P3

HALAMAN PENGESAHAN

Laporan praktikum Bioproses berjudul Minyak yang disusun oleh:

Kelompok : 2 / Selasa
Anggota :
1. Dhyeta Ulzana Zizi Rahma 21030115130203
2. Emiwati Simanjuntak 21030115120084
3. Hafid Rizki Adinursetya 21030115130146
4. Yuda Kurniawan Argoyuwono 21030115120062

Telah disahkan pada,

Hari :
Tanggal :

Semarang, 2017
Asisten Pengampu

Nur Aini Hamada

NIM 21030114120061

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PRAKATA

Puji syukur kehadirat Tuhan Yang Maha Esa yang telah memberikan rahmat
dan hidayah-Nya sehingga laporan Praktikum Bioproses dapat diselesaikan
dengan lancar dan sesuai dengan harapan.
Laporan ini diperuntukkan untuk memenuhi salah satu tugas mata kuliah
Praktikum Bioproses. Adapun isi laporan ini adalah pembahasan mengenai hasil
percobaan dari praktikum Minyak.
Berbagai dukungan dan doa sehingga penyusun dapat menyelesaikan
laporan ini. Untuk itu penyusun mengucapkan terimakasih kepada:
1. Dr. Ing. Silviana, S.T., M.T. selaku Penanggung jawab Laboratorium
Mikrobiologi Industri,
2. Jufriyah, S.T. selaku PLP Laboratorium Mikrobiologi Industri,
3. Iqbal Ryan R. selaku koordinator asisten Laboratorium Mikrobiologi
Industri,
4. Nur Aini Hamada selaku Asisten Pengampu materi Minyak,
5. Asisten-asisten Laboratorium Mikrobiologi Industri, dan
6. Teman-teman yang telah membantu baik dalam segi waktu maupun
motivasi.
Laporan ini merupakan laporan yang saat ini dapat diajukan. Namun, pasti
masih banyak kekurangan yang mendasar pada laporan ini dan perlu diperbaiki.
Oleh karena itu, kritik dari pembaca sangat diharapkan untuk penyempurnaan
laporan ini. Semoga laporan ini dapat bermanfaat.

Semarang, April 2017

Penyusun

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iv
 P3

RINGKASAN

Minyak kelapa sering digunakan sebagai bahan baku industri dan

pembuatan minyak goreng. Teknik pembuatan minyak kelapa yang baik dapat
meningkatkan dan menjaga kualitas dan kuantitas minyak yang dihasilkan.
Dewasa ini dikembangkan pembuatan minyak dengan menggunakan
Saccharomyces sp untuk memisahkan minyak dari karbohidrat dan protein yang
terdapat dalam sel-sel endosperm biji kelapa. Adapun tujuan praktikum ini
dilakukan antara lain untuk memproduksi minyak kelapa dari santan dengan
metode fermentasi, mengetahui hubungan pH terhadap waktu. mengetahui
hubungan densitas terhadap waktu. menganalisis hasil minyak kelapa yang
didapat secara fisika dan kimia.
Minyak adalah trigliserida yang merupakan ester asam lemak gliserol
serta larut dalam pelarut lemak atau minyak. Cara Pembuatan minyak ada 7
yaitu pressing , ekstraksi , rendering, pengasaman, sentrifugasi , enzimatis, dan
fermentasi. Faktor faktor yang mempengaruhi minyak kelapa yaitu pH 4
4,5 ,waktu 50 jam atau tergantung kadar gula ,suhu, 80 F atau 26,7 oC ,kadar
gula, 10 19%. Manfaat Minyak antara lain Sebagai suplemen dengan nama
Capricidin untuk mengurangi virus HIV, MCFA (Medium Chain Fatty Acide)
dalam minyak kelapa berguna untuk metabolisme tubuh, menurunkan kolesterol,
menetralisir radikal bebas, membersihkan plak penyumbatan pembuluh darah.
Minyak kelapa juga digunakan sebagai biodiesel (bahan bakar berbasis minyak
yang berasal dari sumber terbarukan)
Pada praktikum kali ini bahan yang digunakan antara lain air kelapa,
shim, gula pasir, urea, ragi roti, ragi tempe, ragi tape, sari nanas, sari papaya
dan sari kiwi. Sedangkan alat yang digunakan diantaranya erlenmeyer, pipet
tetes, gelas ukur, cuvet, pengaduk, beaker glass, timbangan. Cara kerja yang
dilakukan dimulai dari pembuatan santan, pembuatan starter, fermentasi santan
dan analisa hasil minyak kelapa.
Hasil percobaan kami adalah pH selama fermentasi semakin menurun
lalu stabil pada rentang 4-5 karena terbentuknya asam lemak bebas. Densitas
campuran semakin bertambah seiring dengan semakin lamanya waktu fermentasi
minyak kelapa karena mikroorganisme mengalami fase pertumbuhan. Warna
pada tiap run bening, tetapi minyak kelapa dengan run 1 yang menggunakan ragi
roti memiliki bau yang tidak normal dibanding sampel lainnya karena
mengalami rancidity.
Kesimpulan dari percobaan kami adalah metode fermentasi dengan
ragi tempe, ragi roti, sari buah pepaya, dan sari buah kiwi dapat diterapkan
untuk menghasilkan minyak kelapa. pH minyak kelapa menurun pada proses
fermentasi menuju ke titik stabil pada rentang pH 4-5 seiring waktu fermentasi
minyak kelapa. Densitas campuran semakin bertambah seiring dengan semakin
lamanya waktu fermentasi minyak kelapa. Saran dalam praktikum kami yaitu
perhitungan pH lebih baik dilakukan menggunakan pH meter dan sebaiknya saat
inkubasi sampel diletakkan dalam ruangan yang telah dikondisikan untuk
pertumbuhan mikroorganisme yang optimum.

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v
 DAFTAR ISI

HALAMAN JUDUL ........................................................................................... i

HALAMAN PENGESAHAN............................................................................. ii
PRAKATA ..................................................................................................... iii
RINGKASAN..................................................................................................... iv
DAFTAR ISI ...................................................................................................... v
DAFTAR TABEL .............................................................................................. vi
DAFTAR GAMBAR ......................................................................................... vii
BAB I PENDAHULUAN
1.1 Latar Belakang ................................................................................... 1
1.2 Perumusan Masalah.............................................................................. 1
1.3 Tujuan Praktikum ............................................................................ 2
1.4 Manfaat Praktikum ............................................................................ 2
BAB II TINJAUAN PUSTAKA
2.1 Kelapa ..................................................................................................3
2.2 Minyak Kelapa......................................................................................4
2.2.1 Kandungan Minyak Kelapa........................................................4
2.2.2 Standar Baku Mutu Minyak Kelapa...........................................5
2.2.3 Pembuatan Minyak Kelapa.........................................................6
2.2.4 Manfaat Minyak Kelapa.............................................................9
2.2.5 Kerusakan pada Minyak Kelapa.................................................9
BAB III METODE PRAKTIKUM
3.1 Rancangan Praktikum ...................................................................... 10
3.2 Bahan dan Alat yang Digunakan ...................................................... 10
3.3 Gambar Rangkaian Alat....................................................................... 11
3.4 Prosedur Praktikum............................................................................. 11
BAB IV HASIL DAN PEMBAHASAN
4.1 Hubungan pH terhadap Waktu Fermentasi Minyak Kelapa............... 14
4.2 Hubungan Densitas terhadap Waktu Fermentasi Minyak Kelapa...... 15
4.3 Analisis Hasil Minyak Kelapa............................................................. 16
BAB V PENUTUP
5.1 Kesimpulan.......................................................................................... 18
5.2 Saran.................................................................................................... 18
DAFTAR PUSTAKA ......................................................................................... 19
LEMBAR PERHITUNGAN.............................................................................. 21

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 DAFTAR TABEL

Tabel 2.1. Komposisi kimia daging buah kelapa segar pada 3 tingkatan umur.......................4
Tabel 2.2 Komposisi asam lemak jenuh..................................................................................4
Tabel 2.3 Komposisi asam lemak tak jenuh............................................................................5
Tabel 2.4 Standar baku mutu minyak kelapa berdasarkan SNI 7381:2008..............................5
Tabel 4.1 Hasil Analisis Minyak Kelapa................................................................................16

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 DAFTAR GAMBAR

Gambar 2.1 Bagian-bagian buah kelapa...................................................................................3

Gambar 3.1 Skema praktikum minyak..................................................................................10
Gambar 3.2 Rangkaian alat titrasi........................................................................................11
Gambar 4.1 Hubungan pH terhadap waktu fermentasi minyak kelapa.................................14
Gambar 4.2 Hubungan densitas terhadap waktu fermentasi minyak kelapa.........................15
Gambar 4.3 Fase pertumbuhan sel mikroorganisme.............................................................15
Gambar 4.4 Reaksi auto-oksidasi minyak kelapa.................................................................17

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 BAB I
PENDAHULUAN

1.1 Latar Belakang

Tanaman kelapa (Cocos nucifera) mendapat julukan pohon kehidupan, karena mulai
dari akar, batang, daun, bunga dan buah dapat di manfaatkan untuk memenuhi sebagian
besar kebutuhan manusia (Banzon and Velasco, 1982). Walaupun manfaat tanaman kelapa
beraneka ragam, namun dalam kenyataannya masih terbatas pada produk tradisional
diantaranya pembuatan minyak goreng atau minyak makan (Somaatmadja, 1984).
Minyak kelapa sering digunakan sebagai bahan baku industri dan pembuatan minyak
goreng. Selain itu, minyak kelapa baik digunakan untuk meningkatkan kesehatan
masyarakat. Maka, tidak heran minyak kelapa atau yang biasa dikenal sebagai virgin
coconut oil ini menjadi incaran banyak orang.
Di Indonesia, umumnya pengolahan minyak kelapa dilakukan dengan menggunakan
kopra sebagai bahan dasar dengan cara tradisional. Minyak yang dibuat dari kopra
mempunyai kandungan asam lemak yang tinggi (sekitar 5%) dengan bau yang kurang
disenangi sehingga perlu diberi perlakuan pemurnian lebih lanjut sebelum dikonsumsi.
Teknik pembuatan minyak kelapa yang baik dapat meningkatkan dan menjaga
kualitas dan kuantitas minyak yang dihasilkan. Minyak kelapa dapat diekstrak dari daging
buah kelapa. Mengekstrak minyak dari daging buah kelapa merupakan teknik pembuatan
tradisional yang masih sering dipergunakan karena mudah dilakukan serta tidak
memerlukan banyak biaya. Namun masih terdapat kelemahan pada teknik tersebut yaitu
rendahnya rendemen yang dihasilkan.

1.2 Perumusan Masalah

Kelapa merupakan salah satu dari sekian banyak biji tanaman yang dapat digunakan
dalam pembuatan minyak. Minyak yang terbuat dari kelapa banyak digunakan
masyarakat sebagai minyak goreng. Pembuatan minyak kelapa secara tradisional
dilakukan dengan pemanasan pada suhu tinggi. Pembuatan minyak kelapa secara
tradisional ini banyak menimbulkan kerugian. Sebagai contoh, pemanasan yang tinggi
dapat mengubah struktur minyak serta menghasilkan warna minyak kurang baik.
Dewasa ini telah ditemukan suatu metode pembuatan minyak kelapa yang dapat
mengurangi kerugian-kerugian tersebut. Metode ini didasarkan pada penemuan
bioteknologi sederhana, yaitu penggunaan Saccharomyces sp untuk memisahkan minyak
dari karbohidrat dan protein yang terdapat dalam sel-sel endosperm biji kelapa. Metode
ini lebih dikenal dengan Pembuatan Minyak Kelapa dengan Menggunakan Ragi atau
Pembuatan Minyak Kelapa secara Fermentasi. Keberhasilan pembuatan minyak dengan
metode ini sangat dipengaruhi oleh jenis subtrat, jenis ragi, dan faktor lingkungan yang
mempengaruhi kehidupan Saccharomyces sp. Oleh sebab itu, perlu dilakukan penelitian
untuk mengetahui bagaimana produksi minyak kelapa dari santan dengan metode

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 fermentasi, bagaimana hubungan pH dan densitas terhadap waktu, serta untuk
menganalisis hasil minyak kelapa yang didapat secara fisika dan kimia.

1.3 Tujuan Praktikum

1. Memproduksi minyak kelapa dari santan dengan metode fermentasi.
2. Mengetahui hubungan pH terhadap waktu.
3. Mengetahui hubungan densitas terhadap waktu.
4. Menganalisis hasil minyak kelapa yang didapat secara fisika dan kimia.

1.4 Manfaat Praktikum

1. Mahasiswa dapat memisahkan minyak kelapa dari santan dengan cara fermentasi.
2. Mahasiswa dapat membandingkan hasil minyak yang diperoleh dengan variabel jenis
ragi dan sari buah.
3. Meningkatkan nilai guna dari tanaman kelapa sebagai bahan baku dari minyak.
4. Mengetahui kondisi optimum pemisahan minyak kelapa dari santan dengan cara
fermentasi

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 BAB II
TINJAUAN PUSTAKA

2.1 Kelapa
Kelapa (Cocos nucifera) termasuk jenis tanaman palma yang mempunyai buah
berukuran cukup besar. Batang pohon kelapa umumnya berdiri tegak dan tidak
bercabang, dan dapat mencapai 10 14 meter lebih. Daunnya berpelepah, panjangnya
dapat mencapai 3 4 meter lebih dengan sirip-sirip lidi yang menopang tiap helaian.
Buahnya terbungkus dengan serabut dan batok yang cukup kuat (Palungkun, 2004). Buah
kelapa berbentuk bulat yang terdiri dari 35 % sabut (eksokarp dan mesokarp), 12 %
tempurung (endokarp), 28 % daging buah (endosperm), dan 25 % air (Syah, 2005).

Gambar 2.1 Bagian-bagian buah kelapa

Buah kelapa yang sudah tua mengandung kalori yang tinggi, sebesar 359 kal per 100
gram; daging kelapa setengah tua mengandung kalori 180 kal per 100 gram 8 dan daging
kelapa muda mengandung kalori sebesar 68 kal per 100 gram. Sedang nilai kalori rata-
rata yang terdapat pada air kelapa berkisar 17 kalori per 100 gram.
Air kelapa hijau, dibandingkan dengan jenis kelapa lain banyak mengandung tanin
atau antidotum (anti racun) yang paling tinggi. Kandungan zat kimia lain yang menonjol
yaitu berupa enzim yang mampu mengurai sifat racun. Komposisi kandungan zat kimia
yang terdapat pada air kelapa antara lain asam askorbat atau vitamin C, protein, lemak,
hidrat arang, kalsium atau potassium. Mineral yang terkandung pada air kelapa ialah zat
besi, fosfor dan gula yang terdiri dari glukosa, fruktosa dan sukrosa. Kadar air yang
terdapat pada buah kelapa sejumlah 95,5 gram dari setiap 100 gram (Direktorat Gizi
Depkes RI, 1981).

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 Tabel 2.1 Komposisi kimia daging buah kelapa segar pada 3 tingkatan umur
Umur buah
No Komposisi per 100 g
Satuan Setengah
. bahan Muda Tua
tua

1. Kalori kal 68,0 180,0 359,0

2. Protein g 1,0 4,0 3,4

3. Lemak g 0,9 15,0 34,7

4. Karbohidrat g 14,0 10,0 14,0

5. Kalsium mg 7,0 8,0 21,0

6. Fosfor mg 30,0 55,0 98,0

7. Besi mg 1,0 1,3 2,0

8. Nilai Vitamin A SI 0,0 10,0 0,0

9. Vitamin B1 mg 0,06 0,05 0,1

10. Vitamin C mg 4,0 4,0 2,0

11. Air g 83,0 70,0 46,9

2.2 Minyak Kelapa

Menurut SNI 7381:2008 minyak kelapa murni adalah minyak yang diperoleh dari
daging buah kelapa (Cocos nucifera L.) tua yang segar dan diproses dengan diperas
dengan atau tanpa penambahan air, tanpa pemanasan atau pemanasan tidak lebih dari
60OC dan aman untuk dikonsumsi. Minyak kelapa murni tidak berwarna (bening), tidak
berasa, serta mempunyai aroma yang harum dan khas.

2.2.1 Kandungan Minyak Kelapa

Menurut Thieme (1969) komposisi asam lemak minyak kelapa:
1 Asam lemak jenuh

Tabel 2.2 Komposisi asam lemak jenuh

Asam lemak Rumus kimia % berat

Asam kaproat C5H11COOH 0,0 0,8

Asam kaprilat C7H17COOH 5,5 9,5

Asam kaprat C9H19COOH 4,5 9,5

Asam laurat C11H23COOH 4,0 5,0

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 Asam miristat C13H27COOH 13,0 19,0

Asam palmitat C15H31COOH 7,5 10,5

Asam stearat C17H35COOH 1,0 3,0

Asam archidat C19H39COOH 0,0 0,45

2 Asam lemak tak jenuh

Tabel 2.3 Komposisi asam lemak tak jenuh

Asam lemak Rumus kimia % berat
Asam palmitoleat C15H29COOH 0,0 1,3

Asam oleat C17H33COOH 5,0 8,0

Asam linoleat C17H31COOH 1,5 2,5

2.2.2 Standar Baku Mutu Minyak Kelapa

a. Berdasarkan SNI 7381:2008
Tabel 2.4 Standar baku mutu minyak kelapa berdasarkan SNI 7381:2008
No Jenis Uji
.
1 Keadaan:
1 Bau
2 Rasa
3 Warna
2. Air dan senyawa yang menguap
3. Bilangan iod
4. Asam lemak bebas (dihitung
sebagai asam laurat)
5. Bilangan peroksida
6. Asam Lemak :
6.1 Asam kaporat (C6 : 0)
6.2 Asam kaprilat (C8:0)
6.3 Asam kaprat (C:10)
6.4 Asam laurat (C12:0)
6.5 Asam miristat (C14:0)
6.6 Asam palmitat (C16:0)
6.7 Asam stereat (C18)
6.8 Asam oleat (C18:1)
6.9 Asam linoleat (C18:2)
Satuan Persyaratan
Khas kelapa segar, tidak
tengik, Normal, khas
minyak kelapa
Tidak berwarna hingga
kuning pucat
% Maks 0,2
g iod/100g 4,1-11,0
% Maks 0,2
mg ek/kg Maks 2,0
% ND 0,7
% 4,6 10,00
% 5,0 8,0
% 45,1-53,2
% 16,8-21
% 7,5-10,2
% 2,0-4,0
% 5,0-10,0

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 6.10 Asam linolenat (C18:3) % 1,0-2,5
% ND-0,2
Koloni/ml Maks 10
7 Cemara mikroba
7.1 Angka lempeng total mg/kg Maks 0,1
8. Cemara Logam : Maks 0,4
8.1 Timbal (Pb) mg/kg Maks 5,0
8.2 Tembaga (Cu) mg/kg Maks 0,1
8.3 Besi (Fe)
8.4 Cadmium (Cd) mg/kg Maks 0,1
9. Densitas kg/m3 915,0-920,0

2.2.3 Pembuatan Minyak Kelapa

a. Pressing
Metode utama untuk mengekstrasi minyak kelapa adalah dengan
pressing menggunakan expeller. Minyak kelapa kemudian dapat difilter dan
dimurnikan untuk menghilangkan asam lemak bebas. Metode ini pada
umumnya merupakan metode pembuatan minyak kelapa yang paling murah
dibanding metode lainnya (Apetrei, 2015).
b. Ekstraksi
Ekstraksi minyak kelapa dapat dilakukan menggunakan pelarut.
Pelarut yang paling efektif dalam metode ini adalah n-hexane. Larutan n-
hexane memiliki titik didih yang rendah sehingga dapat diseparasi dengan
mudah dari minyak kelapa. Akan tetapi, karena sifatnya yang mudah terbakar
dan sedikit beracun metode ini tidak dapat digunakan dalam skala industri.
Selain itu, minyak kelapa yang dihasilkan tidak cocok sebagai bahan baku
dalam industry pangan tanpa treatment lebih lanjut (Apetrei, 2015).
c. Rendering
Metode rendering dapat digunakan dalam memisahkan kelapa ke
bentuk bahan- bahan dasarnya. Metode ini biasanya disertai pemanasan dan
dilakukan bersama pressing dan ekstraksi (Apetrei, 2015).

