TUGAS 1

METODE ILMIAH

OLEH

NAMA : MARIA FEBRIANA MITE

NIM : 1904060098

DOSEN PA : Ir. Titik Sri Harini, MP

UNIVERSITAS NUSA CENDANA

FAKULTAS PERTANIAN

PROGRAM STUDI AGROTEKNOLOGI

KUPANG

2021
ARTIKEL PERTAMA

Judul penelitian Penghambatan Pertumbuhan Gulma Commelina diffusa

oleh Pemberian Ekstrak Segar Daun Mikania micrantha
Tujuan penelitian Penelitian ini bertujuan mengetahui potensi ekstrak daun
M. micrantha sebagai bioherbisida untuk menghambat
pertumbuhan gulma C. diffusa.
Metode penelitian Penelitian dilakukan dengan menggunakan Rancangan
Kelompok Lengkap Teracak (RKLT) dengan faktor
tunggal dan 4 ulangan. Perlakuan yang digunakan adalah
konsentrasi bioherbisida terdiri atas tujuh konsentrasi
yaitu: 0.00 (kontrol), 0.33, 0.67, 1.00, 1.33, 1.67, dan
2.00 g bobot basah mL-1. Sebagai pelarut digunakaan
aquades.
Pengamatan persentase kematian gulma dilakukan pada
5, 10, 15, dan 20 HSA. Nilai persentase kematian gulma
dihitung dengan rumus sebagai berikut:

Kematian gulma =
∑ gulma yang mati x 100 %
∑ gulma yang ditanam
Analisis data dilakukan dengan menggunakan aplikasi
SAS dan Microsoft excel. Data dianalisis dengan uji F
pada taraf 5%, apabila menunjukkan pengaruh nyata
dilanjutkan dengan duncan multi range test (DMRT). Uji
polinomial dilakukan untuk menentukan konsentrasi
optimal ekstrak daun M. micrantha.
Rumusan masalah penelitian C. diffusa diketahui resisten pada beberapa jenis
herbisida, seperti 2.4-dichlorophenoxy-acetic acid (Isaac
et al., 2007) dan dicamba (Mortensen et al., 2012).
M. micrantha dapat menekan pertumbuhan gulma lain,
seperti Cleome rutidosperma D.C dan Paspalum notatum
Flugge (Pebriani et al., 2013), Cyperus iria dan
Ageratum conyzoides (Sahid dan Yusoff, 2014),
 Melastoma affine D.Don (Hamidah et al., 2015) serta
Mimosa pudica L (Adin et al., 2017). Ekstrak daun M.
micrantha diketahui dapat menghambat perkecambahan
tanaman hanjeli (Coix lacryma-jobi) (Li dan Jin, 2010)
dan Arabidobsis thaliana (Xu et al., 2013).
Ringkasan hasil dan pembahasan Kondisi Umum
Gulma C. diffusa yang mengalami keracunan
menunjukkan gejala berupa perubahan warna daun
menjadi kuning hingga kecoklatan (Gambar 2). Setelah
itu, batang gulma C. diffusa juga menjadi layu dan
kering. Tingkat keracunan yang tinggi dapat
menyebabkan kematian pada C. diffusa.
Pertumbuhan C. diffusa
Ekstrak gulma M. micrantha mempengaruhi beberapa
peubah pengamatan hingga 2 MSA (Tabel 1). Hal ini
diduga karena gulma C. diffusa memiliki kemampuan
yang baik dalam melakukan pertumbuhan kembali
(regrowth).
Aplikasi ekstrak daun M. micrantha tidak mempengaruhi
panjang gulma secara nyata (Tabel 2). Gulma C. diffusa
yang diberi ekstrak daun M. micrantha dengan
konsentrasi 0.33 g mL-1 memiliki rata-rata panjang gulma
yang 15 % lebih pendek dibandingkan dengan kontrol,
sedangkan rata-rata panjang gulma yang diberi perlakuan
konsentrasi 2 g mL-1 yaitu 13 % lebih panjang
dibandingkan dengan kontrol pada 1 MSA. Menurut
Pebriani et al. (2013), ekstrak daun M. micrantha
mengandung senyawa alelokimia yang dapat
mengganggu aktivitas hormone sitokinin sehingga
pembelahan dan pemanjangan sel terhambat dan pada
gilirannya menghambat pertumbuhan panjang. Fariba et
al. (2007) menyatakan bahwa senyawa alelopati dapat
merangsang atau menghambat pertumbuhan tanaman
tergantung dengan konsentrasi yang diberikan.
Aplikasi ekstrak daun M. micrantha memengaruhi jumlah
daun gulma C. diffusa pada 1-2 MSA, tetapi tidak
mempengaruhi pada 3 MSA (Tabel 3). Hal ini
menunjukkan bahwa ekstrak daun M. micrantha efektif
untuk mengendalikan gulma C. diffusa hingga 2 MSA.
Jumlah cabang gulma C. diffusa tidak dipengaruhi oleh
pemberian ekstrak daun M. micrantha (Tabel 4).
Aplikasi ekstrak daun M. micrantha menyebabkan
keracunan pada gulma C. diffusa pada 1 MSA (Tabel 5).
Perbedaan konsentrasi ekstrak daun M. micrantha tidak
memengaruhi tingkat toksisitas pada 2 dan 3 MSA.
Ekstrak daun M. micrantha meningkatkan persentase
kematian gulma C. diffusa secara nyata pada 10 dan 15
HSA. Pemberian ekstrak daun M. micrantha dengan
konsentrasi terendah yaitu 0.33 g mL-1 telah
menyebabkan kematian gulma C. diffusa pada 10 dan 15
HSA.
Aplikasi ekstrak daun M. micrantha tidak mempengaruhi
bobot kering gulma C. diffusa secara nyata (Tabel 7).
Bobot kering C. diffusa pada kontrol yaitu 1.85 g,
sedangkan pemberian ekstrak daun M. micrantha dengan
konsentrasi terendah yaitu 0.33 g mL-1 menghasilkan
bobot kering C. diffusa sebesar 1.69 g pada 10 HSA.
Konsentrasi Optimal
Penentuan konsentrasi optimal didapat dengan cara
menurunkan persamaan regresi kurva respon peubah skor
toksisitas gulma C. diffusa. Berdasarkan persamaan
regresi y = -0.8646x2 + 1.9811x + 0.3826, konsentrasi
 optimal ekstrak daun M. micrantha yang dapat
menyebabkan toksisitas pada gulma C. diffusa sebesar
1.15 g mL-1.
Kesimpulan Gulma M. micrantha memiliki potensi sebagai
bioherbisida yang ditandai oleh penghambatan
pertumbuhan daun gulma C. diffusa sampai 2 MSA dan
bersifat toksik bagi pertumbuhan gulma C. diffusa pada 1
MSA. Aplikasi ekstrak daun M. micrantha menyebabkan
kematian gulma C. diffusa mulai 10 HSA sampai 15
HSA. Konsentrasi optimal ekstrak daun M. micrantha
yang menyebabkan toksisitas pada gulma C. diffusa
adalah 1.15 g mL-1.

