THE POTENTIAL OF FOREST HONEY (Apis spp.) FROM TIMOR ISLAND AS ANTIBACTERIAL AGAINST PATHOGENIC BACTERIA IN FISH CULTURE

This study aims to assess the potential of forest honey Apis spp. from Timor Island as an antibacterial on pathogenic bacteria, Aeromonas hydropilla and Vibrio alginolyticus in cultured fish. There were six types of honey tested in this study of which active compound, total glucose, pH, and water content of each honey were determined. The chemical contents of the honey were examined for alkaloid using Culvenor-Fitzgerald method, saponin with foam test, terpenoid and steroid with Lieberman-Burchard method, tannin with addition of FeCl3, and flavonoid addition of HCl and Mg powder. Total glucose was measured using spectrophotometer, water content with gravimetry, and pH with pH-meter. The antibacterial activity test of the honey was done using disc method without dilution. Results showed that all forest honey from Timor Island contained alkaloid, saponin, steroid, and terpenoid, except Kefa honey which did not have steroid and terpenoid. The water content of the honey ranged from 15.70% to 26.65%, total glucose of 71.16% to 80.58%, and pH of 3.84 to 4.06. The forest honey also had antibacterial activity against A. hydropilla and V. alginoliticus with different inhibition zones.


INTRODUCTION
Honey is a viscous liquid produced by honey bees from flower nectar (Yuliati, 2017). Based on the nectar source, honey can be distinguished as monoflora and polyflora. The former is produced from one major plant and the latter from the nectar of several types of plants. Forest honey is a polyflora one produced by wild bees Apis dorsata. These bees usually live in the forest and depend their various food sources from forest plants such as flower nectar or tree sap. Due to diverse food sources, the honey has better quality due to honey content of more complex compounds. Forest honey produced by the wild bees is also very good for health since it contains natural antibiotics.
Several studies have proved the potential of the forest honey from Indonesia as an antibacterial against pathogenic Gram-positive or Gram-negative bacteria in human. Yuliati (2017) did an in vitro study which revealed the antibacterial superiority of forest honey from Sumbawa against bacteria Staphylococcus aureus and Pseudomonas aeruginosae. Honey from Bandung and Riau Islands have also been proved to have antibacterial activity against Staphylococcus aureus and Escherichia coli (Dewi et al., 2017). Furthermore, bitter and sweet black honey (forest honey from Kalimantan) has antibacterial activity against E. coli and S. aureus (Fitrianingsih et al., 2014).
Honey has been studied for its applications as a pathogenic antibacterial in human. Nevertheless, reports on its utilization as antibacterial in fish culture is currently limited. Considering the antibacterial feature of the honey, Testing the antibacterial activity of forest honey against Aeromonas hydropilla and Vibrio alginolyticus infecting cultured fish is a researchable topic. Salosso (2017) tried to study the antibacterial activity of Timorese honey from Soe against A. hydropilla and V. alginolyticus and its active compounds. Her finding revealed that the honey from Soe contained active compounds such as flavonoid, triterpenoid, and saponin. The compounds can inhibit the growth of A. hydrophilla and V. alginolyticus in all tested concentrations. However, this study was only limited to the forest honey from Soe and not from other places.
The potential of forest honey (Apis spp.) from Timor Island ..... (Yuliana Salosso) Timor Island is one of the large islands in East Nusa Tenggara Province, Indonesia. The forest on the island is known to produce high-quality forest honey, such as forest honey from Soe, Kefa, Ampang, South Ampang, Naikliu, and others. The forest honey from Timor Island is mostly traditionally produced by the local communities and has not been studied about their chemical contents and natural benefits. This study assessed the potential of forest honey Apis spp. from Timor Island as antibacterial on pathogenic bacteria, Aeromonas hydropilla and Vibrio alginolyticus in cultured fish.

Honey Collection
Forest honey was purchased from honey retailers who bought the honey from local honey finders originated from three regencies in Timor Island: North Central Timor (Kefa), South Central Timor (Soe), and Kupang Regency (Ampang, South Ampoang, and Naikliu). Commercial honey, madu rasa, was used as a comparison honey. The paper disc containing 30 µg of nobiocin antibiotic was used as a control.

Alkaloid test
Honey sample of 2 mL was mixed with 3 mL of chloroform and three drops of ammonia. The chloroform fraction was separated and acidified in 10 drops of 2N H 2 SO 4 . Sulphuric acid (H 2 SO 4) fraction was taken and added with Dragendroff reagent. The presence of alkaloid was indicated with formation of brownish (reddish) orange deposition (Hanani, 2014).

Saponin test
Honey sample of 2 mL was put into a flask, then mixed with 10 mL of distilled water, boiled for 2-3 min, and then cooled. Afterwards, the solution was shaken vigorously for 10 min. Formation of stable foam after 10 min indicated the presence of saponin (Hanani, 2014).

Triterpenoid and steroid test
Honey sample was dropped on the drop plate and added with Lieberman-Buchardt reagent. Presence of terpenoid indicated by the red color formation or steroid with green color (Hanani, 2014).

