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LAPORAN AKHIR

PENELITIAN KERJASAMA ANTAR PERGURUAN TINGGI

(PAKERTI)

DANA ITS 2020

Modifikasi Enzimatik Laccase sebagai Alternatif Teknologi Pasca Panen Biji Kakao (Theobroma Cacao Linn)

Tim Peneliti :

Ketua: Dr. techn. Endry Nugroho Prasetyo, S.Si., MT (Biologi/FSAD) Anggota 1 Siti Zullaikah ST.,MT.,Ph.D (Teknik Kimia/F) Anggota 2 Maharani Pertiwi K, Ph.D. (UNUSA Surabaya)

Anggota 3 Isdiantoni,SP.MP. (UNIJA Sumenep) Anggota 4 Dr. Ida Ekawati,MP (UNIJA Sumenep)

DIREKTORAT RISET DAN PENGABDIAN KEPADA MASYARAKAT

INSTITUT TEKNOLOGI SEPULUH NOPEMBER

SURABAYA

2020

Sesuai Surat Perjanjian Pelaksanaan Penelitian No: 960/PKS/ITS/2020

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Daftar Isi

Daftar Isi ............................................................................................................................................ i

Daftar Tabel ...................................................................................................................................... ii

Daftar Gambar ................................................................................................................................. iii

Daftar Lampiran ............................................................................................................................... iv

BAB I RINGKASAN ....................................................................................................................... 1

BAB II HASIL PENELITIAN.......................................................................................................... 2

BAB III STATUS LUARAN............................................................................................................ 8

BAB IV PERAN MITRA (UntukPenelitian Kerjasama Antar Perguruan Tinggi) .......................... 9

BAB V KENDALA PELAKSANAAN PENELITIAN ................................................................. 11

BAB VI RENCANA TAHAPAN SELANJUTNYA ..................................................................... 12

BAB VII DAFTAR PUSTAKA ..................................................................................................... 13

BAB VIII LAMPIRAN................................................................................................................... 19

LAMPIRAN 1 Tabel Daftar Luaran ............................................................................................... 20

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Daftar Tabel

Tabel 2.1 Hasil Produksi dan Karakterisasi laccase………………………………………….2

Tabel 2.2 Hasil Total Fenol, Kapasitas Antioksidan, Kadar Gula Pereduksi dan Nilai Indeks Fermentasi Laccase…………………………………………………………………………….4

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Daftar Gambar

Gambar 2.2 Hasil Ekstraksi Sampel Analisis Warna dengan Indeks Fermentasi (IF)………..6

Gambar 2.1 Warna Biji Kakao Sangrai dengan Berbagai Perlakuan………………………….6

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Daftar Lampiran

LAMPIRAN 1 Tabel Daftar Luaran…………………………………………………………….20

LAMPIRAN 2 DRAFT PAPER…………………………………………………………………22

LAMPIRAN 3 Publisher dari Jurnal……………………………………………………………..33

LAMPIRAN 4 Poster Conference………………………………………………………………..35

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BAB I RINGKASAN Proses pengolahan biji kakao melalui beberapa tahapan salah satunya adalah fermentasi. Fermentasi dilakukan melalui proses oksidasi untuk mengurangi kadar senyawa polifenol yang dapat menurunkan rasa pahit dan astrigensi (sepat) yang tinggi serta meningkatkan aroma pada biji kakao. Oksidasi dapat dilakukan secara alami atau dengan penambahan agen oksidan salah satunya seperti laccase. Tujuan dari penelitian ini adalah untuk mengetahui pengaruh oksidasi enzimatik laccase terhadap kualitas biji kakao (Theobroma cacao) dengan melihat penurunan senyawa polifenol total, kapasitas antioksidan, kadar gula pereduksi, analisis warna dengan indeks fermentasi dan analisis FTIR. Pada penelitian ini dihasilkan laccase Trametes versicolor dengan aktivitas tertinggi 1081,2 U/ml, uji kandungan total protein dengan hasil 0,003 mg/ml, aktivitas spesifik tertinggi 36040 U/mg serta nilai Km dan Vmax masing-masing sebesar 0,98 mM dan 424 U/mg protein. Treatment yang dilakukan dengam oksidasi laccase dan laccase+ABTS dapat meningkatkan kualitas biji kakao yang menunjukkan kadar senyawa fenol terendah sebesar 0,493 mg/l ± 0,28, kadar antioksidan terbesar sebesar 90,77 % ± 0,22, kadar gula pereduksi terendah sebesar 0,756 ppm ± 0,13 dan menunjukan indeks fermentasi (IF)≥1. Analisis uji FTIR mengkonfirmasi terjadinya perubahan gugus aromatik sehingga memungkinkan peningkatan cita rasa dan aroma khas pada kakao. Kata kunci: Fermentasi, Fenol, Kakao, Laccase, Oksidasi.

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BAB II HASIL PENELITIAN

2.1. Produksi dan Karakterisasi Laccase Trametes versicolor

Produksi dan karakterisasi laccase pada penelitian ini dilakukan dengan menggunakan dua

macam medium berbeda berdasarkan Fukushima and Kirk (1995) (medium I) dan Nyanghongo et

al., (2002) (medium II). Perbedaan pada kedua medium ini adalah komposisi medium fermentasi I

terdiri dari CuSO4, sedangkan medium fermentasi II tidak terdiri dari CuSO4. Karakterisasi laccase

yang diproduksi dilakukan dengan uji aktivitas laccase, kadar protein total, aktivitas spesifik, titik

isoelektrik, dan kinetika enzim (Tabel 2.1). Perhitungan nilai aktivitas, protein total dan aktivitas

spesifik laccase tercantum juga pada Lampiran 2.

Tabel 2.1 Hasil Produksi dan Karakterisasi laccase

Pada Tabel 4.1 dapat diketahui bahwa aktivitas laccase tertinggi yaitu sebesar 1081,2 U/ml

tercapai pada medium II dengan total protein sebesar 0,03 mg/ml, aktivitas spesifik 36040 U/mg

dan titik isoelektrik pada pH 3. Hal tersebut sesuai dengan pernyataan Heap et al. (2014) bahwa

laccase memiliki kisaran titik isoelektrik (Ip) pada pH 3 sampai 7. Rendahnya nilai aktivitas laccase

pada medium I (885 U/ml) dipengaruhi oleh penambahan inducer laccase berupa CuSO4. Menurut

Kumar et al. (2011), produksi laccase dapat ditingkatkan dengan menambahkan berbagai inducer

seperti tembaga sulfat (CuSO4) sampai batas tertentu. Penambahan CuSO4 pada medium I sebanyak

0,01 gr atau setara dengan 6 mM menunjukkan aktivitas yang lebih kecil dibandingkan medium II

yang tidak mengandung CuSO4. Hal tersebut kemungkinan disebabkan adanya penambahan

inducer CuSO4 lebih dari 1,5 mM yang dapat bersifat toksik bagi jamur sehingga menghambat

T.versicolor dalam memproduksi laccase (Hanung et al., 2013). Menurut Tychanowicz et al.

