A Research Plan Presentation :

“Identification Peptides and Compounds Contributing to the Umami Taste

on Fermented Product (Terasi) Produced from Varies Regions in Indonesia”

Presented by
Achmad Wildan M Putra
F251190661

At The Scientific Presentation Vol 2

Bogor Science Club
On January 16th 2021

Food Science Study Program

Graduate School
IPB University
2021
An Overview “A Role of Basic Taste
An Overview “Umami”

Umami is a Japanese term for the word “delicious” which is

associated with monosodium glutamate (MSG). It usually
occurs in the beef extract, soy sauce, protein hydro lysate,
and ripened cheese (Hofmann et al. 2004)

Many authors reported that umami compounds are not

limited to free acids, such as glutamic acid and aspartic acid,
but small peptides, 5’nucleotides, and organic acids
 Fermentation of Terasi

Garam 12-20% Fish/shrimp Fermentation 2-4 weeks

Components Terasi (100 % raw Dry)

Koesoemawardhani et al. 2013
Water content 0.23
Ash content 50.07
Fat content 0.30

Protein content 24.00

Carbohydrate 0.20
 Umami Sources in Fishery product

Abalone Shells Sea Urchin Skin’s Crab Crab Squid

Glutamat, glisin, AMP Glutamat, glisin, Alanin, glutamat, AMP, Arginin, AMP, IMP AMP (Kani et al.
(Konsu 1973) alanin, IMP glisin (Hayashi et al. 1981) 2007)
(Chiou dan Lai (Chiou dan Huang 2003)
2002)
 Sources of Umami

Free Amino Acid Peptides

Glutamat, glisin, arginin, alanin a. Asp-Glu, Tyr-Pro, Val-Pro
(Sarower et al. 2012). (Kecap ikan). (Park et al. 2002).
b. Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val
(Zhang et al. 2012)

Nucleotides Mixture of Salt and free amino

Inosine 5’-Monophosphat (IMP) acid (Lioe et al. 2007)
Guuanosine 5’-Monophosphate (GMP)
Adenosine 5’-Monophosphate.
(Sarower et al. 2012)

Zhang et al. (2012): Buntal flesh (836 Da)

Xu et al. (2019): Volvariela volvacea (735, 759, 791 Da)
 Characterization of Peptides contributing Umami Taste

Membrane RP-HPLC LC-ESI-MS

Chromatography
Ultrafiltration
(Sephadex G-15 dan G-25)
3kDa

Lioe et al. (2007) Xu et al. (2019)

Istiqomah et al. (2018) Tamam et al. (2019)
 Research Problems
1. Mapping of Terasi from Various Region in Indonesia

2. Characterization Compounds of Umami Taste

Ultrafiltration Chromatography RP-HPLC LC-ESI-MS

 Objectives and Benefits

Objectives Benefits

Characterizing Water Soluble Extract Giving such an information towards

(WSE) of Terasi that is potent in giving compound contributing umami on Terasi
strongest umami taste for future investigations.

Fractionating WSE of Terasi using Exposing the potential of Terasi as local

Chromatography Ultrafiltration commodities to be utilized as sources of
umami compounds.
Isolating sub-fractions of umami
using RP-HPLC preparative

Characterizing Umami Fraction

Compunds using LC-ESI-MS
 Hypothesis

One product of Terasi obtained from 5 regions

will have the strongest umami using sensory
Analysis
A
Fraction <3kDa obtained from
Ultrafiltration will contribute the
strongest umami rather than B Fractions of Chromatography Ultra-filtration
>3kDa.
C will give fractions with different intensity

D
Identification of peptides LC-ESI-MS will show
peptides contributing umami taste
 Time and Place

Study will be conducted on March 2021 to June 2021

1. Lab of Sensory Analysis, UGM

2. Lab of Food Processing. UGM
3. Laboratory of Flavour Analysis
Balai Besar (BB) Padi, Subang, West Java
 Materials

Identification of peptide
Natrium Analysis: (LC ESI-MS): Sensory Analysis
AgNO3, NaCl, K2Cr2O7 BEH C18, H2O, Terasi, free water ion
Format acid, acetonitrile

Purification of Compound Analysis of Total Amino Acid:

Sephadex Fractionation (HPLC): HCl, Na-asetat, Na- EDTA,
Gel Sephadex G-15 C18, acetonitrile, free ion methanol, THF, methanol,
water free ion water

Analysis of Sugar: Analysis of Total Nitrogen:

Aquades, CaCO3, Pb Analysis of pH: CuSO4, Na2SO4, H2SO4, NaOH,
acetate, Na-oksalate, Aquades, standard buffer H3BO3, Red Methyl, blue
Anthrone methylene ,HCl
 Tools

Ultrafiltration Fractination Isolation Total amino acids Characterization Sensori

a. Membrane a. Kolom Sephadex a. HPLC analysis a. Cup
ultrafiltration MWCO G15 b. micropipettes
LC-ESI-MS
3kDa b. spectrophotometer a. HPLC
b. Vacuum pomp UV-Vis b. Freeze dryer
c. freeze dryer
 Step of study

Fraksinasi
3 Chromatography
Filtration Gel Sephadex Isolating of sub-fractions
umami using RP-HPLC
G15
4 Preparative

Fractinating WSE using

2 ultrafiltration membrane MWCO
3kDa

Collecting 5 samples of Terasi and

making their WSE
1
 Blending 250 gram of Terasi
using blender
Adding free water ion (3000 mL)
1 (1:12)

