1 NANOENCAPSULANT FORMULATION BASED ON

NANOENCAPSULANT FORMULATION BASED ON KECOMBRANG FRUIT (NICOLAIA SPECIOSA) AND ITS USAGE AS ANTIMICROBIAL Rifda Naufalin, Herastuti Sri Rukmini and Rumpoko Wicaksono Fakultas Pertanian, Universitas Jenderal Soedirman. [email protected]

ABSTRACT Kecombrang (Nicolaia spesiosa Horan) has antimicrobial potential because of chemical component such as alkaloid, flavonoid, steroid, saponin and essential oil. This research is purposed to produce nanoencapsulant formulation based on kecombrang fruit as antimicrobial with the filled of soybean protein, gelatin, maltodextrin and hidrocoloid in sort of jelly. This research is shown that the nanoencapsulant formulation of kecombrang fruit (Nicolaia spesiosa) is effective to inhibit the positive gram positive Bacillus cereus compared with negative gram bacteria Eschericia coli, yield Botrytis cinerea, and mold Saccharomyces cereviceae with the inhibit zone B.cereus 27,667-27,958 mm.the lowest inhibited of bacteria Botrytis cinerea with the inhibit zone 6,221-6,433 mm. Stabilizer is used to increase the inhibited for mold Saccharomyces cereviceae with inhibit zone 18,738 mm.

INTRODUCTION Kecombrang (Nicolaia spesiosa Horan) is one of plant used by society generally to give a flavor. Kecombrang has an antimicrobial potential. As antimicrobial it will hold the bacteria then it used as the natural preservative especially for food product. The research of kecombrang antimicrobial activity is begin with Valianty (2002) which is shown that kecombrang flower inhibit the growth of bacteria Escherichia coli dan Bacillus subtilis. In furthermore, Naufalin (2005) show that flower kecombrang extraction from acetic ethyl and ethanol have inhibited seven bacteria growth they are Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella typhimurium, Escherichia coli, Aeromonas hydrophila and Pseudomonas aeruginosa. The inner kecombrang stem from the acetic ethyl and ethanol have the highest inhibit zone into bacteria Bacillus cereus and Escherichia coli than the outer stem (Syukri, 2008). The research of kecombrang fruit is done by Setiyani (2010) which states that kecombrang fruit with the pink colour in the leather with the concentrate 50% has the highest antibacterial on Bacillus cereus and Eschericia coli. The chemical compounds which are included on kecombrang based on Tampubulon et al. (1983) are alkaloid, flavonoid, polifenol, steroid, saponin and essential oil. The use of essential oil as the antibacterial has the weakness such as the loose of volatile compounds in process with high temperature, easier to oxidize, not easier to dispersion in dried material, in thick shape of then it difficult to handle (Koswara, 2007). The process of kecombrang plant formulation needs the substance to protect active compounds from environment and stabilizer to stabilize formula suspension. The substance material generally used from stabilizer such as jelly, alginate, carrageenan, gelatin, cellulose gel, gum arab, and maltodextrin. Kim et al. (1996) stated that protein and whole carbohydrate could interact with water, ion, and polymer oil or water then it can stabilize emulsion since homogenization. The substance materials based on general protein are soybean protein gelatin, whey and chaseinate. Whereas, the substance based on carbohydrate are dextrin and gum (Naufalin dan Herastuti, 2012). The used substances in this research are soybean protein, gelatin and maltodextrin. Soybean protein is one of product which is produced from free fat soybean or low fat which is processed into high protein.Gelatin is a highest protein level and a heavy polypeptida, it came from collagen, the prior protein on animal tissue such as bone, skin, and tendon, Gelatin consists of 84-90% protein, 1-2 % salt mineral, and 8-15% water (Poppe, 1999). Maltodextrin (C6H12O5)nH2O is a polysakaride which has a bound of α(1-4)D-glukose with a molecule weight less than 4000. Maltodextrin is used as stabilizer and combined with other (Poppe, 1999). Kim et al. (1996) stated that protein and a such carbohydrate could interact to water, ion, and polymers oil or water then it can stabilize droplet emulsion which is made when its homogenized. The efficiency of proportion and concentrate of substance is essential to know how elective a bioactive in an antimicrobial activity. 1