(2006), CuSO4 merupakan inducer untuk mengaktifkan gen laccase (lcc) dalam mengekspresikan

protein enzim, namun penambahan CuSO4 berlebih dapat menyebabkan oksidasi protein bagi

Medium

Aktivitas

Enzim

(U/ml)

Protein

Total

(mg/ml)

Aktivita

s

Spesifik

(U/mg)

Km

(mM)

Vmax

(U/mg

protein)

Medium I 885,0 0,11 8045 0,45 159

Medium II 1081,2 0,03 36040 0,98 424

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mikroorganisme dan berlanjut kepada fase kematian. Menurut Hidayati (2013), senyawa logam

berat salah satunya Cu+ dapat menyebabkan mekanisme toksisitas pada organisme tertentu yang

pada akhirnya dapat menghambat aktivitas enzim. Hal tersebut dikarenakan senyawa logam Cu+

dapat berikatan dengan protein yang akan membentuk ikatan protein-logam. Ikatan protein logam

yang terbentuk akan menyebabkan enzim yang berikatan dengan protein menjadi tidak aktif. Telah

diketahui bahwa penghambatan kerja enzim akan mempengaruhi proses-proses fisiologis dan dapat

menggangu struktur sel dalam organisme tersebut (Al-Aziz dan Marianti, 2014).

Kinetika laccase dibuat melalui persamaan laju pembentukan produk, sehingga diperoleh nilai

konstanta Michaelis-Menten (Km) dan laju maksimum katalisis (Vmax) enzim. Perhitungan nilai

Km dan Vmax diperoleh menggunakan persamaan Lineweaver-Burk tercantum pada Lampiran 4

dan Lampiran 5. Berdasarkan persamaan Lineweaver-Burk, laccase T. versicolor pada medium I

diperoleh nilai Km dan Vmax masing-masing adalah 0,45 mM dan 159 U/mg protein. Sedangkan

medium II diperoleh nilai Km dan Vmax masing-masing 0,98 mM dan 424 U/mg protein (Tabel

4.1). Nilai Km yang besar menunjukkan afinitas enzim terhadap subtrat rendah yang berarti

dibutuhkan konsentrasi substrat yang tinggi untuk melangsungkan reaksi enzim. Nilai Km yang

kecil menunjukkan afinitas enzim terhadap substrat tinggi, sehingga reaksi enzim dapat terjadi

meskipun konsentrasi substrat rendah (Rosetaati, 2017). Peningkatan konsentrasi substrat akan

meningkatkan laju katalisis sehingga pada saat tertentu akan tercapai tingkat kejenuhan terhadap

substrat yang menyebabkan laju katalisis mencapai maksimum (Vmax) (Nelson dan Cox 2008).

Laccase yang dihasilkan oleh medium I dan II pada penelitian ini memiliki nilai Km yang

termasuk dalam kategori cukup untuk diaplikasikan dalam enzim industri dengan nilai Km berturut-

turut sebesar 0,45 mM dan 0,98 mM. Nilai tersebut bahkan lebih baik daripada penelitian Castro et

al., (2013) yang memiliki nilai Km sebesar 1,54 mM untuk laccase T. versicolor terimobilisasi.

Tetapi nilai Km pada penelitian ini masih lebih tinggi dibandingkan laccase T. versicolor penelitian

Asgher et al. (2012) sebesar 0,073 mM. Hal ini juga seiring dengan nilai Vmax tertinggi yang

dihasilkan oleh medium II yaitu sebesar 424 U/mg. Nilai Vmax tersebut masih lebih tinggi

dibandingkan dengan penelitian yang dilakukan oleh Ashger et al., (2012). Tetapi nilai Vmax yang

dihasilkan oleh medium I (159 U/mg) masih lebih kecil dibandingkan dengan nilai Vmax yang

dihasilkan oleh Asgher et al. (2012). Hal ini kemungkinan dikarenakan laccase yang dihasilkan

belum mengalami proses pemurnian sehingga nilai Vmax masih tergolong rendah (Ashger et al.,

2012).

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2.2. Oksidasi Laccase Pada Biji Kakao (Theobroma cacao)

Performa oksidasi laccase pada biji kakao diamati berdasarkan analisis fenol total, kapasitas

antioksidan, kadar gula pereduksi, dan nilai indeks fermentasi (Tabel 4.2).

Pada Tabel 4.2 dapat diketahui bahwa nilai fenol total terendah dihasilkan pada sampel biji

kakao dengan perlakuan laccase+ABTS (Azinobis- (3-ethylbenzthiazoline-6-sulfonate) sebesar

0,493±0,28 mg/l. Hal tersebut menunjukkan bahwa laccase mampu mengoksidasi senyawa fenolik

pada kakao. Menurut Jeon et al. (2012), laccase dapat mengoksidasi senyawa fenol dan

menghasilkan radikal fenoksi yang kemudian akan diubah dalam reaksi enzim-katalis berikutnya

sehingga terbentuk kuinon (Jeon et al., 2012; Mishra, 2018). Penambahan ABTS dalam perlakuan

juga dapat menurunkan nilai fenol total pada biji kakao. ABTS berfungsi meningkatkan potensial

oksidasi yang menyebabkan cincin aromatis pada senyawa fenolik berubah menjadi alifatik (Hilgers

et al., 2018). ABTS memiliki berat molekul rendah yang dapat teroksidasi oleh laccase, sehingga

aktif mengoksidasi senyawa non-fenolik yang dapat dioksidasi oleh laccase (Madhavi dan Lele,

2009).

Tabel 2.2 Hasil Total Fenol, Kapasitas Antioksidan, Kadar Gula Pereduksi dan Nilai Indeks

Fermentasi Laccase

Perlakuan Fenol Total

(mg/l)

Kapasitas

Antioksidan

(% Inhibisi)

Gula

Pereduksi

(ppm)

Indeks

Fermentasi

Kontrol Negatif (TF) 3,793±0,10 80,54±6,46 1,106±0,11 1,185±0,15

Kontrol Positif (F) 3,135±0,44 89,68±1,49 1,062±0,06 1,505±0,16

Laccase (TF) 1,025±0,32 88,87±2,45 0,907±0,03 1,948±0,32

Laccase (F) 0,872±0,54 88,96±2,90 0,813±0,08 1,759±0,12

Laccase+ ABTS (TF) 0,540±0,16 90,77±0,22 0,846±0,06 1,987±0,02

Laccase+ ABTS (F) 0,493±0,28 89,23±0,36 0,756±0,13 1,793±0,33

Keterangan :

