Ekstrak Daun Rumput Kebar sebagai Antikapang Aspergillus flavus Toksigen dan Antiaflatoksin serta Aplikasinya Pada Sistem Pangan

EKSTRAK DAUN RUMPUT KEBAR (

Biophytum petersianum

) SEBAGAI

ANTIKAPANG

Aspergillus flavus

TOKSIGEN DAN ANTIAFLATOKSIN

SERTA APLIKASINYA PADA SISTEM PANGAN

MEIKE MEILAN LISANGAN

SEKOLAH PASCASARJANA INSTITUT PERTANIAN BOGOR

BOGOR 2014

PERNYATAAN MENGENAI DISERTASI DAN

SUMBER INFORMASI SERTA PELIMPAHAN HAK CIPTA

Dengan ini saya menyatakan bahwa disertasi berjudul Ekstrak Daun Rumput Kebar (Biophytum petersianum) sebagai Antikapang Aspergillus flavus Toksigen dan Antiaflatoksin serta Aplikasinya pada Sistem Pangan adalah benar karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam bentuk apa pun kepada perguruan tinggi mana pun. Sumber informasi yang berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di bagian akhir disertasi ini.

Dengan ini saya melimpahkan hak cipta dari karya tulis saya kepada Institut Pertanian Bogor.

Bogor, Agustus 2014 Meike Meilan Lisangan NIM F261090071

RINGKASAN

MEIKE MEILAN LISANGAN. Ekstrak Daun Rumput Kebar (Biophytum petersianum) sebagai Antikapang Aspergillus flavus Toksigen dan Antiaflatoksin serta Aplikasinya pada Sistem Pangan. Dibimbing oleh RIZAL SYARIEF, WINIATI P RAHAYU, dan OKKY SETYAWATI DHARMAPUTRA.

Beberapa spesies kapang bersifat toksigen, diantaranya Aspergillus flavus yang dapat memproduksi aflatoksin. Berbagai upaya telah dilakukan untuk mencari bahan antikapang dan antiaflatoksin yang berasal dari bahan alami seperti tumbuhan. Biophytum petersianum adalah salah satu tumbuhan herbal yang dikenal oleh masyarakat di Papua Barat dengan nama rumput kebar, dimanfaatkan sebagai obat penyubur kandungan dan sejauh ini menunjukkan aktivitas antibakteri dan berpotensi sebagai antikapang. Tujuan penelitian ini adalah memperoleh jenis ekstrak B. petersianum yang paling efektif sebagai penghambat pertumbuhan kapang Aspergillus flavus toksigen dan penghambat produksi aflatoksin serta mengidentifikasi komposisi kimianya.

Metode penelitian yang digunakan adalah eksperimen, melalui 5 tahap penelitian yang terdiri atas (1) identifikasi fitokimia dan pengujian aktivitas antikapang berbagai jenis ekstrak daun rumput kebar untuk menghambat pertumbuhan A. flavus toksigen, (2) pengujian mekanisme penghambatan ekstrak daun rumput kebar, (3) pengujian antiaflatoksin B1 ekstrak daun rumput kebar

pada media model pangan, (4) pengujian aktivitas antikapang dan antiaflatoksin B1 ekstrak daun rumput kebar pada media sistem pangan berbasis jagung dan

kacang tanah, dan (5) pengujian sitotoksisitas ekstrak daun rumput kebar terhadap sel Vero secara in-vitro.

Daun rumput kebar diekstraksi dengan pelarut heksana, etil asetat, metanol, dan akuades secara tunggal dan bertingkat. Hasil penelitian menunjukkan bahwa ekstrak bertingkat heksana-etil asetat-metanol (HEM) daun rumput kebar pada konsentrasi 20 mg mL-1 di media kaya karbohidrat, media kaya lemak, dan media kaya protein menyebabkan hambatan pertumbuhan A. flavus tertinggi dari pada jenis ekstrak lainnya, yaitu berturut-turut 96.2, 100, dan 96.1%.

Konsentrasi hambatan minimum ekstrak HEM daun rumput kebar terhadap pertumbuhan A. flavus BCCF0219 pada media kaya karbohidrat, kaya lemak, dan kaya protein bertutur-turut sebesar 12 mg mL-1 (97.9%), 14 mg mL-1 (93.5%), dan 14 mg mL-1 (90.8%). Sedangkan konsentrasi hambatan minimum A. flavus BIO 2236 pada media kaya karbohidrat, kaya lemak, dan kaya protein berturut-turut sebesar 12 mg mL-1 (93.9%), 16 mg mL-1 (99.4%) and 16 mg mL-1 (93.8%). Pada 3 tingkat konsentrasi MIC yaitu 1 MIC, 1.5 MIC, dan 2 MIC, persentase hambatan pertumbuhan A. flavus BCCF0219 dan BIO 2236 pada media kaya karbohidrat, lemak, dan protein berkisar antara 90.8 – 100% dan 93.8 – 100%, sedangkan hambatan produksi aflatoksin B1 berkisar antara 70.9 – 100% dan

83.4 – 98.8%. Hasil analisis SEM memperlihatkan bahwa mekanisme yang terlibat dalam penghambatan pertumbuhan kapang oleh ekstrak daun rumput kebar adalah melalui perubahan morfologi hifa yang mengakibatkan kematian sel.

Pada media dengan konsentrasi jagung 12.5, 25, 50 dan 100%, persentase hambatan pertumbuhan berturut-turut berkisar pada 50.4 – 81.0, 59.3 – 85.1, 52.9 – 83.0, dan 44.0 – 66.5%. Sedangkan pada media dengan konsentrasi kacang

tanah 12.5, 25, 50 dan 100%, persentase hambatan pertumbuhan berturut-turut berkisar pada 75.9 – 100, 72.3 – 100, 63.7 – 100, 64.9 – 100. Reduksi AFB1 pada

media dengan konsentrasi jagung 100% berkisar pada 7.0 – 91.3%, sedangkan pada media dengan konsentrasi kacang tanah 100% berkisar pada 19.3 – 59.4%.

Ekstrak HEM daun rumput kebar menghambat proliferasi sel Vero lebih dari 75% pada tingkat konsentrasi ekstrak 12 – 32 mg mL-1, sedangkan nilai CC50

ekstrak HEM daun rumput kebar adalah 13.41 mg mL-1, jauh lebih besar dari batas yang ditentukan oleh NCI yaitu <20 μg mL-1.

SUMMARY

MEIKE MEILAN LISANGAN. Kebar Grass (Biophytum petersianum) Leaf Extract as Antifungal Toxigenic Aspergillus flavus and Antiaflatoxin and Its Application in Food System. Supervised by RIZAL SYARIEF, WINIATI P RAHAYU, and OKKY SETYAWATI DHARMAPUTRA.

Some fungal species are toxigenic, among others Aspergillus flavus which can produces aflatoxin. Many efforts have been done to investigate the antifungal and antiaflatoxin agents derived from plants. Biophytum petersianum is one of herbs known by the people of West Papua with the name of kebar grass utilized as fertility drugs and showed antibacterial activity and has potency as antifungal. The purpose of this study was to obtain the type of the most effective extract of B. petersianum as growth inhibitors toxigenic Aspergillus flavus and aflatoxin production inhibitor and identify its chemical composition.

The research consisted of 5 research stages i.e. (1) identification of phytochemicals and antifungal activity assay of kebar grass leaf extracts to inhibit the growth of toxigenic Aspergillus flavus, (2) inhibitory mechanism assay of kebar grass leaf extract, (3) antiaflatoxin B1 of kebar grass leaf extract in food

model media, (4) antifungal and antiaflatoxin B1 activity of kebar grass leaf

extract in maize and peanut based media, and (5) in-vitro cytotoxicity of kebar grass leaf extract on Vero cell.

Kebar grass leaf was extracted using hexane, ethyl acetate, methanol, and aquadest in single and successively way. The results showed that kebar grass extract of hexane-ethyl acetate-methanol at concentration of 20 mg mL-1 in carbohydrate-enriched medium, fat-enriched medium, and protein-enriched medium caused higher growth inhibition of A. flavus than the other extracts, i.e. 96.2, 100, and 96.1%, respectevely.

Minimum inhibitory concentration (MIC) of kebar grass HEM leaf extract on the growth of A. flavus BCCF0219 on carbohydrate-enriched medium, fat-enriched medium, and protein-fat-enriched medium were 12 mg mL-1 (97.9%), 14 mg mL-1 (93.5%), dan 14 mg mL-1 (90.8%), respectevely. Meanwhile, MIC of kebar grass HEM leaf extract on the growth of A. flavus BIO 2236 on carbohydrate-enriched medium, fat-carbohydrate-enriched medium, and protein-carbohydrate-enriched medium were 12 mg mL-1 (93.9%), 16 mg mL-1 (99.4%) and 16 mg mL-1 (93.8%), respectevely. At 3 different levels of MIC i.e. 1 MIC, 1.5 MIC and 2 MIC, the percentage of growth inhibition of A. flavus BCC F0219 and BIO 2236 in carbohydrate, fat and protein-enriched medium ranged between 90.8 - 100 % and 93.8 - 100 %, meanwhile, the inhibitory effect of Aflatoxin B1 production ranged between 70.86 - 100 % and 83.42 - 98.84 %. Based on SEM observation results, it was found that the mechanisms involved in fungal growth inhibitory of KGE were by morphological alterations of hyphal development, even the collapse of the entire hyphae.

In maize based media with maize at concentrations of 12.5, 25, 50 and 100%, the growth inhibition ranged between 50.4 - 81.0, 59.3 - 85.1, 52.9 - 83.0, and 44.0 - 66.5%. While in the peanut based media with peanuts at concentrations of 12.5, 25, 50 and 100%, the growth inhibition ranged between 75.9 - 100, 72.3 -

100, 63.7 - 100, 64.9 - 100%. Reduction of AFB1 at 100% maize based media

ranged at 7.0 - 91.3%, while the range for peanuts at 19.3 - 59.4%.

Kebar grass leaf HEM extract can inhibit Vero cell proliferation more than 75% at concentration of 12 – 32 mg mL-1, while CC50 value of kebar grass leaf

HEM extract was 13.41 mg mL-1, more higher than NCI limit of <20 μg mL-1. Keywords: antifungal, antiaflatoxin, cytotoxicity, kebar grass, Py-CMS

© Hak Cipta Milik IPB, Tahun 2014

Hak Cipta Dilindungi Undang-Undang

Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan IPB

Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis ini dalam bentuk apa pun tanpa izin IPB

Disertasi

sebagai salah satu syarat untuk memperoleh gelar Doktor

pada

Program Studi Ilmu Pangan

EKSTRAK DAUN RUMPUT KEBAR (

Biophytum petersianum

) SEBAGAI

ANTIKAPANG

Aspergillus flavus

TOKSIGEN DAN ANTIAFLATOKSIN

SERTA APLIKASINYA PADA SISTEM PANGAN

SEKOLAH PASCASARJANA INSTITUT PERTANIAN BOGOR

BOGOR 2014

Penguji pada Ujian Tertutup: Prof. Dr. Ir. Tun Tedja Irawadi, MS Dr. Nur Wulandari, STP, MSi

Penguji pada Ujian Terbuka: Dr. Nancy Dewi Yuliana, STP, DEA Dr. Raphaella Widiastuti, BSc.

