves 50% ethanol extract nanoemulsion formulation consisted of VCO (Virgin Coconut Oil) as oil, tween 80 as surfactant, and PEG 400 as cosurfactant. There are 3 formulations with variations in the concentration of mangosteen leaves ethanol extract, there are concentrations of 1%, 2%, and 3%. All formulations have a T% of more than 90%. The results of the particles measurement of nanoemulsion using PSA were in formulation 1 amounting to 16.1 nm; formula 2 is 16.7 nm; and formula 3 is 16.6 nm. The zeta potential characterization shows that formula 1 has a zeta value of -40.9 mV. The three formulations had a pH of 5. The largest inhibitory zone in the mangosteen leaf ethanol extract against S.aureus and E. coli bacteria were 11.08 mm and 5.87 mm respectively. Whereas in the S. cerevisiae antifungal test there was no inhibition zone at all concentrations. In the antibacterial and antifungal tests nanoemulsion preparations did not produce inhibitory zones in each concentration. Nanoemulsion preparations can retain the quality of strawberries when compared to the non-nanoemulsion preservative group, both in room storage and refrigerator temperature. The best preservative result is when the fruit is coated with nanoemulsion preservative and stored in refrigerator temperature.


INTRODUCTION
Indonesia has abundant natural resources including agricultural produce in the form of fruits and vegetables. Fruits that are not handled properly will decompose faster. This is because the process of respiration and transpiration in fruit that will continue after the fruit is harvested. This respiration takes place aerobically by requiring oxygen and producing CO2 and H2O. Increased respiration during ripe fruit causes the overhaul of polysaccharides and cell wall compilers to run fast, so that the texture of the fruit will be softer. Besides that, fruit damage can also be caused by mechanical influences, for example due to impact, scratches on the skin or microbiological damage such as decay by microbes, so that the shelf life is relatively short (Purwadi, n.d., 2007). The example of microbial that can damage the fruits quality is bacterial and fungal. Based on that statements, antimicrobial is needed as preservative fruits.
According to Friedman et al. (2002) and Gulmez et al. (2006), the use of plant and herbal extracts as antimicrobial agents in food and soft drinks has also been reported for centuries (Jabeen and Khanum, 2017). Chemical food preservatives have the potential for toxicity, so it will increase the demand on natural food preservatives. Antimicrobial agents used as preservatives are chemical compounds that protect food from spoilage by inhibiting the growth of pathogenic microorganisms and increasing shelf life (Jabeen and Khanum, 2017).
The mangosteen plant has antioxidant compounds consisting of xanthones, tannins, phenolic acids, and anthocyanins. Among these compounds, the ones with the highest levels of antioxidants are xanthones. Xanthones are beneficial for the body, such as anti-inflammatory, anti-diabetic, anticancer, antibacterial, anti-fungal, anti-plasmodial, and are able to increase immunity and are also hepatoprotective (Shiddiqi et al., 2014).
One of antimicrobial agent can be found in mangosteen leaves. Some compound that can be pulled out by 50% ethanolic solvent in mangosteen extract is phenolic, flavonoid, and saponin compounds. The research that has been done by Diniatik and Suparman (2010) reported the antibacterial activity from mangosteen leaves is better than the skin of mangosteen fruit. The first phase of the study show that 50% ethanol extract of mangosteen leaves have the best potential for antioxydant, antifungal, and antibacterial activity.

Plant Material
The part of mangosteen plant that researcher used is mangosteen leaves part. This plant were collected from Kemranjen District, Banyumas Regency, Central Java Province, Indonesia. The mangosteen leaves were indentified in environmental laboratory Faculty of Biology, Jenderal Soedirman University, Indonesia.

Preparation of Ethanol Extract
Mangosteen leaves were collected, dried and pulverized by mechanical grinder. The powder of mangosteen leaves 500 grams were extracted by maseration and remaceration method for 2x24 hours with comparison between the powder and ethanol is 1:5 for the first day, and 1:4 for the second day. Stirring process is needed to improve the effectiveness during extraction. Extracts of mangosteen powder with ethanol were evaporated with pressure at 40 0 C until getting thick consistency.

Preparation of Nanoemulsion
This nanoemulsion consist of nanoemulsion base like Virgin Coconut Oil (VCO) as oil phase 1 ml, Tween 80 as surfactant 5 ml, and PEG 400 as co-surfactant 4 ml. This nanoemulsion base is replicated to 3 formulation, each formula consist of different ethanolic mangosteen leaves extract 1 ml, there are 10mg/ml ; 20 mg/ml; and 30 mg/ml. Total volume for each formulation is 11 ml.
All base composition is mixed in one, then use vortex for 1 minute, sonicated for 10 minutes, use waterbath at 45 0 C for 15 minutes to improve the homogenisation process. Then adding the ethanolic mangosteen leaves extract to the base, vortex it for 1 minute, sonicated for 10 minutes, then put the formulation on waterbath at 45 0 C for 15 minutes. That 100 µl SNEDDS added to 5 ml distilled water and mixed it well using vortex 30 seconds.