TF : Biji kakao tanpa fermentasi

F : Biji kakao terfermentasi

Berdasarkan Tabel 4.2 dapat diketahui bahwa konsentrasi total fenol tidak berkorelasi dengan

kapasitas antioksidan biji kakao. Hal ini dapat terjadi karena aktivitas antioksidan pada biji kakao

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diduga akibat adanya senyawa melanoidin yang merupakan hasil dari reaksi antara fenol teroksidasi

dengan senyawa asam amino, gula sederhana serta aldehida yang terbentuk melalui proses Maillard

setelah biji kakao difermentasi dan disangrai (Bittner et al., 2006; Yashin et al., 2013). Melanoidin

merupakan produk akhir dari reaksi Maillard yang berpotensi sebagai antioksidan untuk menangkal

radikal bebas dan mampu membentuk struktur redukton (enaminol). Hal tersebut memungkinkan

kapasitas antioksidan pada biji kakao semakin meningkat dengan adanya penambahan laccase pada

proses treatment. Penambahan ABTS juga dapat meningkatkan potensial oksidasi dari sistem

laccase-ABTS sehingga menghasilkan lebih banyak senyawa alifatik dari oksidasi fenol untuk

membentuk melanoidin (Christopher et al., 2014).

Hasil analisis gula pereduksi pada Tabel 4.2 menunjukkan bahwa kadar gula pereduksi

terendah dihasilkan pada sampel biji kakao dengan perlakuan laccase+ABTS sebesar 0,755±106,7

ppm. Hal ini disebabkan terjadinya reaksi oksidasi gula pereduksi oleh laccase yang dibantu oleh

ABTS sebagai mediator. Reaksi oksidasi gula pereduksi terjadi karena gula pereduksi seperti

glukosa dan fruktosa memiliki gugus aldehid dan keton yang mampu mereduksi senyawa-senyawa

pengoksidasi. Berdasarkan Marzorati et al. (2005), laccase-ABTS mampu mengkatalis reaksi

oksidasi gugus fungsi (aldehid dan keton) dari gula pereduksi, sehingga menyebabkan kadar gula

pereduksi menurun.

Proses oksidasi enzimatik pada biji kakao juga mempengaruhi perubahan warna yang terbentuk

pada biji ataupun ekstrak bubuk kakao seperti pada Gambar 4.1 dan Gambar 4.2. Pada penelitian

ini perubahan warna akibat oksidasi laccase merupakan parameter terjadinya fermentasi yang dapat

dianalisis melalui hasil nilai indeks fermentasi (IF) seperti yang tercantum pada Tabel 4.2. Hasil

dari semua jenis sampel biji kakao menunjukkan nilai IF ≥ 1 yang menandakan bahwa biji telah

terfermentasi sempurna.

Perubahan warna pada ekstrak sampel biji kakao hasil oksidasi laccase dapat dilihat pada

Gambar 4.2. Warna pada ekstrak biji kakao yang telah mengalami perlakuan laccase terlihat lebih

cerah. Hal ini kemungkinan disebabkan adanya proses bleaching akibat oksidasi senyawa aromatik

sebagai penyusun pigmen warna (Pereira et al., 2005; Fu et al.,2012). Fu et al., (2012) menyatakan

bahwa laccase mampu melakukan proses biobleaching dengan mengoksidasi senyawa fenolik

dengan menghasilkan radikal fenoksi yang tidak stabil dan selanjutnya akan merusak cincin

aromatik senyawa berwarna pada biji kakao.

Treatment yang dilakukan dengam oksidasi laccase dan laccase+ABTS dapat

meningkatkan kualitas biji kakao berdasarkan menurunya kadar senyawa fenol, meningkatnya

kapasitas antioksidan (% imbibisi), menurunnya kadar gula pereduksi dan menunjukan indeks

fermentasi (IF) lebih besar atau sama dengan 1.

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Gambar 2.1 Warna Biji Kakao Sangrai dengan Berbagai Perlakuan A) Biji kakao tanpa fermentasi

b) Biji kakao terfermentasi, laccase c) Biji kakao terfermentasi, laccase+ABTS (LMS).

Gambar 2.2 Hasil Ekstraksi Sampel Analisis Warna dengan Indeks Fermentasi (IF) Kakao a) tidak

terfermentasi b) terfermentasi c) tidak terfermentasi, laccase d) terfermentasi, laccase e) tidak

terfermentasi, laccase+ABTS (LMS) f) terfermentasi, laccase+ABTS (LMS).

A

B

C

A

B

C

D

E

F

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4.1 Analisis FTIR pada Biji Kakao

Analisis FTIR dilakukan untuk mengetahui perubahan gugus fungsi pada biji kakao dengan

berbagai perlakuan yang tercantum pada Lampiran 13. Kisaran absorbansi biji kakao yang telah

mengalami perlakuan berada pada spektra FTIR dengan panjang gelombang 400-4.000 cm-1. Pada

kisaran panjang gelombang 1680-1700 cm-1 ditunjukkan perubahan peak gugus aromatik hasil uji

FTIR pada sampel biji kakao tidak terfermentasi (Lampiran 5) dan sampel biji kakao terfermentasi

(Lampiran 5). Sedangkan gugus fungsi yang lainnya seperti gugus hidroksi (O-H) pada kisaran

panjang gelombang 3200-3600 cm-1 dan gugus amina (N-H) pada kisaran panjang gelombang 660-

900 cm-1 tidak mengalami perubahan peak hasil uji FTIR (Segneanu et al., 2012).

Hasil uji FTIR pada sampel biji kakao terfermentasi dan mengalami perlakuan laccase serta

laccase ABTS menunjukkan peak yang hilang akibat adanya perubahan gugus fungsi aromatik

(1680-1700 cm-1 ) dibandingkan dengan hasil FTIR pada sampel biji kakao yang tidak terfermentasi

dan tidak mengalami perlakuan apapun (Lampiran 5). Hal tersebut menunjukkan bahwa telah terjadi

oksidasi fenol yang dilakukan oleh laccase (Risdianto, 2006). Proses oksidasi tersebut

mengakibatkan berubahnya gugus fungsi senyawa aromatik, sehingga dapat memungkinkan

terjadinya perubahan cita rasa dan aroma khas pada kakao (Utami, 2018).

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BAB III STATUS LUARAN Luaran pada peneltian ini ada dua (Lampiran 1, 2, dan 3) yaitu:

1. International paper pada Jurnal: Applied Biochemistry and Microbiology. (Submitted) 2. IBOC 2020 International Conference (sudah berlangsung)

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BAB IV PERAN MITRA Peran Mitra dalam penelitian ini diuraikan di bawah ini yaitu:

1. Perendaman dan Pencucuian Biji Kakao dilakukan di tempat Mitra

Pencucian terhadap biji kakao dilakukan karena jumlah pulp pada kulit yang masih tebal, sehingga menurunkan kadar kulit biji kering. Sebelum dilakukan pencucian, dilakukan terlebih dahulu perendaman biji kakao dengan air selama 1-3 jam, kemudian dilakukan proses pencucian biji secara manual. Biji kakao dari buah yang telah dikenakan proses pemeraman tidak perlu dicuci karena kadar kulitnya sudah rendah.

