Judul: Pengaruh Paparan Ekstrak Kasar Alkaloid Daun Pepaya Muda Kering Beku terhadap Empat Isolat Staphylococcus aureus dan Ekspresi Gen sea
Abstrak: Staphylococcus aureus produces staphylococcal enterotoxin A (SEA) that causes food poisoning and resistant to the heating process. The presence of S. aureus and SEA on food could be detected by moleculer technique, its conducted quickly and accurately. Molecular detection is based on the genetic information of cell. Alkaloids are bioactive compound in young papaya leaves that can act as antimicrobials. The drying process and extraction method are greatly affecting the total alkaloid content in papaya leaves. Alkaloids as antimicrobial can acts by intercalating into cell wall and double-stranded DNA, and affecting topoisomerase enzymes and DNA-repairing enzymes. The aims of this research were to study the effect of drying methods of young papaya leaves to the yields of crude alkaloid extract, to identify toxin-producing of four S. aureus by molecular technique, and to identify the effect of crude alkaloid extract exposure towards S. aureus cells and relative expression of sea gene of four S. aureus isolated from food origin, i.e. raw milk (S10), balado eggs (TBI), stir-fried chicken intestine (UA13) and chicken offal satay (SJI). Drying of Calina young papaya leaves was conducted with the oven temperature of 55°C and freeze drying. Crude alkaloid extract were extracted by ultrasonication method and detected using Mayer’s reagent. S. aureus isolates that have been stored in ampoules were examined morphologically by growing them on baird parker agar (BPA) and triptyc soy agar (TSA) media. Antibiotic resistance was conducted towards S. aureus isolates using gentamicin, streptomycin, kanamycin, chloramphenicol, tetracycline and oxytetracycline. S. aureus isolates were molecularly detected with conventional PCR. The PCR results were sequenced to see the sequence of bases on each DNA, then the sequence was analyzed with Basic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov. Exposure of crude alkaloid extract with concentrations of 0.25 mg/ml and 0.5 mg/ml towards S. aureus was conducted for 2 hours. The cell number of S. aureus before and after 2-hour exposure was calculated using agar spread-plate method, then the relative expression of sea gene was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Papaya leaves dryed with freeze drying method contained crude alkaloid extract content of 1.34±0.36%. These results were relative higher than the oven drying method that only produced 0.99±0.03% crude alkaloid extract, although statistically they were not significantly different. S10, TBI, UA13 and SJI isolates on BPA media has round-shaped colonies, smooth, convex and gray to black in color. The colonies’ surrounded edge was clear (with bright area). All isolates formed a yellow pigment on TSA media. All isolates were resistant to streptomycin. In addition, there was a different sensitivity of each bacteria toward antibiotic gentamicin, kanamycin, chloramphenicol, tetracycline and oxytetracycline. Analysis of isolates by conventional PCR showed that all isolates were positive for sea gene with the amplicon size 120 bp. S10, UA13 and TBI isolates have similarities with Staphylococcus aureus strain RKI4 and SJI isolate has similarities with S. aureus 08BA02176 genome in GenBank. Exposure of crude alkaloid extract obtained from freeze-dried young papaya leaves at 0.25 mg/ml and 0.5 mg/ml for 2 hours slightly reduced the cell number of S. aureus for approximately
Keyword: alkaloids, S. aureus, sea gene, real time PCR

Judul: Aktivitas ekstrak kasar alkaloid daun pepaya terhadap Staphylococcus aureus dan ekspresi gen Staphylococcal enterotoxina
Abstrak: Papaya plant is widely grown in Indonesia. Papaya leaves are used by people as vegetable, meat tenderizer and traditional herbs. Extracts of papaya leaves have been reported to have antimicrobial activity against both Gram positive and Gram negative bacteria. This antimicrobial activity was related to phytochemical content of papaya leaves. Tannins and terpenoids can make cell membrane disruption, whereas alkaloids have been shown to intercalate into DNA and inhibit DNA synthesis. Therefore, besides as meat tenderizer, papaya leaves can be an alternative natural preservatives in food. Staphylococcus aureus is known pathogen causing intoxication by producing staphylococcal enterotoxins (SEs) in food. Staphylococcal enterotoxin A (SEA) is one of SEs that is commonly implicated in staphylococcal food poisoning. SEA is synthesized from sea gene and expressed from the midexponential phase of growth. In addition to the ability to inhibit S. aureus growth, there is a potential action of papaya leaves extract, i.e the ability to inhibit SEA production through the alkaloids activity. This study was conducted to investigate the ability of crude alkaloid extract from papaya leaves to inhibit SEA expression. Crude alkaloid extract was extracted from grounded dried papaya leaves using ultrasonic-assisted extraction. S. aureus was isolated from ready to eat food and raw cow milk using modified Bacteriological Analytical Manual (BAM) method. Isolates with typical colony of S. aureus on Baird-Parker Agar (BPA) and Mannitol Salt Agar (MSA) and produced coagulase were identified using API Staph test and PCR of 16S rRNA gene, followed by sequencing. Those 16S rRNA gene sequence were analyzed using BLAST. Minimum Inhibitory Concentration (MIC) of crude alkaloid extract were determined by broth macro-dilution method. Furthermore, S. aureus isolate was exposed to crude alkaloid extract at 0, 1, and 2 folds of MIC, and subsequently the expression of sea gene was analyzed using a quantitative reverse transcription PCR (qRT-PCR). The results demonstrated that the yield of crude alkaloid extracts was 0.48% to 1.82% per dry weight of papaya leaves. SEA-producing S. aureus were obtained from some foods, i.e raw milk, fried egg in chilli sauce, sauteed chicken intestine, and chicken liver satay. The MIC of crude alkaloid extract to S. aureus was 0.25 mg/ml. After exposure to the crude alkaloid extract at 0.25 mg/ml and 0.5 mg/ml for 2 h, significant increase in cycle threshold (CT) values of sea gene was observed. These results indicated a decrease of initial amount of sea cDNA which meant lesser mRNA was synthesized from the sea gene. The sea gene was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid extract at 1 and 2 folds MIC, respectively, determined relatively to the calibrator sample and an internal control using comparative CT method. The crude alkaloid extract of papaya leaves possessed antibacterial activity against S. aureus which was not only inhibited the bacteria but also the toxin formation by suppressing the expression of sea gene.
Keyword: crude alkaloid extract, papaya leaves, Staphylococcus aureus, staphylococcal enterotoxin A

Judul: Cycle Hamilton pada Graf Lengkap, Graf Regular, dan Graf 2-Connected 4-Regular Berorder Kurang dari Sepuluh
Abstrak: Suatu graf disebut graf Hamilton apabila graf tersebut memuat cycle Hamilton, yaitu cycle yang melewati setiap vertex tepat satu kali. Menentukan keberadaan cycle Hamilton pada suatu graf dapat menggunakan syarat cukup yang berupa teorema-teorema yang telah diperkenalkan sejak tahun 1950-an. Beberapa syarat cukup yang dibahas dalam karya ilmiah ini adalah syarat cukup yang berhubungan dengan graf lengkap dan graf regular. Suatu graf lengkap berorder 𝑛������ ≥ 3 dan graf r-regular berorder 𝑛������ ≤ 2𝑟������ + 1 merupakan graf Hamilton, Karena kedua graf tersebut memuat cycle Hamilton. Selanjutnya, syarat cukup tersebut digunakan untuk menentukan keberadaan cycle Hamilton pada graf 2-connected 4- regular berorder kurang dari 10.
Keyword: graf Hamilton, graf regular, graf lengkap, graf 2-connected 4-regular