Endrika WIDYASTUTI

Food Science and

Technology Department
2012
FOOD ANALYSIS AND
BIOCHEMISTRY PRACTICE
INTRODUCTION OF PROTEIN
TYPES PROTEIN ANALYSIS
NINHIDRIN TEST
KJEDAHL METHOD
FORMOL TITRATION
THE OUTLINE
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Proteins are made up of amino acids

Food analysts are interested in knowing
the total concentration, type, molecular
structure and functional properties of
proteins in foods

Amino acids are the building blocks of
protein
PROTEIN
Total organic nitrogen = protein + non-protein nitrogen
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PROTEIN
PROTEIN ANALYSIS IN FOODS HAS BEEN MOSTLY DONE
BY DETERMINING NITROGEN CONTENT
Nitrogen is: largely unique to protein only MAJOR
constituent of foods containing N.
Other nitrogenous compounds (NPN): Chlorophyll,
nucleic acids, some vitamins, lecithins, urea, amino
sugars, alkaloids, ammonium ions, etc.
On the average, food proteins contain 16% nitrogen.
100% divided by 16% = 6.25. Therefore multiply N
content by 6.25 to get protein content.
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PROTEIN
Protein is analyzed for:
1. determination of biological activity
2. investigation of functional properties
3. nutritional labeling

Protein analysis is required for you to know:
1. total protein
2. amino acid composition
3. amount of a particular protein in a
mixture
4. protein content during isolation and
purification
5. Nonprotein nitrogen
6. Nutritional value of a protein

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TYPES OF PROTEIN
ANALYSIS
QUALITATIVE
METHOD
QUANTITATIVE
METHOD
Kjeldahl measures
the amount of
nitrogen in a sample
Lowry- measures the
tyrosine/tryptophan
residues of proteins
NINHIDRIN
ELECTROFORESIS
CHROMATOGRAPHY

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PROTEIN ANALYSIS
NINHIDRIN TEST
Primary amino groups on the end of
proteins, peptides, and free amino
acids will react with ninhydrin.
This reaction forms a strongly colored
purple solution referred to as
Ruheman's purple
Sample can be tested for the amount
of primary amino acids currently
present, or sample can be alkaline
hydrolyzed to increase the amount of
these amino acids.

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PROTEIN ANALYSIS
NINHIDRIN TEST
Ninhidrin mengalami deaminasi
oksidatif dan asam amino
dekarboksilasi menjadi CO2,
NH3 dan aldehid
Ninhidrin yang tereduksi akan
bereaksi dengan amonia dan
dengan molekul ninhidrin lain
sehingga terbentuk senyawa
kompleks berwarna ungu ( ungu
Ruhemann)
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PROTEIN ANALYSIS
NINHIDRIN TEST
ADVANTAGES
Faster and more convenient that Kjeldahl

DISADVANTAGES
Large dilutions are necessary for spec. reading
Proteins differ in the dye binding capacity
Make standard curve based on predominant
primary amino acid present in the food
NPN, calcium, or phosphorous constituents will
bind to the dye or to protein, causing interference
Addition of a metal chelator (i.e.. oxalic acid)
may help reduce binding

PROTEIN ANALYSIS
NINHIDRIN TEST
larutan susu skim (10%)
gelatin (5%)
putih telur
enzim
keju,
pemanis sintetik diasweet
MSG (5%)
akuades sebagai (kontrol)
BAHAN
- Pelabelan tabung reaksi
- Pengambilan sampel 2 ml + 2 ml larutan ninhidrin
- Pemasukan tabung reaksi pada air mendidih (15-
20 detik)

Pengamatan warna larutan :
(+ ) jika hasil test positif (berwarna ungu, berarti
sampel mengandung gugus amina bebas)
( ) jika hasil test negatif.

Jika terbentuk warna lain seperti (kuning, orange
dan merah) maka uji negatif
prolin, hydroxyproline, dan 2-, 3-, and 4-asam aminobenzoat
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PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
Crude protein content
Johan Kjeldahl (1883)
developed the basic process
PRINCIPLE: total organic N
released from sample and
absorbed by acid
Digestion: sulfuric acid +
catalyst
Neutralization and
distillation; Sodium
hydroxide
Titration; Hydrochloric acid
(NH
4
)
2
SO
4
+ 2NaOH 2NH
3
+ Na
2
SO
4
+ 2H
2
O

NH
3
+ H
3
BO
3
NH
4
+
: H
2
BO
3
-
+ H
3
BO
3

(boric acid) (ammonium-borate complex)
excess
PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL

Protein (NH
4
)
2
SO
4

(ammonium sulfate)


Protein N NH
4
+
+ H
2
SO
4
(NH
4
)
2
SO
4

NEUTRALIZATION
AND DISTILLATION
DIGESTION
SULFURIC ACID
HEAT, CATALYST
Color change
melepaskan unsur N dari protein yang diubah menjadi amonium sulfat
amonium sulfat diubah menjadi amoniak yang
ditangkap oleh larutan asam standar berlebih
PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
Titration (direct titration)
H
2
BO
3
-
+ H
+
H
3
BO
3

Calculation
moles HCl = moles NH
3
= moles N in the sample


(HCl)
TITRATION
Sisa asam yang tidak bereaksi dengan amoniak dititrasi, sehingga dapat diketahui
jumlah amoniak dari N protein sampel
%N = (ml NaOH blanko ml NaOH contoh) x N HCl x 100 x 14.008
g sampel x 1000
% protein = %N x faktor konversi (tergantung jenis sampel)
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PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
Calculation
%Protein = %N conversion factor
Conversion factor: generally 6.25
most protein: 16% N