2. Pengeringan Biji Kakao dilakukan di tempat Mitra

Pengeringan dilakukan untuk mengurangi kadar air biji kakao sekitar 50-55% menjadi 6-7%. Tahapan pengeringan adalah tahapan lanjutan dari proses fermentasi. Pengeringan dilakukan dengan metode pengeringan artifisial atau pengeringan dengan alat. Pengeringan pada penelitian ini dilakukan dengan menggunakan oven. Suhu yang digunakan diatur pada kisaran 50-60°C. Proses pengeringan dilakukan selama ± 40-50 jam.

3. Sortasi dan Pengemasan dilakukan di tempat Mitra

Sortasi dilakukan untuk memilah biji kakao agar sesuai dengan persyaratan Standar Nasional Indonesia (SNI) biji kakao

dan menghindari biji cacat dan benda asing. Sortasi dilakukan dengan memilah kotoran-kotoran, pecahan biji, biji lengket, biji

berjamur dan biji berkecambah. Setelah itu dilakukan pengelompokkan secara manual untuk mendapatkan ukuran biji yang seragam. Selanjutnya dilakukan pengemasan dengan menggunakan kemasan karung goni yang baru dan bersih, bebas hama dan bebas bau asing.

4. Penyangraian atau Roasting dilakukan di tempat Mitra

Penyangraian atau roasting adalah salah satu tahapan dalam proses pengolahan kakao yang dibutuhkan untuk mengembangkan rasa, aroma dan warna biji kakao yang dihasilkan. Penyangraian dilakukan pada mesin sangrai tipe dengan suhu 190-225°C dan waktu sangrai berkisar antara 10-35 menit. Waktu yang diperlukan untuk proses pendinginan biji (tempering) yang optimum adalah 8-10 menit.

5. Pemecahan Biji dan Pemisahan Kulit dilakukan di tempat Mitra

Pemecahan biji dan pemisahan kulit biji kakao dilakukan di Universitas Wiraraja, Sumenep di Fakultas Pertanian. Pemecahan dan pemisahan kulit biji dilakukan dengan menggunakan mesin pemecah dan pemisah kulit biji desheller tipe pisau putar (rotary cutter). Mesin tersebut menggunkan rancangan geometris pisau putar karena memiliki teknologi

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konstruksi. Pemisahan kulit dari keping biji dilakukan dengan metode perbedaan berat jenis menggunakan embusan udara (Puslit Kopi dan Kakao Indonesia, 2015).

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BAB V KENDALA PELAKSANAAN PENELITIAN

Kendala-kendala di penelitian ini adalah:

1. Peralatan yang harus bergantian dengan peneliti alain di lab yang sama, menyebabkan tertundanya analisis data.

2. Kakao yang dihasilkan dari petani tidak memiliki kualitas yang seragam sehingga perlu dilakuakn sortasi yang membutuhkan waktu yang lebih lama.

3. Peralatan analasis biji kopi yang masih sangat terbatas, sehingga sampel kopi perlu dikirim ke pusat penelitian kopi dan kakao di kota Jermber.

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BAB VI RENCANA TAHAPAN SELANJUTNYA

Penelitian ini diharapkan dilakukan uji lanjutan yaitu uji organoleptik pada biji kakao kontrol

maupun biji kakao yang sudah dilakukan treatment laccase dan laccase+ABTS. Rencana ke depan

penelitian ini adalah perlu dilakukan produksi edible laccase, sehingga sesuai dengan standar bahan

tambahan produk pangan. .

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BAB VII DAFTAR PUSTAKA

Afoakwa, E. O., Quao, J., Takrama, F. S., Budu, A. S. and Saalia, F. K. 2012. Changes in total polyphenols, o-diphenols and anthocyanin concentrations during fermentation of pulp pre-conditioned cocoa (Theobroma cacao) beans. International Food Research Journal, 19 (3): 1071-1077.

Afoakwa, E. O. 2014. Cocoa Production and Processing Technology. New York : CRC Press.

Agustini, L., Irianto, R. S. B., Turjaman, M., dan Santoso, E. 2011. Isolat Dan Karakterisasi Enzimatis Mikroba Lignoselulolitik di Tiga Tipe Ekosistem Taman Nasional. Jurnal Pendidikan Ilmiah dan Konservasi Alam, 8 (2) : 197-210.

Aikpokpodion, P. E. and Dongo, L. N. 2010. Effects of Fermentation Intensity on Polyphenols and Antioxidant Capacity of Cocoa Beans. Int. J. Sustain. Crop Prod. 5(4):66-70

Ainiyah, S., Lisdiyanti, P., Pertiwi, M., dan Nugroho Prasetyo, E. 2014. BIOGROUTING: Produksi Urease Dari Bakteri Laut (Oceanobacillus sp.) Pengendap Karbonat. Jurnal Sains Dan Seni Pomits, 2 (1) : 2337-3520.

Al Aziz, R. dan Marianti, A. 2014. Efek Paparan Kronik Timbal (Pb) Per Oral Pada Struktur Histopatologik Lambung Tikus Putih. Unnes Journal of Life Science, 3(2).

Amir, R. M., Anjun, F. M., Khan, M. I., Pasha, I, and Nadeem, M. 2013. Application of Fourier Transform Infrared (FTIR) Spectroscopy For The Identification of Wheat Varieties. Journal Food Sci Technol, 30 (5) : 1018-1023.

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BAB VIII LAMPIRAN

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LAMPIRAN 1 Tabel Daftar Luaran Program : Penelitian Kerjasama Antar Perguruan Tinggi (PAKERTI) Nama Ketua Tim : Dr. techn. Endry Nugroho Prasetyo, S.Si.,MT. Judul : Modifikasi Enzimatik Laccase sebagai Alternatif Teknologi

Pasca Panen Biji Kakao (Theobroma Cacao Linn) 1.Artikel Jurnal

No Judul Artikel Nama Jurnal Status Kemajuan*) 1. Effect of laccase from White Rot

Fungus Trametes versicolor Oxidation on Roasted Cocoa Bean

Applied Biochemistry and Microbiology

Persiapan

*) Status kemajuan: Persiapan, submitted, under review, accepted, published

2. Artikel Konferensi

No Judul Artikel Nama Konferensi (Nama Penyelenggara, Tempat,

Tanggal)

Status Kemajuan*)