Judul Disertasi : Ekstrak Daun Rumput Kebar (Biophytum petersianum) sebagai Antikapang Aspergillus flavus Toksigen dan Antiaflatoksin serta Aplikasinya Pada Sistem Pangan Nama : Meike Meilan Lisangan

NIM : F261090071

Disetujui oleh Komisi Pembimbing

Prof Dr Rizal Syarief Ketua

Prof Dr Winiati P Rahayu Anggota

Prof Dr Okky Setyawati Dharmaputra Anggota

Diketahui oleh

Ketua Program Studi Ilmu Pangan

Prof Dr Ratih Dewanti-Hariyadi

Dekan Sekolah Pascasarjana

Dr Dahrul Syah

PRAKATA

Puji dan syukur kepada Tuhan Yang Maha Esa karena atas

rahmat-Nya sehingga disertasi dengan judul ” Ekstrak Daun Rumput Kebar

(Biophytum petersianum) sebagai Antikapang Aspergillus flavus Toksigen dan Antiaflatoksin serta Aplikasinya Pada Sistem Pangan” dapat diselesaikan dengan baik. Bagian dari disertasi ini berupa artikel ilmiah berjudul “Antifungal activity of Kebar grass leaf extracts on the growth of aflatoxigenic flavus in food model media” telah dipresentasikan dalam 1st BIOTROP Conference, Oktober 2013 di IPB Convention Center (ICC) Bogor, dan telah diterima untuk diterbitkan pada International Journal Sciences: Basic and Applied Research Vol. 17 No. 2, 2014. Sebuah artikel berjudul “Effect of kebar grass (Biophytum petersianum) leaf extract on the growth and morphological structure of aflatoxigenic Aspergillus flavus” telah dipresentasikan pada International Symposium on Tropical Fungi, September 2013 dan telah dimasukkan pada International Journal of Food Science and Technology. Artikel lainnya berjudul “Aktivitas antiaflatoksin B1 ekstrak daun rumput kebar (Biophytum petersianum) terhadap

Aspergillus flavus” telah diterima untuk dipublikasi pada Jurnal Agritech Vol. 35 No. 1, Februari 2015.

Pada kesempatan ini penulis mengucapkan terima kasih dan penghargaan sebesar-besarnya kepada:

1. Prof. Dr. Rizal Syarief, Prof. Dr. Winiati P Rahayu dan Prof. Dr. Okky Setyawati Dharmaputra selaku komisi pembimbing atas curahan waktu, arahan dan bimbingannya sehingga disertasi ini dapat diselesaikan dengan baik.

2. Prof. Dr. Tun Tedja Irawadi dan Dr. Nur Wulandari selaku penguji pada ujian tertutup, Dr. Nancy Dewi Yuliana dan Dr. Raphaella Widiastuti selaku penguji pada ujian terbuka.

3. Ketua dan Sekretaris Program Studi Ilmu Pangan serta seluruh staf pengajar dan karyawan atas ilmu pengetahuan dan bantuan yang telah diberikan selama menempuh pendidikan.

4. Direktorat Jenderal Pendidikan Tinggi atas dukungan beasiswa BPPS selama menempuh pendidikan.

5. Rektor Universitas Negeri Papua (UNIPA) Manokwari yang telah memberi kesempatan bagi penulis untuk melanjutkan pendidikan di Institut Pertanian Bogor.

6. Southeast Asian Regional Centre for Tropical Biology (SEAMEO BIOTROP) atas pendanaan penelitian dalam bentuk BIOTROP PhD Thesis Grant tahun 2012.

7. PEMDA Provinsi Papua Barat yang telah memberi bantuan beasiswa penelitian dan PT Freeport yang telah memberi bantuan dana penelitian melalui Lembaga Pemberdayaan Masyarakat Amugme Komoro (LPMAK) Timika, Papua.

8. Laboratorium Mikrobiologi PAU dan Laboratorium Kimia PAU IPB beserta teknisinya Mbak Ari dan Mas Taufik, yang telah membantu selama proses penelitian.

9. Teman-teman seperjuangan IPN 2009 (Mbak Mardiah, Bu Aswita, Bu Zita, Bu Siti, Bu Tita, Ai) dan rekan-rekan IMAPA (Ikatan Mahasiswa Papua) Bogor atas suntikan semangatnya.

10. Yang saya hormati dan sayangi kedua orang tua (Bpk. Jaya Taufan Lie dan Ibu Marthina Tan Bonai-Abaa), mertua (Bpk. Welhelmus Talakua dan Ibu Josephine Kayadoe), suami (Richard Talakua), anak-anak yang saya cintai (Hazella Ishera Talakua, Jehefar Dearic Talakua, Ellysia Nathania Talakua, dan Elldora Nathaniela Talakua), serta seluruh keluarga besar atas doa dan dukungannya.

Semoga karya ilmiah ini bermanfaat untuk semua pihak.

Bogor, Agustus 2014 Meike Meilan Lisangan

i

DAFTAR ISI

RINGKASAN SUMMARY

HALAMAN PENGESAHAN KATA PENGANTAR

DAFTAR ISI

1 PENDAHULUAN

Latar Belakang ... 1

Tujuan Penelitian ... 2

Hipotesis Penelitian ... 2

Manfaat Penelitian ... 3

Ruang Lingkup Penelitian ... 3

Daftar Pustaka ... 4

2 ANTIFUNGAL ACTIVITY OF KEBAR GRASS (Biophytum petersianum) LEAF EXTRACTS ON THE GROWTH OF AFLATOXIGENIC Aspergillus flavus IN FOOD MODEL MEDIA Abstract ... 6

Introduction ... 7

Materials and Methods ... 8

Results and Discussion ... 10

Conclusion ... 16

References ... 16

3 EFFECT OF KEBAR GRASS (Biophytum petersianum) LEAF EXTRACT ON THE GROWTH AND MORPHOLOGICAL STRUCTURE OF AFLATOXIGENIC Aspergillus flavus Abstract ... 19

Introduction ... 19

Materials and Methods ... 20

Results and Discussion ... 23

Conclusion ... 31

References ... 32

4 AKTIVITAS ANTIAFLATOKSIN B1 EKSTRAK DAUN RUMPUT KEBAR (Biophytum petersianum) TERHADAP Aspergillus flavus Abstract ... 35

Pendahuluan ... 36

Metode Penelitian... 37

Hasil dan Pembahasan... 39

Kesimpulan ... 44

ii

5 ANTIKAPANG DAN ANTIAFLATOKSIN B1 EKSTRAK DAUN

RUMPUT KEBAR (Biophytum petersianum) PADA MEDIA BERBASIS JAGUNG DAN KACANG TANAH

Abstract ... 49

Pendahuluan ... 49

Bahan dan Metode... 51

Hasil dan Pembahasan... 53

Kesimpulan ... 57

Daftar Pustaka ... 57

6 UJI SITOTOKSISITAS EKSTRAK DAUN RUMPUT KEBAR (Biophytum petersianum Klotzsch) Abstract ... 60

Pendahuluan ... 60

Bahan dan Metode... 61

Hasil dan Pembahasan... 62

Kesimpulan ... 67

Daftar Pustaka ... 67

7 PEMBAHASAN UMUM ... 69

Daftar Pustaka ... 73

1 PENDAHULUAN

Latar Belakang

Pertumbuhan kapang pada bahan pangan selama penyimpanan dapat menyebabkan peningkatan kadar air bahan dan kelembaban relatif tempat penyimpanan. Pada kondisi demikian kerusakan terjadi dengan cepat sehingga sumber nitrogen bahan yang diperlukan kapang untuk tumbuh juga berkurang. Apabila persediaan nitrogen telah habis maka kapang mempertahankan hidupnya dengan memanfaatkan karbon yang ada dalam bahan sebagai sumber energi. Keadaan ini berlangsung dengan cara kapang melakukan metabolisme sekunder yang dapat menghasilkan mikotoksin (Syarief et al. 2012). Kontaminasi kapang dan produksi mikotoksinnya menyebabkan kerugian 25 - 40% atau bahkan lebih, terutama di negara berkembang (Murugan et al. 2013). Beberapa jenis mikotoksin utama yang telah diidentifikasi antara lain aflatoksin, okratoksin, patulin, islanditoksin, zearalenon, sterigmatosistin, trikotesen, asam penisilat, sitrinin, sporidesmin, rubratoksin, dan slaframin (Fernández-Cruz et al. 2010). Dari sekian banyak mikotoksin ini, aflatoksin merupakan jenis mikotoksin yang perlu lebih mendapat perhatian.

Aflatoksin merupakan toksin yang dihasilkan oleh kapang Aspergillus flavus dan A. parasiticus yang bersifat hepatotoksik, mutagenik, teratogenik, dan karsinogenik bagi manusia dan hewan (Alberts et al. 2006). Aflatoksin B1

merupakan jenis aflatoksin yang paling banyak diproduksi dan memiliki sifat mutagenik, karsinogenik, dan teratogenik yang sangat tinggi, baik terhadap manusia maupun hewan. Kontaminasi aflatoksin yang terjadi di Afrika, Tiongkok, dan Asia Tenggara berkorelasi positif dengan tingkat kejadian kanker hati. Selain berbahaya bagi kesehatan manusia dan hewan, kontaminasi aflatoksin pada pangan dan pakan juga mengakibatkan kerugian ekonomi yang cukup signifikan berkisar antara US $85 - 100 juta di USA (Alberts et al. 2009).

Hingga saat ini, telah banyak upaya yang dilakukan untuk meminimalisir paparan mikotoksin, terutama aflatoksin melalui berbagai macam metode. Diantaranya melalui metode fisik seperti teknik ekstruksi (Syarief et al. 2012) dan pemanasan dengan kombinasi penambahan buffer fosfat (Saalia dan Phillips 2010), metode kimia seperti penggunaan amonia (Dharmaputra dan Ambarwati 1999), dan metode biologi dengan menggunakan Bakteri Asam Laktat (Topcu et al. 2010) juga telah dilakukan. Selain itu beberapa peneliti juga telah mencoba mereduksi aflatoksin dengan menggunakan agen botani seperti ekstrak tumbuhan

dari spesies Agave (Sánchez et al. 2005), ekstrak tumbuhan Allium cepa, A. sativum dan A. indica (Reddy et al. 2009), ekstrak tumbuhan Equisetum

arvense dan Stevia rebaudiana (Garcia et al. 2011) yang cukup efektif mereduksi pertumbuhan kapang (86 – 100%) dan produksi aflatoksin.