Conversion factor
egg or meat 6.25
milk 6.38
wheat 5.33
soybean 5.52
rice 5.17
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PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
ADVANTAGES
1. applicable to most food samples
2. simple
3. inexpensive
4. accepted as Official method
5. can measure mg levels of proteins

DISADVANTAGES
1. Measures total N not protein
2. Time consuming at least 2 hrs
3. Poor precision when compared to other
methods
4. Corrosive (dangerous) method
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PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
Asam sulfat pekat, berat jenis 1.84
Air raksa oksida
Kalium sulfat
Larutan natrium hidroksida-natrium
tiosulfat Larutan asam borat jenuh
Larutan asam klorida 0.02 N
NO KELAS A KELAS D KELAS E
1 Susu cair segar Kedelai mentah Ikan
2 Susu bubuk Tempe kedelai Kecap ikan
3 Yoghurt Kecap Bakso ikan
4 Keju Keripik tempe Kacang tanah mentah
5 Yakult Tahu Tempe kacang
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PROTEIN ANALYSIS
CRUDE PROTEIN-KJEDAHL
Pemasukan bahan kedalam labu kjeldahl.
Penambahan 7.5 g K
2
S
2
O
4
dan 0.35 g HgO (Awas: zat ini beracun) ,tambahkan 15 ml
H
2
SO
4
pekat.
Pemanasan semua bahan dalam labu kjeldahl dalam almari asam sampai berhenti
berasap dan cairan menjadi jernih.
Penambahan aquades dalam labu kjeldahl yang didinginkan dalam air es dan beberapa
lempeng Zn, juga tambahkan 15 ml larutan K
2
SO
4
% (dalam air) dan akhirnya tambahkan
perlahan-lahan larutan NaOH 50% sebanyak 50 ml yang sudah didinginkan dalam lemari
es.
Pasanglah labu kjeldahl dengan segera pada alat distilasi.
Panaskan labu kjeldahl perlahan-lahan sampai dua lapisan cairan tercampur kemudian
panaskan dengan cepat sampai mendidih.
Distilat ini ditampung dalam erlenmeyer yang telah diisi dengan 50 ml larutan standar
HCl (0.1 N) dan 5 tetes indikator metil merah. Lakukan distilasi sampai distilat yang
tertampung sebanyak 75 ml.
Titrasilah distilat yang diperoleh dengan standar NaOH (0.1 N) (lampiran) sampai warna
kuning.
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PROTEIN ANALYSIS
BIURET
Cupric ions react with peptide bonds under
alkaline conditions
(copper sulfate + K-Na-tartrate + alkali)
Measure color in SPEC at 520 nm
N
N
O
H
R H
O
H
Cu
2+
OH-
O
N
N
H
H
H
R
O
N
N
O
H
R H
O
H
Cu
2+
Purpl e bi uret compl ex
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PROTEIN ANALYSIS
ADVANTAGES
most sensitive (20-200g)
- Cheaper and faster than Kjeldahl
- Less problem with color deviations
- Few substances interfere
- Does not measure non protein nitrogen (NPN)

DISADVANTAGES
color development not proportional to protein
concentration
color varying with different proteins
interference (sugars, lipids, phosphate buffers, etc)

BIURET
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PROTEIN ANALYSIS
BIURET
Pereaksi biuret
Larutkan 3 gram CuCO
4
.5H
2
O
dan 9 gram Na-K-Tartrat
dalam 500 ml NaOH 0.2 N.
Tambahkan 5 gram KI
kemudian encerkan sampai
1000 ml dengan
menggunakan NaOH 0.2 N.
Larutan protein standar
Larutan bovine serum
albumin atau kasein 5 mg/ml
PEMBUATAN KURVA STANDAR
PERSIAPAN SAMPEL
Penimbangan Sampel
Penghancuran Sampel
Penyaringan dan Pensentrifuga asian
Supernatan didekantasi
Jika supernatan keruh (atau bahan
mengganggu), ada perlakuan tambahan
BAHAN
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PROTEIN ANALYSIS
FORMOL TITRATION
(N-AMINO)
Prinsip: larutan protein dinetralkan
dengan basa (NaOH), kemudian
penambahan formalin akan membentuk
dimethilol
Pembentukan dimethilol ini menunjukkan
gugus amino sudah terikat dan tidak akan
mempengaruhi reaksi antara asam (gugus
karboksil asam amino) dengan basa NaOH
perubahan warna menjadi merah muda
yang tidak hilang selama 30 detik.
Titrasi formol hanya tepat untuk
menunjukkan proses hidrolisis protein dan
kurang tepat untuk menentukan kadar
protein
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PROTEIN ANALYSIS
FORMOL TITRATION
(N-AMINO)
K-oksalat
Fenolftalein 1%
NaOH
Rosanilin klorida
Formaldehid 40 %
Akuades
Sampel seperti analisis protein metode Kjeldahl
BAHAN
titrasi formol
% N = ____________ x N NaOH x 14.008
g bahan x 10
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PROTEIN ANALYSIS
FORMOL TITRATION
(N-AMINO)
Pindahkan larutan protein ke dalam erlenmeyer 125 ml dan tambahkan
20 ml aquades dan 0.4 ml larutan Kalium oksalat jenuh (kalium oksalat
: air = 1:3) dan 1 ml phenolphtalein 1%. Diamkan selama 2 menit.

Titrasilah larutan contoh dengan 0.1 N NaOH sampai mencapai warna
seperti warna standar di bawah ini atau sampai warna merah jambu.

Warna standar : 10 ml susu + 10 ml aquades + 0.4 ml K-oksalat jenuh + 1
tetes 0.01% indikator rosanilin-chlorida.

Setelah warna tercapai, tambahkan 2 ml larutan formaldehid 40% dan
titrasilah kembali dengan larutan NaOH sampai warna seperti warna
standar tercapai lagi.
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