Tannin test
Honey sample of 2 mL was boiled for several mins., then added with 2-3 drops of 1% FeCl 3 . The presence of tannin was indicated by a dark blue or greenishblack color (Hanani, 2014).

Flavonoid test
Honey sample of 2 mL was extracted in 5 mL of methanol. The filtrate was then added with two drops of 2 N HCl and strongly shaken, added with magnesium powder, and strongly re-shaken. Flavonoid was indicated by a high intensity of foam and the solution changes to yellow-orange or dark red (Hanani, 2014).

Analysis of pH, Water Content, and Honey
pH was determined using a pH-meter, total glucose measurement used spectrophotometer, and water content of the honey used gravimetry.

Solid and semi-solid TSA media preparation
Media used for the growth of bacteria A. hydropilla used trypticase soy agar (TSA) with addition of 0.5% NaCl and V. alginolitycus used TSA media with addition of 2% NaCl. These media were used for antibacterial test and made two layers, solid TSA, and semisolid TSA. The former had been made a day before the antibacterial test was done using the TSA as labeled, while the latter was made at the time the test was done using 70% TSA as labeled.
The solid TSA was autoclaved and poured into a petri dish as much as 10 mL and left up to solid and kept in the refrigerator in an upside-down position. The semi-solid media were autoclaved and cooled down to 50°C, then added with A. hydropilla on 0.5% TSA media and V. alginolitycus on 2% TSA at a density of 10 6 cells/mL, and each of them was poured as much as 10 mL into the solid TSA, then left down frozen. Afterward, it was ready to do the disc test (Salosso, 2017).

Disc Test for Antibacterial of Honey
The sterile paper disc was dipped into the original honey (without dilution). After 30 minutes, the disc paper was attached to the TSA media inoculated with A. hydropilla and V. alginolitycus. Measurements were done after 24-hour inoculation at 37°C by observing whether clear zone occurs around the paper disc or not.

The Active Compound of the Honey
Qualitative test of the active compounds of the honey from Timor Island is presented in Table 1. This study demonstrates that all forest honey from Timor Island contain alkaloid, saponin, and steroid and terpenoid, except Kefa honey which does not contain steroid and terpenoid.
Indonesian Aquaculture Journal, 14 (2), 2019, 63-68 Commercial honey contains only alkaloid. The active compound content of the forest honey from Timor Island is not quite different from that of forest honey from Aceh, Seulawah (Aceh Besar) and Trumon (South Aceh) which also contains saponin and terpenoid, but no alkaloid and steroid (Fadhmi et al., 2015). However, Forest honey from Timur Island has different active compound compared to Nigerian honey which did not contain alkaloid and saponin, but contain tannin and flavanoid (Adeyemo et al., 2017).
The chemical compounds of the forest honey from Soe in this study contained an alkaloid, steroid, terpenoid, and saponin. In fact, different from those found by Salosso (2017) which were only flavonoid, triterpenoid, and saponin. This evidence reflects that active compound content of the honey might be influenced by seasons as both types of forest honey from Soe were collected from the same locality but at different times. Francois et al. (2017) also found similar result where honey from Benin Republic holds alkaloid, tannin, and flavonoid in dry season, and alkaloid, tannin, flavonoid, and terpenoid and steroid in rainy season.
The difference in active compounds content of the honey could be the result pf different nectars taken by different bees (Ma'ruf et al., 2018). Moreover, Parwata et al. (2010) stated that honey had different composition of chemical compounds based on the food/nectar sources of the bee. This difference could result in different antibacterial activity of the honey.

Water Content, pH, and Total Glucose
Total sugar, water content, pH of the honey from Timor Island is provided in Table 2.

Water Content
The water contents in the forest honey from Timor Island were ranged from 15.70% to 26.65% (Table 2). This content was the highest compared to that of found by Nadhila (2014) of 15%-21%. Ampoang honey had the lowest water content of 15.70%, which was lower than that of the commercial one since this honey has had water reduction process and well packed in sales. Honey from Kefa, Naikliu, South Ampoang, and Soe have not been treated for water reduction process so that it has higher water content than the commercial one.
The water content of honey could be different with different localities. Honey water content of rubber tree nectar from Central Bangka Regency was 24.25% (Evahelda et al., 2017), while the water contents of Trumon and Seulawah honey from Aceh were 22.05% and 19.81% (Fadhmi et al., 2015), respectively. The water contents of bitter and sweet black honey from Central were 16.19% and 15.40% (Fitrianingsih et al., 2014), respectively. In general, the water content of some types of honey from Indonesia ranged from 17.8% to 21.0% (Dewi et al., 2017). The difference in the water content of the honey is affected by several factors, such as climate, post-harvest handling, nectar type collected, honey maturity, production techniques, and storage (Baroni et al., 2009).