Transmittance Measurements
The sample was prepared by mixing SNEDDS 100 µl with distilled water 5 ml using vortex for 30 seconds. The emulsions were measured the precents transmittance at 650 nm using UV spectrophotometer to determine the level of clarity (Pratiwi, 2017).

pH Measurements
SNEDDS 100 µl was dissolved in 5 mL distilled water then use pH stick to determine nanoemulsion pH for food.

Particle Size and Zeta Potential Analysis
This analysis using a Particle Size Analyzer (PSA) to know the distribution of nanoparticles. A total of 1 mL SNEDDS was mixed with distilled water to 5 mL, then homogenized slowly. Zeta potential measurements were determined with a Zetasizer (Pratiwi, 2017 A total of one ose E. coli and S. aureus from stock were taken with sterile ose needle and put it into test tube containing aseptically solid NA and incubated at 37 0 C for 24 hours.

Bacterial Suspension
A total of one ose E. coli and S. aureus from stock were taken with sterile ose needle and put it into 10 mL NaCl 0,9% sterile. Absorbanced in UV spectrophotometer with wavelength 625 nm. The allowed range of absorbantion is 0,08-0,13 that have same meaning colony level of bacterial 1-2 x 108 CFU/mL (Aristyawan et al., 2018).
Before putting the papper disc, take 100 µl bacterial suspension then put it in MHA when still liquid medium. After all, put the petry dish in incubator at 37°C for 24 hours. The diameter of the inhibitory zone in observed.

Preparation of Medium
Potato Dextrose Agar (PDA) 39 grams dissolved with 1000 mL of aquadest, then heated to dissolved completely. Put the warm PDA solution 5 and 15 mL for each tube, then sterilized in autoclave at 121°C for 15 minutes. 5 mL PDA for fungal culture and 15 ml PDA for antifungal assay.
Potato Dextrose Broth (PDB) 24 grams dissolved with 1000 mL aquadest then heated it. Put 10 mL PDB warm solution into each tube , then sterilized in autoclave at 121°C for 15 minutes.

Culture of Saccharomyces cerevisisae (S. cerevisiae)
A total of one ose S. cerevisiae from stock were taken with sterile ose needle and put it into test tube containing aseptically solid 5 mL PDA and incubated at 28 0 C for 48 hours.

Fungal Suspension
A total of one ose S. cerevisiae from culture was taken with sterile ose needle and put it into 10 mL PDB sterile. Absorbanced in UV spectrophotometer with wavelength 600 nm. The allowed range of transmittance is 20%-80%

Antifungal Activity Assay
Each petry dish contain of 15 mL PDA media, 5 papper disc, then replicated to 3 petry dish.
Each papper disc contain of negative control (DMSO 10%); positive control (Cetoconazole 5µg/1µl); and ethanolic mangosteen leaves extract with 1%, 2%, 3% concentration. Before putting the papper disc, take 100 µl fungal suspension then put it in PDA when still liquid medium. After all, put the petry dish in incubator at 28°C for 48 hours. The diameter of the inhibitory zone in observed.

Application of Nanoemulsion as Fruits Preservative
Fruits were filtered then soaked with nanoemulsion for 5 minutes and dried the fruits well.
Fruits were covered by styrofoam and wrapping plastic. Then keep the fruits in room and refrigerator temperature.

Loosing fruits weight measurements
This measurements is calculated by decreasing of weight since the first day of keeping fruits until the last day during keeping period.

Fruits Organoleptic Acceptable Assay
This assay aims to know the fruits quality after using mangosteen leaves nanoemulsion through the sense of colour, texture, and fragrance. A total of 20 people asked to determine the likely level of each fruits using hedonic scale. There are 7 hedonic scale start from 1 (very not interested), 2 (not interested), 3 (almost not interested), 4 (neutral), 5 (almost interested), 6 (interested), and 7 (very interested). The limit of rejected fruits is scale of under 3.5 (Marpaung, 2015).

Formulation of Self Nanemulsifying Drug Delivery System (SNEDDS)
Nanoemulsions are made in a drug delivery system called the Self-Nanoemulsifying Drug Delivery System (SNEDDS). SNEDDS is a mixture of oil, surfactants, cosurfactants and active substances which when mixed with water will form an oil/water (M/A) type nanoemulsion (Lina et al., 2017). In the development of a drug delivery system based on pharmaceutical technology, a provision is needed that can increase the ability of active compounds to penetrate, one of which is the self-nanoemulsifying drug delivery system (SNEDDS). According to Nazzal (2002)

Transmittance Measurements
Good nanoemulsion must have a clear visual sighting with transmittance more than 90%, so that formula could be said to form a medium nanoemulsion when it was emulsified in water (Pratiwi, 2017). Based on data of transmittance, all formula have transmittance more than 90%. Decreasing of transmittance during keeping process is caused by nanoemulsion characteristic that can not stabile for long time keeping process.

pH Measurements
Nanoemulsion pH testing aims to determine the safety of each formulation especially when it is used for food. Based on table 2, this nanoemulsion has good pH because not include as strong acid food (pH<4,6).

Particle Size
Particle size of nanoemulsion is < 100 nm. Based on Table 3, all particle size is less than 100 nm.