The used stabilizers in this research are jelly, and substance of soybean protein, maltodextrin, and gelatin. This research purposed to find the nanoencapsulant of antimicrobial activity formulation. METHODOLOGY Tools and Material The used tools in this research are cabinet dryer, drilling, digital measure (ohaus), analytics measure (AND), Erlenmeyer glass, reaction tube, screw tube (Pyrex,Germany), rotabit shaker (Selecta), evaporator-rotary (Stuart, Germany), filter fabric 250 mesh and N2 tube. The used tolls to formulate kecombrang formulations are analytics measure, spatula, beaker glass, hot-plate (Maspion), vortex-stirrer (Boeco), micro pipet (Gilson), tip, filter fabric, glass spatula, autoclaf (All American), petri cup, ose needle, spirtus lamp, refrigerator (Panasonic), water heater (PMC), oven (Memmert), incubator 37ºC (Memmert, Japan) and other laboratorium tools to analyze. The other supporting tools are aluminium foil (Klin Pak), wrapping plastics, newspaper, label, plastics of 2 kg (Joyo Boyo), and tissue (Nice). The used materials in this research are kecombrang fruit from Tasikmalaya, West Java. The substances to formulate are maltodextrin (Bogor), gelatin (Bogor), soybean protein (Bogor), stick jelly (AA), akuades (Biology Laboratorium Faculty), N2 gass (Samator Gas), ethanol 96% (Brataco Chemical). The used materials to analyze antimicrobial activity are Nutrient Agar (NA) (Merck), Nutrient Broth (NB) (Merck), Potato Dextrose Agar (PDA) “Merck”, NaCl 0,85% (E-Merck, Germany), pure microbe culture are Escherichia coli, Bacillus cereus, Botrytis cinerea and Saccharomyces cerevisiae. Kecombrang Fruit Extraction Kecombrang fruit powder is measured and added with ethanol on comparing fruit and flower powder : ethanol is 1 : 4. Doing maceration with shaker tools on 2 hours and deposit on 10 hours and separate kecombrang extraction. kecombrang extraction is evaporated in steaming process using evaporator rotary and having a solid solution in concentrate form. Process of Kecombrang Formulation Heat water on 50C, add gelatin or soybean concentrate and vortex on a minute, add maltodextrin and vortex on a minute. A temperature is decreased until 40C and added kecombrang extraction and vortex in a minute. Put jelly and homogenized on 1 second. A ratio for gelatin or soybean concentrate : maltodextrin is 2 : 1, substance concentrate 2 % and 6 %. Antimicrobial test with a diffusion well method (Carson and Riley, 1995) Put 20 µl bacteria test in 20 ml NA media on temperature 45°C and pour into petridis steril let it solid. Make a whole to diffuse in that media, and add extract formula amount 60µl. Incubate on 24 hours in 37C. Gaze on diameter zone. Antimold test with paper disk method (Yang and Ray, 1994) 20 µl mold culture is added in 7 ml PDA media, put these into Petridis which filled with 12 ml PDA media and let it solid. paper disk 6 mm which is dropped on 10 µl kecombrang formula and put on media. petri cup is incubated for 24-48 hours on 30C. Gaze on diameter zone. RESULT AND DISCUSSION Kecombrang Fruit Powder Rendement Based on data analysis, it found the kecombrang fruit rendement powder 16.54%. it indicates the highest water content of kecombrang fruit than leaf, and rhizome from kecombrang (Tabel 1). It has the highest rendement 2

than flower and stem. It caused from epidermis of kecombrang fruit consist more water content, whereas mesodermis consist a few water content. Tabel 1. Rendement Data of kecombrang powders Physical characteristic Rendemen

Fruit*)

Flower*)

Leaf*)

Stem*)

Rhizome*)

16,54%

10,89%

20,51%

13,86%

18.00%

* Rendemen powder of kecombrang according to Istianto (2008) Antibacterial Activity Antibacterial activity test with well diffusion method from negative gram bacteria (Eschericia coli), positive gram bacteria (Bacillus cereus), and paper disk on mold (Botcytis cinerea), and yield (Saccharomyces cereviceae). The effect of formulation into antimicrobial is shown on table 2. Tabel 2. The effect of nanoencapsulation formula of kecombrang fruit on inhibit zone bacteria Nanoencapsulation Formula of kecombrang fruit

E. coli (mm)

B. cereus (mm)

B. cinerea (mm)

S.cereviceae (mm)

Soybean protein : maltodextrin, extract concentrate 2%

25,890

27,823

6,433

16,559

26,082

27,958

6,399

16,841

26,132

27,667

6,255

16,431

26,324

27,802

6,221

16,713

Soybean protein : maltodextrin, extract concentrate 6% Gelatin : maltodextrin, extract concentrate 2% Gelatin : maltodextrin, extract concentrate 6 %