1. White Rot Fungus Trametes versicolor Laccase production using waste based substrate

5th International Biology Conference (IBOC) 2020, 17 Oktober 2020

Persiapan

*) Status kemajuan: Persiapan, submitted, under review, accepted, presented

3. Paten

No Judul Usulan Paten Status Kemajuan

*) Status kemajuan: Persiapan, submitted, under review 4. Buku

No Judul Buku (Rencana) Penerbit Status Kemajuan*)

*) Status kemajuan: Persiapan, under review, published

5. Hasil Lain No Nama Output Detail Output Status Kemajuan*) *) Status kemajuan: cantumkan status kemajuan sesuai kondisi saat ini

21

6. Disertasi/Tesis/Tugas Akhir/PKM yang dihasilkan No Nama Mahasiswa NRP Judul Status*)

*) Status kemajuan: cantumkan lulus dan tahun kelulusan atau in progress

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LAMPIRAN 2 DRAFT PAPER

Effect of laccase from White Rot Fungus Trametes versicolor Oxidation on Roasted Cocoa Bean

Violisa Linanda1,a), Maharani Pertiwi Koentjoro2), Isdiantoni3), Ida Ekawati3), Endry Nugroho Prasetyo1,b)

1Department of Biology, Faculty of Science, Institut Teknologi Sepuluh Nopember (ITS), Gedung H Kampus ITS Keputih, Sukolilo Surabaya 60111 Indonesia

2Study Program of Medical Laboratory Technology, University of Nahdlatul Ulama Surabaya, Indonesia

3Department of Agribusiness, Faculty of Agronomy, Universitas Wiraraja, Jalan Raya Sumenep‒Pamekasan Km. 5 Patean, Sumenep 69451, Indonesia.

a)Corresponding author : endry@bio.its.ac.id Abstract. Cocoa beans (Theobroma cacao) processing consist of several stages including fermentation. Fermentation actually is a process of oxidation to reduce polyphenol compounds which leads lowering of bitter taste, astringency and as well increase cacao beans aroma. Oxidation can be done by artificial oxidation or natural agent such as enzyme. Laccase is an enzyme that has many functions and roles in industrial fields like delignification, coloring or stain bleaching, bioremediation, and also in the food industry. The purpose of this study was to determine the effect of enzymatic oxidation of laccase from Trametes versicolor on the quality of cocoa beans by measuring the total polyphenol compounds, antioxidant capacity, reducing sugar, and fermentation index (IF). The highest activity of laccase showed in medium II (1081.2 U/ml), with the highest specific activity of 36040 U/mg and total protein content of 0.003 mg/ml using ABTS as a substrate. The Km value of the enzyme was obtained as 0.98 mM with the corresponding Vmax as 424 U/mg. The best Cacao bean quality was obtain in the treatment of laccase-ABTS oxidation with the lowest levels of polyphenol compound is 0.493 mg/l ± 0.28, highest antioxidant capacity is 90.77 % ± 0.22, lower reducing sugar level is 0.756 ppm ± 0.13 and showing fermentation index (IF) ≥ 1, thus allowing an increase in distinctive taste and aroma of cocoa. Keywords: Fermentation, Cocoa, Laccase, Oxidation, Polyphenol.

Introduction

The flavours and, in particular, the flavour precursors of cocoa are developed during primary processing of the cocoa beans, i.e., fermentation and drying. Cocoa bean processing consists of several stages, including fermentation, drying and roasting to get the distinctive taste and aroma of cocoa. The process must be carried out because cocoa beans that have not fermentation process, have a high bitter and astringency taste. It caused by polyphenol compounds contained in cocoa beans. Fermentation causes a decrease in the concentration of polyphenols compound due to the diffusion of polyphenols out of the seeds (through the release of water) and enzymatic oxidation of polyphenol compounds [1]. Oxidation can be done by artificial oxidation or natural agent such as enzyme. According to Camu et al., (2008) it is known that the work period of endogenous enzymes in natural fermentation is very short within a 7-day fermentation period, so it requires the addition of oxidant agents such as laccase treatment that can make fermentation more efficient. Enzymatic

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fermentation by giving laccase allows the reduction of bitter taste and sepat and increase the aroma of cocoa beans.

Laccase is a multi-copper oxidoreductase enzyme that oxidizes aromatic compounds such as polyphenols, diphenols, monophenol, and aromatic amines by using O2 as an electron acceptor to form H2O [3]. The laccase that used in this study was the laccase which produced from the white rot fungus Trametes versicolor. The oxidation of phenol compounds by laccase on cocoa bean will change the polyphenol compounds to produce phenoxy radicals which can then produce quinones [4].

The addition of laccase in this study expect to be able effectively to oxidize polyphenol compounds which leads lowering of bitter taste, astringency and as well increase cacao beans aroma [3]. The purpose of this study was to determine the effect of Trametes versicolor laccase enzymatic oxidation on the quality of cocoa becoming less of total polyphenol compounds, increasing antioxidant capacity, reducing sugar content, and color analysis with fermentation index (IF).

MATERIAL AND METHODS

Fungal Culture and Laccase Production Fungal Culture

Laccase that used in this study produced from the Trametes versicolor. T. versicolor is growth in PDA (Potato Dextrose Agar). The PDA medium composed of 39 g PDA powder in 1 liter of distilled water. PDA medium was sterilized by autoclave at 121°C and pressure of 1.5 atm for 15 minutes [5]. T. versicolor on a PDA medium was incubated at room temperature for 12 days at 28 ° C [5][6]. After 12 days, Trametes versicolor was inoculated on a 100 ml fermentation medium and incubated for 3 days, 4 days and 5 days at room temperature using a rotary shaker at a speed of 130 rpm [7]. Production and Isolation of Laccase

The medium used for enzyme production is a modified fermentation medium from the methods of Fukushima and Kirk (1995); Nyanhongo et al., (2002). The following composition of the laccase production medium by Fukushima and Kirk (1995) and Nyanhongo et al., (2002) in TABLE 1.

TABLE 1. Composition of Laccase Production Medium Ingredients variation in the number of

compositions (gram) Medium I Medium II

Rice husk 0.45 0.45 Yeast extract 0.15 0.15

Glukosa 0.1 0.1 Ammonium

chloride 0.5 0.5

KH2PO4 0.2 0.2 MgSO4.7H2O 0.05 0.05 CaCl22H2O 0.01 0.01

KCL 0.05 0.05 CuSO4 0.01 -

Information : Medium I: Fermentation medium based on Fukushima and Kirk (1995). Medium II: Fermentation medium based on Nyanghongo et al. (2002).