ARTIKEL KEDUA

Judul penelitian Novel angiotensin I-converting enzyme inhibitory

peptides from soybean protein isolates fermented by
Pediococcus pentosaceus SDL1409
Tujuan penelitian The objective of this study was to investigate the ACE
inhibitory ability of soybean protein isolates fermented
with Pediococcus pentosaceus SDL1409.
Metode penelitian 2.1. Chemicals and cultures
Soybean protein isolates (ISP YX 2000) were
obtainedfrom Shandong Yuxin Soybean Protein
Company LTD, China. Pediococcus pentosaceus
SDL1409 was obtained from the Department of Food
Science and Biotechnology. The bacteria stock culture
was maintained at -80 °C in de Man, Rogosa and Sharpe
(MRS) broth (Difco), containing 20% glycerol (v/v). The
culture was streaked on MRS agar and cultured at 37 °C
for 24 h. A single colony was then transferred into MRS
broth at 37 °C and harvested at the exponential phase of
growth. The viable bacterial count was determined by
plate count on MRS agar. The basal growth medium for
the lactic acid bacteria consisted of 2% (w/v) soybean
protein isolates (SPI) in distilled water. The SPI growth
media was autoclaved at 121 °C for 15 min prior to
cultivation with lactic acid bacteria.
2.2. Fermentation of SPI
Pediococcus pentosaceus SDL1409 (2 × 108 cfu/mL) in
the starter culture was inoculated into a 500 mL
Erlenmeyer flask containing 200 mL of SPI media (pH
6). Cultivation was carried out at 37 °C with 150 rpm of
agitation. After growth for 48 h, the media was
centrifuged (10,000 × g; 10 min) to remove precipitates
(cell mass, heat-denatured proteins, etc). The supernatant
was freeze-dried using TFD5505 table top freeze dryer
(ilshinBioBase Co. Ltd, South Korea) and the freezedried
samples were stored at -80 °C for further analysis.