Based on inhibit zone, nanoencapsulant formulation of kecombrang fruit affect into antibacterial activity to inhibit the growth of bacteria Eschericia coli and Bacillus cereus. According to inhibit zone measure, kecombrang fruit formula is effective to inhibit bacteria Bacillus cereus if compared to bacteria Eschericia coli. According to Hadioetomo et al. (1988), positive gram bacteria (Bacillus cereus) have a simple structure cell rilem bane, it has not lypopolisakaride layer, and it is easy to find antibacterial in the bacteria cell. Negative gram bacteria have outer membranes which inhibit the entry of antibiotic compounds (Cock, 2009). Besides the different cell rillem structure, acid level (pH) is the essential factor to affect antimicrobial effectivity. Kecombrang fruit formula has pH amount 3-4. Stratford (2000) stated that acid decrease pH cytoplasm, affect membrane cell structure and its fluidity and also stuck ions in cell rilem of bacteria. Microbes inhibit mechanism of antimicrobial compounds is using many ways, they are: 1) break the cell rilem compounds; 2) increase elasticity of cell membrane (cytoplasm membrane) which cause the loss of cell membrane; 3) deactive the essential enzyme in cell metabolism; 4) break the genetics function or DNA (Hadioetomo et al.,1998). Furthermore the inhibite zone into bacteria Bacillus cereus broadly than Eschericia coli. Nanoencapsulant formula of kecombrang extraction inhibits more the activity of antibacterial than antifungal. Fungal are mold and yield. Yield is generally unicellular whereas mold is filament. Fungal can grow in a bad environment form another microbes, it includes acid and a high sugar concentrate (Pelezar dan Chan, 1986). In this research, the small inhibit mark is shown on treatment of Botrytis cinerea. It is shown on medium antimold activity (Stout, 1971 in Priyatmoko, 2008), amount mm. Botrytis cinerea is growth mold and live on strawberry. It has flavonoid compound as same as kecombrang fruit. Strawberry contains anthocyanin, ellagid 3

acid, catechin, quercetin, and kaempferol. The analysis of flavone in strawberry has many variations and identified by kaempferol-rutinoside, quercetin-glucoside, quercetin-glucuronide and kaempferol-glucuronide as main flavone in strawberry (Seeram et al., 2006). Nanoencapsulant formula of Kecombrang extraction is made from addition materials, it purposes to protect kecombrang extraction compounds. These addition materials are affecting the kecombrang extract effective in antimicrobial inhibited. The affection of addition materials into antimicrobial activity is shown on table 2 Tabel 2. The effect of addition materials into nanoencapsulation of kecombrang formula to inhibit zone bacteria. E. coli (mm)

B. cereus (mm)

B. cinerea (mm)

S.cereviceae (mm)

Soybean protein : maltodextrin

27,393

29,448

6,593

16,128

gelatin : maltodextrin

28,121

28,981

6,0585

15,744

Substance concentrate 2 %

25,567

27,553

6,334

14,810

Substance concentrate 6%

26,143

27,957

6,233

15,657

Jelly

24,708

26,468

6,371

18,738

Addition material

The effect of bioactive formula is dominant and kinds of protein did not affect anything because of lowest soluble capacity. Protein soluble is affected by pH. Carboxyl and amin in amino acid will cause amphoteric protein, in acid and alkali based on pH. In isoelectric point, total protein content is zero then the interaction of protein compounds is maximal. In addition, the protein is in minimal point of soluble. The addition of miscible non-polar solution (could mix in water on any ratio) into protein solution will decrease protein solubility. These solutions are ethanol, methanol, and acetone. They will decrease dielectric constant solution or media that cause the increase of trade-off protein molecules. Finally, the interaction between proteins are increased and protein solution is decreased (Kusnandar, 2010). Soybean protein is one of free fat soybean or low fat soybean that processed into high protein. The soybean protein concentrate is continue product from soybean powder, which is made from throwing a whole carbohydrate and minerals, then the protein fraction is increased at least 70% in dried measure. According to Poppe (1999) gelatin is soluble in water on 71C and does not soluble on ethanol. In ethanol solution test, gelatin is clotted. It caused the lowest ethanol soluble. Protein and others material in a formula is bounded to maltodextrin. According to Winarno (2002) maltodekstrin has a characteristic which bounds hydrophobic essences. The more matodextrin contain in a formula, the more compounds that bound each other. (Anwar, 2002). The highest substance concentrate will decrease antimicrobial activity. Gelatin and soybean protein are substances which contains many proteins and will be an energy source and growth media for microbes, and it protects microbes from antimicrobial compounds (Dwijoseputro, 1982). But in substance material 6%, the antibacterial activity is increased 28,203-30,109 mm. According to Stout (1971) in Priyatmoko (2008), the strength of antibacterial from inhibits area 20 mm or more strength. This is caused from the highest substance concentrate 6%, therefore it is bounded each other. Gelatin which is homogenized with maltodextrin and water in formula processing is being cold and solid because it decreases until 40°C. In this temperature, gelatin is not a liquid but it has to be solid (Sesella, 2010). The compounds of gelatin-maltodekstrin which is cold and gel structure when it is added kecombrang fruit concentration does not mix completely. It is caused from heating temperature is not enough to melt gelatin-maltodextrin therefore it will mix well with kecombrang fruit concentrate. In addition, Anonim (1995) in Sesella (2010) stated that gelatin dissoluble in cold water, fat oil and evaporated oil. Most of compounds in kecombrang fruit concentrate is oily, then it guessed that substance material with concentration 6% did not mix completely in kecombrang fruit concentrate. Jelly stabilizer gives a high inhibit effect into mold Saccharomyces cereviceae compared with other treatments. Gel characteristic is affected with concentrate, temperature, pH and sugar compound. Jelly solution 4