Rumput kebar merupakan tumbuhan yang tergolong dalam famili Oxalidaceae yang ditemukan di Distrik Kebar, Papua Barat. Rumput kebar umumnya tumbuh secara alami dan tersebar hampir di seluruh wilayah Distrik Kebar (Santoso et al. 2007). Selain di Distrik Kebar, tumbuhan ini juga ditemukan di daerah Mali dan beberapa wilayah Afrika lainnya. Di Indonesia, tumbuhan ini juga dapat ditemukan di pulau-pulau lainnya kecuali Sumatra, Kalimantan, dan Jawa Barat (Veldkamp, 1976 dalam Imbiri et al. 2000). Masyarakat Mali menggunakan tumbuhan ini untuk pengobatan luka, inflamasi,

luka lambung, malaria, dan demam (Inngjerdingen et al. 2008). Masyarakat Papua sendiri, terutama di Distrik Kebar sering menggunakan tumbuhan ini sebagai obat penyubur kandungan. Berdasarkan pengalaman empiris masyarakat setempat, tumbuhan ini telah terbukti mampu meningkatkan kesuburan wanita sehingga selain digunakan oleh masyarakat setempat, tumbuhan ini bahkan telah diperdagangkan hingga keluar wilayah Papua. Santoso et al. (2007) dan Karamang (2010) mengemukakan bahwa rumput kebar memiliki kandungan alkaloid, saponin, tanin, fenolik, flavonoid, triterpenoid, steroid, dan glikosida. Komponen-komponen tersebut banyak diteliti sebagai komponen bioaktif yang dapat berperan sebagai antioksidan dan antimikrob. Keberadaan kelompok bioaktif yang cukup lengkap dalam rumput kebar membuat tumbuhan ini berpotensi sebagai antimikrob. Beberapa kajian yang dilakukan memperlihatkan aktivitas antibakteri dari ekstrak daun Rumput kebar terhadap bakteri patogen Gram positif (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae) dan Gram negatif (Klebsiella pneumonia, Salmonella Typhii, Proteus vulgaris, Escherichia coli) (Natarajan et al. 2010), namun sejauh ini belum ada laporan tentang aktivitas antikapang dari rumput kebar.

Jenis substrat merupakan salah satu faktor yang dapat menentukan kandungan aflatoksin yang dihasilkan oleh A. flavus aflatoksigen. Substrat dalam bahan pangan dapat terdiri dari karbohidrat, lemak dan protein, sehingga perlu mengkaji pengaruh ekstrak rumput kebar terhadap pertumbuhan A. flavus aflatoksigen dan produksi aflatoksin B1 (AFB1) pada berbagai jenis substrat bahan

pangan. Potensi penggunaan rumput kebar sebagai antikapang diharapkan dapat menghambat pertumbuhan kapang terutama kapang aflatoksigen serta produksi aflatoksin B1 pada bahan pangan.

Tujuan Penelitian

Tujuan penelitian secara umum adalah untuk mengkaji aktivitas antikapang dan antiaflatoksin B1 ekstrak daun rumput kebar pada media model pangan dan

media sistem pangan.

Secara khusus, penelitian ini bertujuan untuk :

1. Menentukan jenis ekstrak rumput kebar yang paling efektif menghambat pertumbuhan kapang A. flavus toksigen dan mengetahui mekanisme kerjanya.

2. Mengetahui potensi rumput kebar sebagai antikapang A. flavus dan antiaflatoksin B1 pada media model pangan dan sistem pangan.

3. Mengetahui sitotoksisitas ekstrak daun rumput kebar terhadap sel Vero secara in vitro

Hipotesis Penelitian Hipotesis yang diajukan pada penelitian ini adalah :

1. Ekstrak rumput kebar memiliki potensi sebagai antikapang A. flavus toksigen pada media model pangan.

2. Ekstrak rumput kebar memiliki potensi sebagai agen penghambat produksi aflatoksin B1 dalam sistem pangan.

Manfaat Penelitian

Hasil penelitian ini diharapkan dapat memberi informasi baru mengenai komponen aktif yang terdapat pada rumput kebar dan kemampuannya sebagai antikapang A. flavus toksigen dan agen penghambat produksi aflatoksin. Diharapkan rumput kebar dapat digunakan sebagai bahan tambahan pangan yang berfungsi sebagai antikapang A. flavus toksigen dan antiaflatoksin B1.

Ruang Lingkup Penelitian

Penelitian ini dilakukan melalui lima tahapan yaitu: (1) Identifikasi fitokimia dan pengujian aktivitas antikapang berbagai jenis ekstrak daun rumput kebar untuk menghambat pertumbuhan A. flavus toksigen, (2) Pengujian mekanisme penghambatan ekstrak daun rumput kebar, (3) Pengujian

antiaflatoksin B1 ekstrak daun rumput kebar pada media model pangan,

(4) Pengujian aktivitas antikapang dan antiaflatoksin B1 ekstrak daun rumput

kebar pada media sistem pangan berbasis jagung dan kacang tanah, (5) Pengujian sitotoksisitas ekstrak daun rumput kebar terhadap sel Vero secara in vitro.

Pelarut yang digunakan untuk ekstraksi menggunakan tiga jenis pelarut yaitu pelarut non-polar, semi-polar dan polar yang masing-masing diwakili oleh n-heksana, etil asetat dan metanol. Isolat yang digunakan merupakan isolat aflatoksigen yaitu isolat BCC F0219 yang dicirikan dengan pertumbuhan yang sangat cepat dan produksi konidium yang berlimpah, dan BIO 2236 yang bercirikan produksi konidium sedikit tetapi produksi aflatoksin B1 tinggi.

Gambar 1 Bagan alir pelaksanaan penelitian Daftar Pustaka

Alberts JF, Engelbrecht Y, Steyn PS, Holzapfel WH, van Zyl WH. 2006. Biological degradation of aflatoxin B1 by Rhodococcus erythropolis

cultures. Int J Food Microbiology. 109:121 – 126

Alberts JF, Gelderblom WCA, Botha A, van Zyl WH. 2009. Degradation of aflatoxin B1 by fungal laccase enzymes. Int J Food Microbiol. 135:47– 52Fernández-Cruz ML, Mansilla ML, Tadeo JL. 2010. Mycotoxins in fruits and their processed products: Analysis, occurrence and health implications. J Adv Res. 1:113–122

Dharmaputra OS, Ambarwati S. 1999. Detoxification effects of ammonium hydroxide on aflatoxin and total nitroge content of maize. Hayati 6(2):25-28.

Garcia D, Garcia-Cela E, Ramos AJ, Sanchis V, Marín S. 2011. Mould growth and mycotoxin production as affected by Equisetum arvense and Stevia rebaudiana extracts. Food Control. 22: 1378–1384.

Persentase hambatan pertumbuhan, persentase hambatan

produksi AF

Rumput Kebar

Ekstraksi dengan pelarut organik dan akuades

Ekstrak Etil asetat Ekstrak Metanol Ekstrak Heksana Ekstrak Akuades Ekstrak Heksana-etil asetat Ekstrak Heksana-Etil asetat-Metanol

Uji fitokimia kualitatif Uji Aktivitas antikapang

Media model pangan Media sistem pangan

Diameter koloni Produksi konidium

Diameter koloni

Identifikasi golongan senyawa

Py/GCMS

Uji sitotoksisitas Mekanisme antikapang

Ekstraksi Tunggal Ekstraksi Bertingkat

Data fitokimia Jenis ekstrak_terpilih

Diameter koloni Produksi AF

Persentase hambatan pertumbuhan, data MIC Produksi AF Golongan senyawa Persentase hambatan pertumbuhan, persentase

hambatan produksi AF

CC50 Tahap V Tahap III Tahap IV Tahap II Tahap I

Imbiri ANNH, Wanggai F, Maturbongs RA. 2000. An ecological aspect of Biophytum petersianum Klotzsch in Kebar district, Manokwari, Irian Jaya. Beccariana. 2 (2):44 -47.

Inngjerdingen M, Inngjerdingen KT, Patel TR, Allen S, Chen X, Rolstad B, Morris GA, Harding SE, Michaelsen TE, Diallo D, Paulsen BS. 2008. Pectic polysaccharides from Biophytum petersianum Klotzsch, and their activation of macrophages and dendritic cells. Glycobiology. 12:1074–1084.

Karamang S. 2010. Studi Morfologi, Agrobiofisik dan Produksi Saponin Rumput Kebar (Biophytum petersianum Klotzsch) Asal Papua [tesis]. Bogor (ID): Fakultas Teknologi Pertanian, Institut Pertanian Bogor.

Murugan K, Anandaraj K, Al-Sohaibani S. 2013. Antiaflatoxigenic food additive potential of Murraya koenigii : An in vitro and molecular interaction study. Food Res Int. 52:8 – 16

Natarajan D, Shivakumar MS, Srinivasan R. 2010. Antibacterial activity of leaf extracts of Biophytum sensitivum (L.) DC. J Pharm Sci Res. 11:717-720 Reddy KRN, Reddy CS, Muralidharan K. 2009. Potential of botanicals and

biocontrol agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains. Food Control. 20:173–178

Saalia FK, Phillips RD. 2010. Degradation of aflatoxins in aqueous buffer in the presence of nucleophiles. Food Control. 21:1066–1069

Sa´nchez E, Heredia N, Garcia S. 2005. Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergillus parasiticus by extracts of Agave species. Int J Food Microbiol. 98:271 – 279

Santoso B, Kilmaskossu A, Sambodo P. 2007. Effects of saponin from Biophytum petersianum Klotzsch on ruminal fermentation, microbial protein synthesis and nitrogen utilization in goats. Animal Feed Sci Tech. 137:58–68.

Syarief R, La Ega, Nurwitri CC. 2012. Mikotoksin Bahan Pangan Ed.2. Bogor (ID): IPB Pr.

Topcu A, Bulat T, Wishah R, Boyac IH. 2010. Detoxification of aflatoxin B1 and

patulin by Enterococcus faecium strains. Int J Food Microbiol. 139:202– 205

2 ANTIFUNGAL ACTIVITY OF KEBAR GRASS

(

Biophytum petersianum

) LEAF EXTRACTS ON THE

GROWTH OF AFLATOXIGENIC

Aspergillus flavus

IN FOOD MODEL MEDIA

1,2)

ABSTRACT

Some fungal species are toxigenic, for example Aspergillus flavus which can produces aflatoxin. Various methods have been conducted to reduce aflatoxin contamination in foods, among others using antimicrobial compounds derived from natural plant extract. Kebar grass (Biophytum petersianum) is one of herbs that has potency as antimicrobe. The objective of this study was to investigate the effect of three types of model media i.e. carbohydrate- enriched media, fat-enriched media, and protein- enriched media containing kebar grass leaf extract on the growth of aflatoxigenic A. flavus. Kebar grass leaf extract at concentrations of 5, 10, and 20 mg mL-1 was extracted using various solvents, i.e. hexane, ethyl acetate, methanol, hexane-ethyl acetate, hexane-ethyl acetate-methanol, and aquadest. The extracts were tested on the growth of A. flavus. The result showed that kebar grass leaf extract of hexane-ethyl acetate-methanol at concentration of 20 mg mL-1 in carbohydrate-enriched medium, fat-enriched medium, and protein-enriched medium caused highest growth inhibition of A. flavus compared to extracts, i.e. 96.2, 100, and 96.1%, respectively. The other extracts caused growth inhibition less than 90%. The results showed that kebar grass leaf extract can inhibit the growth of toxigenic A. flavus.