2.3. Recovery of low molecular weight peptides (≤7000

Da)
The freeze-dried SPI supernatant (700 mg) was dissolved
in 2 mL distilled water and dialyzed (7000 Da cut-off;
Viskase Corporation, Chicago, Illinois) against water for
48 h to recover low molecular weight peptides ≤ 7000
daltons.

2.4. In-vitro assay for ACE inhibitory activity

For each assay, 20 μL of ACE inhibitor solution with 50
μl of 5mM HHL in 100 mM sodium borate buffer (pH
8.3) containing 0.3 M NaCl was incubated at 37 °C for 5
min. To initiate the reaction, 10 μL of 0.1 U/mL ACE
solution was added and the mixture was incubated at 37
°C for 30 min. The reaction was terminated by adding
100 ìL of 1M HCl and the reaction mixture was mixed
with 1.0 mL ethyl acetate. The mixture was vortexed for
60 s and centrifuged at 2000 × g for 5 min. An aliquot
(0.8 mL) of the ethyl acetate layer was transferred to a
clean tube and evaporated on a water bath. Distilled
water (0.8 mL) was then added to dissolve the hippuric
acid (HA) in the tube. The amount of HA formed was
measured at 228 nm using biospectrometer (Eppendorf
Biospectrometer fluorescence, Eppendorf Korea Ltd.
Korea). The amount of HA liberated from Hip-His-Leu
underthis reaction conditions without an inhibitor was
used as a control. Theextent of inhibition was calculated
as 100% × [(B− ∕ A) B] where A is the optical density
(OD) in the presence of ACE and ACE inhibitory
component, B is the OD without ACE inhibitory
component. For the determination of IC50, series of
dilutions containing 5000 μg/mL, 500 μg/mL, 50 μg/mL,
5 μg/mL, 0.5 μg/mL, and 0.05 μg/mL were prepared. The
amount of peptides required to suppress 50% ACE
activity was calculated from the regression curves
observed for each fractionnalysis.

2.5. Identification of peptides by mass spectrometry

1.6 g of the LMW peptides was dissolved in 50 mL of
double distilled water. Fractions (2 μL) of the samples
were injected into the LC-nano ESI-MS/MS system. The
samples were trapped on a ZORBAX 300SB-C18 trap
column (300-μm i. d × 5 mm, 5-μm particle size, 100
pore size, Agilent Technologies, part number 5065-9913)
and washed for 6 min with gradient with 98% solvent A
[water/acetonitrile (98:2, v/v), 0.1% formic acid] and 2%
solvent B [Water/acetonitrile (2:98, v/v), 0.1% formic
acid] at a flow rate of 5 μL/min. The peptides were
separated on a ZORBAX 300SB-C18 capillary column
(75-μm i. d × 150 mm, 3.5 μm particle size, 100 pore
size, part number 5065-9911) at a flow rate of 300
nL/min with gradient at 2% to 35% solvent B over 30
min, then from 35% to 90% over 10 min, followed by
90% solvent B for 5 min, and finally 5% solvent B for 15
min. Electrospray was performed at an ion spray voltage
of 2000 eV through a coated silica tip (FS360-20-10-
N20-C12, PicoTip emitter, New Objective). The peptides
were analyzed automatically using Analyst QS 2.0
software (Applied Biosystems, USA). The range of m/z
values was 200–2000.

2.6. In vitro simulated gastrointestinal digestion and

analysis of digests by LC-ESI-TOF-MS/MS
A two-stage simulated gastrointestinal digestion was
carried out similar to an earlier report (You & Wu, 2011)
with modifications. Pepsin (0.2 mg) was added to 10 mL
of 1 mM peptide solutions and adjusted to pH 2.0 using 1
M HCl. The sample was incubated at 37 °C. After 120
min, the pH was adjusted to 7.5 by adding 1 M NaOH.
0.2 mg of Pancreatin was added and the sample was
further incubated at 37 °C for 180 min. The reaction was
stopped by heating at 80 °C for 10 min in a water bath,
followed by cooling at room temperature. Each sample
was stored at −20 °C till further analysis by LC-ESI-