with concentrate 1,5% formed a gel in 32-39°C, and did not melt on 85°C. pH value affect jelly gel. Though jelly has a highest gel, where agarose compounds in jelly have double helix structure which is aggregated in three dimensions, bound with water molecule make a themoreversible gel (Venugopal, 2009 in Ramadhan, 2011), decreasing of pH caused the weak gel (Imeson, 2010). Optimal pH revolution in making gel of jelly is 3-10. The highest of pH, the more stabilization of gel in jelly. Kecombrang fruit formula has pH acid amount 3-4, then in pH, gel of jelly is not strong and antimicrobial essences in kecombrang fruit formula did not bound each completely from jelly stabilizer. This is the matter of stabilized formula which is given a spread inhibit zone of Sacharomyces cerevisiae. CONCLUSSION Nanoencapsulant of kecombrang fruit formula (Nicolaia spesiosa) is effective to inhibit positive gram bacteria Bacillus cereus than negative gram bacteria Eschericia coli, mold Botrytis cinerea, and yield Saccharomyces cereviceae with inhibit zone of B.cereus 27,667-27,958 mm. The lowest inhibit is bacteria Botrytis cinerea with inhibit zone 6,221-6,433 mm. Stabilizer of jelly could increase inhibit activity yield activity Saccharomyces cereviceae with inhibit zone 18,738 mm. REFERENCES Anwar, E. 2002. Pemanfaatan maltodekstrin dari pati singkong sebagai bahan pengikat lapis tipis tablet. Makara Sains 6 (1) : 1-5. Cock, I.E. 2009. Antimicrobial Activity of Eucalyptus major and Eucalyptus baileyana Methanolic Extracts. The Internet Journal of Microbiology. Vol 6 : 1. Biomolecular and Physical Sciences, Griffith University. Australia. Dwijoseputro. 1982. Dasar-Dasar Mikrobiologi. Penerbit Djambatan, Jakarta. 205 hal. Imeson A. 2010. Food Stabilisers, Thickeners, and Gelling Agent. United Kingdom: Willey Blackwell Publishing Ktd 31-47 p. Hadioetomo, R.S., T.I.S.S. Tjitrosomo, dan S.L. Agka. 1988. Dasar-Dasar Mikrobiologi I. Terjemahan. UI Press, Jakarta. 234 hal. Kim, Y.D., C.V. Moor, and T.W. Schenz. 1996. Microencapsulation properties of gum arabic and several food proteins: liquid orange oil emulsion particles. J. Agric. Food Chem. 44:1314-1320. Koswara, S. 2007. Teknologi Enkapsulasi Flavor Rempah-Rempah (On-line) http://www. ebookpangan.com Diakses 15 April 2012. Kusnandar, F. 2010. Kimia Pangan Komponen Makro. Dian Rakyat, Jakarta Naufalin, R dan S.R, Herastuti. 2012. Pengawet Alami pada Produk Pangan. UPT Percetakan dan Penerbitan Universitas Jenderal Soedirman. Purwokerto. 131 hal. Pelezar, M.J. dan E.C.S. Chan. 1986. Dasar-Dasar Mikrobiologi. UI Press, Jakarta. 443 hal. nd