Keywords: antifungal activity, aflatoxigenic Aspergillus flavus, Biophytum petersianum, food model media

ABSTRAK

Beberapa spesies kapang bersifat toksigen, diantaranya Aspergillus flavus yang dapat memproduksi aflatoksin. Berbagai metode telah dilakukan untuk mereduksi kontaminasi aflatoksin pada bahan pangan, diantaranya menggunakan komponen antimikrob yang berasal dari ekstrak tumbuhan alami. Rumput kebar (Biophytum petersianum) adalah salah satu tumbuhan herbal yang berpotensi sebagai antimikrob. Tujuan penelitian ini adalah untuk mengkaji pengaruh tiga jenis media model yaitu media kaya karbohidrat, media kaya lemak, dan media kaya protein yang mengandung ekstrak daun rumput kebar terhadap pertumbuhan A. flavus aflatoksigen. Ekstrak rumput kebar pada konsentrasi 5, 10, dan 20 mg mL-1 yang diekstraksi menggunakan heksana, etil asetat, metanol, heksana-etil asetat, heksana-etil asetat-metanol, dan akuades diuji terhadap pertumbuhan A. flavus.

1)

Has been presented in 1st BIOTROP International Conference, October 3-4th 2013, Bogor-Indonesia.

2)

Has been accepted in International Journal of Sciences: Basic and Applied Research, Vol. 17 No. 2, 2014

Hasil penelitian menunjukkan bahwa ekstrak bertingkat heksana-etil asetat-metanol rumput kebar pada konsentrasi 20 mg mL-1 di media kaya karbohidrat, media kaya lemak, dan media kaya protein menyebabkan hambatan pertumbuhan A. flavus tertinggi dibandingkan dengan jenis ekstrak lainnya, yaitu berturut-turut 96.2, 100, dan 96.1%. Jenis ekstrak lainnya menyebabkan hambatan pertumbuhan kurang dari 90%. Hasil yang diperoleh dari penelitian ini mengindikasikan bahwa ekstrak daun rumput kebar dapat digunakan untuk menghambat pertumbuhan A. flavus toksigen.

Kata kunci: aktivitas antikapang, Aspergillus flavus aflatoksigen, Biophytum petersianum, media model pangan

1. Introduction

Foods are easily spoiled by either bacteria or fungi. During pre and post -harvest handling foodstuffs can be infected by fungi. Some fungal species among others Aspergilus flavus can produce toxins (aflatoxins) which can cause liver cancer on human and animal [1]. Many efforts have been done to extend the shelf life of foods and its safety, for example using thermal and non-thermal processing techniques or the use of preservatives such as synt hetic or natural antioxidants and antimicrobes. Plant metabolites have been known to have antimicrobial and antioxidant activities and some of them are classified as Generally Recognized as Safe Substances (GRAS) [2,3].

One of the biological material that could be used as antimicrobe is kebar grass (Biophytum petersianum), a plant belong to family Oxalidaceae. In Indonesia, the plant is found in Kebar District, West Papua. The plant grows naturally and spread almost all over Kebar District [4]. Besides in Kebar District, this plant is also found in Southeast Asia and Africa, especially in Mali region. Mali community use this plant for the treatment of wounds, inflammation, ulcers, malaria, and fever [5]. Papuan community, especially in Kebar District often use this plant as a fertility drug.

It was reported that kebar grass contains bioactive compounds such as alkaloids, saponins, tannins, phenolic, flavonoids, triterpenoids, steroids and glycosides compounds [4,6]. As kebar grass contains fairly complete bioactive groups, therefore this plant is potential could be used as antimicrobe. [7] reported that the leaf extract of kebar grass has antibacterial activity against Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae) and Gram-negative bacteria (Klebsiella pneumonia, Salmonella Typhii, Proteus vulgaris, Escherichia coli), but there are no reports concerning the effect of kebar grass against toxigenic mold.

Foodstuff can consist of carbohydrate, fat and protein. Kind of substrates is a factor which will determine the growth and aflatoxin content produced by aflatoxigenic A. flavus. In order to be applied as A. flavus growth inhibitor on food, it is necessary to study the effects of kebar grass leaf extract on the growth of aflatoxigenic A. flavus in food model. The objective of this study was to study the effect of three types of model media i.e. carbohydrate-enriched media, fat-enriched media, and protein-enriched media containing kebar grass extract on the growth of aflatoxigenic A. flavus.

2. Materials and Methods

2.1 Extraction and Determination of Phytochemicals Content of Kebar Grass Leaf Extracts

Kebar grass leaves and aflatoxigenic A. flavus

Kebar grass were collected from Kebar district, Manokwari county, West Papua province, in February, 2013. Identification of kebar grass was confirmed by Indonesian Institute of Science (LIPI). Leaves of kebar grass were then air-dried for about 2 weeks until their colour turned into golden brown and they are easily broken. The leaves were then crushed into 40 mesh coarse powder using a blender (Philips). One strain of aflatoxigenic A. flavus BCCF0219 obtained from Indonesian Research Center for Veterinary Science culture collection was used in this study.

Chemicals

All chemicals were of analytical grade purchased from JT. Baker, USA. Single extraction using organic solvents

A method of [8] was used to obtain the extract of kebar grass leaves. Powder of kebar grass leaves was extracted using maceration method and various organic solvents, i.e. a non-polar (n-hexane), semi-polar (ethyl acetate), and polar organic solvent (methanol). The kebar grass powder (200 g) was placed in a glass jar (volume 1000 mL), then 800 mL n-hexane was added. Its mixture was macerated for one hour at room temperature (28 – 30 °C), stirred in a shaker that combined with sonification for 30 minutes, followed by further maceration for one hour then filtered using Whatman No.1 filter paper. The extraction was conducted three times. The extract was collected and dried using a rotary evaporator to form a thick concentrate. The remaining solvent was removed using nitrogen gas. The yield of extract was calculated as the percentage of extract (mg extract/100 g kebar grass powder). Extraction using ethyl acetate and methanol was conducted with the same way. The extraction process was performed in triplicate. The extracts stored at 4°C until used.

Extraction using distilled water

Kebar grass leaves were extracted in distilled water (60 0C) for one hour. The extract was centrifuged at 5000 xg for 10 minutes, the supernatant was collected and dried using freeze drier to form a powder and the dry weight of extract was determined. The yield of extract was calculated as the percentage of extract (mg extract/100 g kebar grass powder). The extraction process was performed in triplicate. The extract was stored at 4°C until used [9].

Successively extraction by using n-hexane-ethyl acetate-methanol

Powdered kebar grass (200 g) was extracted using maceration (1 hr) method combined with sonication (30 min) at room temperature (28 – 30 °C) using three types of organic solvent, i.e. n-hexane, ethyl acetate, and methanol solvents, followed by Whatman No.1 paper filtration. Maceration was repeated for 2 times and the filtrate was collected. The remaining residue of the plant material was dried for 24 hours and extracted using ethyl acetate and methanol solvent

sequentially in a similar manner. The filtrate was collected and dried using a rotary evaporator to form a thick concentrate. The hexane-ethyl acetate-methanol filtrate was called as HEM extract. Residual solvent in HEM extract was removed using nitrogen gas [8]. The extract was then stored at 4 °C until used.

Phytochemical test

Phytochemical tests were performed on crude organic extract and aqueous extract of kebar grass leaves. The tests were conducted to determine the content of flavonoids, tannins, alkaloids, phenylhydroquinon, triterpenoids, and saponins [10].

2.2.Antifungal Activity Screening of Kebar Grass Extract in Food Model Media

Preparation of agar medium enriched with carbohydrate, fat, and protein Carbohydrate-enriched medium was prepared by the addition of 30% (w/v) glucose on Bacto Agar. The medium was sterilized in an autoclave at 121 °C for 15 minutes [11].

Medium enriched with fat was prepared based on [12]. One hundred grams of grated coconut flesh was homogenized using 300 mL of hot distilled water for five minutes. The mixture was filtered adjusted its pH into 7.0 using NaOH 2M. Agar powder was added (20 g L-1), and sterilized in an autoclave at 121°C for 15 minutes.

Protein-enriched medium was prepared using the method of [13] with a slight modification on the type of protein source. As much as 30% (w/v) skim milk obtained from Difco Laboratory was added to Bacto Agar medium and sterilized in an autoclave at 121°C for 10 minutes.

Preparation of A. flavus conidia

The conidia of A. flavus were prepared using the method of [14] with a slight modification. The fungus was cultured in PDA slant and incubated at 28°C for 5 days. The conidia were harvested by adding 10 mL of sterile distilled water and collected aseptically. Conidial suspension was dissolved in Tween 80 (0.5%) and centrifuged (2000xg) at 28 – 30 °C for five minutes. Conidial concentration was calculated using a haemacytometer and adjusted to 106 CFU mL-1.

Mycelial Growth Assay

The effect of kebar grass extract on mycelial growth of A. flavus was tested using [15] and [16] methods. Each kebar grass extract dissolved in their solvent to reach a concentration of 50 mg mL-1, pipetted aseptically onto the food model media to produce concentration 5, 10, 20, 50 mg mL-1. Ten mililitres model medium containing extract was poured into each Petri dish (9 cm in diameter) and left it until solid. Five microlitres of conidial suspension of A. flavus was inoculated on the surface of each test media, and then incubated at room temperature (28 –30 °C) for 10 days. The growth of mycelia was determined by measuring the diameter of colony (in mm) every 24 hours. As control, the fungus was cultured in food model media without kebar grass extract. Two replicates were

used for each treatment. The percentage of inhibitory growth was determined using the following formula:

(dc - dt)

Growth Inhibition = X 100%, dc

where dc = diameter of fungal colony in medium without kebar grass extract (mm)

dt = diameter of fungal colony in medium containing the extract of kebar grass (mm)

2.3.Statistical Analyses

The results were analyzed using Microsoft Excel 2007. All the values are expressed as means ± standard deviation (n=2).

3. Results and Discussion

3.1.Yield and Physical Properties of Kebar Grass Extracts

Yield and physical properties of kebar grass extracted with single solvent and the combination of various solvents is presented in Table 2.1. Extraction using hexane-ethyl acetate produced the lowest extract yield (1.55%), while a single extraction of methanol produced the highest yield (14.17%). Extract yield of hexane-ethyl acetate was low, because some components were soluble in nonpolar and semipolar solvents, such as steroids were extracted by nonpolar solvent, therefore the less that can be extracted by a semipolar solvent. The yield of a polar extract, either extracted using a single solvent (methanol) or graded solvent (hexane-ethyl acetate-methanol) was high, because of the more polar components were able to be extracted well using polar solvents.