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d. Pengasaman
e. Pengasaman merupakan salah satu upaya pembuatan minyak
kelapa dengan cara membuat suasana emulsi (santan) dalam keadaan asam.
Asam memiliki kemampuan untuk memutus ikatan lemak-protein dengan cara
mengikat senyawa yang berikatan dengan lemak. Namun asam yang
dicampurkan ke dalam santan hanya bisa bekerja dengan maksimal bila
kondisi pH (derajat keasamannya) sesuai. Pada proses pembuatan minyak
kelapa, pH yang paling optimal yaitu 4,3.
f. Sentrifugasi
g. Sentrifugasi merupakan salah satu pembuatan minyak kelapa
dengan cara mekanik. Pembuatan minyak kelapa dengan sentrifugasi
dikelompokan menjadi tiga, yaitu : pembuatan santan, pembuatan minyak
kelapa, serta penyaringan. Pada cara ini krim dimasukan dalam tabung ke
dalam sentrifuse. Pemutusan ikatan lemak protein pada santan dilakukan
dengan pemutaran (pemusingan), yaitu dengan gaya sentrifugal karena berat
jenis minyak dan air berbeda. Setelah dilakukan sentrifugasi, keduanya akan
terpisah dengan sendirinya. Berat jenis minyak lebih ringan dibanding air
sehingga minyak akan terkumpul pada lapisan atas. Kunci dari pembuatan
minyak kelapa dengan sentrifugasi yaitu kecepatan pemutaran, yaitu 20.000
rpm. Di samping itu faktor waktu juga ternyata menjadi pembatas dalam
pemutaran tersebut. Waktu yang dibutuhkan untuk memutus ikatan lemak-
protein dari santan dengan kecepatan 20.00 rpm yaitu sekitar 15 menit. Alat
yang digunakan untuk memutar santan dinamakan dengan sentrifuge.
h. Enzimatis
i. Pembuatan minyak kelapa dengan cara enzimatis merupakan
pemisahan minyak dalam santan tanpa pemanasan melainkan dengan bantuan
enzim. Enzim bisadisintetis atau disuplai dari alam. Beberapa jenis enzim
yang bisa digunakan untuk memecah ikatan lipoprotein dalam emulsi lemak
yaitu papain (pepaya), bromelin (nanas), dan enzim protease yang berasal dari
kepiting sungai. Enzim papain banyak yang terdapat dalam getah daun pepaya.
Sementara enzim bromelin banyak terdapat pada bagian bonggol (hatinya)
nanas.
j. Dengan rusaknya protein maka ikatan lipoprotein dalam santan
juga akan terputus dengan sendirinya. Kemudian, minyak yang diikat oleh
ikatan tersebut akan keluar dan mengumpul menjadi satu. Karena minyak
memiliki masa (berat) jenis lebih rendah dibandingkan dengan air, maka
posisinya kemudian berada paling atas, disusul dengan protein, dan terakhir
(bawah) yaitu air.

7
k. Fermentasi
l. Pembuatan minyak secara fermentasi pada prinsipnya adalah
pengrusakan protein yang menyelubungi globula lemak menggunakan
menggunakan enzim proteolitik. Enzim yang dimaksud adalah enzim yang
dihasilkan oleh mikroorganisme atau tanaman sebagai inokulum. Pada
pembuatan minyak kelapa dengan fermentasi, krim yang didapatkan
dicampurkan dengan ragi. Mikroba yang ada dalam ragi (Saccharomyces
cerevisiae dan Rhyzopus oryzae) ini mempunyai kemampuan menghasilkan
enzim protease dan lipase yang dapat menghidrolisis minyak dengan didukung
oleh kadar air yang tinggi
m. Kondisi optimum yang diperlukan agar mikroba dapat tumbuh
antara lain suhu berkisar antara 25 30 C, keadaan asam yaitu pada Ph 4
4,5. Mikroba, khususnya khamir, dapat tumbuh dengan baik pada kondisi
anaerob karena berdasarkan sifat metabolismenya, termasuk kelompok khamir
fermentatif yang dapat melakukan fermentasi alkohol yaitu memecah glukosa
melalui jalur glikolisis (Fardiaz, 1989; Buckle et al., 1987; Pelczar et al.,
1981).
n. Ada beberapa faktor yang mempengaruhi mikroba dalam
melakukan fermentasi yaitu:
1) Waktu
o. Semakin lama waktu fermentasi, hasil yang diperoleh semakin besar
sampai titik optimum dimana bahan telah habis terfermentasi. Pada fase ini
khamir mengalami kematian masih ada sel-sel yang dihasilkan akan tetapi
kecepatan pertumbuhannya lebih rendah dari sel-sel yang mati.
2) Cahaya
p. Mikroorganisme kebanyakan tidak mampu untuk melakukan
fotosintesis, oleh karena itu mikroorganisme pada umumnya tidak
memerlukan cahaya, bahkan cahaya ini dianggap sebagai faktor
penghambat bagi kehidupannya. Radiasi yang timbul akibat cahaya
merupakan bahaya bagi kehidupannya.
3) Suhu
q. Suhu optimum untuk pertumbuhan dan perkembangbiakan adalah 28
30 oC. Pada waktu fermentasi terjadi kenaikan panas.
4) Perbandingan jumlah ragi
r. Dalam proses fermentasi, perbandingan yang tepat antara jumlah ragi
dengan skim sebagai nutrisi akan berpengaruh terhadap hasil fermentasi.
Hal ini disebabkan oleh optimalnya jumlah dan waktu sel ragi

8
 s. mengekstrak skim sebagai nutrisi sehingga menghasilkan minyak yang
optimal (Zubaedah, 2010).
t.
2.2.4 Manfaat Minyak Kelapa
u. Menurut Enig (1996), minyak kelapa mengandung rantai triglyceride
yang dapat bermanfaat bagi kesehatan manusia, manfaat yang dimiliki minyak
kelapa antara lain sebagai berikut:
v. Meningkatkan sistem kerja pencernaan dan absopsi nutrisi.
w. Mengurangi penyakit jantung.
x. Membantu sistem kerja neuron.
y. Meningkatkan sistem imun.
z. Meningkatkan aktivitas anti-mikroba.
aa. Mengandung efek anti kanker.
ab. Membantu memperkuat tulang
ac.
2.2.5 Kerusakan pada Minyak Kelapa
ad. Kerusakan minyak dapat disebabkan oleh air, cahaya, panas, oksigen,
logam, asam, basa, dan enzim. Kerusakan minyak terutama terjadi ketika
pemanasan bahan, pengolahan, dan penyimpanan. Minyak kelapa yang belum
dimurnikan biasanya mengandung kaotoran-kotoran seperti air, protein, karbohirat,
asam lemak bebas dan komponen-komponen yang tidak tersebutkan. Asam lemak
bebas sudah terdapat pada minyak atau lemak sejak bahan itu mulai dipanen dan
jumlahnya akan terus bertambah selama proses pengolahan dan penyimpanan.
Penurunan mutu minyak karena ketengikan, ditandai dengan timbulnya bau dan
rasa yang tidak enak. Walaupun demikian, adanya bau dan rasa tidak enak tersebut
tidak menjadi faktor penentu dalam menilai suatu jenis minyak (Yakub, 2014).

9
 ae. BAB III
af...............................................................................................................................................M
ETODE PRAKTIKUM
ag........................................................................................................................................
3.1 Rancangan Praktikum
3.1.1 Skema Rancangan Percobaan
ah.
ai. Kelapa parut Diperas
Air Santan
aj.
ak.
al. Santan Didinginkan Santan
2 jam
am. Krim
an.
ao. Skim Dicampur dan
Campuran
Air Kelapa disterilkan dalam
ap. steril
autoclave
aq. urea dan gula
ar.
as.
at. Campuran steril
Ditutup al. foil Starter
au. Ragi atau sari buah
av. dan diinkubasi
sesuai variabel
aw.
KrimDiatur pH, ukur densitas,
ax. Campuran hasil
Starter ditutup al. foil dan
ay. fermentasi
diinkubasi
az. Minyak
Campuran hasil sentrifugasi Protein
ba.Gambar 3.1 Skema praktikum minyak
fermentasi Air
bb.
3.1.1 Variabel Praktikum
bc. Variabel kontrol : volume skim, volume air kelapa, pH awal, waktu
fermentasi,
bd. volume krim, volume starter, kecepatan centrifuge,
waktu sentrifugasi, penambahan urea dan gula pasir
be. Variabel bebas : ragi roti, ragi tempe, ragi tape
bf. Variabel terikat : Densitas, pH, bau, warna, kadar asam lemak bebas
bg.
3.2 Bahan dan Alat
3.2.1 Bahan
1. Kelapa parut 1,5 kg 7. Urea 12,28 gr
2. Air 1,5 L 8. NaOH 0,1 N 100 ml
3. Ragi roti 20,48 gr 9. Etanol 95% 200 ml
4. Ragi tempe 20,48 gr 10. Indikator PP
5. Aquadest 100 ml 11. Sari buah pepaya 5 ml
6. Air kelapa 240 ml 12. Sari buah kiwi 5 ml
13.
3.2.2 Alat
3.2.3
1. Erlenmeyer 250 ml 8 buah 14.............................................

10
2. Pipet tetes 2 buah
3. Beaker glass 100 ml 1 buah
4. Gelas ukur 100 ml 1 buah
5. Inkubator goyang 1 buah
6. Cuvet 5 buah
7. Neraca analitik 1 buah
8. Autoclave 1 buah
9. Alumunium foil
10. Indikator pH
11. Pengaduk kaca 1 buah
12. Centrifuge 1 buah
13. Statif 1 buah
14. Klem 1 buah
15. Buret 1 buah
16. Picnometer 50 ml 1 buah

10
3.3 Gambar Rangkaian Alat

Keterangan:
1. Klem
2. Statif
3. Buret
4. Erlenmeyer

Gambar 3.2 Rangkaian alat titrasi

3.4 Prosedur Praktikum

3.4.1 Pembuatan Santan
a Kelapa tua yang sudah diparut ditambah air dengan perbandingan 1:1 diperas
sehingga dihasilkan santan.
b Santan didinginkan selama 2 jam pada suhu ruang.
c Setelah 2 jam akan terbentuk 2 lapisan (krim dan skim) pada santan.

3.4.2 Pembuatan Starter

a. Skim yang telah dipisahkan dari krim kemudian ditambah dengan air kelapa ke
dalam erlenmeyer sebanyak 60 ml, kemudian tambah nutrient urea sebanyak
3,07 gr dan gula sebanyak 2,05 gr.

12
 b. Aduk campuran hingga homogen dan sterilisasi dalam autoclave pada suhu
121C selama 15 menit.
c. Setelah steril, campuran didinginkan.
d. Campuran diinokulasikan dengan mikroba atau enzim dalam erlenmeyer steril
pada ruang aseptis. Menambahkan 5,12 gr ragi roti ke variabel I, 5,12 gr ragi
tempe ke variabel II, ,5 ml sari pepaya ke variabel III, dan 5 ml sari kiwi ke
variabel IV.
e. Campuran ditutup dengan alumunium foil, diinkubasi dalam ikubator goyang
pada suhu kamar selama waktu yang ditentukan.
f.
3.4.3 Fermentasi Santan
a Krim santan sebanyak 55 ml dicampur dengan starter sebanyak 45 ml
g. ke dalam erlenmeyer pada ruang aseptis.

12
 b Campuran diatur pH-nya menggunakan asam asetat dan ditutup dengan
alumunium foil.
c Campuran diukur densitasnya menggunakan picnometer.
d Campuran diinkubasikan selama waktu tertentu.
e
3.4.4 Analisis Hasil Minyak Kelapa
a Menghitung volume minyak kelapa yang didapat.
Campuran yang telah selesai difermentasi akan terlihat menjadi 3 lapisan
f (minyak, protein, dan air).
Campuran dituang ke dalam cuvet untuk disentrifugasi pada putaran
g tertentu selama waktu tertentu.
Minyak kelapa dapat diambil dari cuvet dan diukur volumenya, minyak
h kelapa selanjutnya dapat dikenakan analisis yang lain.
i
b Uji secara organoleptik menggunakan indera penciuman.
Minyak kelapa yang didapat dituangkan ke dalam beaker glass.
Minyak kelapa diuji pada jarak kira-kira 5 cm dari hidung dan kemudian
j dikebaskan ke arah hidung agar baunya dapat diketahui.
Analisis dilakukan oleh minimal 3 orang.
Jika tercium bau khas minyak kelapa segar dan tidak tengik maka hasil
k dinyatakan normal. Jika tercium bau asing maka hasil dinyatakan
tidak
l normal.
m
c Uji secara organoleptik menggunakan indera penglihat.
Minyak kelapa yang didapat dituangkan ke dalam beaker glass.
Warna minyak kelapa diamati oleh minimal 3 orang.
Jika tidak terlihat warna lain atau kuning pucat maka hasil dinyatakan
n normal. Jika terlihat warna lain maka hasil dinyatakan tidak
normal.
o
d Menghitung densitas minyak kelapa
Picnometer kosong dihitung massanya menggunakan neraca analitik (m0).
Minyak kelapa dengan volume yang sudah diketahui (V) selanjutnya
p dimasukkan ke dalam picnometer.
Picnometer yang berisi minyak kelapa kemudian dihitung massanya (m1)
Menghitung densitas minyak kelapa dengan rumus:

13
 m1m0
q =
V
r

13
 s Menghitung kadar asam lemak bebas.
Membuat larutan standar NaOH 0,1 N.
Menimbang 30 gr minyak kelapa ke dalam erlenmeyer 250 ml.
Tambahkan 50 ml etanol 95%.
Tambahkan 3 tetes indikator PP dan dititrasi dengan larutan standar
NaOH 0,1 N.
Hitung kadar asam lemak bebas (dihitung sebagai asam laurat),
dinyatakan
t sebagai persen asam lemak dengan rumus berikut:
V x N x 200
u Asam lemak bebas=
m x 10
v dengan:
wV = volume NaOH yang diperlukan dalam penitaran (ml)
x N = normalitas NaOH
y m = bobot contoh (g)
z 200 = berat molekul asam laurat
aa

15
ab..............................................................................................................................................B
AB IV
ac HASIL DAN PEMBAHASAN
ad
4.1 Hubungan pH terhadap Waktu Fermentasi Minyak Kelapa
ae Dalam praktikum kami, run 1 menggunakan ragi roti, run 2 menggunakan ragi
tempe, run 3 menggunakan sari papaya, dan run 4 menggunakan sari kiwi.
Berdasarkan praktikum yang telah dilakukan, didapatkan hasil pengukuran pH
terhadap waktu fermentasi minyak kelapa sebagai berikut:
7

4
Run 1
pH
3 Run 2
Run 3
2
Run 4
1

0
Hari ke-1 Hari ke-2 Hari ke-3

Waktu Fermentasi
af
ag Gambar 4.1 Hubungan pH terhadap waktu fermentasi minyak kelapa
ah Dari gambar 4.1, dapat dikatakan bahwa nilai pH masing-masing run
fermentasi minyak kelapa mengalami penurunan setiap harinya. Run 1 mengalami
penurunan dari pH 6 pada kondisi awal menjadi 5,5 pada hari ke-2 dan 5 pada hari ke-
3. Run 2 mengalami penurunan dari pH 6 pada kondisi awal menjadi 5,5 pada hari ke-
2 dan 4,5 pada hari ke-3. Run 3 dan 4 mengalami penurunan dari pH 6 pada kondisi
awal menjadi 4 pada hari ke-2 dan cenderung stabil di pH 4 pada hari ke-3. sehingga
dapat disimpulkan masing-masing run mengalami penurunan pH dari 6 pada kondisi
awal menjadi pH 4-5,5 pada hari ke-2, dan cenderung stabil pada rentang nilai pH 4-5
di hari ke-3.
ai Penurunan pH ini menunjukkan proses terbentuknya asam lemak bebas dan
minyak kelapa. Air kelapa dan krim santan merupakan media yang sangat baik bagi
pertumbuhan mikroba karena mengandung senyawa untuk tumbuh.
aj Menurut Herman Meisner and Karen Tenney (1997), penurunan pH pada suatu
medium menunjukkan pelepasan asam lemak bebas dari suatu sampel yang ditandai
dengan peningkatan ion H+ yang terkandung dalam asam lemak bebas tersebut.
Pelepasan molekul asam lemak bebas sebanding dengan jumlah mol ion H+ yang
terlepas dari sampel. Pada percobaan juga terdapat perbedaan pH setiap run, hal ini
karenakan perbedaan jenis ragi dan sari buah yang ditambahkan.