Rumusan masalah penelitian
Ringkasan hasil dan pembahasan
TOF-MS/MS as already described above (2.3).
2.7. Statistical analysis
All experiments were carried out in triplicates and the
results were expressed as the mean ± standard deviation.
The statistical analysis of data was performed using
GraphPad Prism 5.0 (2007) statistical software system
(GraphPad Software Inc. CA 92037 USA). P ≤ 0.05 was
considered significant.
In this study, Pediococcus pentosaceus SDL1409 was
used to ferment soybean protein isolates for 48 h at 37
°C. Low molecular weight peptides (≤7 KDa) from the
fermented sample showed strong angiotensin I-
converting enzyme inhibitory ability of 65.1 ± 0.78%
(IC50 = 0.123 ± 0.02 mg protein/ml).
3.1. In vitro ACE inhibitory activity of low molecular
weight (LMW)
peptides (≤7kDa) During fermentation, lactic acid
bacteria hydrolyze proteins to release peptides and amino
acids required for their growth. In the process, some of
the peptides generated may be bioactive. In this study, the
LMW peptides recovered from the fermentate showed a
strong ACE inhibitory activity of 65.1 ± 0.78% with an
IC50 of 0.123 ± 0.02 mg/ mL while captopril (a positive
control) showed an inhibitory activity of 90.82 ± 5.3%
(IC50 = 0.005 mg/mL).
3.2. ACE inhibitory peptides from fermented SPI
Out of 88 peptides identified in the peptide profile (Table
1), EDEVSFSP was most abundant and since it fulfilled
some of the common structural features described for
many ACEI peptides derived from food proteins
(Wijesekara & Kim, 2010), it was chemically
synthesized. Three other peptides in the profile
(SRPFNL, RSPFNL and ENPFNL) which only differed
in their last two N-terminal amino acids were also
chemically synthesized despite not being abundant. With
the aim of establishing sequence-inhibitory potency
relationships, the peptides EVSFSP, SFSP, PFNL and
FNL were also synthesized. All the synthesized peptides
showed ACE inhibitory activities at 5 mg/mL. Further
concentration response curves enabled the determination
of IC50 values (Table 2). FNL was found to have the
higher potency as it had the lowest IC50 value.
The novel sequences identified in this study could at least
in part contribute to the observed ACE inhibitory effects
of the fermented SPI. To the best of our knowledge, this
is the first time the peptides EDEVSFSP, SRPFNL,
RSPFNL and ENPFNL produced by soybean protein
fermentation have been reported as ACE inhibitory
peptides. Also, this is the first time the peptides EVSFSP,
SFSP, PFNL and FNL are being reported as ACE
inhibitory peptides.

3.3. Resistance of SPI- derived peptides to

gastrointestinal enzymes
For bioactive peptides to function after ingestion, they
must resist gastrointestinal digestion (Daliri, Lee, & Oh,
2017b; Daliri, Oh, & Lee, 2017) or yield other active
fragments after digestion. We therefore tested the
resistance of the pure peptides (EDEVSFSP, SRPFNL,
RSPFNL and ENPFNL) to gastrointestinal enzymes by
subjecting them to pepsin digestion at pH 2 followed by
 pancreatin at pH 7.5. The hydrolysates were then
analyzed by LC-ESI-TOF-MS/MS to identify the peptide
fragments generated (Table 3). Yet, after the peptides
were subjected to pepsin and pancreatin digestion,
SRPFNL, RSPFNL and ENPFNL were hydrolyzed to
yield SRPFN, RSPFN and ENPFN respectively. The
hydrolyzed fragments showed no ACE inhibitory activity
except ENPFN whose inhibitory activity was
significantly reduced (p < 0.05). These results show that
the presence of Leucine at the C-terminal of peptides
SRPFNL, RSPFNL and ENPFNL was important for ACE
inhibition.
Kesimpulan We have shown that the peptides EDEVSFSP, SRPFNL,
RSPFNL and ENPFNL generated during soybean protein
fermentation using P. pentosaceus SDL1409 for 48 h at
37 °C have potent ACE inhibitory activities. Although
the isolated ACE-inhibitory peptides in our study
havelower activities than synthetic drugs such as
captopril(IC50 = 0.005 mg/mL), our results show that P.
pentosaceus SDL 1409may be useful for developing
antihypertensive functional foods from soybean proteins.
Whether only the 4 sequences identified in the peptide
profile are responsible for the observed ACE inhibitory
effects require further characterization studies.
Meanwhile, further studies on the in-vivo
antihypertensive ability of these SPI-derived peptides are
warranted.