Table 2.1 Yield and physical properties of kebar grass extract

Physical properties

Type of extracts n-Hexane extract Ethyl acetate extract Methanol extract n-Hexane-ethyl acetate extract n-Hexane-ethyl acetate-methanol extract Aquadest extract

Colour Greenish brown

Brown Dark

brown

Greenish brown

Dark brown Murky brown Appearance Viscous,

oily

Viscous Fluid, somewhat viscous

Viscous Fluid, somewhat viscous Brown powder Yield (% w/w)

2.94 6.15 14.17 1.55 12.05 3.28

The difference of solvent polarity resulted different phytochemical components of extract. Phytochemical components produced by various organic solvents is presented in Table 2.2.

Hexane can extract nonpolar components such as waxes, fats and oils, including essential oils. However, extraction using hexane also resulted in less

flavonoids. Other components that could be extracted using hexane solvent are steroids and triterpenoids. Extraction using ethyl acetate can extract alkaloids, flavonoids, phenylhydroquinon, steroids and triterpenoids, whereas extraction using methanol can extract components such as alkaloids, flavonoids, phenolhydroquinon, steroids, triterpenoids, tannins and saponins. According to [10], alkaloids are components of semipolar alkaline containing one or more nitrogen atoms and are widely used in medicine. Alkaloids are also often used as an antibacterial ingredient that is bactericidal [17,8].

Table 2.2 Phytochemicals of kebar grass extracts

Group Compound

Type of extracts Kebar grass powder Hexane extract Ethyl acetate extract Methanol extract Hexane-ethyl acetate extract Hexane-ethyl acetate-methanol extract Aquadest extract

Alkaloids + - + + - - +3

Flavonoids +2 + +2 +3 +2 +3 +2

Phenolhydroquinon +2 - +2 +3 + +3 +2

Steroids + +2 +3 + +3 +2 +

Triterpenoids +2 + + + + + +

Tannins +3 - - +2 - + +3

Saponins +3 - - +2 - +2 +

Note : + = weak positive; +2 = positive; +3 = strong positive ; - = not detected

Another class of compounds extracted, either using nonpolar, semipolar or polar solvents are flavonoids and phenolhydroquinon, but more strongly extracted in polar solvents, due to these compounds are polar [18]. As an antioxidant [19], flavonoids and phenolhydroquinon also have antimicrobial properties [18,20]. Steroids strongly extracted by hexane and ethyl acetate solvents. This is because most of the steroid is nonpolar and semipolar, so it can be extracted using nonpolar and semipolar solvents. Triterpenoids extracted using hexane, ethyl acetate, and methanol solvent, can be divided into four groups, namely true triterpene, steroids, saponins and cardiac glycosides [10]. Triterpenoids are non-volatile terpenoid compounds. Terpenoids are the building blocks of essential oils. Besides having biological activity, triterpenoids also have antimicrobial properties [21]. Tannins and saponins are highly polar compounds, they can be extracted only using polar solvents. Both classes of compounds have antimicrobial activity [18]. Saponins are surface active compounds and it is like soap, which can be detected by their ability to form foam and hemolyzed blood cells. Saponin has expectorant action, which is very useful in the management of upper respiratory tract inflammation. Saponins was also reported to have antidiabetic properties [22].

3.2.Antifungal Activity of Kebar Grass Single Extract Extracted Using Single Solvent

Antifungal activity of kebar grass extracts at single extraction solvents (hexane, ethyl acetate, methanol, and distilled water) and at single concentrations (5, 10, 20, and 50 mg mL-1) in three types of media

(carbohydrates-enriched, fat-enriched and protein-enriched medium) is presented in Figure 2.1.

Figure 2.1 Effect of type and concentration of a single extract of kebar grass on growth inhibitory of A.flavus BCC F0219 in three types media models

The growth inhibition of A. flavus at concentration 50 mg mL-1 of methanol and ethyl acetate extract in carbohydrate-enriched medium was 100%, while hexane extract at concentration of 50 mg mL-1 caused 89.8% of growth inhibition. At a lower extract concentrations (5, 10, and 20 mg mL-1) all types of extracts caused growth inhibition of less than 50%. At concentration of 20 mg mL-1 ethyl acetate extract caused growth inhibitory higher than n-hexane and methanol extracts. Observation on mycelila growth of A. flavus on carbohydrate-enriched medium after 10x24-hour incubation after being treated with extract is presented in Figure 2.2(a-c).

(a) (b) (c)

Figure 2.2 The growth of A. flavus BCC F0219 on carbohydrate-enriched medium treated using 20 mg mL-1 of ethyl acetate extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation Figure 2.1 showed that on fat-enriched medium, methanol and ethyl acetate extracts at a concentration of 50 mg mL-1 caused growth inhibition of

-20 -10 0 10 20 30 40 50 60 70 80 90 100 110

Gr

o

wt

h

In

h

ib

ition

(

%

)

Type of Single Extracts

Carbohydrate Fat

A. flavus more than 90%, i.e. 100 and 96.3% respectively, while hexane and water distilled extracts caused only 81.3 and 45.4% growth inhibition, respectively. At lower concentrations of 5, 10, and 20 mg mL-1, all types of extracts caused growth inhibition less than 90%. At concentration of 20 mg mL-1 methanol extract caused growth inhibition higher than n-hexane and ethyl acetate extracts. Mycelial growth of A. flavus on fat-enriched medium treated using methanol extract after 10x24-hours of incubation are presented in Figure 2.3(a-c).

(a) (b) (c) Figure 2.3 The growth of A. flavus BCC F0219 on fat-enriched medium treated

using 20 mg mL-1 of methanol extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation

Inhibitions of kebar grass extract using methanol, ethyl acetate and hexane at concentration of 50 mg mL-1 each in protein-enriched medium were 100, 98.6, and 100%, respectively, while at a lower concentration of 5, 10, and 20 mg mL-1, all types of extracts caused growth inhibition less than 90%. At concentration of 20 mg mL-1 methanol extract caused growth inhibition higher than n-hexane and ethyl acetate extracts. Mycelial growth of A. flavus after 10x24-hours of incubation and after treated using extract is presented in Figure 2.4(a-d).

(a) (b) (c)

Figure 2.4 The growth of A. flavus BCC F0219 on protein-enriched medium treated using 20 mg mL-1 of methanol extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation

Apart from the type of extract, it is assumed that the difference of the pattern of growth inhibition is caused by the different content of active compounds in each kind of extract. In methanol extract, all of antimicrobial active components can be extracted, while in non-polar and semi-polar extracts only specific components were extracted. In methanol extract, flavonoids and phenolhydroquinons were detected in larger amount compared to other kinds of extracts, whereas tannins and saponins were only detected in methanol extract.

Tannins was reported to have physiological astringent properties, which hasten wound healing and ameliorate inflamed mucus membrane, it also has hemostatic properties [22]. Saponin has the property of precipitating and coagulating red blood cells. It is known that saponins can change the membrane permeability leading to the rupture of the cell [23].

3.3.Antifungal Activity of Kebar Grass Graded Solvent Extract

In carbohydrate-enriched medium, hexane-ethyl acetate (HE) extract caused growth inhibition of less than 90%, but the percentage of inhibition was higher than other medium models, on the concentration of extract 5, 10, or 20 mg mL-1 (Figure 2.5). Hexane-ethyl acetate-methanol (HEM) extract at concentrations of 10 and 20 mg mL-1 caused growth inhibition 96.4 and 100%, respectively (Figure 2.5 and 2.6).

Figure 2.5 Effect of type and concentration of kebar grass extract on growth inhibitory of A. flavus BCC F0219 in various media models

(a) (b) (c)

Figure 2.6 The growth of A. flavus BCC F0219 on carbohydrate-enriched medium treated using 10 mg mL-1 of n-hexane-ethyl acetate-methanol extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation

-30 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110

Gr

o

wt

h

In

h

ib

ito

ry

(

%

)

Type of Graded Extracts

Carbohydrate Fat

In fat-enriched medium, HE extract did not cause any inhibition on the growth of A. flavus, and tended to stimulate the growth of A. flavus (Figure 2.5). According to [10] steroid compounds belonging to the class of triterpenes, some of which include plant sterols that are only found in low level plants such of yeasts and molds. Ergosterol is a major component of the molds cell wall. The presence of high steroid in hexane-ethyl acetate extract did not inhibit the growth of A. flavus, otherwise a supporter of the A. flavus cell wall synthesis.

Hexane-ethyl acetate-methanol (HEM) extract (polar extract) at concentration of 20 mg mL-1 caused 96.2% stunted growth of A. flavus which was higher than the single extract. It is assumed that compounds extracted in graded extraction was more pure and has stronger activity. Mycelial growth of A. flavus on fat-enriched medium after 10x24-hours of incubation and after treated using extract is presented in Figure 2.7.

(a) (b) (c)

Figure 2.7 The growth of A. flavus BCC F0219 on fat-enriched medium treated using 20 mg mL-1 of n-hexane-ethyl acetate-methanol extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation

Experiment on the protein-enriched medium showed that the kind of hexane-ethyl acetate extract with a concentration of 20 mg mL-1 caused growth inhibition less than 90%, while the kind of hexane-ethyl acetate-methanol extract caused growth inhibition of 96.1%. The lower concentration of ethyl acetate-hexane-methanol extract produces less than 90% inhibition. Mycelial growth of A. flavus on protein-enriched medium after 10x24-hours of incubation and after treated using extract is presented in Figure 2.8.

(a) (b) (c)

Figure 2.8 The growth of A. flavus BCC F0219 on protein-enriched medium treated using 20 mg mL-1 of n-hexane-ethyl acetate-methanol extract (a), negative control (b), and solvent control (c) after 10x24 hours of incubation

Extract HEM rich of flavonoids, phenolhydroquinon, steroids, triterpenoids, tannins, and saponins. The antifungal property in this study could be due to flavonoids and tannins contents of the extract. They are known to have appreciable antimicrobial activities [22]. Other while triterpenoids can reduces the synthesis of ergosterol fungal cell membrane component and cause defective cell wall formation and leakage of cellular contents [24].

4. Conclusion

Kebar grass leaf extract was able to inhibit the growth of A. flavus in food model media. The most effective extract tested (20 mg mL-1) with growth inhibitory 96.1 – 100% was extract of hexane-ethyl acetate-methanol (HEM). Therefore, the kebar grass leaf could be recommended as antifungal of aflatoxigenic A. flavus.