14
 ak Sehingga dapat disimpulkan bahwa 4 run dari hasil percobaan kami sesuai
dengan teori yaitu pH mengalami penurunan selama fermentasi karena terbentuknya
asam lemak bebas sehingga menurunkan pH run tersebut.
al
4.2 Hubungan Densitas terhadap Waktu Fermentasi Minyak Kelapa
amDalam praktikum kami, run 1 menggunakan ragi roti, run 2 menggunakan ragi
tempe, run 3 menggunakan sari papaya, dan run 4 menggunakan sari kiwi.
Berdasarkan praktikum yang telah dilakukan, didapatkan hasil pengukuran densitas
terhadap waktu fermentasi minyak kelapa sebagai berikut :
1.01
1.01
1
1
Run 1
Densitas (gr/ml) 0.99
Run 2
0.99 Run 3
0.98 Run 4

0.98
Hari ke-1 Hari ke-2 Hari ke-3
Waktu Fermentasi
an
ao Gambar 4.2 Hubungan densitas terhadap waktu fermentasi minyak kelapa
ap Dari gambar 4.2, dapat dikatakan bahwa nilai densitas pada sampel dengan
perlakuan run 1, 2, 3 dan 4 mengalami peningkatan setiap harinya. Run 1 mengalami
kenaikan densitas dari 0,98785 gr/ml pada kondisi awal menjadi 0,9918 gr/ml pada
hari ke-2 dan 0,9928 gr/ml pada hari ke-3. Run 2 mengalami kenaikan densitas dari
1,00044 gr/ml pada kondisi awal menjadi 1,00247 gr/ml pada hari ke-2 dan 1,00618
gr/ml pada hari ke-3. Run 3 mengalami kenaikan densitas dari 1,00155 gr/ml pada
kondisi awal menjadi 1,00286 gr/ml pada hari ke-2 dan 1,00395 gr/ml pada hari ke-3.
Run 4 mengalami kenaikan densitas dari 1,00266 gr/ml pada kondisi awal menjadi
1,00287 gr/ml pada hari ke-2 dan 1,00357 gr/ml pada hari ke-3.

aq
ar Gambar 4.3 Fase pertumbuhan sel mikroorganisme

15
 as Menurut Hamzah M. Al-Qadiri (2007), densitas dari suatu sampel meningkat
karena adanya pertumbuhan sel mikroogranisme pada fase pertumbuhan. Pada sampel
run 1, 2, 3, dan 4 pertumbuhan mikroorganisme ini meningkatkan densitas dari
minyak kelapa seiring terbentuknya produk minyak kelapa dan pelepasan asam lemak
bebas (Al-Qadiri, 2007).
at Sehingga dapat disimpulkan bahwa 4 run dari hasil percobaan kami sesuai
dengan teori yaitu densitas mengalami kenaikan selama fermentasi karena adanya
pertumbuhan sel mikroogranisme pada fase pertumbuhan.
au
4.3 Hasil Analisis Minyak Kelapa
av Dalam praktikum kami, run 1 menggunakan ragi roti, run 2 menggunakan ragi
tempe, run 3 menggunakan sari papaya, dan run 4 menggunakan sari kiwi.
Berdasarkan praktikum yang telah dilakukan, didapatkan hasil analisis minyak kelapa
sebagai berikut :
aw Tabel 4.1 Hasil Analisis Minyak Kelapa

ax Run ay Bau az Warna

ba 1 bb Tidak Normal bc Bening

bd 2 be Normal bf Bening
bg 3 bh Normal bi Bening
bj 4 bk Normal bl Bening

bm
4.3.1 Bau Minyak Kelapa
bn Dari tabel 4.1 dapat dikatakan bahwa pada minyak kelapa perlakuan
run 2, 3, dan 4 memiliki bau yang normal, berbeda dengan minyak kelapa
perlakuan run 2 yang memiliki bau yang tidak normal.
bo Fenomena ini disebabkan karena minyak kelapa perlakuan run 1
mengalami rancidity yang disebabkan adanya reaksi auto-oksidasi. Kerusakan
minyak sering disebut dengan ketengikan (rancidity) yaitu kerusakan atau
perubahan bau dan flavor dalam lemak atau bahan pangan berlemak (Maharani,
2012). Reaksi tersebut menyebabkan bau minyak kelapa menjadi tidak sedap
dan warna menjadi lebih gelap dari warna minyak kelapa pada umumnya
(Ketaren, 2008).

16
 bp
bq Gambar 4.4 Reaksi auto-oksidasi minyak kelapa
br
4.3.2 Warna Minyak Kelapa
bs Dari tabel 4.1, dapat dikatakan bahwa pada minyak kelapa perlakuan
run 1, 2, 3, dan 4 memiliki warna yang normal yakni bening hingga kuning
keruh. Minyak kelapa secara fisik berwujud cairan bening hingga kuning
kecokelatan dan beraroma khas. Zat warna yang termasuk golongan ini terdapat
secara alamiah dalam bahan yang banyak mengandung minyak dan dalam
proses ekstraksi ikut terekstrak bersama minyak. Warna pada minyak kelapa
disebabkan oleh zat warna dan kotoran-kotoran lainnya. Zat warna tersebut
berupa betakaroten yang merupakan hidrokarbon tidak jenuh dan tidak stabil
pada suhu tinggi. Proses pengolahan minyak kelapa dengan udara panas
menyebabkan warna minyak menjadi kuning akibat karoten terdegrasi
(Suhardijono dan Syamsiah, 1987).

bt

17
 bu BAB V
bv PENUTUP
bw
5.1 Kesimpulan
1. Metode pembuatan minyak kelapa secara fermentasi dengan ragi roti, ragi tempe,
sari buah pepaya, dan sari buah kiwi dapat diterapkan untuk menghasilkan minyak
kelapa.
2. pH setiap run semakin menurun dari pH awal 6 kemudian stabil pada rentang pH 4-5
karena terbentuknya asam lemak bebas selama proses fermentasi.
3. Densitas minyak kelapa semakin bertambah seiring dengan semakin lamanya waktu
fermentasi minyak kelapa karena mikroorganisme mengalami fase pertumbuhan.
4. Minyak kelapa didapatkan pada perlakuan run 2, 3, dan 4 memiliki warna dan bau
minyak kelapa yang normal. Minyak kelapa pada run 1 didapatkan memiliki warna
yang normal tetapi baunya tidak normal karena mengalami rancidity.
bx
by 5.2 Saran
1 Perhitungan pH lebih baik dilakukan menggunakan pH meter.
2 Neraca analitik sebaiknya jumlahnya ditambah untuk meningkatkan kecepatan
pekerjaan.
3 Sebaiknya dalam inkubasi, sampel diletakkan dalam ruangan yang telah
dikondisikan untuk pertumbuhan mikroogranisme yang optimum.
bz

18
 ca DAFTAR PUSTAKA

Al-Qadiri, Hamzah M et.al. 2007. Studying of The Bacterial Growth Phases Using Fourier
Transform Infrared Spectroscopy and Multivariate Analysis. J. of Rapid Methods
& Automation in Microbiology, 16, 73-89.
Apetrei, Constantin. 2015. Corn and Coconut Oil Antioxidant Properties, Uses and Health
Benefits. New York: Nova Publisher.
Badan Standardisasi Nasional.(2008). SNI 7381:2008, Minyak Kelapa Virgin (VCO). Jakarta :
Badan Standardisasi Nasional.
Banzon, J.A. and Velasco J.R. 1982. Coconut Production and Utilization. PCRDF Manila.
Buckle, K.A. 1987. Ilmu Pangan. Universitas Indonesia Press. Jakarta.
Direktorat Gizi Depkes R.I 1981. Daftar Komposisi Bahan Makanan. Bhratara Karya Aksara,
Jakarta.
Enig, M.G. 1996. Health and Nutritional Benefits from Coconut Oil: An Important
Functional Food for the 21st century, AVOC (ASEAN Vegetable Oils Club) Lauric
Oils Symposium, Ho Chi Min, Vietnam, 25 April 1996.
Fardiaz, S. 1989. Mikrobiologi Pangan. Jurusan Teknologi Pangan dan Gizi. Fateta IPB.
Bogor.
Ketaren, S. 2008. Pengantar teknologi minyak dan lemak pangan. Jakarta: UI-Press.
Maharani, D.M., Bintoro, N., dan Rahardjo, B. 2012. Kinetika Perubahan Ketengikan
(Rancidity) Kacang Goreng Selama Proses Penyimpanan. AGRITECH, Vol. 32, No. 1.
Meisner, Herman dan Karen Tenney. 1997. pH As an Indicator of Free Fatty Acid Release
from Adipocytes. J. Lipid Research, 18, 774-776.
Palungkun, R., 2004. Aneka Produk Olahan Kelapa. Penebar Swadaya, Jakarta
Pelczar, M.J. & E.C.S. Chan. 1986. Penterjemah , Ratna Siri Hadioetomo dkk. Dasar-Dasar
Mikrobiologi 1. Universitas Indonesia Press. Jakarta.
Somaatmadja, Dardjo. 1984. Perkembangan Mutu Minyak Atsiri. Prosiding Seminar Minyak
Atsiri II. Balai Penelitian Kimia : Bogor.
Suhardijono dan Syamsiah, S. 1987. Bioproses Dalam Industri Pangan. PAU Pangan & Gizi
dan Penerbit Liberty, Yogyakarta.
Syah et al. 2005. Manfaat dan Bahaya Bahan Tambahan Pangan. Himpunan Alumni
Fakultas Teknologi Pertanian IPB. Bogor.
Thieme, J.G. 1968. Coconut Oil Processing. Paper, Food Agriculture Organization of The
United Nation. Rome.
Yakub, Nurhidayati Agustin Arifah. 2014. Pengaruh Pemberian Minyak Goreng Deep Frying
Terhadap Gambaran Histologi Ginjal Tikus Putih Strain Wistar. Tugas Akhir. Fakultas
Kedokteran, Universitas Muhammadiyah, Malang.

19
Zubaedah, S. 2010. Bakteri: Kajian tentang Beberapa Aspek Biologis. Jurusan Pendidikan
Biologi, FMIPA Universitas Negeri Malang, Malang.

20
 LEMBAR PERHITUNGAN

Menghitung W
m picno kosong = 30,42 gr
m picno + aquadest = 82,78 gr
m aquadest = 52,36 gr
Aquadest = 0,996 gr/ml
m ( 82,7830,42 ) gr
=
= 0,996 gr /ml
= 52,57 ml

Massa picno + air kelapa = 83,72 gr

m ( 83,7230,42 ) gr
Air Kelapa = = 1,012 gr/ml
52,57 ml

Air Kelapa x basis = 1,012 gr/ml x 60 gr = 60,72 gr

Massa picno + skim = 83,52 gr

m ( 83,5230,42 ) gr
skim = = = 1,044 gr/ml
52,57 ml

skim x basis = 1,044 gr/ml x 40 gr = 41,76 gr

m total = 60,72 gr + 41,76 gr = 102,48 gr W

Menghitung Kebutuhan Nutrient

Urea 3 % x 102,48 gr = 3,07 gr
Gula pasir 2 % x 102,48 gr = 2,05 gr
Ragi roti 5 % x 102,48 gr = 5,12 gr
Ragi tempe 5 % x 102,48 gr = 5,12 gr

Menghitung Densitas
Hari ke-I
m picno kosong = 17,55 gr
m picno + aquadest = 62,01 gr
m aquadest = 44,46 gr
Aquadest = 0,996 gr/ml
m ( 44,4617,55 ) gr
= = 27,018 ml
= 0,996 gr /ml
m ( 44,2417,55 ) gr
Densitas Run 1 = = = 0,98785 gr/ml
27,018 ml

21
 m ( 44,5817,55 ) gr
Densitas Run 2 = = 27,018 ml
= 1,00044 gr/ml

22
 m ( 44,6117,55 ) gr
Densitas Run 3 = = = 1,00155 gr/ml
27,018 ml
m ( 44,6417,55 ) gr
Densitas Run 4 = = = 1,00266 gr/ml
27,018 ml

Hari ke-2
m picno kosong = 30,45 gr
m picno + aquadest = 82,75 gr
m aquadest = 52,3 gr
Aquadest = 0,996 gr/ml
m 52,3 gr
=
= 0,996 gr /ml = 52,51 ml
m ( 82,5330,45 ) gr
Densitas Run 1 = = = 0,9918 gr/ml
52,51 ml
m ( 83,0930,45 ) gr
Densitas Run 2 = = = 1,002474 gr/ml
52,51 ml
m ( 83,1130,45 ) gr
Densitas Run 3 = = = 1,00286 gr/ml
52,51 ml
m ( 83,1030,45 ) gr
Densitas Run 4 = = = 1,00267 gr/ml
52,51 ml

Hari ke-3
m picno kosong = 17,69 gr
m picno + aquadest = 44,55 gr
m aquadest = 26,76 gr
Aquadest = 0,996 gr/ml
m 26,76 gr
=
= 0,996 gr /ml = 26,867 ml

m ( 44,3617,69 ) gr
Densitas Run 1 = = = 0,9928 gr/ml
26,867 ml
m ( 44,7217,69 ) gr
Densitas Run 2 = = = 1,00618 gr/ml
26,867 ml
m ( 44,6617,69 ) gr
Densitas Run 3 = = = 1,00395 gr/ml
26,867 ml
m ( 44,6517,69 ) gr
Densitas Run 4 =
= 26,867 ml
= 1,00357 gr/ml

23
80 H.M. AL-QADIRI ET AL.

might be due to: (1) cell lysis leading to leakage of the cellular materials
outside the cytoplasm causing a net increase in absorbance; or (2) the
presence of the dead cells on the membrane filter; these could not be
enumerated on TSA but at the same time would have contributed to the
microbial cell density on the membrane. However, in spite of the higher
absorbance during the death phase, the spectral pattern for death phase was
also characterized by a relative depletion of major cellular components
(particularly in region II) including proteins, nucleic acids and most
predominantly polysaccharides (1100

950 cm-1) due to the destruction of the cell wall and cell membrane. The
depletion of the cellular components might be associated to the reduction in
the metabolic activity during the death phase.
Spectral binning, smoothing and second derivative transformation were
performed before multivariate statistical analysis to: (1) detect more details
about bacterial biochemical structure by increasing the number of discrimina-
tive features associated with the bacterial spectra (Pedone et al. 2003; Sandt
et al. 2003); (2) reduce spectral pattern overlapping; and (3) characterize and
amplify the related variations among bacterial FT-IR spectra since unproc-
essed spectral patterns look similar (Kansiz et al. 1999; Lin et al. 2005).
Spectral normalization was conducted to balance the differences in
absorbance as a prerequisite to PCA and SIMCA analysis (Holt et al.
1995; Lin et al.
2003).
The composition and distribution of the biochemical components in bac-
terial cells are different during growth phases and FT-IR spectroscopy
provides a method and an indication of which specific biochemical changes
occur during different growth phases. The 15-point second order
derivatization of region I for both strains is shown in Figs. 3 and 4. After
transformation, the spectral variations (18001300 cm-1) among the four
growth phases become more distinctive for both strains. Major variations
could be observed in the log, the stationary and the death phases which
makes these stages obviously dis- tinguishable from each other and from the
lag phase because of unique amide I and amide II bands (proteins) in the
range of ~1650 and ~1540 cm-1. However, relatively minor variations
could also be observed in the range of

~1455 and ~1398 cm-1 from CH3 and CH2 asymmetric and symmetric defor-
mation of proteins.
Figs. 5 and 6 show the 15-point second derivative transformation of
bacterial spectra in the fingerprint region (region II). The major differences
might be seen in the P=O asymmetric stretch of the phosphodiester backbone
of nucleic acids (~1242 cm-1), P=O symmetric stretch of the nucleic acid
ribose or deoxyribose moieties (~1080 cm-1) (Kansiz et al. 1999; Filip and
Hermann 2001), and COC stretching vibration of cell wall and cell mem-
brane polysaccharides (Kansiz et al. 1999; Filip and Hermann 2001). There
were very clear differences in polysaccharide spectral features between the
log
dan habitus tanaman lebih tinggi. Jenis kelapa dalam merupakan jenis kelapa yang
paling banyak di Indonesia. Berdasarkan warna buahnya, jenis kelapa dalam yang
paling banyak terdapat di Indonesia adalah kelapa hijau (varietas Viridis), kelapa
merah cokelat (varietas Rubescens) dan kelapa kelabu cokelat (varietas Macrocaps).

b. Buah Kelapa

Buah kelapa berbentuk bulat memanjang dengan ukuran kurang lebih sebesar
kepala manusia. Berdasarkan umurnya, buah kelapa dapat dibagi menjadi tiga
golongan, yaitu kelapa muda, kelapa setengah tua dan kelapa tua. Buah kelapa muda
berumur 6-8 bulan, kelapa setengah tua berumur 10-11 bulan, dan kelapa tua
berumur 11-13 bulan (Nainggolan dan Sitinjak 1977). Komposisi buah kelapa tua
terdiri dari 35 persen sabut, 12 persen tempurung, 28 persen daging buah, dan 25
persen air buah (Djatmiko 1983).

Gambar 2. Buah Kelapa Tua Varietas Kelapa Dalam

Komposisi kimia daging buah kelapa bervariasi menurut tingkat kematangan dan
varietas buah kelapa. Daging buah kelapa kaya akan lemak dan karbohidrat, serta
protein dalam jumlah sedang. Kadar lemak tertinggi terdapat pada daging buah
kelapa tua. Adapun komposisi buah kelapa terdapat pada Tabel 1.

Tabel 1. Komposisi Buah Kelapa

Kandungan Satuan Muda Setengah Tua Tua

Kalori kal 68 180 359
Air g 83.3 70 46.9
Protein g 1 4 3.4
Lemak g 0.9 15 34.7
Karbohidrat g 14 10 14
Kalsium mg 7 8 21
Fosfor mg 30 55 98
Besi mg 1 1.3 2
Vitamin A mg 0 10 0
Vitamin B1 mg 0.06 0.05 0.1
Vitamin C mg 4 4 2

4
 Quality Analyses and Authentication of Coconut Oil 133

The various TAG of oils, may be separated and quantified by high-performance liquid
chromatography (HPLC), according to their carbon numbers (Moreda et al., 2003;
Aitzetmller et al., 1988). The values obtained can then serve to distinguish them from other
oils or to interpret their properties. For example, the percentage of trilinolein (LLL) was
adopted as criterion to detect the presence of seed oils in olive oils (EEC 2568/91). TAGs are
effectively separated byHPLC on reversed-phase columns, containing silica with chemically
bonded to octadecyl groups (RP-18) as stationary phase. Using acetonitrile as mobilephase,
separation of TAGs occurs according to the chain length and degree of unsaturation of the
fatty acids in the glycerol moiety (Schulte 1981). Other solvents were used as mobile phase.
One of the most frequently employed is acetone indifferent proportions. In the standard
official methods, acetone/acetonitrile (1 volume: 1 volume) is used as mobile phase (IUPAC
1987; EEC 2472/97).
Regarding the differences between oils, only oils that are rich in one fatty acid contain
much monoacid triacylglycerol, for example, olive oil, linseed oil and sunflower oil
containing triolein (OOO), trilinolein (LLL) and trilinolenin (LnLnLn) (Reske et al., 1997).

The triacylglycerol compositions of several oils are presented in the Table 1.