Homogenizing

Boiling 100°C 10 minutes

Chilling 50 minutes

Filtrating supernatant using cheese-cloth

Centrifuging 3200 in 30 minutes

Total of: Filtrating using cellulose membrane

pH 0.45 µm.
Sugar TDA
Natrium freeze-dried
Nitrogen

Chemical Analysis Sensory Analysis Istiqamah et al. 2018

 Fractionations WSE using Ultra filtrations
Membrane MWCO 3kDa

2 Ultrafiltration Membrane
Chemical analysis

MWCO 3 kDa
Fractions
(< 3kDa dan > 3kDa) Chosen Ultra-filtrated Fraction

WSE of Chosen Terasi

+

Chemical Analysis
pH
Total Acids
Sensory Analysis
Total Sugar
Total Natrium Sensory Analysis:
Total Nitrogen Test Dilution Analysis (TDA) Istiqamah et al. 2018
 Fractionation using chromatography ultrafiltration
Gel (Sephadex G-15)

3 Chemical Analysis

Selected Fraction Fractionation using Analysis of Fraction

Fraction Umami
of Ultrafiltration Sephadex G-15 (214 nm dan 254 nm)

Analisis kimia:
Asam Amino
Total garam Analisis sensori:
Total gula Uji Taste Dilution Sensory Analysis
Nukleotida Analysis (TDA)
Asam organik
Istiqamah et al. 2018
Asam glutamat
 Isolasi sub-fraksi umami
dengan RP-HPLC Preparatif

4
Injecting to RP-HPLC Collecting Sub-Fraction Peptide Characterization of
Preparative of RP-HPLC Umami sub-fractions using LC-
Sub-Fractions of umami ESI-MS

Fraction of Umami

Sensory Analysis: Sensory Analysis

Taste Dilution
Analysis (TDA)
Tamam et al. 2019
 Analisis Sensori (Taste Dilution Analysis)

a. Sensory Characterization: To know the taste intensity level of sample using trained
panelist
b. Criteria:
• Anyone who does not have any limitation in consuming foods
• Passing selection of panelist test ISO 8586 (2012).

1 Panelist Screening

2 Panelist Training

3 Sample Analysis
Frank et al. 2003
 Panellist Selection

a. Matching test

50 of Panelist
Candidates
Form pre-screening + 14 Panelists

b. Detection of 5 basic tastes

Matching test Detection test

Flavor Stimulus Concentration g/L Flavor Stimulus Concentration g/L

Sweet Sucrose 10 Sweet Sucrose 6.0

Sour Citrate Acid 0.3 Sour Citrate Acid 0.2

Bitter Caffeine 0.3 Bitter Caffeine 0.2

ASTM (2011) Salty NaCl 2.0 Salty NaCl 1.3

ISO 8586 (2012)
Umami MSG 0.6 Umami MSG 0.3
Meilgaard et al. (2007)
 Panellist Selection

Factor of Taste Dillution Compound to be tested

256 WSE of Terasi

256 WSE of Terasi
128 WSE of Terasi
128 WSE of Terasi
64 WSE of Terasi
64 WSE of Terasi Chosen
32 WSE of Terasi Panellist Training
32 WSE of Terasi FD
16 WSE of Terasi
16 WSE of Terasi
8 WSE of Terasi
8 WSE of Terasi
4 WSE of Terasi
4 WSE of Terasi
2 (1:1) WSE of Terasi
2 (1:1) WSE of Terasi

Frank et al. 2003

 Analysis of Statistic

One Way Analysis of Varian is used to know differences of Terasi and WSE of 5 Samples of
result of Chromatography and RP-HPLC.

Once it is found differences then cont’d to Duncan Test (p<0.05) to know which one different
treatment. T-test is used to know the differences among ultrafiltration fractions. (SPSS version
22)

PCA (Principal Component Analysis) using software XL Stat 2014 Pearson edition is used to
know mapping of average point of Terasi and its WSE
Thanks for Listening

Umami
 Produksi Umami

Saus Udang o   𝑁𝐻 3

 𝐶 − 𝐶𝑂𝑂 −  𝐻 − 𝐶 −𝐶𝑂𝑂 −

 𝑁𝐻 3 +  𝐻 − 𝐶 − 𝐻  𝐻 − 𝐶 − 𝐻

 𝐻 − 𝐶 − 𝐻  𝐻 − 𝐶 − 𝐻
Glutamat
dehidrogenase
  𝐶𝑂𝑂 −   𝐶𝑂𝑂 −

Amonia α-ketoglutarat glutamat

Hajeb dan Jinab (2012)

Form Seleksi panelis
Form pelatihan panelis
Kondisi LC-ESI-MS
Kondisi HPLC
 Analisis Nukleotida

a. Persiapan larutan standar

Larutan standar ATP (605), ADP (507), AMP (409), IMP (424), dibuat menjadi konsentrasi
10 mm dan larutan standar Hipoxantin (288), Xanatin (136) dibuat menjadi konsentrasi 20
mm. dari larutan tersebut masing-masing diambil 1 ml dan dicampurkan kedalam larutan
buffer fosfat dengan pH 7 sampai volume 25 ml. Kemudian di injeksi ke HPLC.

b. Persiapan sampel
1 gr sampel ditambah 10 ml asam perklorat 6% kemudian diekstraksi sehingga menjadi
campuran yang homogen membentuk endapan dan cairan sampel. Cairan sampel diambil
dan di netralisir dengan KOH 10% hingga pH nya menjadi 7-7.5 dan membentuk endapan
berwarna putih. Larutan sampel dipindahkan dalam gelas ukur dan ditambahkan aquades
sehingga menjadi 20 ml, lalu disaring dengan kertas whatman no. 40. Sampel sebanyak 20
microliter di injeksikan ke HPLC