5. Acknowledgment

The authors acknowledge the financial support of the Southeast Asian Regional Centre for Tropical Biology (SEAMEO BIOTROP). 6. References

1. J.I. Pitt and A.D. Hocking. Fungi and Food Spoilage. New York: Springer, 2009.

2. C. Proestos, N. Chorianopoulos, G.J. Nychas and M. Komaitis. RP-HPLC analysis of the phenolic compounds of plant extracts : Investigation of their antioxidant capacity and antimicrobial activity. J Agri and Food Chem., Vol. 53, 1190–1195, 2005.

3. M. Trigui, A.B. Hsouna, S. Tounsi and S. Jaoua. Chemical composition and evaluation of antioxidant and antimicrobial activities of Tunisian Thymelaea hirsuta with special reference to its mode of action. Indust Crops and Prod., Vol. 41, 150-157, 2013. 4. B. Santoso, A. Kilmaskossu and P. Sambodo. Effects of saponin from

Biophytum petersianum Klotzsch on ruminal fermentation, microbial protein synthesis and nitrogen utilization in goats. Animal Feed Sci and Tech., Vol. 137, 58–68, 2007.

5. M. Inngjerdingen, K.T. Inngjerdingen, T.R. Patel, S. Allen, X. Chen, B. Rolstad, G.A. Morris, S.E. Harding, T.E. Michaelsen, D. Diallo and B.S. Paulsen. Pectic polysaccharides from Biophytum petersianum Klotzsch, and their activation of macrophages and dendritic cells. Glycobiol., Vol. 12, 1074–1084, 2008.

6. S. Karamang. Studi morfologi, agrobiofisik dan produksi saponin Rumput Kebar (Biophytum petersianum Klotzsch) asal Papua [tesis].Bogor: Fakultas Teknologi Pertanian, Institut Pertanian Bogor, 2010.

7. D. Natarajan, M.S. Shivakumar and R. Srinivasan. Antibacterial activity of leaf extracts of Biophytum sensitivum (L.) DC. J Pharm Sci and Res., Vol. 11, 717-720, 2010.

8. Y. Deng, Y. Yu, H. Luo, M. Zhang, X. Qin and L. Li. Antimicrobial activity of extract and two alkaloids from traditional Chinese medicinal plant Stephania dielsiana. Food Chem. Vol. 124, 1556–1 560, 2011.

9. S.H. Lee, K.S. Chang, M.S. Su, Y.S. Huang and H.D. Jang. Effects of some Chinese medicinal plant extracts on five different fungi. Food Control, Vol. 18, 547–1554, 2007.

10. J.B. Harborne. Metode Fitokimia : Penuntun cara modern menganalisis tumbuhan. Terbitan kedua. Bandung: Penerbit ITB, 2006.

11. C.N. Shih and E.H. Marth. Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus. App Microbiol., Vol. 27, 452-456, 1974.

12. A. Riba, N. Boura, S. Mokrane, F. Mathieu, A. Lebrihi and N. Sabaou. Aspergillus section Flavi and aflatoxins in Algerian wheat and derived products. Food and Chem Toxicol., Vol. 48, 2772-2777, 2010.

13. J.E. Mellon and P.J. Cotty. Effects of oilseed storage proteins on aflatoxin production by Aspergillus flavus. JAOCS, Vol. 75, 1085-1089, 1998.

14. G.S. Joseph, G.K. Jayaprakasha, A.T. Selvi, B.S. Jena and K.K. Sakariah. Antiaflatoxigenic and antioxidant activities of Garcinia extracts. Int J Microbiol.,Vol. 101, 153-160, 2005.

15. V.G. de Billerbeck, C.G. Roques, J.M. Bessière, J.L. Fonvieille and R. Dargent. Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger. Can J Microbiol. Vol. 47, 9–17, 2001.

16. J. Tian, X. Ban, H. Zeng, J. He, B. Huang and Y. Wang. Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. Int J Food Microbiol., Vol. 145, 464– 470, 2011.

17. D.E. Okwu and M.E. Okwu. Chemical composition of Spondias mombin Linn plant parts. J Sustain Agric Environ., Vol. 6, 140-147, 2004.

18. H. Jaberian, K. Piri and J. Nazari. Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants. Food Chem., Vol. 136, 237 –244, 2013.

19. M.B. Sghaier, W. Bhouria, I. Bouhlela, I. Skandrania, J. Boubakera, L. Chekir-Ghediraa and K. Ghediraa. Inhibitory effect of Teucrium ramosissimum extracts on aflatoxin B 1, benzo(a)p yrene, 4 -nitro-o-phen ylenediamine and sodium azide induced mutagenicity : Corelation with antioxidant activity. South African J Botany, Vol. 77, 73 0- 740, 2011.

20. E.O. Sousa, J.B.T. Rocha, L.M. Barros, A.R.C. Barros and J.G.M. Costa. Phytochemical characterization and in vitro antioxidant properties of Lantana camara L. and Lantana montevidensis Briq. Indust Crops and Prod., Vol. 43, 517–522, 2013.

21. P.J. Houghton, A.Y. Mensah, N. Iessa and L.Y. Hong. Terpenoids in Buddleja: relevance to chemosystematics, chemical ecology and biological activity. Phytochem., Vol. 64, 385–393, 2003.

22. S. Sanni, P.A. Onyeyili and F.S. Sanni. Phytochemical analysis, elemental determination and some in vitro antibacterial activity of Ocimum basilicum L. leaf extracts. Res J Phyto., Vol. 2, 77-83, 2008.

23. G. Brusotti, S. Tosi, A. Tava, S. Picco, A.M. Grisoli, P.I. Cesari and G. Caccialanza. Antimicrobial and phytochemical properties of stem bark extracts from Piptadeniastrum africanum (Hook f.) Brenan. Indust Crops and Prod, Vol.43, 612–616, 2013.

24. E. Pinto, L. Vale-Silva, C. Cavaleiro and L. Salguerro. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol., Vol. 58, 1454-1462, 2009.

3 EFFECT OF KEBAR GRASS (

Biophytum petersianum

) LEAF

EXTRACT ON THE GROWTH AND MORPHOLOGICAL

STRUCTURE OF AFLATOXIGENIC

Aspergillus flavus

3,4) ABSTRACT

The objective of this study was to investigate antifungal activity of kebar grass extract (KGE) and its effect on the mycelial growth, conidiation and morphological structure of aflatoxigenic Aspergillus flavus BCCF0219 and A. flavus BIO 2236 in three types of model media, i.e. carbohdyrate-enriched medium, fat-enriched medium and protein-enriched medium. Each type of model medium containing five concentrations of KGE, i.e. 12, 14, 16, 18, and 20 mg mL-1. The best A. flavus BCCF0219 growth inhibition activity of KGE for each media was as followed : 12 mg mL-1 was 97.9% (carbohydrate-enriched medium), 14 mg mL-1 was 93.5% (fat-enriched medium) and 14 mg mL-1 was 90.8% (protein-enriched medium). The percentage of growth inhibition of A. flavus BIO 2236 caused by KGE at a concentration of 12 mg mL-1 was 93.9% (carbohydrate-enriched medium), 16 mg mL-1 was 99.4% (fat-enriched medium) and 16 mg mL

-1

was 93.8% (protein-enriched medium). Based on SEM observation results, it was found that the mechanisms involved in fungal growth inhibitory of KGE were by morphological alterations of hyphal development, even the collapse of the entire hyphae. These findings indicated the potential of KGE as natural anti aflatoxigenic agent.

Keywords: aflatoxigenic, Aspergillus flavus, Biophytum petersianum, food model media, kebar grass

Introduction

Kebar grass (Biophytum petersianum), belonging to the family Oxalidaceae, is a native plant of Kebar District, West Papua, Indonesia. The plant grows naturally and distributed almost all over Kebar District (Santoso et al., 2007). This plant is also found in Southeast Asia and Africa, especially in Mali region. Mali community uses this plant for treatment of wounds, inflammation, ulcers, malaria, and fever (Inngjerdingen et al., 2008). Papuan community, especially in Kebar District, often uses this plant as a fertility drug.

Several studies reported that kebar grass has potential as antibacterial, hypoglycemic agent, immunomodulatory agent, chemoprotective agent, hypocholesterolemic agent, apoptosis, anti-inflammatory, antitumor and prostaglandin biosynthesis (Natarajan et al., 2010). Furthermore, the use of methanol extract of kebar grass can affect the expression of COX-2 (Guruvayoorappan and Kuttan 2008) and stimulate immune system cells in mice (Inngjerdingen et al., 2008). Some phenolic components that affect the system COX-1/COX-2 also have been isolated by Bucar et al. (1998). Kebar grass extract also shows the effect of apoptosis on B16F-10 cells and is able to regulate NO production and cytokine-associated macrophages to tumor formation (Guruvayoorappan and Kuttan, 2007).

3)

Has been presented in International Symposium on Tropical Fungi, September

10-11th 2013, Bogor-Indonesia

During storage fungal growth on foodstuffs can increase the moisture content and humidity of the storage material. In this condition, the damage occurs so fast, consequently nitrogen source materials needed to grow will be reduced and fungi should maintain their life by utilizing the carbon source in the substrate as a source of energy. This situation cause fungi produce secondary metabolite, among others mycotoxins (Syarief et al., 2012).

Fungal contamination and their mycotoxin production causes 25 – 40% or even more post-harvest economic losses in developing country. About 4.5 billion people in under developed countries are exposed to mycotoxins (Murugan et al., 2013). Aflatoxins are mycotoxins produced by A. flavus and A. parasiticus that are hepatotoxic, mutagenic, teratogenic, and carcinogenic to humans and animals (Alberts et al., 2006). Besides harmful to human and animal health, aflatoxin contamination in food and feed also resulted in loss of significant economic ranged from U.S. $ 85-100 million in the USA (Alberts et al., 2009), so that contamination of aflatoxigenic fungi received worldwide attention (Sohaibani et al., 2011 ).

Type of substrates is a factor which determines aflatoxin content produced by aflatoxigenic A. flavus. Since it is difficult to avoid aflatoxin contamination completely, a technology is needed to reduce aflatoxin contamination without affecting the flavor and nutritional value of food. As foodstuff can consist of carbohydrate, fat and protein, therefore it is necessary to investigate the effects of kebar grass in inhibiting the growth of aflatoxigenic A. flavus in food system model and the mechanism of the growth inhibition itself.

Selection on the type of kebar grass extract that caused the highest percentage of inhibition of A. flavus growth has been conducted. In our preliminary study, the fungal growth inhibitory activity of six kebar grass extracts (KGE) has been examined, i.e. four extracts from single solvent extraction using n-hexane, ethyl acetate, methanol, and water. The other two extracts were obtained from successively extracted of EtOAc-MeOH and n-hexane-EtOAc. The result showed that n-hexane-EtOAc-MeOH of KGE caused the highest percentage of inhibition of A.flavus BCCF0219 and A.flavus BIO 2236 growth. In this study, a minimum inhibitory concentration (MIC) of KGE against the growth of aflatoxigenic A. flavus and the mechanism involved were determined. The chemical profile of KGE was also analyzed using pyrolisis GC-MS.