Table 1. Triacylglycerol composition of several oils (molecular species (wt%))

Coconut oil Olive oil Soybean oil

Triacylglycerol wt % Triacylglycerol wt % Triacylglycerol wt %
12, 12, 8 12 OOL 11 LnLL 7
12, 12, 10 6 OOO 43 LnLO 5
12, 12, 12 11 POP 3 LLL 15
12, 12, 14 11 POL 4 LLO 16
14, 12, 8 9 POO 22 LLS 13
StOO 5 LOO 8
LOS 12
OOS 5
where: L-linoleic acid; Ln-linolenic acid; O-oleic acid; P-palmitic acid; S-saturate acids; St-stearic; 8-
caprylic acid (8:0); 10-capric acid (10:0); 12-lauric acid (12:0); 14-myristic acid (14:0). (Laureles
et al., 2002).

Determination of Fatty Acids

Fatty acids are usually analyzed by gas-liquid chromatography (GLC). This method is
applicable to oils containing fatty acids with chain length in the range C14 to C24. GLC
analysis of fatty acids is performed following their conversion to methyl ester derivatives.
Columns with polar phases are used, for example polyethylene glycol stationary phase
(Carbowax) (Jennings 1987).
When samples contain short-chain fatty acids, methods of derivatization have been
proposed, for example benzylation and ter-butyldimethyl silylation (Monseur et al., 1981;
Burger et al., 1990).
 Analysis of free fatty acids can be carried out by Gas Chromatography: Flame Ionization
Detection (GC-FID), this technique being an alternative method of analysis (Hajimahmoodi et
134 Irina Mirela Apetrei and Constantin Apetrei

al., 2005; Regulation (EEC) N8 2568/ 91, annex X; Lerma Garca 2012). Furthermore, other
analytical methods, such as High-Performance Liquid Chromatography (HPLC) (Shahidi
31
2005; Kotani et al., 2002), or P-NMR (Dayrit et al., 2008) were also developed for this
purpose.
The use of pre-column HPLC method for the analysis of fatty acid with 2-
nitrophenylhydrazine hydrochloride was reported (Miwa & Yamamoto 1990). After alkaline
hydrolysis of coconut oil free fatty acids are reacted with 2-nitrophenylhydrazine
hydrochloride and then derivatized to corresponding fatty acid hydrazides. Each of the
derivatives was separated on reversed-phase HPLC with isocratic elution and detected at VIS
400 nm (Miwa & Yamamoto 1996). In Table 2 is presented the typical fatty acid composition
of coconut oil.

Table 2. Fatty acid composition of coconut oil as determined by gas liquid

chromatography from coconut oil samples (expressed as percentage of total fatty acids)

Fatty acid Fatty acid concentration (%wt)

C6:0 ND-0.7
C8:0 4.6-10.0
C10:0 5.0-8.0
C12:0 45.1-53.2
C14:0 16.8-21.0
C16:0 7.5-10.2
C18:0 2.0-4.0
C18:1 5.0-10.0
C18:2 1.0-2.5
C18:3 ND-0.2
C20:0 ND-0.2
C20:1 ND-0.2
Adapted from Codex 2003.

Determination of Sterols

The methods for the analysis of total sterols in coconut oil involve sample preparation
steps such as saponification, extraction of the neutrals from the soap solution, pre-separation
by preparative thin-layer chromatography, silylation of sterols and GC-FID analysis (ECCR
N8 2568/1991, annex V; Ham et al., 2000).
The major disadvantage of GC is the requirement of thermally stable columns and the
need of chemical derivatization prior to analysis. For this reason, alternative methods were
described based on the use of liquid chromatography-mass spectrometry (HPLC-MS) (Santos
et al., 2011; Itoh et al., 1973). In Table 3 are presented the levels of desmethylsterols in crude
coconut oil samples.
 Production Methods and Coconut Oil Quality 107

EXTRACTION OF COCONUT OIL

Pressing Copra

The main method of extraction of coconut oil from copra is by pressing copra using
expellers. Copra is pressed in large expeller presses that generate heat and pressure. The
resultant crude coconut oil is brown and turbid in appearance. This oil can be further purified
by filtering and refining to remove free fatty acids (a breakdown product from the oil), any
remaining moisture, any bad flavor or smell. Coconut oil made this way is the least expensive
of all coconut oils, which is used in food preparations. Pressing is also used to produce virgin
coconut oil in the dry extraction process. The quality of copra used in the extraction of virgin
coconut oil is of high quality and free of fungal contaminations. Special driers can be used to
prepare copra for this purpose and the brown portion of copra is not used to prepare virgin
coconut oil by pressing copra. Here, the pressing can be considered as cold pressing, since the
temperature is controlled.

Solvent Extraction

A solvent can be used in the extraction of coconut oil from copra. n-Hexane is considered
to be the most efficient solvent for oil extraction as oils easily dissolve in hexane. It is the
most suitable solvent also because of the low boiling point, which makes it easier to remove
from the oil. It is also a relatively low cost solvent. However, its flammability, mild toxicity,
explosiveness and environmental impacts are the concerns of industrial scale solvent
extraction of coconut oil. The solvent extraction leaves low levels of solvent residue in the
oil, which is safe but undesirable for food purposes. During the extraction, the oil in copra is
leached out with the solvent and the insoluble copra meal is retained unaffected. The
efficiency of extraction depends upon the temperature of the solvent, the ratio of the solvent
to copra meal, size and the porosity of the copra particles and contact time with the solvent.
Oil extraction by solvent extraction is more suitable for oil seeds containing relatively low
amounts of oil. As copra contains about 70% oil, mechanical extraction by pressing is more
efficient and economical. In addition to the full solvent extraction, prepress solvent extraction
can also be used. In the mechanical extraction by pressing under moderate pressure, the oil
content of copra can be reduced to nearly 15%. This remaining oil can be further extracted by
solvent extraction. The resultant mixture of solvent extraction includes solvent, oil solution
and extracted coconut meal. The solvent in the meal is removed by heating to boil off the
volatile solvent and the solvent is recovered by condensation. The remaining solvent in the oil
solution is removed by distillation. Traces of solvent left in the meal and the oil are removed
by steam-stripping under reduced pressure. Various high temperature steps in the solvent
extraction process may thermally degrade the oil to a very small extent and about 500-1000
ppm concentrations of solvent will also remain in the oil after purification.
 Production Methods and Coconut Oil Quality 109

Wet Extraction by Boiling Coconut Milk

One traditional way of extracting coconut oil from the coconut milk emulsion is by
prolonged heating of the emulsion. Heating breaks down and deposits proteins at the bottom
of the container. When the heating is continued, water in the emulsion evaporates. Due to its
high boiling temperature, coconut oil does not evaporate significantly during this process.
Finally, the coconut oil can be separated by decanting from the residue containing proteins,
carbohydrates and other substances. The resultant coconut oil gives a nice coconut aroma and
the oil is free of water. This oil can be kept for a very long time without forming oxidation
products that cause rancidity. However, due to caramelization and other reactions such as
Maillard reaction, the coconut oil produced by this method has a light yellowish color. One
disadvantage of this method is the high amount of energy needed and relatively longer period
of time taken to evaporate water from the coconut milk emulsion. In addition, there are no
machines designed to produce coconut oil in industrial scale using this method. Therefore,
this method is limited to the preparation of coconut oil in small scale for household
consumption.

Centrifugation Process

The coconut milk emulsion prepared by pressing fresh coconut kernels contains
approximately 40% oil. In the production of high quality virgin coconut oil, the pressing
should be done using a special machine of which both the pressing plate and the sleeve are
cooled by chilled water. Using a centrifuge, the cream is then concentrated to yield a higher
percentage of oil while the proteins and water-soluble substances are separated out. Coconut
oil produced by this method has a very light coconut flavor and the texture of coconut oil is
extremely mild and smooth. In some instances, coconut milk is chilled at 10C for 10 h to
solidify the lipids. Then the aqueous layer is discarded and the lipid block is allowed to stand
at 30C until it dissolves completely. Then the mixture is centrifuged and the oil layer is
separated. The coconut oil produced by this centrifugation method is considered to be one of
the highest quality coconut oils. These oils are expensive and usually labeled as extra virgin
coconut oil.

Fermentation Process

Fermentation method is the least consistent of all the coconut oil production processes.
Therefore, the quality of coconut oil produced by this method varies for different producers.
The oil has to be further purified for food purposes. In the preparation of coconut oil by this
method, fresh coconut kernel is first grated and then coconut milk or cream is pressed out
from the white flesh. This milk is placed into vats or buckets and allowed to ferment at about
37C. The enzymes and bacteria break the proteins in emulsion and separate the milk into
different layers which include a top protein curd layer, a coconut oil layer underneath, another
curd layer and a layer of water. The protein curd on the top can be removed and then the oil
layer can be siphoned.
Comparative Physicochemical Characteristics of Virgin Coconut Oil
al.
MATERIALS AND METHODS
Materials
Coconuts from two varieties of coconut, namely,
La- guna tall (LT) and Catigan green dwarf (CGD),
and their hybrid were obtained from the Philippine
Coco- nut Authority, Zamboanga Research
Center, Zamboanga City. These coconuts were
used to pro- duce VCO by the desiccated coconut
meat-40 C in- cubation dry method and the coconut
milk-40 C incu- bation wet method described
below. The nuts used were mature (from 11 to 13
mo old after pollination). The coconuts were taken
at random from 25 trees and were carefully chosen
so that spoiled nuts would not be included in the
preparation of VCO. Spoiled nuts were
differentiated from sound and mature nuts by a
softening that occurs in the eye of the kernel and by
foul odor (Banzon et al. 1990). Samples of coco-
nuts for the coconut milk-freeze-and-thaw cold pro-
cessing method were obtained from a local market
in Los Baos, Laguna. Six commercial VCO
and RBDCO products were obtained from different
manu- facturers. The commercial samples were
packaged in PET bottles; in some of the samples,
the fatty acid composition of the oil had been
printed on the labels. Manufacturing date was not
printed on the label of the commercial samples.
All the chemicals used were of analytical grade.
Production of Virgin Coconut Oil
Three processing methods representative of
methods commonly used by commercial VCO
producers were used to produce VCO in the
laboratory. For all pro- cesses, the coconut was
deshelled and pared using the coconut processing
equipment available at the pi- lot plant of the
Institute of Food Science and Technol- ogy, College
of Agriculture, University of the Philip- pines Los
Baos. The coconut meat was carefully removed
from the shell so that no part of the testa was
included in the sample. The coconut meat was
soaked in a 1% sodium metabisulfite solution for
about
1 h to prevent browning (Fennema 1996) and
ground
to pass a 10-mesh sieve. The stainless steel and
elec- tric-operated coconut meat grinder and
coconut oil extractor were fabricated by the
Prinsena Machine Shop in San Pablo, Laguna.
Vermont P. Dia et
The dry method involved the following steps:
The ground coconut meat was dried at 40 C using a
cabi- net dryer for 24 h. The dried coconut meat was
stored inside a freezer for 1 wk before use and the
coconut oil was extracted from the dried meat using
a coconut oil extractor. The maximum temperature
of the oil as it got out of the expeller was measured
to be 47.0 C. The extracted oil was centrifuged at
8000 x g. The water clear virgin oil was collected
by decantation. This method is termed as
desiccated coconut meat-
40 C incubation
method.
For the first wet method, coconut milk was first
extracted from freshly ground meat. Coconut milk
was incubated for 24 h in a 40 C water bath to
hasten separation of the cream from the skim milk
following the procedure described by Del Rosario
and Mabesa (1976), Del Rosario (1979) and Banzon
and Velasco (1982). The oil layer that separated
was further cen- trifuged at 8000 x g for 15 min to
separate water clear virgin coconut oil from the non-
oil components. This method is termed coconut
milk-40 C incubation method.
For the second wet method, the coconut milk was
extracted as before and subjected to approximately
4 cycles of freeze and thaw to obtain the oil from
the coconut milk. The freeze and thaw method
involved refrigeration of coconut milk at 4 C for 4 h
and allow- ing the frozen coconut milk to thaw at
ambient tem- perature for 2 h. The oil that separated
was scooped using a spoon and was centrifuged at
8000 x g to further remove suspended particles.
This method is termed coconut milk-freeze-and
thaw method.
Physicochemical Analysis
The melting point, specific gravity, saponification
num- ber and iodine value of the laboratory
produced and commercial VCO and RBDCO were
determined fol- lowing standard procedures (AOAC
2000). All analy- ses were done in triplicate.
Quality Characteristics
The moisture content, free fatty acid number and
per- oxide value of VCO and RBDCO were
determined following standard procedures (AOAC
2000). All analyses were done in triplicate.
464 The Philippine Agricultural Scientist Vol. 88 No. 4 (December
2005)

