PROTEIN ANALYSIS FOOD ANALYSIS AND BIOCHEMISTRY PRACTICE. Endrika WIDYASTUTI. Food Science and Technology Department 2012

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Endrika WIDYASTUTI

Food Science and

PROTEIN

ANALYSIS

FOOD ANALYSIS AND

BIOCHEMISTRY PRACTICE

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INTRODUCTION OF PROTEIN

TYPES PROTEIN ANALYSIS

NINHIDRIN TEST

KJEDAHL METHOD

FORMOL TITRATION

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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

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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

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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

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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

DISADVANTAGE

S

• 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

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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

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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

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(

NH₄)₂SO₄ + 2NaOH  2NH₃ + Na₂SO₄ + 2H₂O

NH₃+ H₃BO₃  NH₄+_{: H}

2BO3- + H3BO3

(boric acid) (ammonium-borate complex) excess

PROTEIN ANALYSIS

CRUDE PROTEIN-KJEDAHL

Protein

(NH

₄

)

₂

SO

₄ (ammonium sulfate) Protein N  NH₄+_{+ H} 2SO4  (NH4)2SO4 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

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PROTEIN ANALYSIS

CRUDE PROTEIN-KJEDAHL

Titration (direct titration)

H

₂

BO

₃-

+ H

+



_H

3

BO

3

– Calculation

moles HCl = moles NH₃ = 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

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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

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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

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PROTEIN ANALYSIS

CRUDE PROTEIN-KJEDAHL

Pemasukan bahan kedalam labu kjeldahl.

Penambahan 7.5 g K₂S₂O₄ dan 0.35 g HgO (Awas: zat ini beracun) ,tambahkan 15 ml H₂SO₄ 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 K₂SO₄% (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.

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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 C u 2 + O H -O N N H H H R O N N O H R H O H C u 2 + P u r p l e b i u r e t c o m p l e x

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PROTEIN ANALYSIS

• ADVANTAGES

– most sensitive (20-200g)

- 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)

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PROTEIN ANALYSIS

BIURET

Pereaksi biuret

Larutkan 3 gram CuCO₄.5H₂O 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

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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

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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

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