Materials and Methods Plant materials

Kebar grass leaves was collected from Kebar district, Manokwari county, West Papua province, in February, 2013. Identification of kebar grass was confirmed by Indonesian Institute of Science (LIPI).

Chemicals

All solvents were analytical grade and purchased from JT. Baker, USA. Plant extracts preparation

Kebar grass leaves were air-dried for about 14 days or until the colour turned into golden brown and they were easily broken. The leaves were then

crushed into coarse powder about 40 mesh using a blender. As much as 200 g powdered kebar grass was extracted using maceration (1 hr) method combined with sonication (30 min) at room temperature (28 °C) using three types of organic solvent, i.e. n-hexane, ethyl acetate, and methanol solvents, followed by Whatman No.1 paper filtration. Maceration was repeated 2 times and the filtrate was collected. The remaining residue of the plant material was dried for 24 hours and extracted using ethyl acetate and methanol solvents sequentially in a similar manner. The filtrate was collected and dried using a rotary evaporator. The hexane-ethyl acetate-methanol filtrate was called as HEM extract. Residual solvent in HEM extract was removed using nitrogen gas (Deng et al., 2011). The extract was then stored at 4 °C before being used for chemical composition analysis, antifungal activity and morphological changes of fungal structure.

Fungal isolates

Isolates of A. flavus BCCF0219 and A. flavus BIO 2236 were obtained from the Research Institute for Veterinary Science (BBLITVET) and SEAMEO BIOTROP Culture Collections, respectively.

Preparation of fungal conidia

Fungal conidia were prepared using a method by Joseph et al. (2005) with a slight modification. Each fungal isolates was cultured in Potato Dextrose Agar (PDA; Oxoid, England) slant and incubated at 28°C for 5 days. The conidia were harvested by adding 10 mL of sterile distilled water and collected aseptically using a sterile inoculation loop. Conidial suspension was dissolved in Tween 80 (0.5%) and centrifuged (2000xg) at 28°C for 5 minutes. Conidial concentration was calculated using a haemacytometer and adjusted to 106 CFU mL-1. The suspension was used to test the effect of kebar grass extract in inhibiting the growth and morphological changes of A. flavus isolates.

Preparation of agar media enriched with carbohydrate, fat, and protein

Carbohydrate-enriched medium was prepared by the addition of 30% (w/v) glucose (E Merck, Germany) on Bacto Agar (Difco, USA) (Shih and Marth, 1974).

Fat-enriched medium was prepared using a method of Riba et al. (2010). As much as 100 g of grated coconut flesh was homogenized using 300 mL of heat distilled water for five minutes. The mixture was filtered using four layers of cheesecloth. The filter obtained was adjusted its pH into 7.0 using NaOH 2N. Agar powder (Difco, USA) was added (20 g L-1). Both of media were sterilized in an autoclave at 121°C for 15 minutes.

Protein-enriched medium was prepared using a method of Mellon and Cotty (1998) with a slight modification. As much as 30% (w/v) skim milk (Difco, USA) was added to Bacto Agar medium and sterilized in an autoclave at 121°C for 10 minutes.

Py-GC/MS analyses of kebar grass extract

Samples of kebar grass polar extract was pyrolyzed by using a CDS Pyroprobe 2020iS connected on line to a gas chromatograph with a Shimadzu GC–MS mass detector (GCMS-QP2010). Samples were introduced into a quartz capillary tube in each experiment. The capillary was automatically introduced into

the center of a platinum resistor which heats up in an inert atmosphere. The products generated in pyrolysis were rapidly removed from the reaction zone (quartz capillary surrounded by the resistor) using a flow of helium, through a transfer line at the temperature of 280 °C until introduced into the gas chromatograph provided with a mass spectrometry detector for analysis. Pyrolysis temperature was 400 °C. GC analysis was carried out using a Rtx-5MS fused silica capillary column (60 m×0.25 mm×0.25 µm). Column temperature was 50 – 280 °C. Helium was used as a carrier gas at a constant pressure of 100 kPa. Identification of different compounds obtained in this analysis was performed by using the Wiley 7 mass spectral library (Valdés et al., 2013).

Mycelial Growth Assay

The effect of kebar grass extract on mycelial growth of the two A. flavus isolates was conducted using Tian et al. (2011) methods. The concentrations of kebar grass extract added to food model media were 12, 14, 16, 18, and 20 mg mL-1. Ten milliliters of each test media containing each extract concentration was poured into medium in a Petri dish (9 cm in diameter) and left until it was solid. Five microliters of conidial suspension of each A. flavus isolate was inoculated on the surface of each test media, and incubated at 28 °C for 10 days. The diameter of colony (in millimeter) was observed every 24 hours. Fungal cultured in food model media without kebar grass extract was used as negative control. Two replicates were used for each treatment combination, including the control. The percentage of growth inhibition was calculated using following formula:

Growth inhibition = (dc - dt)/dc X 100%,

where dc = diameter of control colony (in mm)

d t = diameter of fungal colony in treatment set (in mm)

The concentration of extract that causing >90% of growth inhibitory activity was expressed as Minimum Inhibitory Concentration (MIC) and it was used in subsequent test (Manso et al., 2013).

Sample preparation for scanning electron microscopy analysis

A mycelial disc (2-mm diameter) was taken at the periphery of the colony grown on the cellophane disc placed on the model media with 0, 1, 1.5, 2 MIC with and without kebar grass polar extract after ten days of incubation. It was then dried at 60 °C on a small glass slide (1 cm×1 cm). Samples were fixed in 2.5% glutaraldehyde for 30 min at room temperature. They were rinsed in distilled water (three times, 15 min each). The specimens were then dehydrated in a graded ethanol series (30, 50, 70%, 10 min for each ethanol dilution and 30 min in 100% ethanol). The samples were then coated with gold palladium using an ion sputter-cutter (JEOL JFC 1100). The preparations were observed using a SEM Zeiss EVO 50 (Germany) (Billerbeck et al., 2001).

terdapat dalam ekstrak daun rumput kebar merupakan asam lemak tidak jenuh yang dilaporkan mampu menghambat pertumbuhan kapang (Yong 2009).

Komponen lainnya yang terdeteksi pada ekstrak daun rumput kebar berasal dari golongan senyawa alifatik hidrokarbon. Senyawa alifatik sering terdeteksi pada kultur endofit dan memperlihatkan aktivitas biologis sebagai antikapang (Yu et al. 2010). Senyawa alkohol merupakan komponen minor yang juga ditemukan pada ekstrak daun rumput kebar. Senyawa alcohol dilaporkan dapat menghambat pertumbuhan khamir dan kapang (Sacchetti et al. 2005).

Hasil penelitian juga memperlihatkan bahwa ada korelasi langsung antara pertumbuhan kapang dengan produksi AFB1. Sejauh ini mekanisme degradasi atau pengikatan belum dapat digambarkan dengan pasti, namun diduga bahwa pertumbuhan miselium berkorelasi dengan sintesis enzim yang berperan dalam produksi AFB1. Bila pertumbuhan miselium terhambat, produksi AFB1 juga terhambat. Namun anggapan ini tidak sepenuhnya benar. Tatsadjieu et al. (2009) mengemukakan bahwa penghambatan produksi AFB1 tidak selalu disebabkan oleh pertumbuhan kapang yang tereduksi tetapi dapat disebabkan oleh penghambatan katabolisme karbohidrat A. flavus dengan cara mempengaruhi beberapa enzim kunci yang pada akhirnya menurunkan kemampuan A. flavus untuk memproduksi AFB1. Ditambahkan oleh Pitt (1993) bahwa kerusakan aflatoksin dapat terjadi

pula akibat enzim-enzim yang dilepaskan selama lisis miselium yang berakibat pada penurunan produksi aflatoksin.

Sejauh ini, mekanisme penghambatan ekstrak tumbuhan terhadap pertumbuhan kapang belum secara lengkap dipahami. Dugaan mekanisme

penghambatan yang terjadi disebabkan oleh beberapa aspek, antara lain (1) kehadiran gugus hidroksil yang dapat membentuk ikatan hidrogen yang

akhirnya dapat mempengaruhi enzim-enzim pertumbuhan, dan memodifikasi berbagai fungsi intraseluler; (2) perubahan morfologi kapang yang disebabkan oleh interaksi ekstrak dengan enzim-enzim pada membran sel yang mengakibatkan hilangnya ketegaran sel dan integritas dinding sel kapang; (3) perubahan permeabilitas membran sel, granulasi sitoplasma, dan pecahnya membran sitoplasmik (da Cruz Cabral 2013). Sifat hidrofobisitas dari komponen-komponen aktif tersebut menyebabkan komponen-komponen-komponen-komponen tersebut mampu menyeberangi membran sel dan berinteraksi dengan komponen-komponen sel sehingga dapat mempengaruhi baik enzim-enzim di membran sel maupun intraseluler. Dugaan bahwa beberapa komponen hidrofobik yang terdapat dalam ekstrak tumbuhan dapat merubah permeabilitas membran mikrob dan menyebabkan perubahan aliran proton, memodifikasi pH sel dan mempengaruhi komposisi kimia sel dan aktivitasnya. Kemampuan komponen hidrofobik untuk larut dalam fase lemak membran sitoplasma merupakan kunci aktivitas penghambatannya, namun kelarutan yang tinggi tidak selalu menghasilkan aksi antimikrob yang tinggi (Samapundo et al. 2007).

Pengamatan dengan SEM (Scanning Electron Microscopy) memperlihatkan bahwa hifa kedua isolat kapang mengalami kerusakan, dinding sel kehilangan integritas dan ketegaran sel, dan dinding sel terlipat. Hasil ini mendukung dugaan bahwa mekanisme penghambatan pertumbuhan oleh ekstrak HEM daun rumput kebar merupakan hasil intervensi komponen aktif ekstrak dengan reaksi enzimatik sintesis dinding sel yang akhirnya mempengaruhi morfogenesis dan pertumbuhan kapang.

Pencarian antikapang alternatif merupakan salah satu hal yang mendapat perhatian penting di bidang industri pangan, terutama karena kerugian akibat kontaminasi kapang. Selain itu, bahaya yang mengintai di balik perkembangan kapang di bahan pangan adalah produksi mikotoksin oleh kapang toksigen. Kemungkinan penggunaan komponen yang diekstrak dari tumbuhan untuk mengendalikan kontaminasi mikotoksin merupakan alternatif yang menjanjikan dan perlu dipelajari lebih jauh. Meskipun ekstrak tumbuhan digunakan sebagai bumbu atau rempah, ada kemungkinan aplikasi ekstrak ke bahan pangan harus ditambahkan dengan konsentrasi yang tinggi. Oleh karenanya harus diketahui toksisitasnya terhadap manusia dan hewan. Dalam uji sitotoksisitas ekstrak HEM daun rumput kebar terhadap sel Vero secara in vitro diperoleh nilai CC50 ekstrak HEM daun rumput kebar terhadap sel Vero adalah sebesar 13.41 mg mL-1. Hasil ini mengindikasikan bahwa ekstrak HEM daun rumput kebar aman untuk digunakan sebagai bahan antikapang alami.