The lauric acid in coconut oil
is used by the body to make
the same disease-fighting fatty
acid derivative monolaurin
that babies make from the
lauric acid they get from their
mothers milk. The
monoglyceride monolaurin is
the substance that keeps
infants from getting viral or
bacterial or protozoal
infections.
Health and nutritional benefits
from coconut oil and its
advantages over competing oils
Mary G. Enig, Ph.D., F.A. C.N.
Introduction
wo important areas will be
T covered in this paper. In the
first
part, a review of the major health
challenge facing coconut oil today
has been included. This challenge
is based on a supposed negative
role played by saturated fat in
heart disease. It is proposed to
dispel any acceptance of this
notion with the information that
will be presented here.
In the second part some new
directions where important positive
health benefits are seen for coconut
oil are suggested. These benefits
stem from coconut oils use as a
food with major antimicrobial and
anti- cancer benefits. The rationale
for this effect and some of the
literature will be reviewed here.
The health and nutritional
benefits derived from coconut
oil are unique and
compelling. They are under-
appreciated today by both the
producer and the consumer. Better
recognised are the functional
advantages coconut oil has, over
competing oils, in many food
products. Historically, coconuts and
their extracted oil have served man
as important foods for thousands of
years. The use of coconut oil as a
shortening was advertised in the
United States in popular cookbooks
Director, Nutritional Sciences Division, Enig Associates Inc., Maryland, USA
HEALTH
at the end of the 19th century. Note
that both the health-promoting
attributes of coconut oil and those
functional properties useful to the
house maker were recognized 100
years ago. These same attributes, in
addition to some newly discovered
ones, should be of great interest to
both the producing countries as
well as the consuming countries.
Origins of the Diet/ Heart
Hypothesis
The literature of epidemiological
studies usually attribute an in-
creased risk of coronary heart
disease (CHD) to elevated levels of
serum cholesterol, which in turn are
thought to be derived from a
dietary intake of saturated
fats and cholesterol. But,
saturated fats may be considered a
major culprit for CHD only if the
links between serum cholesterol and
CHD, and between saturated fat
and serum cholesterol are each
firmly established. Decades of large
scale tests and conclusions there
from have purported to
establish the first link. In fact, this
relationship has reached the level of
dogma. Through the years
metabolic ward and animal
studies have claimed that dietary
saturated fats increase serum
cholesterol levels, thereby
supposedly establishing the second
link. But the scientific basis for
these relationships has now been
l 9 Indian Coconut
Journal
1724 Metabolism and Function of Bacterial Lipids. II Vol. 239, No. 6
rate was observed at about 5 X 10-4 to 10-3 M CDP-dipalmitin, serine is the precursor of phosphatidylethanolamine in E. coli,
while higher concentrations were somewhat inhibitory. according to Equation 3.
pH Optimum-When the pH is varied with Tris-phosphate
buffers, prepared as described in "Materials and Methods," Phosphatidylserine -+ phosphatidylethanolamine + C02 (3)
the enzyme is active over a broad range of pH between 6.9 and In keeping with this conclusion, we have found an enzyme in
9.0, with an optimum at about pH 7.9. E.coli which catalyzes the decarboxylation of phosphatidylserine.
Ejf ects of Inhibitors-The enzyme is unaffected by iodoaceta The enzyme is specific for the phospholipid form of serine, since
mide, N-ethylmaleimide, hydrazine, or hydroxylamine when L-serine itself is not attacked.
added to the enzyme incubation mixture at a concentration Active preparations of phosphatidylserine decarboxylase were
of prepared from freshly harvested or frozen cells of E. coli in the
0.004 M. (Mercaptoethanol was omitted from the incubation following manner. Suspensions of cells (l.O g of cells (wet
mixture in these experiments.) The enzyme was likewise un weight) in 40 ml of 0.1 M phosphate buffer of pH 6.8 containing
affected by KF at a final concentration of 0.04 M. 0.01 M mercaptoethanol) were subjected to ultrasonic disintegra
DivalentCations-No requirement for divalent cations for the tion in a Gilson Medical Electronics apparatus for 10 minutes.
activity of the L-serine-CMP phosphatidyltransferase was The container was immersed in an ice-salt bath during this
detected. The enzyme incubation mixture routinely contained procedure, during which the temperature of the suspension rose
EDT A. The addition of calcium, magnesium, or manganese to about 10. The suspension was cooled to 0 and centrifuged
ions did not stimulate the reaction; at high concentrations a at 35,000 X g for 40 minutes in a refrigerated centrifuge. The
distinct inhibition was noted, probably the result of precipitation supernatant solution contained little phosphatidylserine decar
of the CDP-diglyceride. boxylase activity, and was set aside for the preparation of other
Decarboxylation of Phosphatidylserine-We have previously enzymes. The precipitate was washed twice with the same
reported that phosphatidylethanolamine is the principal glycero loa
buffer used for suspending the cells, and the wash solutions were de
phosphatide found in this strain of E. coli. This fact, together discarded. The precipitate was then extracted by stirring at 0 d
with the finding that only traces of phosphatidylserine are found for 30 minutes with buffer containing 5% (w /v) of the nonionic fro
in the organism after exposure of growing cultures to orthophos detergent Cutscum, in a volume about one-fifth of that of the m
phate labeled with 32P (1), strongly suggests that phosphatidyl- original cell suspension. The precipitate was removed by cen htt
p:/
trifugation at 35,000 X g for 40 minutes and was discarded. /w
16000 Cold ethanol was then added dropwise to the extract with good w
stirring, in a container immersed in an ice-salt bath, until a final w.j
concentration of 30% was reached. The precipitate was re bc.
~ or
u moved by centrifugation in a refrigerated centrifuge. The g/
I 12000 supernatant solution was discarded. The precipitate was by
A
r. :i
dissolved in a small volume of 0.1 M phosphate buffer of pH 6.9 gu
en containing 0.02 M EDTA, and was dialyzed against the same est
< on
~ buffer. The enzyme could be stored frozen for several weeks
M
~ arc
N
8000 with no detectable loss of activity.
0 The activity of phosphatidylserine decarboxylase can most h
~ 8,
.-j readily be followed by measuring the release of radioactive C02 20
u from phosphatidyl-t-serine-l-v'C as shown in Fig. 4. It is 17
4000
seen that the release of radioactive C02 is a linear function of
enzyme concentration. Since phosphatidylserine and serine
itself are somewhat unstable substances, it is necessary to make
sure that nonenzymatic decarboxylation is not taking place
under the conditions of the experiment. This is done by ap
1
propriate controls from which enzyme is omitted, or in which
2 3
boiled enzyme is used.
ENZYME ADDED - MICROGRAMS The properties of the phosphatidylserine decarboxylase will
be described in greater detail in a later publication.
Fm. 4. Phosphatidylserine decarboxylase of E. coli. The in Synthesis of Phosphatidylglycerol-The biosynthesis of phos
cubations were carried out in 25-ml Erlenmeyer flasks provided phatidylglycerol in animal tissues has been shown by work in
with a center well and rubber serum bottle caps. Each vessel
contained phosphate buffer of pH 6.8 (0.10 M), Cutscum (1 mg), this laboratory (13) to proceed according to Equations 4 and 5.
phosphatidyl-t-serine-Lo-C (2 X 10-4 M), and enzyme (prepared
as described in the text) in the amounts indicated. The final CDP-diglyceride + L-a-glycerophosphate -+ (4)
volume was 0.50 ml. The center wells contained 0.1 ml of 2 N phosphatidylglycerophosphate + CMP
KOH on folded pieces of Whatman No. 1 filter paper, 2 X 3 cm.
The vessels were shaken in a bath at 37 for 30 minutes, after which Phosphatidylglycerophosphate-+ phosphatidylglycerol + Pi (5)
0.5 ml of 0.5 N H2S04 was added through the rubber cap by means
of a hypodermic needle. Shaking was continued for 1 hour longer, Extracts of E. coli catalyze analogous reactions. Reaction
after which the radioactivity in the collected carbon dioxide was
determined by direct counting by the method of Buhler (14). 4
The specific activity of the phosphatidylserine-1-14C in this ex was demonstrated by measuring the CDP-diglyceride-requiring
periment was 4.8 X 105 c.p.m. per mole.
conversion of 1ra-glycerophosphate-1,3-14C into lipid (Table V).
As mentioned above, Reaction 4 was also detected in enzyme
fractions derived from E. coli by spectrophotometric measure-
138 Biopendix, Volume 1, Nomor 2, Maret 2015, hlm. 137-142
tua, sedangkan kandungan kalori dan lemak
mencapai maksimal pada buah kelapa tua
(Heyana, dkk, 2000).
Minyak kelapa memiliki sejumlah fungsi
yang dapat dimanfaatkan dalam kehidupan
manusia. Disamping sebagai bahan pangan
yang penting dalam penggorengan, minyak
kelapa juga baik digunakan untuk
meningkatkan kesehatan masyarakat. Maka
tidak heran minyak kelapa selalu menjadi
incaran banyak orang. Selain itu minyak
juga merupakan sumber energi dimana
satu
gram minyak dapat menghasilkan 9 kkal
(Winarno, 2004).
Meskipun penggunaan minyak kelapa
yang diolah secara modern sejauh ini masih
aman bagi kesehatan namun sebagian
masyarakat Maluku terutama yang
berdomosili didaerah pedesaan masih
tergantung pada minyak yang diolah secara
tradisional karena mudah dilakukan serta
tidak memerlukan banyak biaya. Disamping
alasan ekonomi karena harga minyak
kelapa yang diolah secara modern memiliki
harga lebih mahal, tindakan ini juga untuk
memanfaatkan buah kelapa yang
bertumpuk dan sulit dipasarkan pada waktu
musim panen. Minyak mengandung asam
lemak tak jenuh dan beberapa asam lemak
esensial seperti asam oleat, linoleat dan
linolenat (Sulistyo et al, 2006).
Pembuatan minyak kelapa dengan
fermentasi merupakan salah satu alternatif
untuk mengatasi masalah pada pembuatan
dengan cara tradisional. Beberapa faktor
mempengaruhi produksi minyak kelapa
secara fermentasi diantaranya pH,
konsentrasi inokulum, suhu, bahan baku
kelapa, dan lamanya fermentasi. Sehingga
perlu dilakukan pengkajian untuk
mendapatkan kondisi optimal proses
sehingga dihasilkan jumlah dan kualitas
minyak kelapa yang lebih optimal. Selain itu
dalam proses pembuatan minyak kelapa
secara fermentasi minyak yang dihasilkan
lebih banyak dan warnanya lebih jernih
(Sukmadi dan Nugroho, 2002).
Pembuatan minyak kelapa dengan
fermentasi lebih kurang di diamkan selama
beberapa jam. Selama proses fermentasi
berlangsung terjadi suksesi (pergantian)
mikroba dalam emulsi santan dan terjadi
perubahan keasaman (pH). Perubahan pH
merupakan salah satu faktor yang
mempercepat oksidasi minyak. Oksidasi
Paulus Valentino Patty, Pengaruh Lama Fermentasi 138
minyak adalah reaksi asam-asam lemak
tidak jenuh yang menyusun lemak sebagai
radikal bebas dengan molekul oksigen
sehingga menyebabkan minyak menjadi
lebih cepat tengik dan menyebabkan
penurunanan kualitas minyak kelapa,
Winarno (2004).
Menurut ketaren (2008), ada tiga
penyebab ketengikan dalam lemak yaitu
ketengikan oleh oksidasi, (oxidative
ranciditas), ketengikan oleh enzim
(enzymatic ranciditas), dan ketengikan oleh
proses hidrolisa (hidrolitic ranciditas).
Ketengikan oleh oksidasi terjadi karena
proses oksidasi oleh oksigen udara
terhadap asam lemak tidak jenuh
dalam lemak. Proses oksidasi dapat terjadi
pada suhu kamar, dan selama proses
pengolahan menggunakan suhu tinggi. Hasil
oksidasi lemak dalam bahan pangan tidak
hanya mengakibatkan rasa dan bau tidak
enak, tetapi juga dapat menurunkan nilai
gizi, karena kerusakan vitamin (karoten dan
tokofenol) dan asam lemak esensial dalam
lemak.
Kerusakan lemak yang utama adalah
timbulnya bau dan rasa tengik yang disebut
proses ketengikan. Hal ini disebabkan oleh
otooksidasi radikal asam lemak tidak jenuh
dalam lemak. Radikal bebas dengan O2
membentuk peroksida aktif yang dapat
membentuk hidroperoksida yang bersifat
tidak stabil dan mudah pecah menjadi
senyawa dengan rantai karbon yang lebih
pendek oleh radiasi energi tinggi, energi
panas, katalis logam, atau enzim. Senyawa-
senyawa dengan rantai C lebih pendek ini
adalah asam-asam lemak, aldehida-
aldehida, dan keton yang bersifat volatile
dan menimbulkan bau tengik pada lemak
(Winarno, 2004).
Mutu dari suatu minyak dapat diketahui
dari rasa dan aromanya. Salah satunva
adalah ketengikan atau adanya Asam
Peroksida. Peroksida merupakan suatu
tanda adanya pemecahan atau kerusakan
pada minvak karena terjadi oksidasi (kontak
dengan udara) yang menyebabkan bau
aroma tengik pada minyak. Ukuran dari
ketengikan dapat diketahui dengan
menentukan bilangan peroksida. Semakin
tinggi bilangan peroksida maka semakin
tinggi pula tingkat ketengikan suatu minyak
(ASA, 2000).
 Copyrighted Materials
Copyright&'.: 2012 AOCS Press
Retrievedfromwww.knovel.com

Palm Oil: Production,Processing, Characterizationa, nd

Uses

Preface
Palm oil is now the world's largest vegetable oil by volume. With proven nutritional
benefits in addition to the presence of nutraceutical components, such as tocotrienols
and carotenoids, palm oil is consumed in over 150 countries worldwide. It plays a
pivotal role in the socioeconomic development of Asian, Latin American, and African
regions. In 2010, global production of oils and fats stood at 172.1 million tonnes.
Palm oil combined with palm kernel oil was the largest contributor to these, account
ing for 51.6 million tonnes, or 30% of the total output. Of the 65 million tonnes of
oils and fats entering global trade, palm oil and palm kernel oil accounted for close to
61% of the total volume, or 39.6 million tonnes, with Malaysia having 45% of the
market share, dominating the international trade in palm oil.
The success of the palm oil industry in Malaysia, Indonesia, and elsewhere has
never been easy and has only gotten harder in recent years. Its success has often been
made more difficult by mistrust and misconceptions about the oil, its composition,
and its nutritional benefits. Thus, with this critical issue in mind, this book has been
assembled with the hope of being an authoritative, comprehensive, and highly infor
mative compilation of recent advances and issues in various important areas of palm
oil industry-s-from the production, processing, and characterization to the utilization
of palm oil and its components.
The topics herein deal with both the production and processing of palm oil and
kernels, and more importantly, the editors attempted to provide an international per
spective to this area by drawing experts from around the world, especially Malaysia,
Brazil, and Nigeria. Thus, this book has amassed diverse topics from these experts
in specific areas from across the globe. This book begins with a chapter on the his
tory of oil palm and subsequently three chapters related to oil palm breeding, oil
palm genomics, tissue culture and genetic engineering of oil palm. The book also
includes several topics related to postharvest activities, pests, and diseases of oil palm.
Information on production of palm oil and palm kernel oil; physical, chemical, and
polymorphic properties of palm oil and its components; as well as the measurement
and maintenance of palm oil quality are included in the book, which may be of inter
est to researchers, scholars, technologists, and food manufacturers. To understand the
importance of this plant-based oil, the variety of applications of palm and kernel oil,
and their fractions in food, nutritional and oleochemical products are included. The
potential use of palm oil as an alternative to trans fats in foods is also highlighted.
There are also special chapters dedicated to the use of palm biomass in various wood
based products and as sources of bioenergy and biofuels. We have also included in
depth accounts of three current issues on oil palm/palm oil that are garnering atten
tion and becoming globally relevant, namely waste and environmental management,
trade and traceability, together with other issues relating to sustainable development
vii
 A.M. Marina et al. / Trends in Food Science & Technology 20 (2009) 481e487
483

combination of these treatments (Seow & Gwee, 1997).
These techniques were possible due to the fact the coconut
milk proteins were easily coagulated and precipitated at
pH 4 (Tangsuphoom & Coupland, 2008).
Enzymatic extraction technique
Extraction of oil can also be carried out by the use of en-
zymes in aqueous extraction process. This is due to the fact
that plant cell walls consists of complex carbohydrate mol-
ecules such as cellulose, hemicellulose, mannans, galacto-
manans, arabinogalactans, pectin substances and protein
(Christensen, 1991). Coconut meat contains about 10% of
carbohydrate, in which 50% of this is cellulose and 75%
of the cellulose is made up with a-cellulose (Rosenthal
et al., 1996).
Oil can be found inside plant cells, linked with proteins
and wide range of carbohydrate such as starch, cellulose,
hemicellulose, and pectins. Cell-wall degrading enzymes
can be used to extract oil by solubilizing the structural
cell wall components of the oil seed. Che Man, Suhar-
diyono, Asbi, Azudin, and Wei (1996) also successfully
extracted coconut oil with 1% enzyme mixture of cellulase,
a-amylase, polygalacturonase, and protease with an oil
yield of 74%. The polygalacturonase hydrolyses a-linkages
of polygalacturonic acid of the polymer randomly from the
ends. An a-amylase randomly hydrolyzed a-linkages to liq-
uefy starch and produced maltose, whereas bacterial prote-
ases were used to hydrolyze the plant protein. The study
showed that different enzymes were required to degrade
components of the structural cell wall including mannan,
galactomannan, arabinoxylogactan and cellulose.
Physicochemical properties of virgin coconut oil
Since its appearance in the market, VCO is well ac-
cepted by consumer as functional food oil and the demand
for this oil continues to increase. Due to that, the number of
commercial VCO was increasing in the market. Marina,
Che Man, Nazimah, and Amin (2009a) described the chem-
ical properties of commercial VCO available in Malaysia
and Indonesia. The results revealed that chemical properties
of VCO did not vary much from the RBD coconut oil. The
iodine, peroxide, saponification and free fatty acid values
obtained for commercial VCO samples were well within
the specification limit of Codex standard (2003) for refined
coconut oil. The fatty acid composition was dominated by
lauric acid with percentage ranging from 46 to 48%, which
was within the limit of VCO standard according to Malay-
sian Standard (2007) and Asian and Pacific Coconut Com-
munity (APCC, 2003) (Table 1). According to the study,
medium chain fatty acids ranged from 60 to 63%. The ma-
jor triacylglycerol (TAG) in VCO samples consisted of 22e
25% of LaLaLa, 14e16% of CCLa, 19e21% of CLaLa,
13e15% of LaLaM and 7e9% of LaMM with La, C and
M are lauric, capric and mysristic acids, respectively. Gen-
erally, VCO from Malaysia had relatively higher contents
of CpCpLa (Cp: caproic), CpCLa and LaOO while Indone-
sian VCO had more of LaMP.
Dia, Garcia, Mabesa, and Tecson-Mendoza (2005) com-
pared the physicochemical properties of VCO produced by
different methods. Three different processing methods were
applied, namely incubated dessicated coconut meat, incu-
bated coconut milk and freeze-thaw coconut milk. The re-
sults showed that there were some differences in the
physicochemical properties of the VCO samples produced
by different methods but the differences were not large
enough to significantly affect the overall quality of the
VCO. The levels of the physical and chemical qualities
were within the Codex standard for coconut oil and the
Philippine standards for VCO. In addition, the authors
also determined the a-Tocopherol in the studied VCO.
The result revealed that a-Tocopherol was actually found
in the coconut testa (the thin brown layer that clings to
the white coconut meat) and only trace amount in the
VCO samples. Since coconut testa was removed in the pro-
duction of VCO, it was logical that the VCO samples con-
tain only trace amount in a-Tocopherol.
The quality of VCO is very much determined by its
physicochemical properties. However, as established by
Dia et al. (2005) and Marina et al. (2009a), the physico-
chemical quality of VCO was comparable to RBD coconut
oil. An understanding of descriptive quality of VCO in
terms of sensory evaluation was highly needed to better

Table 1. Fatty acid composition of virgin coconut oil (VCO) and refined, bleached and deodorized (RBD) coconut oil from various sources.
a
Fatty acid Codex standard APCC standard Malaysian standard Marina et al. (2009a) Dia et al. (2005)
for RBD coconut oil for VCO for VCO
C6 nde0.70 0.40e0.60 0.80e0.95 0.52e0.69 nde0.60
C8 4.60e10.0 5.00e10.00 8.00e9.00 7.19e8.81 5.98e10.44
C10 5.0e8.0 4.50e8.00 5.00e7.00 5.65e6.59 5.37e6.60
C12 45.10e53.20 43.00e53.00 47.00e50.00 46.89e48.03 47.63e52.55
C14 16.80e21.00 16.00e21.00 17.00e18.50 16.23e18.90 16.79e20.08
C16 7.50e10.20 7.50e10.00 7.50e9.50 7.41e9.55 6.38e10.17
C18:0 2.00e4.00 2.00e4.00 2.50e3.50 2.81e3.57 7.45e10.73
C18:1 5.00e10.00 5.00e10.00 4.50e6.00 5.72e6.72
C18:2 1.00e2.50 1.00e2.50 0.70e1.50 0.90e1.60 nde0.12
C18:3 nde0.20 <0.5 nd nd nd
a
Asian and Pacific Coconut Community.

7.40
6.80
6. 80 ... 7.40
0 Any theory will have to take into consideration the
cE
20
30
Fig. 4. Effect of pH on H+ release in adipocytes. Cells (39.2
mg lipid) from 130-g rats were added to Krebs-Ringer phos
phate medium, pH 7.40 or 6.80. After 10 min, I g of epi
nephrine was added. At the arrow, the pH of the medium was
raised from 6.754 to 7.387 by addition of NaOH. Solid lines are
:::c
u
a:::
<(
cells incubated with epinephrine; dotted lines are controls.
w release from 113 to 217 nmol tt+mg lipid-1.
(/) hr ", which is identical to the rate of H+ release
w
a::: in cells maintained at pH 7.4.
o w
-0.. Discussion
...J The stoichiometric release of protons and FFA
during activated triglyceride breakdown in adipocytes
LL
0 provides a convenient method to measure lipid
...J mobilization by monitoring changes in medium pH. In
addition, the system can be perturbed multiple times
<(
z
a:::
by addition of a- and /3-adrenergic agonists, antago
::, nists, or by antilipolytic drugs, and initial changes in
0 the rate of appearance of protons (FFA) can be
determined. The well-known variation in the FFN
glycerol ratio, which is dependent on the extent of
-, FFA re-esterification, limits the method to providing
an index of FFA release and not of total lipolytic
activity. Nonetheless, under the conditions described
here, as well as in the presence of insulin and glucose1,
H+ and FFA appearance remain stoichiometric. The
proposed role of FFA as a feedback regulator of
lipolysis ( 12) lends importance to the determination of
this parameter.
To the extent that pH can be used to measure
FFA efflux, the data confirm the experiment of
Rudman and Shank (3), who followed pH changes in
the extracellular medium of perirenal and epididymal
adipose tissue slices at half-hour intervals over a 3 hr
period. By using suspensions of adipocytes, we have
I
Meisner, H. Unpublished data.
776 Journal of Lipid Research Volume 18, 1977 Notes on Methodology
been able to measure rates of FFA release that are
30-60 times greater than were obtained in tis
sue slices, and to do so in a shorter time period.
The mechanism by which acidosis depresses lipoly
sis has been discussed ( l l) and is thought to be
related to cyclic AMP and adenosine levels, although
as yet no causal relationship has been established.
observation reported here that FFA release is immedi
ately affected by pH, presumably before changes in
these cellular metabolites can occur. Certainly, large
pH changes in the circulation do not occur in vivo,
under physiological conditions, although it remains
possible that significant H+ is released into the micro
circulation of the fat organ to affect lipolysis.DIII
This research was supported by grants from the Cleveland
Diabetes Foundation, N .E. Ohio Heart Association, and
N.I.H. HL 18887.
Manuscript received 27 January 1977 and accepted 21 April 1977.
oa
de
REFERENCES d
fro
I. Rudman, D., and P. W. Shank. I 966. Observations
on the production of hydrogen ions during mobilization
m
of fatty acids from adipose tissue. Endocrinology. 79:
1100-1108.
2. Sallee, V., and ]. Dietschy. 1973. Determinants of
w
w.j
lr.
intestinal mucosal uptake of short and medium chain or
fatty acids and alcohols.]. Lipid Res. 14: 475-484. g
3. Campbell,]., A. Martucci, and G. Green. 1964. Plasma by
albumin as an acceptor of free fatty acids. Biochem.]. 93: gu
183-189. es
4. Rodbell, M. 1964. Metabolism of isolated fat cells. I. t,
on
Effects of hormones on glucose metabolism and lipoly
sis. ]. Biol. Chem. 239: 375-380.
M
5. Gliemann,]. 1965. Insulin-like activity of dilute human
ar
serum assayed by an isolated adipose cell method.
ch
8,
Diabetes. 14: 643-649. 20
6. Ho, R. J. 1970. Radiochemical assay of long-chain fatty 17
acids using 63N i as tracer. Anal. Biochem. 36: l 05-113.
7. Wieland, 0. 1957. Eine Enzymatische Methode zur
Bestimmung von Glycerin. Biochem. Z. 329: 313-319.
8. Rod bell, M. 1965. Modulation of lipolysis in adipose
tissue by fatty acid concentration in fat cell. Ann. N.Y.
Acad. Sci. 131: 302-314.
9. Hjemdahl, B., and P. Fredholm. 1974. Comparison of
the lipolytic activity of circulating and locally released
noradrenaline during acidosis. Acta Physiol. Scand. 92:
l -11.
l 0. Poyart, C., and G. Nahas. 1968. Inhibition of activated
lipolysis by acidosis. Mol. Pharmacol. 4: 389-410.
11. Hjemdahl, P., and B. Fredholm. 1976. Cyclic AMP
dependent and independent inhibition of lipolysis by
adenosine and decreased pH. Acta Physiol. Scand. 96:
170-179.
12. Fain,]., and R. Shepherd. 1975. Free fatty acids as
feedback regulators ofadenylate cyclase and cyclic 3':5'
AMP accumulation in rat fat cells.). Biol. Chem. 250:
6586-6592.