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Furthermore, medium was dissolved in 100 ml of distilled water and conditioned at pH 5. Then, the medium was sterilized using an autoclave with a temperature of 121 °C and pressure of 1.5 atm for 15 minutes [3][8]. Trametes versicolor laccase was produced using a modified fermentation medium from the methods of Fukushima and Kirk (1995) and Nyanhongo et al., (2002). Fungal culture from the PDA medium were taken as much as 1x1 cm and inoculated in 100 ml of the fermentation medium. Fungal cultures was incubated at room temperature using a rotary shaker at 130 rpm. Laccase isolation was carried out on the 3rd day, 4th day and 5th day. After incubation, the culture was filtered with Whatman paper no.1 [9][10]. Laccase Characterization Laccase Acticity Assay

Measurement of laccase activity was carried out by adding 1 ml of 10 mM citrate buffer solution pH 4.5, 10 μl crude laccase and 300 μl ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonate)) as a substrate. Then sample mixture was measured using a spectrophotometer with a wavelength of 436 nm [11]. Laccase activity was calculated based on the ABTS standard curve [9]. Isoelectric Point Assay

The isoelectric point (pI) is the value of the pH of a solution at which amphoteric molecules, such as proteins, have a vanish- ing net charge and hence no effective electrophoretic mobility. It shows amount of positive and negative charge of proteins in the same position, so that the protein in the enzyme does not move when placed in an electric field. At isoelectric pH (pI), protein has minimal solubility, causing protein to precipitate [12]. The isoelectric point test is carried out by preparing 6 test tubes. 1 ml crude laccase was adding into each test tube, then a phosphate buffer was added with a pH of 3, 4, 5, 6, 7, and 8. Furthermore, the test tube was homogeneous with vortex and recorded turbidity. The formation of the fastest turbidity sediment is the pH of isoelectric point. The isolelectric point is reached when the sum of the positive and negative charges of the protein is equal so that the precipitation occurs at that pH [13]. Total Protein Assay

Measurement of total protein content in laccase was determined using the Bradford method (1976) with Bovine Serum Albumin as a standard. The total protein assay by Bradford method is a test to measure the concentration of total protein in a solution. In the Bradford test involving Coomassie Brilliant Blue (CBB) dyes that bind to proteins in an acidic solution so as to provide a bluish color that can be measured by absorbance using spectrophotometry at wavelength of 595 nm [14]. The total protein content assay was carried out by making a Bradford reagent by dissolving 10 mg of Coomassie Brilliant Blue G-250 in 5 ml of 95% ethanol, then adding 10 ml of 85% phosphoric acid. After that, added with distilled water to a volume of 100 ml. The total protein test was carried out by adding 0.1 ml of crude laccase in a test tube, then adding 5 ml of Bradford reagent. After that, absorbance was measured using a spectrophotometer with a wavelength of 595 nm [14]. The blank solution used is 0.1 ml of distilled water as an enzyme replacement. The absorbance results are compared with the BSA (Bovine Serum Albumin) standard curve. The standard solution is prepared by dissolving 10 mg of BSA in 50 ml distilled water, then a concentration of 0.1-1 mg / ml is made. The value obtained is graphed with the equation Y = Ax + b, Y is the absorbance value and x is the value of protein concentration. Determination of Laccase Kinetic Parameter

The Michaelis–Menten kinetic parameters Km and Vmax were determined by measuring the laccase activity using various concentrations of ABTS as substrate. The parameter values were obtained by curve fitting of the reciprocal plot of reaction rate versus substrate concentrations using the Lineweaver-Burk plot transformation of the Michaelis-Menten rate equation [15].

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Enzimatic Oxidation of Laccase on Cocoa Bean (Theobroma cacao) The fermentation of Cocoa Bean

Fermentation process is done by traditional fermentation. It is done by hoarding/stacking fresh cocoa beans on a banana leaf to form a cone. The top surface of the seeds is covered in burlap sacks that allow air to enter. After that, cocoa beans are stirred after 48 of fermentation by transferring to another place or stirring in the same place then closed again and left until the fermentation is complete. Fermentation is carried out for 5-7 days [16]. Treatment of Cocoa Bean by Laccase

Cocoa treatment was done by adding 12 U / ml of crude laccase and Azinobis- (3-ethylbenzthiazoline-6-sulfonate) (ABTS) to fermented and unfermented cocoa beans [17]. The treatment in this study was carried out on a variation of 6 different samples, including laccase treatment on unfermented cocoa beans, treatment of laccase on fermented cocoa beans, treatment of laccase + ABTS on unfermented cocoa beans, treatment of laccase + ABTS on fermented cocoa beans and positive control treatment (fermented cocoa beans) ) as well as negative controls (unfermented cocoa beans). The composition of the laccase treatment on cocoa beans is shown in TABLE 2. TABLE 2. Composition of Laccase reatmentn Cocoa Beans

Composition Cocoa Beans Sample S1 S2 S3 S4 S5 S6

Cocoa bean (gram) 10 10 10 10 10 10 Crude laccase (ml) - 6.7 6.7 - 6.7 6.7 ABTS 0,1 µM (ml) - - 5 - - 5

Information: S1: Sample 1 (Unfermented Sample) S2: Sample 2 (Unfermented sample added laccase) S3: Sample 3 (Unfermented sample + laccase and ABTS) S4: Sample 4 (Fermented Sample) S5: Sample 5 (Fermented Sample + laccase) S6: Sample 6 (fermented Sample + laccase and ABTS)

After treatment of the cocoa beans, each sample was washed by immersing the cocoa beans in water for 1-3 hours, then washing the beans manually. After the washing process, the cocoa beans are dried using an oven. The temperature sets in the range of 50-60°C. The drying process is carried out for ± 40-50 hours. After the cocoa beans are dried, cocoa beans are selecting and grouping. The cocoa beans which don’t have impurities, broken seeds, sticky seeds, moldy seeds and germinated seeds are carried out for the roasting process. Roasting is one of the stages in the processing of cocoa needed to develop the taste, aroma and color of the cocoa beans produced. Roasting is done conventionally at a temperature of 190-225°C and time ranges around 10-30 minutes. Furthermore, cocoa beans go through the process of seed cooling (tempering) for 8-10 minutes. After roasting and tempering, cocoa beans are mashed to get cocoa powder samples by the method of pounding and grinding using a blender [16]. Total Fenol Analysis

Measurement of total phenol in this study serves to determine changes in total phenol total. Samples were prepared according to the technique described by [18], in which 10 gram samples were extracted using pure methanol (100 mL), then filtered using Whatman filter paper No. 1. Measurement of total phenols refers to the Folin-Ciocalteu method. Furthermore, measurement of

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total phenol levels was carried out based on [19] and modified. Total phenol analysis was performed with the Folin-Ciocalteu reagent. 1 ml of cocoa extract was taken and then 1 ml of Follin Ciocalteu reagent was added and homogenized and incubated for 10 minutes. After 10 minutes, add 1 ml Na2CO3. The mixture was stored at room temperature for 45 minutes. Blank was prepared by replacing reagents with aquades. After incubation, the absorbance value of the sample was measured by a spectrophotometer with a wavelength of 750 nm. Total phenol was calculated based on gallic acid standard curves. Preparation of a standard gallic acid curve was preceded by making a standard solution of 1 mg/ml by dissolving 10 mg of gallic acid in 10 ml of methanol 100%. Furthermore, standard solutions made various kinds of concentrations, such as 0.005; 0.01; 0.015; and 0.02. The absorbance was measured with a UV-Vis spectrophotometer with a wavelength of 750 nm using methanol 100% as a blanks instead of gallic acid. Antioxidant Analysis