Zeta Potential
Zeta potential values which were less than -30 mV or greater than 30 ,V indicated a stable nanoemulsion (Ujilestari, 2018). Based on the result, zeta potential in 1 % concentration extract is -40 mV. The zeta surface has potential to produce an electric repulsion between oil droplets which can inhibit droplet incorporation (Haryani et al. 2017). This zeta potential is less stable because electric repulsion not enough to refuse between each dominant droplets in nanoemulsion system and can cause droplets aglomeration (Ariviani et al., 2015).

Antibacterial Assay of Ethanolic Mangosteen Leaves Extract
According to Furukawa et al. (1996) and  This antibacterial assay is used on disc diffusion method through calculate the inhibitory zone around papper disc. The result on Table 4  Some compound that can be pulled out by 50% ethanolic solvent in mangosteen extract is phenolic, flavonoid, and saponin compounds. The research that has been done by Diniatik and Suparman (2010) reported the antibacterial activity from mangosteen leaves is better than the skin of mangosteen fruit. The first phase of the study show that 50% ethanol extract of mangosteen leaves have the best potential for antioxydant, antifungal, and antibacterial activity.
Another study showed that the optimized mangosteen peel extract contained alpha-mangostin compounds with strong antibacterial activity against gram-positive Staphylococcus aureus. The     1250 mg/ml at 1, 071 mg/ml, but has not been able to inhibit at a concentration of 500 mg/ml and 750 mg/ml. It can be said that the ethanol extract of mangosteen leaves at a concentration of 10 mg/ml (1%), 20 mg/ml (2%), and 30 mg/ml (3%) has not been able to inhibit the growth of S. cerevisiae fungi because the concentration is too little.

Antibacterial Assay of nanoemulsion of ethanolic mangosteen leaves extract
This test aims to confirm the presence of antibacterial activity or not if the ethanol extract of mangosteen leaves is made for nanoemulsion preparations. That is because the mangosteen leaf extract itself has antibacterial activity.
Based on table 7 and 8, naoemulsion formulation of mangosteen leaves extract with consentration 1%, 2%, 3% can not inhibit the bacterial of S. aureus and E. coli, and also S. cerevisiae fungal. This nanoemulsion is made to keep the fresh fruits have longer save time than before not using nanoemulsion. Although this formula does not have antibacteria and antifungal activity, this formulation can keep long save time for the fruits after harvesting.

Weight Loss Assay
This is one of parameter that can show the decreasing of fruits quality and levels of fruits freshness. The decreasing of weight in fruits is caused by losing water from transpiration and respiration process. During storage there is an increase in the weight loss of strawberries, both at room temperature and refrigerator temperature ( Table 9). The loss of water causes strawberries have decreasing in weight. In addition, the process of respiration or the breakdown of complex compounds into simple compounds with low molecular weight also causing weight loss. Water loss during storage not only decreases weight loss, but also reduces quality and causes fruits damage so that the fruit withers and wrinkles (Marpaung, 2015   On the treatment of strawberries on the first day to the fifth day, strawberries with a nanoemulsion layer of 1%, 2%, 3%, and the control group (non-preservative) could not be accepted by panelists on the fifth day with room temperature storage. This is because the fruit has experienced a decreased quality in color, aroma, and texture. These three indicators can reflect the taste in fruit. In contrast to the storage at the refrigerator temperature that can still survive its quality until day 5 (Aliya et al., 2016).
When viewed from the fruit rating scale when at room temperature, there is a slight difference between the control group and the group given nanoemulsion preservatives. The rating scale of the control group (non preservative) is lower than that of the group given preservatives. Although the results of antibacterial and antifungal test of nanoemulsion preparations cannot provide inhibitory zones, these preparations can still help maintain fruit quality. The defense mechanism of fruit quality from nanoemulsion preparations can be through a process of minimization from loss of moisture, gas exchange, respiration, and oxidation. Wahyudi (2018) reported that mangosteen leaves have very strong antioxidant properties with an average IC50 at a concentration of 1% which is 13,551 ± 0.034. Antioxidants act as donors of hydrogen atoms in free radicals to reshape fat molecules. If antioxidants are given it will slow down the auto-oxidation process. Therefore, the antioxidants contained in mangosteen leaves can increase the shelf life of strawberries at room temperature and refrigerator when compared to the nonpreservative group of nanoemulsion (Table 11 and 12).

CONCLUSION
Ethanol extract of mangosteen leaves have different antibacterial activity for each extract concentration. Antibacterial activity of mangosteen leaves extract for S. aureus is bigger than E. coli because of different cell membrane compound between them. In same concentration, mangosteen leaves extract can not inhibit S. cerevisiae as fungal that caused by the extract consentration is too small. Nanoemulsion of mangosteen leaves extract can not inhibit S. aureus, E. coli, and S. cerevisiae on the same extract concentration that used in extract antibacterial assay. But, nanoemulsion of ethanolic mangosteen leaves extract can keep the quality of strawberries better than non nanoemulion strawberries.

Conflicts of interest:
The authors declare no conflict of interest.