Daftar Pustaka

Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils – A review. Food and Chem Toxicol 46:446–475

Boligon AA, Achwanz TG, Froeder ALF, Athayde ML. 2014. GC-MS analysis of dichloromethane fractions of Scutia buxifolia Reissek leaves and stem bark. Res J of Phytochem 8:16-20.

da Cruz Cabral L, Pinto VF, Patriarca A. 2013. Application of plant derived compounds to control fungal spoilage and mycotoxin production in foods (Review). Int. J. of Food Microbiol. 166:1-14.

de Lau LML, Bornebroek M, Witteman JCM, Hofman A, Koudstaal PJ, Breteler MMB. 2005. Dietary fatty acids and the risk of Parkinson disease. Neurology. 64:2040-2045.

Fernández-Cruz ML, Mansilla ML, Tadeo JL. 2010. Mycotoxins in fruits and their processed products: Analysis, occurrence and health implications. J of Adv Research 1:113–122

Griffin GH. 1994. Fungal Physiology, 2 nd edn. Wiley-Liss, New York.

Houghton PJ and Raman A. 1998. Laboratory Handbook for The Fractionation of Natural Extracts. London: Chapman and Hall.

Jaberian H, Piri K, Nazari J. 2013. Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants. Food Chem. 136:237 –244.

Moss MO. 1991. The environmental factors controlling mycotoxin formation. In: Smith JE, Anderson RA (Eds), Mycotoxins and Animal Foods. CRC Press, Boca Raton, FL, 37-56.

Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. 2009. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol. 58:1454-1462.

Pitt RE. 1993. A descriptive model of mold growth and aflatoxin formation as affected by environmental conditions. J Food Protect. 56:139-146.

Pitt JI, Hocking AD. 2009. Fungi and Food Spoilage, 3rd edn. Springer, New York.

Riba A, Bouras N, Mokrane S, Mathieu F, Lebrihi A, Sabaou N. 2010. Aspergillus section Flavi and aflatoxins in Algerian wheat and derived products . Food and Chem Toxicol. 48:2772-2777

Sacchetti G, Maietti S, Muzzoli M, Scaglianti M, Manfredini S, Radice M, Bruni R. 2005. Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91:621-632.

Samapundo S, De Meulenaer B, Osei-Nimoh D, Lamboni Y, Debevere J, Devlieghere F. 2007. Can phenolic compounds be used for the protection of corn from fungal invasion and mycotoxin contamination during storage? Food Microbiol. 24:465–473

Shephard GS. 2008. Impact of mycotoxins on human health in developing countries. Food Addict Contam. 25(2):146–51.

Siranidou E, Kang Z, Buchenauer H. 2002. Studies on symptom development, phenolic compounds and morphological defence responses in wheat cultivars differing in resistance to Fusarium head blight. J Phytopathol. 150:200-208.

Sousa EO, Rocha JBT, Barros LM, Barros ARC, Costa JGM. 2013. Phytochemical characterization and in vitro antioxidant properties of Lantana camara L. and Lantana montevidensis Briq. Indust. Crops and Prod. 43:517–522

Tatsadjieu NL, Dongmo PMJ, Ngassoum MB, Etoa FX, Mbofung CMF. 2009. Investigations on the essential oil of Lippia rugosa from Cameroon for its potential use as antifungal agent against Aspergillus flavus Link ex. Fries. Food Control 20:161-166.

Yong JWH, Ge L, Ng YF, Tan SN. 2009. The chemical composition and biological properties of coconut (Cocos nucifera L) water. Molecules 14:5144-5164.

Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L. 2010. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res 165:437-449

Zwenger S, Basu C. 2008. Plant terpenoids: applications and future potentials. Biotech and Mol Biol Rev 3: 001-007.

8 SIMPULAN DAN SARAN

Simpulan

1. Ekstrak daun rumput kebar dapat menghambat pertumbuhan Aspergillus flavus aflatoksigen pada media model pangan. Jenis eksrak yang paling efektif adalah ekstrak bertingkat heksana -etil asetat-metanol (HEM) dengan tingkat aktivitas antikapang moderat. Ekstrak bertingkat heksana-etil asetat-metanol (HEM) daun rumput kebar memiliki aktivitas antikapang terhadap pertumbuhan A. flavus aflatoksigen yang mengakibatkan perubahan morfologi hifa kapang.

2. Ekstrak HEM daun rumput kebar dapat menghambat pertumbuhan dan produksi AFB1 dari A. flavus BCC F0219 dan BIO 2236 baik pada media model pangan yaitu media kaya karbohidrat, lemak dan protein, maupun pada media sistem pangan yaitu media jagung dan kacang tanah.

3. Persentase hambatan pertumbuhan A. flavus BCC F0219 pada media kaya karbohidrat, lemak dan protein berturut-turut sebesar 97.9, 93.5, dan 90.8%, sedangkan persentase hambatan produksi AFB1 berturut-turut sebesar 100, 99.2, dan 70.9%. Persentase hambatan pertumbuhan A. flavus BIO 2236 pada media kaya karbohidrat, lemak dan protein berturut-turut sebesar 93.9, 99.4, dan 93.8%, sedangkan persentase hambatan produksi AFB1 berturut-turut sebesar 83.4, 91.3, dan 98.8%. Persentase hambatan pertumbuhan A. flavus

BIO 2236 di media dengan konsentrasi jagung 100% pada 1MIC (12 mg mL-1), 1.5MIC (18 mg mL-1) dan 2MIC (24 mg mL-1) berturut-turut

sebesar 44.0, 53.7, dan 66.5%, sedangkan persentase hambatan produksi AFB1 berturut-turut sebesar 7.0, 18.9, dan 91.3%. Persentase hambatan pertumbuhan A. flavus BIO 2236 di media dengan konsentrasi kacang tanah

100% pada 1MIC (16 mg mL-1), 1.5MIC (24 mg mL-1) dan 2MIC (32 mg mL-1) berturut-turut sebesar 64.9, 82.0, dan 100.0%, sedangkan

persentase hambatan produksi AFB1 berturut-turut sebesar 19.3, 59.4, dan 100%.

4. Nilai CC50 ekstrak HEM daun rumput kebar terhadap sel Vero adalah sebesar 13.41 mg mL-1. Nilai CC50 ekstrak HEM daun rumput kebar jauh lebih tinggi daripada kriteria sitotoksisitas yang ditetapkan NCI yaitu kurang dari

20 μg mL-1 .

Saran

Secara umum, aktivitas antikapang dan antiaflatoksin yang dihasilkan oleh ekstrak tumbuhan disebabkan oleh adanya efek sinergisme antara beberapa komponen aktif yang terkandung dalam ekstrak tumbuhan. Ekstrak tumbuhan tersusun atas berbagai komponen kimia sehingga sulit untuk memastikan hubungan antara satu komponen dengan target tunggal pada sel. Berdasarkan hasil penelitian, hal-hal yang perlu dilakukan lebih lanjut antara lain :

1. Penelitian lebih lanjut perlu dilakukan untuk mengetahui secara pasti apakah konsentrasi minimum yang dapat menghambat pertumbuhan kapang bersifat fungistatik atau fungisidal.

3. Identifikasi komponen apa yang berperan terhadap penghambatan pertumbuhan dan produksi aflatoksin baik pada media model pangan maupun pada media sistem pangan.

4. Mengisolasi dan mengidentifikasi komponen tunggal yang memiliki aktivitas antikapang dan antiaflatoksin.

5. Metode aplikasi yang tepat dan pengaruhnya terhadap nilai sensori bahan pangan.

6. Jenis pelarut yang sesuai untuk diaplikasikan pada pangan.

7. Selanjutnya perlu dianalisis keamanan ekstrak melalui uji in vivo dan aplikasinya secara luas di bidang pangan.

RIWAYAT HIDUP

Penulis dilahirkan di Serui pada tanggal 20 Mei 1978. Penulis merupakan anak pertama dari tiga bersaudara dalam keluarga bapak Jaya Taufan Lie dan ibu Marthina Tan Bonai-Abaa. Penulis menempuh pendidikan Strata 1 pada tahun 1996 di Program Studi Agronomi, Fakultas Pertanian-Universitas Cenderawasih dan lulus pada tahun 2000. Pada tahun 2003 penulis berkesempatan melanjutkan pendidikan Strata 2 pada Program Studi Teknologi Pasca Panen, Program Pasca Sarjana Institut Pertanian Bogor dan lulus tahun 2005. Tahun 2009 penulis kembali diberi kesempatan melanjutkan pendidikan Strata 3 pada Program Studi Ilmu Pangan, Sekolah Pasca Sarjana IPB melalui program beasiswa BPPS.

Penulis bekerja sebagai dosen di Jurusan Teknologi Pertanian, Fakultas Pertanian dan Teknologi Pertanian (Fapertek), Universitas Negeri Papua (UNIPA), Manokwari, Provinsi Papua Barat sejak tahun 2001 sampai sekarang. Bidang penelitian yang ditekuni sejak menjadi dosen mencakup kajian pasca panen dan antimikrob dari bahan pangan lokal antara lain buah merah (Pandanus conoideus) dan kayu akway. Penulis aktif melakukan penelitian dan memperoleh pendanaan dari DIKTI (Hibah Bersaing 2008/2009 dan 2012/2013) bersama tim peneliti di Fapertek UNIPA. Selama menempuh pendidikan S3, penulis mendapatkan bantuan dana penelitian berupa PhD Thesis Grant dari Southeast Asian Regional Centre for Tropical Biology (SEAMEO BIOTROP) tahun 2012/2013.

Karya ilmiah yang merupakan bagian dari disertasi ini telah diajukan

sebagai artikel ilmiah pada beberapa jurnal, yaitu: 1) “Antifungal activity of Kebar grass leaf extracts on the growth of aflatoxigenic Aspergillus flavus in food model media” telah dipresentasikan dalam 1st BIOTROP Conference, Oktober 2013 di IPB Convention Center (ICC) Bogor, dan telah diterima untuk diterbitkan pada International Journal Sciences: Basic and Applied Research Vol. 17 No. 2, 2014; 2) artikel “Effect of kebar grass (Biophytum petersianum) leaf extract on the growth and morphological structure of aflatoxigenic Aspergillus flavus” telah dipresentasikan pada International Symposium on Tropical Fungi, September 2013 dan telah dimasukkan pada International Journal of Food Science and Technology, dan 3) artikel “Aktivitas antiaflatoksin B1 ekstrak daun rumput kebar (Biophytum petersianum) terhadap Aspergillus flavus” telah diterima untuk dipublikasi pada Jurnal Agritech Vol. 35 No. 1, Februari 2015.