Analysis method of antioxidant capacity is a quantitative measurement of antioxidant activity by measuring the capture of DPPH radicals using compounds that have antioxidants. This method was measured by using UV-Vis spectrophotometry, so that the value of free radical scavenging activity will be known [20]. Cocoa samples used in antioxidant analysis were extracted based on the techniques described by [18], in which 10 g samples were extracted using pure methanol (100 mL), then filtered using Whatman No. 1. filter paper. Supernatant of cocoa extract was used as a sample for antioxidant testing. Furthermore, the determination of antioxidant activity was carried out based on [21] and modified. Cocoa extract sample was taken as much as 300μl then 900μl of DPPH (2,2-diphenyl-1-picrylhydrazyl) solution was added and homogenized using vortex. Then the solution was incubated for 30 minutes (no light). Absorbance was measured using a UV-VIS spectrophotometer at a wavelength of 517 nm. Blank in this method uses 300μl pure methanol and 900μl DPPH. The absorbance results were analyzed the percentage of antioxidant capacity with the equation: Percentage of Antioxidant Activity:

DPPH Absorbance−Sample AbsorbanceDPPH Absorbance

X 100% [21] Reducing Sugar Analysis by DNS Method

The DNS method is a colorimetric technique that consists of a redox reaction between the 3,5- dinitrosalicyclic acid and the reducing sugars present in the sample for the determination of reducing sugars [22]. The analysis of this test is when the DNS (yellow) is reduced to 3-amino-5-nitrosalicylic acid (red- brown) which can be quantified by spectrophotometry at 540 nm, using glucose as an analytical standard [23][24]. DNS reagents were made based on the method developed by Miller (1959) by mixing 100% NaOH 1 ml with 1 gram DNS reagent, 200 mg crystalin phenol and 50 mg NaSO4. Then put into Erlenmeyer which has been coated with aluminum foil and homogeneous with vortex. To get extraction of total sugar samples in cocoa beans, the cocoa sample was extracted by homogenizing 2 grams of cocoa powder with 10 ml of boiling water for 1-3 minutes and then filtered using filter paper. The supernatant extract was used as a sample for the test of reducing sugar content. Reducing sugar level analysis is done by adding 3 ml of cocoa extract sample with 3 ml of reagent DNS. The sample is homogeneous and heated in boiling water for 5 minutes until the color changes and is immediately cooled at room temperature. Reducing sugar levels in cocoa extract samples were measured using a spectrophotometer at a wavelength of 540 nm. A total of 3 ml of pure methanol plus 3 ml of DNS reagent was used as a blank. The absorbance results are then compared with the standard glucose curve.

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Seed Color Analysis With Fermentation Index Seed color analysis of cacao was carried out to determine whether the seeds have complete

fermentation by measuring the fermentation index (IF) value. The color of the cocoa beans after fermentation is brown and the purple color are disappears. Complete fermented beans that are brown can be measured using the value of the Fermentation Index (IF) using a spectrophotometer to ensure the success rate of fermentation. The cocoa sample was extracted by dissolving 0.5 grams cocoa powder in 50 ml of a mixture of methanol 100% with HCl in a ratio of 97: 3. The sample mixture with the solution was then left homogeneous in the refrigerator (8°C) for 16-19 hours. Furthermore, sampel was filtered using Whatmann paper No. 1. Spectrum absorption was observed using a UV-Vis spectrophotometer. The fermentation index is calculated based on the ratio of absorbance values at a wavelength of 460 nm and absorbance of 530 nm [25]. The brown color of perfect fermented cocoa beans will read absorption (OD) at a wavelength of 460 nm, while the purple color reads at a wavelength of 530 nm, so that the fermentation index is formulated as a comparison of OD values at 460 nm with OD values at 530 nm (IF = A460 / A530). If the value of IF ≥ 1, the seeds are fully or complete fermented [16].

RESULTS AND DISCUSSION

Production and Characterization of Trametes versicolor Laccase Laccase production and characterization in this study were carried out using two different

types of mediums based on Fukushima and Kirk (1995) (medium I) and Nyanghongo et al., (2002) (medium II). The fermentation medium I consist of CuSO4, while the fermentation medium II does not consist of CuSO4. Characterization of laccase was carried out by measuring the laccase activity, total protein content, specific activity, isoelectric point, and enzyme kinetics (TABLE 3).

TABLE 3. Result of Laccase Production and Characterization

The highest laccase activity was 1081.2 U/ml (medium II) with a total protein was 0.03

mg/ml, specific activity was 36040 U/mg and isoelectric point at pH 3. This results agree with Heap et al. (2014) that laccase has an isoelectric point range (IP) at pH 3 to 7. The low value of laccase activity in medium I (885 U / ml) was affected by the addition of laccase inducer in the form of CuSO4. According to [26], laccase production can be increased by adding various inducers such as copper sulfate (CuSO4) with a certain concentration. Medium I (contain CuSO4 0.01 gram) produced smaller enzyme activity than medium II (without CuSO4). This is probably due to the addition of CuSO4 more than 1.5 mM which can be toxic to the fungus so that it inhibits T.versicolor in producing laccase [27]. According to [28], CuSO4 is an inducer to activate the laccase (lcc) gene in expressing enzyme proteins, but the addition of CuSO4 can cause oxidation of proteins to microorganisms and continue to the death phase. According to [29], one of the heavy metal compounds is Cu+, which can cause a mechanism of toxicity to certain organisms that can ultimately inhibit enzyme activity. It is because the metal compound of Cu+ can bind to proteins that will form protein-metal bonds. Protein-metal bonds will cause enzymes that bind to proteins inactive. It is known that the inhibition of enzyme will affect physiological processes and can interfere with cell structure in the organism [30].

Medium Enzyme Activity (U/ml)

Total Protein (mg/ml

)

Specific Activity (U/mg)

Km (mM)

Vmax (U/mg

protein)

Medium I 885.0 0.11 8045 0.45 159 Medium

II 1081.2 0.03 36040 0.98 424

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The kinetic parameters (Km and Vmax values) of crude T. versicolor laccase enzyme was determined by using ABTS as a substrate and calculated from Lineweaver and Burk plots. The results indicated that the Km and Vmax value of laccase T. versicolor on medium I respectively 0.45 mM and 159 U / mg protein. Whereas medium II obtained values of Km and Vmax respectively 0.98 mM and 424 U / mg protein (TABLE 3). A large Km value indicates the affinity of the enzyme for low substrate which means that a high substrate concentration is needed to carry out the enzyme reaction. The small Km value indicates the affinity of the enzyme for the high substrate, so that the enzyme reaction can occur even though the substrate concentration is low [31]. Increasing the concentration of the substrate will increase the rate of catalysis so that at a certain moment a level of saturation of the substrate will be reached which causes the catalysis rate to reach a maximum (Vmax) [32].

