Potential of encapsulated rambutan peel extract (Nephelium lappaceum) as a food preservative

Rambutan peel is one of the agricultural wastes that is rarely used and contains several active compounds with antimicrobial properties. The antimicrobial properties of rambutan peel can be used as a food preservative. However, these active compounds are unstable and degraded when exposed to air, heat, light, and water. This problem can be prevented by encapsulating the extract. There are two specific objectives of this study, knowing the types of active compounds contained in rambutan peel extract and determining the best coating material for encapsulated rambutan peel extract. The encapsulation process of rambutan peel extract was done by using thin layer drying method using maltodextrin, gum arabic, and gelatin as coating material. The encapsulated extracts were stored for one month and tested weekly. Test parameters of encapsulated rambutan peel extract were moisture content and antibacterial activity. The result showed that rambutan peel extract contains flavonoids, tannins, and saponins. The use of maltodextrin and gum arabic as coating material was able to maintain bacterial inhibition against Staphylococcus aureus during storage, while the use of gelatin as coating material can only maintain up two weeks of storage. The result also showed that rambutan peel extract could not maintain bacterial inhibition against Escherichia coli during storage.


Introduction
Rambutan (Nephelium lappaceum) is tropical plants whose use is limited to the fruit.Rambutan peel waste is rarely used, even though rambutan peel contains antimicrobial substances which are commonly used as drugs and antibiotics.Kusumaningrum's (2012) states that rambutan peel extract has antimicrobial activity against gram-positive bacteria and supported by Alina et al (2017), who investigated the antimicrobial activity of rambutan peel extract against gram-negative bacteria Eschericia coli [1,2].Kusumaningrum (2012), Zulhipri et al (2012) dan Monrroy et al (2020), revealed that rambutan peel contains a lot of active compounds such as steroids, terpenoids, phenolics, flavonoids, tannins and saponins [1,3,4].These compounds have antimicrobial properties that can be used as drugs, antibiotics, pesticides, larvicides and food preservatives.Several previous studies were more directed at the 1230 (2023) 012154 IOP Publishing doi:10.1088/1755-1315/1230/1/012154 2 manufacture of drugs and antibiotics, even though the use of antimicrobial substances was not limited to antibiotics.Antimicrobial substances can also be applied as preservatives in food products, considering that Indonesia still has many cases regarding the use of harmful preservatives in several food products.
There are several matter in utilizing active compounds, one of them is easily damaged due to reactions with water, air, heat and light.One method to prevent that is by encapsulation.Encapsulation is a technique of coating the core material with a special filler to maintain the quality of the core material both physically and chemically.The success of encapsulation is influenced by the type of coating material, the principle and the medium [5].

Procedure
This research consisted of two stages, the first stage was the rambutan peel extraction and the second stage was the rambutan peel extract encapsulation.In the first stage, the first step is the preparation of rambutan peel waste (sorting, washing and chopping).After that, the rambutan peel was dried at a temperature of 60 0 C for ±16 hours and then mashed to form a powder.Furthermore, extraction was carried out using maceration method and 96% ethanol solvent with a ratio of 1:10 (w/v) for 3x24 hours and then filtered and evaporated under vacuum to separate the extract from the solvent.Evaporation was carried out until the solvent was completely separated from the extract and a dry extract was produced.The yield was calculated and then the dried extract was grind and mashed to obtain a rambutan peel extract powder.
The next step is rambutan peel extract encapsulation using thin layer drying method.encapsulation using three types of coating materials (maltodextrin, gum arabic and gelatin) with a ratio between extract and coating material was 1:5.Each coating material was weighed and dissolved with distilled water in a ratio of 1:2 and then stirred at 6000 rpm for 15 minutes.After that, the weighed rambutan peel extract powder was put into a solution of coating material and homogenized at 6000 rpm for 15 minutes.The encapsulated solution was put into a wide glass container with a thickness of ±3 mm and then dried at 50 0 C for ±16 hours.The encapsulation was then grind and crushed and then sieved through an 80 mesh sieve.At this stage, measurement of yield of each encapsulation and measurement of water content and quantitative test of secondary metabolites were carried out once a week.The yield of the encapsulated extract was measured using the formula [6]: Weight of Encapsulated Extract (g) Weight of Extract+Weight of Coating Material (g) x 100% (1)