Poppe, J. 1999. Gelatin. In: Imeson A. (eds). Pp 145-167: Thickening and gelling agents for food. 2 edition. Aspen publisher, Inc. Maryland. 320 p. Priyatmoko, W. 2008. Aktivitas Antibakteri Karang Lunak Hasil Transplantasi (Sinularia sp) pada Dua Kedalaman Berbeda di Perairan Pulau Pramuka Kepulauan Seribu, DKI Jakarta. Skripsi. Fakultas Perikanan dan Ilmu Kelautan, Institut Pertanian Bogor. Bogor (On-line) http://www.google.com/url?sa=t&rct=j&q= &esrc=s&source=web&cd=9&ved=0CHkQFjAI&url=http%3A%2F%2Frepository.ipb.ac.id%2Fbitstream%2Fhandle% 2F123456789%2F5416%2FC08wpr.pdf%3Fsequence%3D4&ei=qcoCUK-sOZCXmQX6rN3qCQ&usg=AFQjCNEt MG8GyE1xrDaqsAMODFmrcP0t9w&sig2=uaH4gn9DzA4Clvqq5yEyEg Diakses 15 Juli 2012. Ramadhan, W. 2011 Pemanfaatan Agar-Agar Tepung sebagai Texturizer pada Formulasi Selai Jambu Biji Merah (Psidium Guajava L.) Lembaran dan Pendugaan Umur Simpannya. Skripsi. Fakultas Perikanan dan Ilmu Kelautan, Institut Pertanian Bogor. Bogor (On-line) http://www.google.co.id/url?sa=t&rct=j&q=&esrc= s&source=web&cd=1&ved=0CDIQFjAA&url=http%3A%2F%2Frepository.ipb.ac.id%2Fbitstream%2Fhandle%2F123 456789%2F48167%2FC11wra.pdf%3Fsequence%3D1&ei=IubiT8jBCsLsrAe3j_WYAw&usg=AFQjCNE1h0i0VsV8H Fhn8_EDXS-baCjvLw&sig2=KprXQHKbFCGbVQ09JosVtQ Diakses 17 Juni 2012. Sesella, A.D. 2010. Formulasi Chewable Lozenges yang Mengandung Ekstrak Kemangi (Ocimum sanctum L.). Skripsi. Fakultas Farmasi, Universitas Muhammadiyah Surakarta. Surakarta. (On-line) http://www.google.co.id/url?sa=t&rct= j&q=&esrc=s&source=web&cd=1&ved=0CEYQFjAA&url=http%3A%2F%2Fetd.eprints.ums.ac.id%2F9541%2F2%2F K100050098.pdf&ei=VOvzT_j7M5DPrQeJ8ujRBg&usg=AFQjCNFhS-hybtwxg3vs_EIaEVlXQapaMQ&sig2=C7 ZsT6NFkDdNCyEI83yQ_A Diakses 3 Juli 2012. Setiyani, T. 2011. Uji Aktivitas Antibakteri Ekstrak Buah Kecombrang (Nicolaia Speciosa Horan): Pengaruh Jenis, Bagian Buah dan Konsentrasi Ekstrak Buah Kecombrang. Skripsi. Fakultas Pertanian, Universitas Jenderal Soedirman. Purwokerto. (Tidak dipublikasikan). 83 hal.

5

Seeram, N.P.,Adams, L.S.,Zhang, Y., Lee R., Sand, D.Scheuller, H.S., et.al.2006a. Blackberry, Black raspberry, Blueberry,Cranberry, Red raspberry, and Strawberry Extracs Inhibit Growth and Stimulate Apoptosis of Human Cancer Cells in Vitro. Journal of Agricultural and Food Chemistry. Stratford M. 2000. Traditional preservatives organic acids. In : Robinson RK, Batt CA, Patel PD, editor. Encyclopedia of food microbiology. volume 1. London : Academic Press. Syukri, A. 2008. Mikroenkapsulasi Hasil Fraksinasi Batang Kecombrang (Nicolaia speciosa Horan) Bagian Dalam: Penetapan Sifat Fisikokimia Mikrokapsul. Skripsi. Fakultas Pertanian, Universitas Jenderal Soedirman. Purwokerto. (Tidak dipublikasikan). 54 hal. Tampubolon, O. T., S. Suhatsyah, dan S. Sastrapradja. 1983. Penelitian Pendahuluan Kimia Kecombrang (Nicolaia speciosa Horan). Risalah Simposium Penelitian Tumbuhan Obat III. Fakultas Farmasi, UGM. Yogyakarta. 63 hal. Valianty, K. 2002. Potensi Antibakteri Minyak Bunga Kecombrang. Skripsi. Fakultas Pertanian, Universitas Jenderal Soedirman. Purwokerto. 38 hal (Tidak dipublikasikan). 38 hal. Winarno, F.G. 2002. Kimia Pangan dan Gizi. PT Gramedia Pustaka Utama, Jakarta. 253 hal.

6