a<11,
HASILDANPE!dBARASAN

4.1 lsolasi Bal'tfflAsam Laktat

Seb3nysl. 3 isobt b3lmri as:m lut3t berlmildiisolas:i cbri yogunplain (I,, It, dm IJ). Isobsi ookteri asam laktat benujum
1.Ultllk mag_etabuidm memastibn ~ bsktm yang tercbpcupad\ bockslop.~!ediumy.mg d:igw:wtm sast isobsiadllah df .\Ian
&gosa Sltt.rpe Q.iR.S). Pemilillm medium tffiebut dibl.i.lkan kareD:S medium didupsesuai umnk pe:n:u:ul:,ww>.b311eri asam lakut
)'3llg bms3.l dari bou.slc,p.Zahoor dtt. (2003: 47) me:ayatakrulbah\\'S ~iR.5 me:rup:;km medium y.mg paling un:mm digu:Dakan
triltuk mmtmlbuhk:'l!l ooktai asam bktat, metiputi pm..--:L- aaobaciUus,Slrqxo,(,(>(.,QJS,PJ:ia::. dm
L,,,,:q,,c,cwc.Atlas (2010, 1231) menyatakm bahwa MRS ,,,_..iu.g ~ IIU!rieo, meliputi btl>oD, tti1Joge>, ,-. <1"1mmeral
y.mg1ltellltllj,mg penumbuban b3kteri asa.m W..1at Beldasa!:ksmBectoo, Dic:kisan & Ccmpeny (2003:
l).~~~sodium31SE'l3fdm3IDOlllllmsittaty.mg
bemmgs:i sebagai ilw:Oitcl'tm..,abakteri sehinlaaobadllus.S. ehiD iru, sodium
aset3t ~ seb:,pi baf!OJ' ~ pH y.mg m>d,h<bpat ~
K.<idu' pH y:mg raxhb W'Sebu! tidak d..."'Pdf i~oleta!LSioleh m:ikroorganisme seb:in
Loctobadl:ie.
831.te.."l asai:n bkta:: ~ b3.i.1m yaug_pada Ulllllmll)'3lllell)'\lkai su.,.sma esera WltUk dapat mm.bub. Oleb..sebab itu,
pembuatan medium dengm pH y:mg seswi perlu clw.l1*..._ ~- de Man &g"' Shop, Apr (!>!RSA) )>ng diassmk:m s::,;mpai
pR S didup dap3r digtm3kao Wlftlk matunbu!ik:m gems Lactobad!lus~. !edium~. fRSAtatlp3 pe,ps,nn'> diduga d3pru di~
tmtuk
mmm:ibubkan gm:as Sr,qraccus. Benbsarum Hutkins & N:moeo (1993:
2354-2355), Laaoba<Jll.u.m: emil.iki pH m,ksinnnn unmk tumbub 5,S sed3llpan Sirf,p:(1,(,0(,QJSDl=lDiliki pH umsmsuu 7.,j tmruk
d:lpat rumbub. Saccaro dkt. (2011: 3903) melapotbnbah~~ti<bk ""'3pat pertumi,ctb3DI.actobocil/usprub medium ~iRS.<\ denpn

pH 6,.2 tettpi pe.mcubub3ll terlih.1.tpe.dl mediumdelgan pHS,2.

Pembwtao yogi.It..., Ob 'la Nov1a.. FMIPAUI, 2012

 E. Onsaard et al. / Food Research International 39 (2006) 7886
3. Results and discussion
3.1. Composition of coconut cream proteins
The proximate composition of the freeze-dried coco-
nut cream proteins is presented in Table 1. The protein
content was higher in CCP1 than in CCP2, but the fat,
ash and carbohydrate contents were higher in CCP2
than in CCP1. These dierences in composition could
partly account for dierences in the observed emulsify-
ing properties and stability of O/W emulsions produced
by these proteins. In addition, the composition and
structure of the proteins present in the dierent fractions
could be dierent, which would also aect their func-
tional properties. A detailed characterization of the
composition, aggregation and denaturation state of the
dierent protein fractions isolated from coconut is cur-
rently being carried out in our laboratory, which will
be reported in a later study.
3.2. Solubility characteristics of coconut cream proteins
The inuence of pH and salt on the solubility of
CCP1, CCP2 and WPI is shown in Table 2. The general
trends in the pH-solubility proles were similar for all
proteins, with the solubility having a U-shaped curve
in which the minimum was around pH 45, which is
close to the reported isoelectric points (pI) of these pro-
teins (Punsri, 1995). The relatively low protein solubility
81
near their pI values can be attributed to the fact that
they have a low net charge, so that there is little electro-
static repulsion between them. In addition, there may
even be an electrostatic attraction between positively
charged patches on one protein molecule and negatively
charged patches on another. Protein solubility increases
at pH values above and below the pI because of the
increase in electrostatic repulsion and ionic hydration
forces between the protein molecules (Damo
daran,
1997). The solubility of the WPC was relatively indepen-
dent of salt concentration at all pH values. The solubil-
ity of the coconut cream protein fractions decreased
with increasing NaCl at pH values far from the pI,
which can be attributed to screening of the electrostatic
repulsion between the protein molecules. On the other
hand, coconut cream protein solubility either stayed
the same or slightly increased with increasing NaCl at
pH values close to the pI, which can be attributed to
screening of the electrostatic attraction between protein
molecules. The precise form of the pH-solubility proles
depended strongly on protein type, with the solubility
decreasing in the following order: WPI > CCP2 >
CCP1. These dierences in solubility could have been
due to dierences in the type of proteins present, the
intrinsic solubility characteristics of these proteins, the
denaturation states of the proteins and the presence of
any impurities that could aect solubility (such as min-
erals or polar lipids).

3.3. Interfacial characteristics of coconut cream proteins

Table 1
a
In this section, we investigated the interfacial behav-
Proximate composition (%) of freeze-dried coconut cream protein 1
ior of CCP1, CCP2 and WPI at an air-water interface at
(CCP1) and coconut cream protein 2 (CCP2)
30 C and pH 7 (Fig. 1). At high protein concentrations
Constituents CCP1 CCP2
(0.11.0 wt%), the surface pressure of the coconut
Moisture 3.58 0.02 3.37 0.16 proteins was higher than that of WPI, suggesting that
Proteinb 88.94 0.23 75.27 0.97
they were more eective at minimizing unfavorable
Fat 1.90 0.07 2.45 0.24
Ash 3.95 0.04 4.24 0.10 interactions at the air-water interface. On the other
Carbohydrate
c
1.64 0.15 14.67 0.67 hand, at protein concentrations less than 0.01%, the p
a
Value are from triplicate determination.
value for WPI was higher than those for CCP1 and
b
6.25 was use as the nitrogen conversion factor. CCP2, indicating that it is more surface active. The
c
Estimated by dierence. dierences of surface activity between dierent proteins

Table 2
A
Inuence of pH and NaCl concentration on protein solubility of coconut cream protein 1 (CCP1), coconut cream protein 2 (CCP2) and whey
protein isolate (WPI)
pH WPI CCP1 CCP2
0 mM 100 mM 200 mM 0 mM 100 mM 200 mM 0 mM 100 mM 200 mM
a ab a bc ab cd a a a
3 84 4 87 2 87 3 16 3 17 3 12.9 0.3 58 5 42 3 36 1
4 81 3a 84 4bc 86 4a 7 2c 11.3 0.2b 8 1e 8 3c 10 3c 6 2c
a c a c ab d c c c
5 80 3 81 5 84 5 8 2 16 3 11.0 0.4 92 10 2 6 2
6 82 4a 87 3ab 88 3a 14 4bc 17 1ab 15 1bc 13 2c 18 4b 17.1 0.1b
7 84 3a 89 2ab 87 4a 20 6ab 19 2ab 17 3b 45 1b 37.9 0.7a 32 2a
8 85 3a 89 2a 87 4a 28 6a 20 3a 20 1a 59 5a 39 1a 36 4a
In a column, mean values followed by the same superscript are not signicantly dierent at P < 0.05.
A
The protein solution was determined using the Lowry method using whey protein isolate as a standard.
 3

Limbah screening impuritas

selokan

Filtrat limbah
selokan

Analisis Awal
(COD, pH)

Fermentor
Anaerob

Analisis hasil
( COD)

Hitung volume
biogas

Gambar 2. Skema percobaan

Bahan baku limbah selokan mula-mula disaring menggunakan screening (penyaring). Filtrat hasil saringan
kemudian dianalisis sesuai dengan parameter yang diinginkan dan impuritas hasil saringan dibuang. Setelah
dianalisis, bahan baku dimasukkan kedalam reaktor dan dikondisikan agar berada pada pH 7 (netral), serta
ditambahkan nutrisi sesuai kebutuhan. Proses pengolahan secara anaerob berjalan, dilakukan pengambilan sampel
pada periode tertentu untuk mengetahui kadar COD dan volume biogas.

3. Hasil dan Pembahasan

3.1. Pengaruh Waktu Tinggal terhadap Penurunan COD
Penelitian ini dilakukan dengan memasukkan 15 L limbah selokan dan ditambah 9 L lumpur aktif, dengan
penambahan nutrient berupa NPK dan urea sebagai makanan bagi bakteri agar tetap bisa hidup. Penelitian ini
berlangsung selama 30 hari, dan di analisis kadar COD setiap 6 hari sekali.
Proses tersebut memerlukan bakteri untuk menguraikan glukosa sampai terbentuk biogas. Terbentuknya
biogas diindikasikan dengan turunnya kadar COD. Semakin besar penurunan kadar COD, maka volume biogas yang
dihasilkan juga semakin banyak. Penurunan kadar COD pada penelitian ini ditampilkan dalam bentuk grafik (lihat
Gambar 3).
 seeds. The recovery of cotton oil increased by up to 5%
with
previous enzymatic action while sunflower oil recovery in
creased by 4.2% after using another enzyme originating
from mold. In the same study, the oil yield was greater
when the enzymatic treatment was carried out in the
presence of a solvent instead of an aqueous medium; this
was attributed to the greater solubilization in organic
solvents of plant tissues and proteins to which oil may
remain bound even after the normal extraction procedures.
Tana-Debrah and Ohta41.42 obtained an increase of around
20% in the solvent

Review

Use of enzymes in solvent-based and

pressing extraction

The possibility of partially releasing the oil from a full fat,

heat-treated soya meal by using a hydrolytic enzyme was
initially investigated by Sherba et al. 35 as reported by Adler
Nissen.36 The oil was more easily recovered by extraction
with petroleum ether. This approach was also followed by
10
Fullbrook, who investigated aqueous hydrolysis of oil
seeds followed by solvent extraction and also tried hydro
lysis in the presence of a solvent to simultaneously extract
the released oil. It was observed that yields could be con
siderably improved if hydrolysis of the finely ground flour
of soybean and rapeseed was carried out in the presence of
solvent. In the case of rapeseed, 50% more oil was thus
obtained. For soybeans, the increase in yield was even
higher, thereby resulting in a net extraction of about 90% of
the original extractable oil; an enzymatic mixture obtained
from Aspergillus niger at a concentration of 3% was used. 10
Olsen31 also described aqueous hydrolysis of dehulled rape
seed followed by extraction of the residual oil with petro
leum ether. The hydrolysis mixture included three cell wall
degrading enzymes: pectinase, cellulase, and hemicellulase.
This partial hydrolysis increased the permeability of the cell
wall, thus allowing more efficient percolation of the solvent
and a more efficient extraction of the oil. It was also found
that an enzyme mixture containing all three active groups
together gave the most extensive hydrolysis. Sosulski et
al. 11 evaluated the effect of different carbohydrases on the
extraction time and yield of canola oil. The enzyme reaction
was carried out on previously autoclaved and moisture
adjusted canola flakes, and followed by drying and hexane
extraction. The enzyme efficiency based on oil yield en
hancement was: mixed activity enzyme > (3-glucanase >
pectinase > hemicellulase > cellulase. Enzymatic treatment
before Soxhlet extraction for a given time gave 45% higher
yields. Furthermore, the time to extract the total extractable
oil comparatively decreased indicating increased rates of
extraction. The maximum extraction yield obtained for the
control in a Goldfish apparatus was approximately 5%
lower compared to the ones obtained with enzyme action. 1
1

Similarly, Dominguez et al. 37 reported an increase in soy

bean extractibility by 8-10% of the extractable oil and up to
4% in the case of sunflower oil38 after enzymatic treatment
with different commercial enzymes. The resulting higher
extraction rates enabled shorter o_Reration to obtain a given
9
percentage of the extractable oil.
Bhatnagar and Johari'" also verified that treatment with
enzymes originating from thermophilus molds increased the
amount of Soxhlet extractable oil in the case of some oil

oil extraction yield by pretreating kernels of shea tree with
a mixture of protease and carbohydrases.
In relation to the pressing process, Cheah et at:P ex
tracted 97.7% of the oil from the pectinase-treated
mesocarp of palm by using a hydraulic press compared
to 91. I% obtained from the untreated material. In the same
way, Bou vier and Entressangles44 used a cellulase
preparation to re duce by 3% and 18%, respectively, the
palm oil losses from press fibers and crude juice during
clarification compared with the traditional process without
use of enzymes.
Smith et al.45 enhanced the yield of expelled oil from
soybean by up to 2.78% of moisture-free oil with a previous
Aspergillusfumigatus mixed-activity crude enzymatic treat
ment which corresponded to an increase of about 11. 7% in
oil recovery; this was equivalent to 63.5% of the total ex
tractable oil.
Aqueous process
Process description and unit operations
The aqueous extraction process (AEP; i.e., extraction using
water alone as medium) was originally suggested as an al
ternative to the solvent oil extraction process in the 1950s.
It was thought to be safer and cheaper. Moreover, the si
multaneous recovery of oil and protein (concentrates or iso
lates) from oil-bearing materials was possible.46
AEP uses a very distinctive principle compared to the
usual solvent extraction processes which are based on the
capacity of the oil to dissolve and be extracted by the sol
406 Enzyme Microb. Technol., 1996, vol. 19, November 1
vent. In AEP, the oil does not have a high chemical affinity
for the extraction medium and, therefore, there is no chemi
cal potential for oil dissolution. Although solvent extraction
theory cannot explain AEP, it certainly helps to understand
the mechanisms involved in the process. It is well known
that the spontaneous dissolution of any solute in a solvent is
always accompanied by a negative charge in free energy
(AG)47 which is related by the Gibbs equation to enthalpy
(!J.H), absolute temperature tT), and entropy (AS) as fol
lows:
AG= AH-TAS (l)
During dissolution, thermal energy is consumed to separate
solute molecules and dissociate the solvent molecules, and
it is released when the dispersed solute molecules interact
with neighboring solvent molecules. The overall enthalpy
change will be more negative (exothermic) if the energy
released during solute-solute and solvent-solvent interac
tions is greater than the energy absorbed in solute-solvent
interactions; however, if the solute molecules are strongly
bound to each other as in water, the solute dissolves well
only if dissolution results in stronger solute-solvent interac
tions. This is not the case for triglyceride-water interactions
which are weak and cannot offset the large amount of en
ergy required to break hydrogen bonds in water. On the
other hand, solubility of oil in hexane is high because the
stronger solute-solvent interactions compensate the energy
losses.47
Extraction of oil by AEP is, consequently, based more on
the insolubility of oil in water than on the dissolution of
oil.47 In this case, the water soluble components of oil crops
 10

BAB 2

LANDASAN TEORI

2.1.Kelapa

2.1.1. Pengertian Kelapa

Menurut Sutardi, Santoso dan Anggia (2008: 3), tanaman kelapa

(Cocos nucifera L.) merupakan tanaman serbaguna yang memiliki nilai

ekonomi tinggi. Seluruh bagian tanaman mulai dari akar, batang, daun dan

buah dapat dimanfaatkan untuk pemenuhan kebutuhan manusia, sehingga

disebut sebagai pohon kehidupan (tree for life).

Menurut Soekardi (2012: 2), Kelapa (nyiur) atau Cocos nucifera,

adalah tumbuhan palma pantai yang pohonnya tinggi, tanaman yang berusia

cukup tua, yang banyak tersebar di seluruh daerah tropika, dan pada

permulaan tarikh Masehi sudah dikenal dan dimanfaatkan orang dalam

kehidupan sehari-hari

Dari teori diatas dapat diambil kesimpulan bahwa buah kelapa

merupakan sumber kehidupan yang dapat dipergunakan untuk keperluan tata

boga, industri dan produk obat - obatan. Tanaman kelapa merupakan

tumbuhan efisien yang sangat bermanfaat bagi memenuhi kebutuhan manusia

sehari hari, karena seluruh bagian pohon kelapa dapat dimanfaatkan untuk

keperluan manusia.
 ABSTRAK

Yakub, Nurhidayati Agustin Arifah, 2014, Pengaruh Pemberian Minyak Goreng

Deep Frying Terhadap Gambaran Histologi Ginjal Tikus Putih (Rattus
Novergicus) Strain Wistar, Tugas akhir, Fakultas Kedokteran Universitas
Muhammadiyah Malang, Pembimbing: (1) Diah Hermayanti (2)
Mochamad Aleq Sander.