Test parameters
2.2.1.Qualitative tests of secondary metabolites compound.This tests was performed for detected the presence of Alkaloids, Tannins, Flavonoids, Steroids, Saponins, and terpenoids.a. Alkaloids [7] Two ml of the extract was added with 2 ml of HCl and 4 ml of methanol and then heated at 95°C for 5 minutes then cooled and filtered.one ml of the filtrate was added with two drops of Mayer's reagent and one ml of the filtrate was added with two drops of Dragendroff's reagent.Positive results using Mayer's reagent were indicated by the formation of a white precipitate, while in Dragendorff's reagent a positive result was the formation of an orange-brown precipitate.b.Flavonoids [7] Two ml of the extract was heated for 5 minutes and then added with 5 drops of concentrated HCl and 0.1 grams of Magnesium.Positive result indicated by the formation of a yellow-orange to red color.c.Saponins [8] Two ml of the extract was added to 10 ml of boiling water, cooled and shaken vigorously for 10 seconds.If foam is formed as high as 1-10 cm which is stable for approximately 10 minutes and does not disappear with the addition of 1 drop of 2 M hydrochloric acid, it indicates the presence of saponins in the extract.d.Tannins [8] Two ml of the extract was heated for 5 minutes.After that, a few drops of 1% FeCl3 were added.
Positive result ndicated by the formation of greenish brown or blue-black color.e. Terpenoids/Steroids [7] Extract was taken 2 ml each and then added with 3 drops of concentrated HCl and 1 drop of concentrated H2SO4.Positive containing terpenoids if a sample forming red or purple color, if a green color is formed, it is positive for steroids.[9].Analysis of moisture content was carried out by drying the material using an oven (T = 1050C) for 4 hours then the material was weighed and dried again until it reached a constant weight (<0.005).percentage of moisture content is calculated using the formula:

Moisture content
(2) [10].20% encapsulated extract solution using distilled water were made and 0.02 ml was injected into the disc then waited for the disc to dry.Each of bacterial isolate Staphylococcus aureus and Eschericia coli were inoculated on a nutrient agar (NA) media using spread plate method.After that, the dried discs were placed on the surface of the media containing the bacterial isolate.Samples were incubated for 24 hours at 37°C and the diameter of the inhibition zone was measured using caliper.

Qualitative tests of secondary metabolites compound for rambutan peel extract
Qualitative test of Secondary Metabolites Compound for Ethanol Extract of The Rambutan Peel was conducted to determine the type of secondary metabolite compounds contained in the rambutan peel extract.Qualitative testing for ethanol extract of rambutan peel gave positive results on flavonoids, tannins, saponins and negative results on alkaloids, terpenoids, and steroids.Steroids are non-polar, so it is difficult to dissolve in ethanol.Terpenoid compounds tend to be non-polar, but triterpenoids can be soluble in polar solvents such as ethanol.Alkaloids are polar, so they can dissolve in polar solvents such as ethanol.Although the solvent used was in accordance with the polarity of the alkaloids, the compound remained undetectable.So it can be concluded that there are no alkaloids in the ethanol extract of rambutan peel.
No detected alkaloids in rambutan peel extract were also reported by Zulhipri et al. (2012) and Monroy et al. (2020) [3,4].Phytochemical screening of rambutan peel ethanol extract conducted by Kusumaningrum (2012), showed negative results for steroids but positive for alkaloids and terpenoids [1].Tjandra et al. (2011) also carried out a phytochemical screening of rambutan peel methanol extract and the results were negative for alkaloids, terpenoids and saponins but positive for steroids [11].Previous studies have shown the diversity of the secondary metabolite content of rambutan peel.This diversity can be caused by geographical conditions and environment, chemical characteristics of the soil, as well as the age and species of rambutan plants [4,12].

Rambutan peel extract encapsulation
Encapsulation is a technique of coating the core material with special fillers to maintain the quality of the core material both physically and chemically [5,13].According to Gharsallaoui et al., (2007), types of coating materials commonly used in the food sector are carbohydrates, gums and proteins [14].This study used three types of encapsulants, maltodextrin from the carbohydrate group, gum arabic from the gum group and gelatin from the protein group.Ethanol extract of rambutan peel encapsulation using thin layer drying method.The solution containing a mixture of extract and coating material were dried in a form of a thin layer to obtain the final product, which is encapsulated rambutan peel extract.The ratio between the coating material and the core material is 1:5 (5 grams of extract and 25 grams of coating material).After encapsulation, the sample weight decreased.Each type of coating material has a different amount of weight reduction.Based on the table, the highest encapsulate yield is the one used maltodextrin, while the lowest yield is the one used gelatin.One of the properties differences between that three coating material is the level of viscosity.The viscosity level of the encapsulant affects the yield of encapsulate.The higher the viscosity of the coating material, the lower the yield of the encapsulate [15].High viscosity causes the atomization process to be disrupted so that will form large and long droplets which causes the drying longer, so that the yield of encapsulated material is reduced [16].