Total Glucose
Honey holds high total sugar, and dominantly consists of glucose and fructose (Nayik & Nanda, 2015). Forest honey from Timor Island has high total sugar, 71.16%-80.58%. Total sugar content of Timorese honey was not quite different from the total sugar content of honey from rubber tree nectar of Central Bangka regency, 74.77% (Evahelda et al., 2017). Honey has also different chemical compounds, including total sugar based upon food source of the nectar (Parwata et al., 2010).
According to Evahelda et al. (2017), the high sugar content will make the honey to be more viscous or concentrated so that it has hygroscopic feature. This honey characteristic is determined by fructose since fructose is more soluble than glucose (Buba et al.,  The potential of forest honey (Apis spp.) from Timor Island ..... (Yuliana Salosso) 2013). Moreover, according to Erguder et al. (2008), sugar content of honey consisting of mixture of glucose and fructose causes the honey to osmotic feature and can inhibit bacterial growth.

Honey pH
The pH of forest honey from Timor Island ranged from 3.84 to 4.06. This range tends to be the same as that of from other places in Indonesia, such as randu honey with pH of 3.8, rambutan honey with pH of 4.21, longan honey with pH of 4.48, kaliandara honey with pH of 4.37 (Chayati, 2008), and rubber tree honey from Bangka, 3.92 (Evahelda et al., 2017). Similarly, honey from India has pH of 4.1 (Veeraputhiran et al., 2013).
Despite having a similar pH range, there is a difference in honey pH among honey from Timor Island. This difference could result from differences in mineral and acid content of the honey (Gulfraz et al., 2010). According to Buba et al. (2013), the mineral content of the honey is influenced by soil condition, geographic position, climatic condition where the nectar source plants grow.
Honey of low pH can inhibit the growth of various bacteria since bacteria can grow in neutral or alkaline pH conditions. Acidity gives high impact on the growth and the survival of bacterial cells. Each species has an optimum range of acidity for growth. When pH declines to the lowest limit for bacterial growth, not only bacterial cells stop growing, but the bacteria will lose their ability to survive as well.

Antibacterial Activity
Antibacterial test of the forest honey from Timor Island against V. alginoliticus and A. hyropilla can be seen in Figure 1. It is apparent that all types of forest honey from Timor Island possess antibacterial activity against A. hydropilla and V. alginoliticus, but the diameters of inhibition zone are different.
Against V. alginoliticus, the inhibition zones of all honey types from Timor Island were all higher than that of commercial honey, madu rasa (11 mm), except that south Ampoang honey has lower inhibition zone (9 mm). All these inhibition zones are still lower than that of antibiotics (22.5 mm). Similarly, against A. hydropilla, the inhibition zones produced by all forest honey from Timor Island were higher than that of the commercial honey (8 mm), but lower than that of antibiotics (16.5 mm). Thus, all types of forest honey from Timor island are potential to be considered as antibacterial against A. hydropilla and V. alginoliticus. The bigger the inhibition zone produced by the honey is, the higher the antibacterial activity.
Comparing among honey types from Timor Island, the highest antibacterial activity against V. alginolitycus was found in the forest honey from Naikliu (13 mm), while the highest antibacterial activity against A. hydropilla was found in the forest honey from Kefa (12 mm). It reveals that the antibacterial activity of each honey is different depending upon the type of honey, geographic position, and flower where the end product comes (Rio et al., 2012).
The potential of antibacterial in the honey could result from several mechanisms, i.e. presence of radical hydrogen peroxide (H 2 O 2 ) compound that can kill the pathogenic microorganisms (Johnston et al., 2018;Nadhilla, 2014;Carina et al., 2014), high acidity of the honey to reduce growth and survival of the bacteria (Johnston et al., 2018;Carina et al., 2014;Nadhilla, 2014;Erguder et al., 2008), high sugar content inhibiting the bacteria to grow and develop (Carina et al., 2014;Erguder et al., 2008), and presence of antibacterial organic compounds (Carina et al., 2014).
Antibacterial activity of forest honey against pathogens in fish is also shown by previous researchers. Stratev et al. (2015) has proved that rapa honey and royal jelly from Bulgaria are potential as an antibacterial against A. hydropilla. Ramalivhana et al. (2014) also found that honey from South Africa has antibacterial activity against A. hydropilla. Salosso (2017) reveals the antibacterial activity of Timor honey from Soe against A. hydropilla and V. alginolyticus.

CONCLUSION
Different forest honey from five areas in Timor Island contained alkaloid, saponin, steroid, and terpenoid, except that Kefa honey which did not hold steroid and terpenoid. The water content of all honey ranged from 15.70% to 26.65%, total glucose 71.16% to 80.58%, and pH 3.84 to 4.06. It also had antibacterial activity against A. hydropilla and V. alginoliticus with different inhibition zones.

ACKNOWLEDGEMENT
The author would like to thank the Dean of the Faculty of Marine and Fisheries, Nusa Cendana, University Kupang, for his encouragement to do this study. My gratitude is also given to the Staff of Fish Quarantine Microbiology Laboratory for their analytical assistance which has made possible to finish this study one time.