Laccase produced by medium I and II in this study has a Km value that is included in the category sufficient to be applied in industrial enzymes with Km values respectively of 0.45 mM and 0.98 mM. This value is even better than that of [33] which has a Km value of 1.54 mM for immobilized T. versicolor laccase. But the value of Km in this study is still higher than the Laccase T. versicolor study by Asgher et al. (2012) of 0.073 mM. This is also in line with the highest Vmax value produced by medium II which is 424 U / mg. The Vmax value is still higher than the study conducted by [15]. But the Vmax value produced by medium I (159 U / mg) is still smaller than the Vmax value produced by Asgher et al. (2012). This is likely because the resulting laccase has not undergone a refining process so that the Vmax value is still relatively low [15]. Oxidation of Laccase on Cocoa Bean (Theobroma cacao)

Laccase oxidation performance on cocoa beans was observed based on analysis of total phenol, antioxidant capacity, reducing sugar content, and fermentation index values (TABLE 4). In Table 4 shows that the lowest total phenol value was produced in cocoa bean samples by laccase + ABTS (Azinobis- (3-ethylbenzthiazoline-6-sulfonate) treatment (0.493±0.28 mg / l) which shows that laccase was able to oxidize phenolic compounds in cocoa. According to [34], Laccase can oxidize phenol compounds and produce phenoxy radicals. It will change in subsequent enzyme-catalyst reactions to form quinones [34][35]. The addition of ABTS in this experiment can reduce the total phenol value in cocoa beans. ABTS has the function of increasing the oxidation potential which causes the aromatic ring of the phenolic compound to turn into aliphatic [36]. ABTS has a low molecular weight that can be oxidized by laccase, so it actively oxidizes non-phenolic compounds, so it can be oxidized by Laccase [36].

TABLE 4. Result of Total Phenol, Antioxidant Capacity, Reducing Sugar Content and Laccase

Fermentation Index Value

Treatment Total Fenol (mg/l)

Antixidant Capacity

(% Inhibisi)

Reducing Sugar

Content (ppm)

Fermentation Index

Negative Control (unfermented) 3.793±0,10 80.54±6,46 1.106±0,11 1.185±0,15 Positive Control (fermentetd) (F) 3.135±0,44 89.68±1,49 1.062±0,06 1.505±0,16 Laccase (TF) 1.025±0,32 88.87±2,45 0.907±0,03 1.948±0,32 Laccase (F) 0.872±0,54 88.96±2,90 0.813±0,08 1.759±0,12 Laccase+ ABTS (TF) 0.540±0,16 90.77±0,22 0.846±0,06 1.987±0,02 Laccase+ ABTS (F) 0.493±0,28 89.23±0,36 0.756±0,13 1.793±0,33

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Based on TABLE 4 it can be seen that the total concentration of phenol does not correlate with the antioxidant capacity of cocoa beans. It was because the antioxidant activity of cocoa beans is thought to be due to the presence of melanoidin compounds which are the result of the reaction between oxidized phenols and amino acid compounds, simple sugars and aldehydes formed through the Maillard process after the cocoa beans were fermented and roasted [38][39]. Melanoidin is the final product of the Maillard reaction which has the potential as an antioxidant to counteract free radicals and is able to form redukton (enaminol) structures. This allows the antioxidant capacity of cocoa beans to increase with the addition of laccase in the treatment process. The addition of ABTS can also increase the oxidation potential of the laccase-ABTS system, so it produces more aliphatic compounds from the oxidation of phenols to form melanoidin [40]

The results of the analysis of reducing sugars in Table 4 shows that the lowest levels of reducing sugars produced in the sample of cocoa beans with laccase + ABTS treatment (0.7551±06.7 ppm). This is because the oxidation of the reducing sugar by laccase which is assisted by ABTS as a mediator. The oxidation reaction of reducing sugars occurs because reducing sugars such as glucose and fructose have aldehyde and ketone groups which are capable of reducing oxidizing compounds. Based on [41], Laccase-ABTS is able to catalyze the oxidation reaction of functional groups (aldehydes and ketones) from reducing sugars, which causes a decrease in reducing sugar levels [41].

The enzymatic oxidation process in cocoa beans also affects the color changes that are formed in the cocoa beans or the powder extracts as in Fig 1. In this study the change in color due to laccase oxidation is a parameter of the fermentation that can be analyzed through the results of the fermentation index (IF) as showed in TABLE 4. The results of all types of cocoa bean samples showed an IF value ≥ 1 indicating that the cocoa beans were fully fermented.

The color change in the extract of the cocoa bean from the laccase oxidation results can be seen in Fig 1. The color of the cocoa bean extract which has been given a laccase look brighter. This is due to the bleaching process due to oxidation of aromatic compounds as constituents of color pigments [6][42]. Fu et al., (2012) states that Laccase is able to conduct biobleaching processes by oxidizing phenolic compounds by producing unstable phenoxy radicals and will further damage the aromatic rings of colored compounds in cocoa beans. Treatment carried out by oxidation of laccase and laccase + ABTS can improve the quality of cocoa beans based on reduced levels of phenol compounds, increased antioxidant capacity (% imbibisi), decreasing reducing sugar levels and showing a fermentation index (IF) greater or equal to 1.

A

B

C

D

E

F

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FIGURE 1. Results of Color Analysis Sample Extraction with Fermented Index (IF) Cocoa a) unfermented b) fermented c) unfermented, laccase d) fermented, laccase e) unfermented, laccase + ABTS (LMS) f) fermented, laccase + ABTS (LMS).

CONCLUSIONS

Based on the research that has been done, it can be concluded that the production and characterization of laccase activity of the Trametes versicolor produces the highest activity of 1081.2 U / ml, the total protein content of 0.003 mg/ ml, the highest specific activity of 36040 U/mg. The Km value of the enzyme was obtained as 0.98 mM with the corresponding Vmax as 424 U/mg. Treatment carried out by oxidation of laccase and laccase + ABTS can improve the quality of cocoa beans with the lowest levels of polyphenol compound is 0.493 mg/l ± 0.28, highest antioxidant capacity is 90.77 % ± 0.22, lower reducing sugar level is 0.756 ppm ± 0.13 and showing fermentation index (IF) ≥ 1, thus allowing an increase in distinctive taste and aroma of cocoa.

ACKNOWLEDGEMENT

The authors are grateful acknowledge for the grant provided by Biomaterial and Enzyme Technology Research Group.

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