Characteristic of encapsulated rambutan peel extract 3.3.1. Moisture Content.
The sample data of moisture content showed that the moisture content of unencapsulated extract would rapidly increase and encapsulation was able to suppress the increase in moisture content during storage.The best coating material that can suppress to the max the increase in water content is maltodextrin.This is because maltodextrin can bind free water to the core material.During the drying process, the free water that bound to the maltodextrin will evaporate so that the moisture content of the encapsulate is low [15].Maltodextrin has non-hygroscopic properties, depending on the Dextrose Equivalent (DE) maltodextrin used.The lower the DE maltodextrin, the stronger the non-hygroscopic properties.However, if the DE of maltodextrin is high, the maltodextrin will be hygroscopic [17].While for gum arabic, although it can suppress the water content of the extract during storage, gum arabic encapsulated extract has the highest overall water content among the other three types of encapsulated extracts.Gum arabic has many hydroxyl groups so that it can bind free water contained in the core material.Different from maltodextrin, free water bound by gum arabic is difficult to evaporate during drying process.This is because gum arabic has a complex molecular structure and a larger molecular weight than maltodextrin (±500,000 g/mol) so that the bond between water and gum arabic is stronger [16].During storage, the water content of the gelatin encapsulated extract increased significantly compared to the maltodextrin and gum arabic encapsulated extracts.This is because gelatin is hygroscopic.The hygroscopic nature of gelatin depends on the guanidine and arginine groups.The higher the content of guardinin and arginine groups, the higher the hygroscopic nature of gelatin [18].

Antibacterial Activity. The analysis of variance of the inhibition test on gram-positive
Staphylococcus aureus showed that the type of coating material, storage time and interactions between treatments had a very significant effect (p < 0.05) on the diameter of inhibition zone rambutan peel extract.With the same sample concentration, the secondary metabolite contents of each sample are different.Encapsulated extracts consist of extracts and coating materials, so the contents of secondary metabolites are lower than non-encapsulated extracts.This causes the inhibition zone of the non-encapsulated extract to be higher than the encapsulated extract.The main reason for measuring antibacterial activity was to see the effect of encapsulation in maintaining the extract's ability to inhibit bacterial growth during storage.The diameter of inhibition zone were fluctuated during storage, both unencapsulated and encapsulated extracts.In gelatin encapsulated extract, the inhibitory activity of Staphylococcus aureus bacteria was no longer present in the 3 rd week.This is probably due to the secondary metabolites in the gelatin encapsulated extract had been damaged and significantly reduced so that the extract had no ability to inhibit bacterial growth.Duncan's test results on the type of encapsulation factor showed that the encapsulated extract of gum arabic had the best inhibition against Staphylococcus aureus.Despite fluctuations in the data, there was no change in the inhibitory strength of the encapsulated extract during storage, which was low because the diameter of the inhibition zone formed was less than 5 mm (≤ 5mm).The fluctuations during storage possibly caused by spontaneous transformation in extract.Spontaneous transformation that occurs can be from an inactive precursor in the extract to an active compound due to auto-oxidation [19,20].

Figure 3. Inhibitory zone of bacterial extracts against Escherichia coli during storage
Inhibition of Eschericia coli bacteria from rambutan peel extract, both encapsulated and unencapsulated, was weak (≤ 5 mm) and did not last during storage.This is because the secondary metabolites in the extract have decreased significantly so that the extract is no longer able to inhibit bacterial growth.The use of arabic gum and gelatin as coating materials could not maintain the extract's ability to inhibit bacterial growth.for gelatin, the inhibition ability of the extract against gram-positive and negative bacteria did not last during storage.Gelatin has hygroscopic properties, so the possibility of the extract being damaged due to reaction with water is high.The ratio between the core material and the coating material also matters [21,22].The ratio used in this study may not be sufficient to protect the active ingredients contained in rambutan peel extract so that the inhibition of the extract decreases rapidly and disappears during storage.
The ability of ethanol extract of rambutan peel to inhibit the growth against Staphylococcus aureus was more sensitive than Eschericia coli.This is influenced by differences in the structure of the cell walls.The cell wall of gram-positive bacteria contains 40-80% peptidoglycan and contains teichoic acid.Teichoic acid is hydrophilic and functions as a positively charged ion transport medium [23,24].The cell wall of gram-positive bacteria is polar, making it easier for polar active compounds to enter the

Unencapsulated
Maltodextrin Arabic Gum Gelatin bacterial cell.Meanwhile, the cell wall structure of gram-negative bacteria is not as simple as that of gram-positive bacteria.The cell wall of gram-negative bacteria consists of three layers, the first layer or outer membrane consists of lipoproteins, the middle layer is composed of lipopolysaccharide and the third layer is composed of peptidoglycan.The peptidoglycan layer of gram-negative bacteria is thinner than that of gram-negative bacteria.Lipoproteins and lipopolysaccharides are lipophilic and can block the entry of polar active compounds into the inner layer [1,10].

Conclusion
The ethanol extract of rambutan peel contains flavonoids, tannins and saponins.The use of maltodextrin as an coating material was able to reduce and suppress the increase in moisture content during storage, but the use of gum arabic was better for maintaining the antibacterial activity of rambutan peel extract during storage.
The difference in the ability of the three types of coating material reported in this study can be utilized for further research by combining two or three different types of coating material to obtain more optimal encapsulation results.In addition, it is necessary to test the efficiency and the morphology of the encapsulate to determine the characteristics of encapsulate.the ratio between coating material and core material in this study also needs to reconsidered.

Figure 1 .
Figure 1.Extract moisture content based on type of coating material during storage.

Figure 2 .
Figure 2. Inhibitory zone of bacterial extracts against Staphylococcus aureus during storage.