Latar Belakang: Proses menggoreng minyak menggunakan metode deep frying

dapat menyebabkan terjadi kerusakan minyak goreng disebabkan oleh proses
oksidasi, degradasi, oksidasi dan dehidrasi. Proses ini dapat meningkatkan kadar
peroksida dan pembentukan radikal bebas serta menyebabkan terjadinya inflamasi
sistemik sehingga dapat merusak sel. Salah satu organ tubuh yang rentan adalah
tubulus ginjal.

Tujuan: Mengetahui pengaruh pemberian minyak goreng deep frying terhadap

gambaran histologi ginjal tikus putih strain wistar.

Metode: Eksperimental, The Post Test Only Control group Design. Sampel di
bagi menjdi 4 kelompok. 1 kelompok kontrol dan 3 kelompok perlakuan diberi
minyak goreng deep frying 0,25 ml/gr BB/hari dengan frekuensi 4x, 8x dan 16x.
Diberikan kepada tikus selama 28 hari. Dianalisis dengan Kruskal Wallis dan
Mann-Whitney.

Hasil Penelitian dan diskusi: pemberian minyak goreng deep frying 0,25
ml/100gr BB/hari dengan frekuensi penggorengan 4x, 8x dan 16x menunjukkan
hasil yang signifikan dapat menyebabkan terjadinya hipertrofi tubulus ginjal.
Jumlah tubulus yang hipertrofi meningkat seiring dengan peningkatan frekuensi
penggorengan akibat peningkatan radikal bebas dan terjadinya inflamasi sistemik.

Kesimpulan: pemberian minyak goreng deep frying dapat menyebabkan

perubahan histologi ginjal pada tikus putih strain wistar yaitu hipertrofi tubulus.

Kata kunci: Minyak deep frying, hipertrofi, tubulus

ix
 A.M. Marina et al. / Trends in Food Science & Technology 20 (2009) 481e487
483

combination of these treatments (Seow & Gwee, 1997).
These techniques were possible due to the fact the coconut
milk proteins were easily coagulated and precipitated at
pH 4 (Tangsuphoom & Coupland, 2008).
Enzymatic extraction technique
Extraction of oil can also be carried out by the use of en-
zymes in aqueous extraction process. This is due to the fact
that plant cell walls consists of complex carbohydrate mol-
ecules such as cellulose, hemicellulose, mannans, galacto-
manans, arabinogalactans, pectin substances and protein
(Christensen, 1991). Coconut meat contains about 10% of
carbohydrate, in which 50% of this is cellulose and 75%
of the cellulose is made up with a-cellulose (Rosenthal
et al., 1996).
Oil can be found inside plant cells, linked with proteins
and wide range of carbohydrate such as starch, cellulose,
hemicellulose, and pectins. Cell-wall degrading enzymes
can be used to extract oil by solubilizing the structural
cell wall components of the oil seed. Che Man, Suhar-
diyono, Asbi, Azudin, and Wei (1996) also successfully
extracted coconut oil with 1% enzyme mixture of cellulase,
a-amylase, polygalacturonase, and protease with an oil
yield of 74%. The polygalacturonase hydrolyses a-linkages
of polygalacturonic acid of the polymer randomly from the
ends. An a-amylase randomly hydrolyzed a-linkages to liq-
uefy starch and produced maltose, whereas bacterial prote-
ases were used to hydrolyze the plant protein. The study
showed that different enzymes were required to degrade
components of the structural cell wall including mannan,
galactomannan, arabinoxylogactan and cellulose.
Physicochemical properties of virgin coconut oil
Since its appearance in the market, VCO is well ac-
cepted by consumer as functional food oil and the demand
for this oil continues to increase. Due to that, the number of
commercial VCO was increasing in the market. Marina,
Che Man, Nazimah, and Amin (2009a) described the chem-
ical properties of commercial VCO available in Malaysia
and Indonesia. The results revealed that chemical properties
of VCO did not vary much from the RBD coconut oil. The
iodine, peroxide, saponification and free fatty acid values
obtained for commercial VCO samples were well within
the specification limit of Codex standard (2003) for refined
coconut oil. The fatty acid composition was dominated by
lauric acid with percentage ranging from 46 to 48%, which
was within the limit of VCO standard according to Malay-
sian Standard (2007) and Asian and Pacific Coconut Com-
munity (APCC, 2003) (Table 1). According to the study,
medium chain fatty acids ranged from 60 to 63%. The ma-
jor triacylglycerol (TAG) in VCO samples consisted of 22e
25% of LaLaLa, 14e16% of CCLa, 19e21% of CLaLa,
13e15% of LaLaM and 7e9% of LaMM with La, C and
M are lauric, capric and mysristic acids, respectively. Gen-
erally, VCO from Malaysia had relatively higher contents
of CpCpLa (Cp: caproic), CpCLa and LaOO while Indone-
sian VCO had more of LaMP.
Dia, Garcia, Mabesa, and Tecson-Mendoza (2005) com-
pared the physicochemical properties of VCO produced by
different methods. Three different processing methods were
applied, namely incubated dessicated coconut meat, incu-
bated coconut milk and freeze-thaw coconut milk. The re-
sults showed that there were some differences in the
physicochemical properties of the VCO samples produced
by different methods but the differences were not large
enough to significantly affect the overall quality of the
VCO. The levels of the physical and chemical qualities
were within the Codex standard for coconut oil and the
Philippine standards for VCO. In addition, the authors
also determined the a-Tocopherol in the studied VCO.
The result revealed that a-Tocopherol was actually found
in the coconut testa (the thin brown layer that clings to
the white coconut meat) and only trace amount in the
VCO samples. Since coconut testa was removed in the pro-
duction of VCO, it was logical that the VCO samples con-
tain only trace amount in a-Tocopherol.
The quality of VCO is very much determined by its
physicochemical properties. However, as established by
Dia et al. (2005) and Marina et al. (2009a), the physico-
chemical quality of VCO was comparable to RBD coconut
oil. An understanding of descriptive quality of VCO in
terms of sensory evaluation was highly needed to better

Table 1. Fatty acid composition of virgin coconut oil (VCO) and refined, bleached and deodorized (RBD) coconut oil from various sources.
a
Fatty acid Codex standard APCC standard Malaysian standard Marina et al. (2009a) Dia et al. (2005)
for RBD coconut oil for VCO for VCO
C6 nde0.70 0.40e0.60 0.80e0.95 0.52e0.69 nde0.60
C8 4.60e10.0 5.00e10.00 8.00e9.00 7.19e8.81 5.98e10.44
C10 5.0e8.0 4.50e8.00 5.00e7.00 5.65e6.59 5.37e6.60
C12 45.10e53.20 43.00e53.00 47.00e50.00 46.89e48.03 47.63e52.55
C14 16.80e21.00 16.00e21.00 17.00e18.50 16.23e18.90 16.79e20.08
C16 7.50e10.20 7.50e10.00 7.50e9.50 7.41e9.55 6.38e10.17
C18:0 2.00e4.00 2.00e4.00 2.50e3.50 2.81e3.57 7.45e10.73
C18:1 5.00e10.00 5.00e10.00 4.50e6.00 5.72e6.72
C18:2 1.00e2.50 1.00e2.50 0.70e1.50 0.90e1.60 nde0.12
C18:3 nde0.20 <0.5 nd nd nd
a
Asian and Pacific Coconut Community.
482 A.M. Marina et al. / Trends in Food Science & Technology 20 (2009) 481e487
Wet extraction
Wet processing or aqueous processing is the term used
for the extraction of coconut oil directly from coconut
milk. This method eliminates the use of solvent which re-
portedly may lower the investment cost and energy require-
ments. Furthermore, it eliminates the RBD process
(Villarino et al., 2007). Even though the concept appears
potentially attractive, however, the method yields compara-
tively low content of oil, which has discouraged its com-
mercial application (Rosenthal, Pyle, & Niranjan, 1996).
The wet processing can only be carried out by means of
coconut milk by breaking the emulsion. This is rather
difficult due to the high stability emulsion of the coconut
milk. Desta- bilization can be done through three
mechanisms. The first stage is creaming by the action of
gravitational force resulting in two phases, with the higher
specific gravity takes place at the top phase and the lower
specific gravity phase moves downward. The second stage
is flocculation or clustering in which the oil phase moves as
a group which does not involve the rupture of the
interfacial film that normally surrounds each globule and
therefore does not change the original glob- ule. The last
stage, coalescence is the most critical phase in
destabilization. During this stage, the interfacial area is rup-
tured; the globules joined together and reduced the
interfacial area (Onsaard, Vittayanont, Srigam, &
McClements, 2005).
The wet process appears more desirable due to the free
usage of chemical solvents, thus more environmental
friendly than the solvent extraction. The method is also
much simpler, which can be carried out at home by anyone
who are interested in producing their own natural oil.
Chilling, freezing and thawing techniques
Attempts have been made before to break the protein
stabilized oil-in-water emulsion including heating and cen-
trifugation, freezing and thawing, chilling and thawing the
coconut cream obtained after centrifugation (Seow &
Gwee, 1997). Emulsion was centrifuged before chilling
and thawing to allow better packing of the coconut oil glob-
ules. According to the studies, the temperature used were
10 C and 4 C for chilling and freezing process, respec-
tively while the thawing process was carried out in a water
bath at 40 C until the coconut cream reached room temper-
ature (25 C). In addition, this action also helps in remov-
ing undissolved solids after extraction. The removal of
solids present in high percentages in the dispersion of oil
seed was important for efficient recovery of oil by centrifu-
gation (Rosenthal et al., 1996). The centrifugation step was
followed to enable the packing of cream oil globule to crys-
tallize on lowering the temperature. Centrifugation process
was carried out from 2000 to 5000 G up to 6 min. During
thawing, the oil coalesced due to loss of spherical shape
and formed large droplets of varying sizes.
Robledano-Luzuriage and Krauss-Maffei were two pro-
cesses known to apply freeze and thaw operation in the
extraction of coconut oil (Marina, 2008). In the Roble-
dano-Luzuriage process, fresh coconut kernel was
comminuted and pressed to obtain approximately equal
amounts of emulsion and residue. The residue was pressed
again to obtain more emulsion and residue. The emulsion
was centrifuged to obtain cream, skim milk and some solids
or protein. The cream was subjected to enzymatic action
un- der closely controlled temperature and pH conditions.
After freeze-thaw operation, the cream was centrifuged
again to obtain the oil. The protein in the skim milk was
coagulated by heating, subsequently filtered and dried to
produce pro- tein concentrate.
The oil recovery reported in the Krauss-Maffei was 89%,
which was less than the conventional expeller process
(95%). In this technique, husked coconuts were autoclaved,
shelled, and the coconut meat (the white solid endosperm
inside the coconut fruit) first sent through cutter and
subsequently through a roller mill. Then it was pressed in a
hydraulic press and the emulsion was centrifuged to obtain
the cream and skim milk. The cream was heated to 92 C
and then filtered to obtain high quality oil. The skim milk is
heated to 98 C in a flow heater to coagulate protein,
which was separated by centrifuging and then drying. The
residue from the hydraulic press was dried and ground to
obtain edible coconut flour. The study shows that quite high
recovery of oil was obtained but the temperature employed
was slightly high which might destroy some of its minor
components such as phenolic compounds.
Fermentation technique
Che Man, Abdul Karim, and Teng, (1997) investigated
the use of pure culture, Lactobacillus plantarum 1041 IAM
to extract coconut oil, which was able to extract as much
as 95% of the oil. In the study, grated coconut meat and
water at 30, 50 and 70 C were mixed in ratio of 1:1, 1:2
and 1:3 and allowed to settle for 2 to 6 h. The most efficient
coconut cream separation was obtained at the 1:1 ratio of
grated co- conut meat to water at 70 C followed by 6 h
settling time. L. plantarum has the ability to ferment sugar
with the produc- tion of considerable amount of lactic acid.
Coconut milk with L. plantarum strain IAM 1041
inoculation indicated a rapid breaking of the emulsion and
the liberation of the oil compared to Lactobacillus
delbrueckii. This was because L. plantarum strain could
multiply faster in coconut milk at
40e50 C under microaerophilic conditions which in-
creased the fermentation process.
Coconut milk emulsion can also be separated by
adjusting pH of the coconut milk emulsion between pH 3
and 5.6 and inoculated with bacteria cultures (Chen &
Diosady, 2003). Che Man, Suhardiyono, Ali, and Azudin
(1992) used acetic acid treatments to destabilize coconut
cream in coconut oil extraction. Treatment of 25% of
acetic acid at level of 0.1,
0.2, 0.3 and 0.4% on coconut cream from 10 to 14 h of
reac- tion time at room temperature had improved the
quality of the oil extracted, with oil recovery up to 60%.
Other treat- ments to break the coconut milk emulsion
included the use of heat, refrigeration, action of enzymes,
acidification, the use of salt or brine, electrolyte action,
short waves and
ELSEVIER

Aqueous and enzymatic processes for

edible oil extraction
A. Rosenthal, D. L. Pyle, and K. Niranjan
Department of Food Science and Technology, University of Reading, Whiteknights,
Reading, United Kingdom

Industrial processes for the extraction of edible oilfrom oilseeds generally involve a solvent extraction step
which may or may not be preceded by pressing. Hexane is the preferred solvent; hexane-based processes have
been in commercial operation for a long time. For such processes, it is possible to achieve oil yields in excess
of 95% with a solvent recovery of over 95%. In the past, the main concern of this process has been the safety
implications surrounding the use of hexane. This prompted attempts to develop processes based on the use
of aqueous extraction media which were unsuccessful mainly due to low oil yields.
The scenario at present appears to be changing. Interest in aqueous extraction processes has been revived by
increasing environmental concern. An aqueous process is looked upon as an environmentally cleaner alternative
technology for oil extraction. Organic solvents such as hexane, in particular, can contribute to the industrial
emissions of volatile organic compounds (VOes). The production of voes in the conventional process is
particularly worrisome since these can react in the atmosphere with other pollutants to produce ozone and other
photochemical oxidants which can be hazardous to human health and can cause damage to crops. Besides this,
the voes are themselves ''greenhouse gases"; some are carcinogenic and have toxic properties. Other advan
tages of the aqueous process compared with solvent-based processes include: (I) simultaneous production of
edible oil and protein isolate or concentration in the same process, (2) lower protein damage during extraction,
and (3) improved process safety due to the lower risk of fire and explosion. It is also reported that aqueous
extraction processes may be more cost effective since the solvent recovery step is eliminated. The main
limitations of this process appear to be: (I) lower efficiency of oil extraction as evident in earlier studies, ( 2)
demulsification requirements to recover oil when emulsions are formed, and (3) treatment of the resulting
aqueous effluent.
With the objective of improving the yield of aqueous processes, enzymes have been used to facilitate oil
release. Selected enzymes have been tried on different types of oilseeds, resulting in extraction yields much
higher than the original aqueous process (in some cases of over 90%). These enzymes mainly hydrolyze the
structural polysaccharides whichform the cell wall of oilseeds or the proteins which form the cell and lipid body
membrane.
This article aims to review aqueous and enzyme-based processes and discuss related issues.

Keywords: Aqueous oil extraction; enzymatic treatment of oilseeds; oil recovery; protein recovery

Introduction
Aqueous enzymatic oil extraction is undoubtedly an emerg
ing technology in the fats and oil industry since it offers
many advantages compared to conventional extraction. For
instance, it eliminates solvent consumption which report
1
ments. Also, it enables simultaneous recovery of oil and
protein from most oilseeds and the process yields oil of
good quality complying with Codex specifications. The
need for further degumming operations is eliminated and
the process allows ready removal of some toxins or antinu
3

edly may also lower investment costs12 and energy require-
Address reprint requests to Dr. K. Niranjan, Food Science and Technology,
University of Reading, P.O. Box 226, Whiteknights. Reading, Berkshire
RG6 6AP, United Kingdom
Received 29 June 1995; revised 22 January 1996; accepted 31 January
1996
Enzyme and Microbial Technology 19:402-420, 1996
1996 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
tritional compounds from certain oilseeds. In this sense,
some of the needs triggering technology innovation in the
oil extraction sector such as cost savings, environmental
and
safety concerns, and nutrition issues seem to be achievable
by successful development of aqueous enzyme-based pro
cesses.
Over the last four decades, several studies have been
carried out on aqueous processing of oilseeds.t "
Although
0141-0229/96/$15.00
PJI 50141-0229(96)00053-1
Silage microbiology and its control through additives consequence, their fermentation is less desirable than that
of lactic acid bacteria.

silage . With very good management , m ycotoxin

concentrations in silage are generally similar to those
present on the crop at ensiling. The development of
mycotoxins in the silo is an indication of excessive
oxygen exposure.
Acetic acid bacteria
Acetic acid bacteria are aerobic bacteria that are
capable of growing at low pH. They grow on ethanol,
producing acetic acid. However, once ethanol has been
exhausted, they can grow on acetic acid, producing
carbon dioxide and water. This will raise pH and permit
other aerobic microorganisms to grow. Consequently,
acetic acid bacteria can be initiators of aerobic
deterioration. At present, acetic acid bacteria have only
been reported in corn silage, silage that usually has a low
buffering capacity so that lactic and acetic acid
concentrations are low and a high sugar content that
could permit high ethanol concentrations. They have not
been found in C3 grass silages or legume silages. Sugar
cane silages and perhaps other similar C4 grass silages
may be candidate silages to look for acetic acid bacteria,
i.e., crops with characteristics similar to corn.
Bacilli
There are two major groups of bacteria that can be
active aerobically and anae robically, bacilli and
enterobacteria. Some bacilli can ferment sugars and
organic acids in the silo. However, their activity under
anaerobic conditions is considered to be of relatively rare
importance. Their more significant activity is in
advancing the spoilage of silage when exposed to oxygen.
After yeasts or acetic acid bacteria have raised pH
(approximately 4.5 or above), and temperature has
increased modestly (40C), a second wave of heating
usually occurs with temperatures rising to
50C or higher. This often is the activity of bacilli (Muck
& Pitt, 1994), producing a silage that has a slimy feel.
Enterobacteria
In contrast to the bacilli, the primary effects of the
enterobacteria, facultative anaerobes, on silage comes
under anaerobic conditions. Various species of
enterobacteria can use nitrate as an electron acceptor in
place of oxygen, reducing nitrate to nitrite or nitrogen
oxide. These bacteria
are the principal source of silo gas (a mixture of various
NOx
gases) emanating from silos.
Enterobacteria are also the principal competitors of
the lactic acid bacteria for the sugars in the crop. Their
principal fermentation product is acetic acid, not lactic.
Other fermentation products in silage that are signs of
their presence are succinic acid and 2,3-butanediol. As a
 185
Finally some enterobacteria may produce endotoxins
in silage. Little is really known about production in the
silo. Anecdotally, high acetic acid silages with no butyric
acid have been associated with poor intake. We do know
that high acetic acid silage produced by Lactobacillus
buchneri treatment has not reduced intake by cattle
(Kleinschmit & Kung, 2006). This suggests that
enterobacteria are the cause of the poor intake due to
some other compound that they produce, not specifically
the acetic acid.
Clostridia
Clostridia are obligate anaerobes, and their effects on
silage quality usually occur long after the lactic acid
bacteria have stopped actively growing in the silo. The
most important silage species are divided into three
groups by the principal compounds they ferment:
proteolytic clostridia that ferment amino acids primarily,
the Clostridium butyricum group that ferment
carbohydrates, and C. tyrobutyricum that ferments
some sugars but more importantly ferments lactic acid.
The proteolytic clostridia produce various compounds in
catabolizing amino acids; the most significant are
ammonia, amines and carbon dioxide. The primary
products of the C. butyricum group and C.
tyrobutyricum are butyric acid, acetic acid, hydrogen and
carbon dioxide.
Clostridial activity is undesirable for several reasons.
First and foremost is the reduced intake by livestock of
silages with evidence of substantial clostridial activity
(i.e., butyric acid at > 5 g/kg DM). The cause of the
reduced intake is uncertain. It does not appear to be
butyric acid, a common acid in the rumen that ruminants
routinely taste as they chew their cud. Amines have
been associated with reduced intake in small ruminants
but not necessarily in cattle. Whatever the cause, the
practical realities are that farmers have to reduce the
proportion of clostridial silage in a ration as butyric
acid increases during storage in order to keep intakes
constant. Secondly, lactating dairy cows, particularly
those in transition, may be more susceptible to ketosis
from the added butyric acid from consuming clostridial
silage. Thirdly, the fermentation of lactic acid to
butyric acid is one of the most wasteful anaerobic
fermentations in silage, producing a 51% loss of DM
and 18% loss of gross energy (McDonald et al., 1991).
How ensiling preserves a crop
Most silages are made at a dry matter content between
200 to 500 g/kg. Within this DM range, many enzymes in
the plant are still active at ensiling. Also the wide variety
of bacteria, yeasts and molds described above can all
grow
within this range. So it is a substantial challenge to bring
R. Bras. Zootec., v.39, p.183-191, 2010 (supl. especial)
 Evaluation Of Nutritional Components Of Carica Papaya L.At Different Stages Of Ripening.

IV. Result And Discussion

Variation exits in the proximate, mineral and vitamin composition among the different stages of
ripening. Papaya moisture content ranged between 5.91-89.21% (Table 1).The highest moisture content was
observed in the pulp at a very ripe stage (89.21%) and lowest in seed at unripe stage (5.91%). Results of
nutritional analysis at the three stages of ripening in papaya showed that the moisture content was highest at
the very ripe stage; with the pulp at 89.21, the seed 6.45 and the peel 68.39 and lowest at the unripe stage;
with the pulp at 81.39; seed at 5.91 and the peel at 54.48. This agrees with Hunt et al.[13] who reported that
the moisture content of very ripe pawpaw to be higher than the hard ripe and unripe ones. There was an
increase in moisture content from one ripening stage to another (Table 1). In the study conducted by Nwofia et
al [14], they recorded high moisture content in the pulp. These results agree with Ahuja et al.[15] statement
that the moisture content in papaya fruits ranges from 85-92%. Also the QECD [16] asserted that unripe
papaya fruits ranges between
86.5 -89.3% and the ripe 92.16-92.60%.
Dry matter varies between 13.32-94.09% (Table 1). The highest percentage dry matter was observed in
the seed at the unripe stage (94.09%) and lowest in pulp at the hard ripe stage. It has been reported that
dry matter content of Carica papaya increases during fruit development from unripe to ripe stages [16].
Crude fibre content was highest in the unripe followed by hard ripe and lastly very ripe (Table 1).The
highest crude fibre content was found in the peel at the unripe stage (5.25%). The crude fibre content in pulp
obtained in this study is higher compared to the values obtained by Nwofia et al [14]. Papayas contain
soft, easily digestible flesh with a good amount of soluble dietary fibre that helps to have normal bowel
movements; thereby reducing constipation and aids nutrient absorption [17].
Crude protein variation is between 0.29-14.41% (Table 1). The highest amount of crude protein was
found in the seed at an unripe stage, while the lowest was found in the pulp at a very ripe stage (0.29%).
Very ripe papaya is not a very good source of protein and has its highest value at 0.29% for the pulp, 12.37%
for the seed and 6.89% for the peel, which is in line with Jari [18] report that very ripe papaya is not a
recognized high source of protein. There was reduction in protein content from one ripening stage to
another. OECD [16] reported that protein ranged between 3.74-8.26% and 5.48-10.8% in the ripe and unripe
pawpaw fruit respectively.
The percentage ash varies between 2.83 -7.35% (Table 1).The highest percentage ash was found
in seed at very unripe stage and lowest in the pulp at a very ripe stage. Fat ranged between 0.23- 5.24%. The
seed recorded the highest amount of fat at very ripe stage (5.24%) and peel the lowest at an unripe stage
(0.23%).
Data recorded for carbohydrate ranged between 9.65-32.18 %. The highest percentage was found in
the seed at the unripe stage, while the pulp in the very ripe stage recorded 9.65% (Table 1). There was a
reduction in carbohydrate content as the papaya fruit moves from one ripening stage to another (Table 1). Data
recorded for carbohydrate in the different ripening stages ranged between 18.47-32.18%, 14.63-29.03% and
9.95-27.50% for Unripe, Hardripe and Very Ripe respectively. These values are lower compared to range of
mean values (64.5
87.8%) for ripe obtained by USDA [19]; Saxholt et al [20] and Wills et al [21] as well as for unripe
carbohydrate values (81.1-87.5%) obtained by Ahuja, et al [15] and Puwastien et al
[23].
The mineral composition of pulp, seeds and peels of papaya at different stages of ripening is
as outlined in Table 2. Calcium content was recorded highest in the pulp at the unripe stage (58.78mg) Table 2,
while the lowest value was recorded in pulp at the very ripe stage. Calcium plays an important role
in photosynthesis and in teeth development [24].
Sodium ranged between 13.68-27.25mg and was highest in pulp (27.25mg) at very ripe stage, while
the lowest value of 13.6mg was observed in the seed at the unripe stage. Potassium (98.93mg) was highest in
the peel and lowest (18.15mg) in the seed at a very ripe stage. Data on riboflavin, thiamine and niacin content
in the pulp, seed and peel of pawpaw at the different stages of ripening were very low (Table 3).
Phosphorus and magnesium ranged from 3.10- 33.33mg and 6.40-28mg respectively. The highest
phosphorus
(33.33mg) and magnesium content (28.00) was found in the seed at the unripe stage, while the lowest
value
phosphorus (3.10mg) and magnesium (6.40mg) was recorded at the pulp at the very ripe stage. Papaya from
the data at the three stages are rich sources of mineral elements (calcium, magnesium, sodium and
potassium), which explains the reason why pawpaw added to its other benefits is a much sought after food
crop because potassium contributes to fight against bacteria and cleanses the digestive system, sodium takes
part in the metabolism of water, promotes digestion, assimilation, osmosis and alkalizes the blood; then
calcium helps to strengthen the bones and magnesium assist in the assimilation of phosphorus. This is
in line with the contribution of Claude and Paule [25].
The pulp had higher content of vitamin A than the seed and peel at the different stages of ripening. It
varies between 0.73 1.36mg in the unripe, 0.14 2.31mg in the Hard Ripe and 0.13-2.09mg in the very ripe.
(Table 3). The unripe value is below USDA [26] obtained value of 9.79mg for unripe pawpaw fruit. Low
vitamin C content was observed in the seed and peel, but the pulp had relatively higher vitamin C (0.11-
0.15mg) at all the stages of ripening. The result also showed that low content of hard ripe papaya and very
ripe papaya

www.iosrjournals.org 15 | Page
 International Journal of Nutrition and Food Sciences 2015; 4(1): 84-88 85

are true proteins. Out of this proportion, Ca. 80% has reducing sugar (10.5 %) is observed in pineapple pulp. The
proteolytic activity due to a protease known as Bromelin. total sugar content of Indian variety was higher in waste
Fresh pineapple contains minerals as Calcium, Chlorine, (9.75%) than in pulp (8.66%).The total sugar content of pearl
Potassium, Phosphorus and Sodium [11]. pineapple of Brazil had a total sugar content of 14.5%. TSS
varies from 10% to 14% brix depending upon the stage of
Table 1. Nutrients in 100 grams (g) pineapple [3].
maturity and season [14]. The reducing sugar content of
Nutrients Amount pineapple was lower in Indian Varieties (10-12.5) [13]. But
Energy 52 calories Ghana pineapples contain higher amount of reducing sugar
Dietary fibre 1.40g
(16.5%) [15].The TSS value of Indian pineapple was higher
Carbohydrate 13.7 g
protein 0.54 g in pulp (13.3%) than waste (10.2%). TSS Value of Malaysian
Iron 0.28 mg Josapine pineapple was 13.5% [16]. Ascorbic acid content
Magnesium 12 mg slightly decreased in ripening stage of pineapple fruits [17].
Calcium 16 mg The pineapple waste had high moisture content (91.35%) and
Potassium 150 mg
moderate titratable acidity [14]. Moisture content of
Phosphorus 11 mg
Zinc 0.10 mg pineapple range from 69 to 89.5%.But it decreased during
Vitamin A 130 I.U room temperature storage and ripening period [18].
Vitamin B 1 0.079 mg
Vitamin B 2 0.031 mg
Vitamin B 3 0.489 mg 3. Uses as Food
Vitamin B 6 0.110 mg
Vitamin C 24 mg Pineapple fruits exhibit high moisture, high sugars, soluble
solid content ascorbic acid and low crude fibre. Thus
Pineapple juice contains ascorbic acid and is a good source pineapple can be used as supplementary nutritional fruit for
of Vitamin C. Ascorbic acid or vitamin C fights bacterial and good personal health [14].The pineapple fruits are normally
viral infections which is an effective antioxidant and helps consumed fresh or as fresh pineapple juice. Field ripe fruits
the body absorb iron. Half a cup of pineapple juice provides are best for eating fresh, and it is only necessary to remove
50 percent of an adult's daily recommended amount of the crown, rind, eyes and core. Pineapple may be consumed
vitamin C [4]. Several essential minerals exist in pineapples, fresh, canned, juiced, and are found in a wide array of food
including manganese, a trace mineral instrumental to the stuffs - dessert, fruit salad, jam, yogurt, ice cream, candy,
formation of bone, as well as the creation and activation of and as a complement to meat dishes [8]. In Panama, very
certain enzymes. Pineapples also include copper, another small pineapples are cut from the plant with a few inches of
trace mineral. It assists in the absorption of iron and regulates stem to serve as a handle. The flesh of larger fruits is
blood pressure and heart rate [8]. cut up in various ways and eaten fresh, as dessert, in salads,
compotes and otherwise, or cooked in pies, cakes, puddings,
Table 2. Physical and chemical constituents of pineapple pulp and waste or as a garnish on ham, or made into sauces or preserves.
[14]
Malayans utilize the pineapple in curries and various meat
Parameters Pineapple pulp Pineapple waste dishes. In the Philippines, the fermented pulp is made into a
Moisture (%) 87.3 91.35 popular sweetmeat. The pineapple does not lend itself
Ash content (mg/100g) 1.8 0.04
well to freezing, as it tends to develop off flavours.
Total soluble solids (%) 13.3 10.2
Crude fibre (g/100g-fw) 0.41 0.60 Canned pineapple is consumed throughout the world. In
Total sugars (%) 8.66 9.75 Africa, young, tender shoots are eaten in salads. The terminal
Reducing sugars (%) 10.5 8.2 bud or "cabbage" and the inflorescences are eaten raw or
Non-reducing sugars (%) 7.4 8.8 cooked. Young shoots, called "hijos de pina" are sold on
Titratable acidity (%) 2.03 1.86
vegetable markets in Guatemala [3].
Ascorbic acid (mg/100g) 21.5 26.5

The Honey Queen Variety is superior in nutritional content 4. Medicinal Value

as well as sweetness than the Giant Kew variety of
pineapple. The Giant Kew variety contained 6% Total
soluble solids (TSS), 3.88% total sugar and 1.75% non
reducing sugar. On the contrary, the Honey Queen variety
contains 10% TSS,
4.84% total sugar and 1.59% non reducing sugar. Honey
Queen contains all the minerals in higher amount than the
Giant Kew variety but Giant Kew contains higher Vitamin C
than Honey Queen. The Honey Queen contains higher
amount of calcium than the Giant Kew [12]. Ascorbic acid
value of pineapple in Nigeria is 22.5 -33.5 mg/100g-fw [13].
The reducing sugar was higher in pineapple pulp when
compared with pineapple waste. The maximum amount of
Pineapple can be used as supplementary nutritional fruit
for good personal health. Pineapple fruits are an excellent
source of vitamins and minerals. One healthy ripe pineapple
fruit can supply about 16.2% of daily requirement for vitamin
C [14]. Vitamin C is the body's primary water soluble
antioxidant, against free radicals that attack and damage
normal cells. A powerful antioxidant, vitamin C supports the
formation of collagen in bones, blood vessels, cartilage and
muscle, as well as the absorption of iron. Vitamin C also
retards the development of urinary tract infections during
pregnancy and reduces the risk of certain cancers, including
174

Bulgarian Journal of Agricultural Science, 19 (No 1) 2013, 174-180

Agricultural Academy

The physical and chemical properTies of

kiwifruiT
harvesTed aT four
sTages
J. HosseinzadeH1, M. FeyzollaHzadeH2* and a. H. aFkari3
1
Saba University, Department of Agricultural Machinery, Faculty of Agriculture, Urmia,
Iran
2
Urmia University, Department of Agricultural Machinery, Faculty of Agriculture, Urmia,
Iran
3
Mohaghegh Ardabili University, Department of Agricultural Machinery, Faculty of Agriculture, Ardabil,
Iran

abstract
HosseinzadeH, J., M. FeyzollaHzadeH and a. H. aFkari, 2013. The physical and chemical
properties of kiwifruit harvested at four stages. Bulg. J. Agric. Sci., 19: 174-180

Comprehensive information on properties of agricultural crops facilitates the design of modern machinery and
processing equipment with modified quality specifications. Hence, mechanics of agricultural materials due to its special
importance has quite developed despite being young. in this research some physical properties of the kiwifruit (Hayward
and Bruno cultivars), soluble solid content and pH were investigated for two fruit cultivar during four harvest stages. The
objective of this study was to investigate the effect of harvest time and cultivar on some physical properties of kiwifruit
varieties, typically cultivated in iran. Physical properties of kiwifruit were determined at a moisture content of 80% to 86%
(wet basis). The considered param- eters were geometric mean diameter, arithmetic mean diameter sphericity, true density
and aspect ratio. results showed that geometric mean diameter and sphericity were significantly affected by fruit cultivar.
However, there was no significant effect on true density. soluble solid content and pH values increased in both cultivars
with increasing harvest time.

Key words: kiwifruit; harvest stage; physical properties,

variety
Abbreviations: M - moisture content (% d. b.); l - length of the seed (mm); W - width of the seed (mm); T -
thickness of the seed (mm); - arithmetic mean diameter (mm); - sphericity; m - mass of the fruit (g); V -
volume of fruit (mm3); - true density (g/cm3); - geometric mean diameter (mm)

introduction climates of Manchuria, Japan, and eastern siberia.

kiwifruit has high levels of vitamin C and citric acid.
Commercial kiwifruit cultivars are part of the Vitamin C content is at least twice that of the orange.
genus Actinidia, which has species that express diverse starch is high at harvest, but it
skin characteristics (Ferguson, 1990). Commercial
cultivars of kiwifruit are large-fruited selections of
Actinidia de- liciosa (a. Chevalier, C.F. liang et al.,
a. r. Ferguson). The genus Actinidia is solely of
asian origin; plants are found ranging from northeast
india through China to tropical Java and into the cold
is converted to soluble sugars during storage and
ripen- ing (Carlos et al., 1999). A. arguta has diploid,
tetraploid, hexaploid, and octoploid cytotypes, and
characteristical- ly has small smooth green-skinned
fruit that grow singly or in multiples. The fruit has
been reported to contain a high vitamin C content,
good flavour, and to be more cold resistant during
growth than commercial kiwifruit. Therefore, the
species is of commercial interest in sev- eral countries
(e.g., kabaluk et al., 1997). The estimated world
production is about 1313827 tonnes of kiwifruit from
an area of 82547 ha land (Fao, 2008). The estimat- ed
iran production is about 30000 tonnes of kiwifruit over
an area of 23000 ha (Fao, 2008). Physical proper-

E-mail: Hosseinzadehtjhz@gmail.com, mazynas@gmail.com, ahafkari@gmail.com

 KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED 313

results obtained with the latter varieties indicate that the effect of moisture
is not confined to cottonseeds of any one variety or those produced in a par
ticular locality. Only the work on the Delfos seeds, however, is reported
here in detail. These seeds were obtained from cotton harvested and ginned
in the fall of 1941 and had an original moisture content of 10.5 per cent.
when received in December of that year.
Lots of 3 pounds each of the cottonseed were conditioned to the desired
moisture content and stored in air-tight, 2-quart glass jars at 25 C. The
moisture content of the seed was changed under conditions which minimized
any other activity during the process, and did not permanently injure the
seed. An increase in moisture was obtained by spreading the seeds in a

1-
z MOISTURE CONTENT
Lu
u
a:: 10 (PERCENT)
Lu
e, e-----e 7.5
.. .. 10 5
v, ~ 130
0
i3
ci
~ 155
0---0 18 0
>
I
c>------() 20 5
I-
~
i.u
Lu
5
a::
II.

----4----
50 100 150 200
LENGTH OF STORAGE (DAYS)

Frn. 2. Effect of moisture content of cottonseed on rate of formation of free fatty

acids.

thin layer in a room maintained at 3 C. and 100 per cent. relative humidity
until the desired moisture content was reached. One three-pound lot of
seed was dried at room temperature in vacuum desiccators until its moisture
content dropped to 7.5 per cent.
Analyses of the stored seeds were made at regular intervals. The results
of the determination of the free fatty acids are shown in graphical form in
figure 2.
Each curve represents seed of a particular moisture content and the
points on the curve represent the content of free fatty acids of the seed at
various times during a storage period of 200 days. Mold formation was
visible after 100 days on the seeds of high moisture content (18 and 20.5
per cent.) and th experiments with these lots were, therefore, discontinued
at that point.