analisis protein - unila

ANALISIS PROTEIN

Bahan Bacaan: • Nielsen, S. 2010. Food Analysis 4th Ed. Hal. 135-146. • Nielsen, S. 2010. Food Analysis: Laboratory Manual 2nd

Ed. Hal.: 17-28. • Pomeranz, Y dan C. E. Meloan. 2000. Food Analysis:

Theory and Practice. Hal. : 575-601.

Jurusan Teknologi Hasil Pertanian Fakultas Pertanian Universitas Lampung

ANALISIS HASIL PERTANIAN1

PENDAHULUAN


• Protein merupakan komponen terbesar kedua dalam sel.

• Komponen penting untuk fungsi biologis dan struktur sel.

• Tersusun terutama oleh: H, C, N, O, dan S. • Tersusun oleh 20 jenis asam amino yang dihubungkan

oleh ikatan peptida. • Nitrogen (N) merupakan unsur yang mencirikan

senyawa protein, persen N dalam protein 13,4 - 19,1%. • Analisis protein didasarkan pada Total Organic Nitrogen

(TON) dalam bahan (pada umumnya).

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TUJUAN ANALISIS PROTEIN1. Persyarataan Labeling

• Kadar nutrisi wajib dicantumkan dalam kemasan (total protein, komposisi asam amino, nitrogen non protein, nilai gizi protein).

2. Penentuan Harga Produk • Harga komoditi ditentukan oleh kandungan

proteinnya (gandum, susu, dan daging) 3. Penentuan Sifat Fungsional

• Beberapa komoditi mengandung jenis protein yang unik (gliadin dan glutenin untuk gandum, casein untuk susu, dan albumin untuk telur)

4. Penentuan Aktivitas Biologis • Beberapa jenis protein bersifat aktif (Enzim). (enzim

proteolitik, pektinase, dan tripsin).

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METODE ANALISIS PROTEIN

1) Prinsip • Dikembangkan oleh Johann Kjeldahl, tahun 1883. • Menentukan kadar Total Organic Nitrogen (TON)

dalam sample. Kadar nitrogen selanjutnya di konversi ke kadar protein.

• Metode resmi: AOAC International, USEPA, ISO, dan EN (European Norms/Standard).

• Terdiri dari 3 tahapan: a). Digestion/destruction b). Neutralization/destillation c). Titration/analysis

A. METODE KJELDAHL

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2) Digestion (Destruction) • Sampel dimasukan dalam labu Kjeldahl, tambahkan

asam dan katalis, supaya terjadi proses digestion. • Pada proses digestion, H2SO4 mengoksidasi bahan

organik, nitrogen (protein) dilepaskan dalam bentuk ion ammonium, terbentuk (NH4)2SO4.

• Karbon dan Hydrogen diubah menjadi CO2 dan H2O.

A. METODE KJELDAHL ………………………Chapter 9 • Protein Analysis 137

Semiautomation, automation, and modification formicrogram nitrogen determination (micro Kjeldahlmethod) have been established by AOAC in Methods976.06, 976.05, and 960.52, respectively.

9.2.1.3 General Procedures and Reactions

9.2.1.3.1 Sample Preparation Solid foods are gro-und to pass a 20-mesh screen. Samples for analysisshould be homogeneous. No other special prepara-tions are required.

9.2.1.3.2 Digestion Place sample (accurately weigh-ed) in a Kjeldahl flask. Add acid and catalyst; digestuntil clear to get complete breakdown of all organicmatter. Nonvolatile ammonium sulfate is formed fromthe reaction of nitrogen and sulfuric acid.

Protein Sulfuric acid−−−−−−−→Heat, catalyst

(NH4)2SO2 [1]

During digestion, protein nitrogen is liberated to formammonium ions; sulfuric acid oxidizes organic matterand combines with ammonium formed; carbon andhydrogen elements are converted to carbon dioxideand water.

9.2.1.3.3 Neutralization and Distillation The digest isdiluted with water. Alkali-containing sodium thio-sulfate is added to neutralize the sulfuric acid. Theammonia formed is distilled into a boric acid solutioncontaining the indicators methylene blue and methylred (AOAC Method 991.20).

(NH4)2SO4 + 2NaOH→ 2NH3 + Na2SO4 + 2H2O

[2]

NH3 + H3BO3 (boric acid) → NH4 + H2BO3− [3]

(borate ion)

9.2.1.3.4 Titration Borate anion (proportional to theamount of nitrogen) is titrated with standardized HCl.

H2BO3− + H+ → H3BO3 [4]

9.2.1.3.5 Calculations

Moles of HCl = moles of NH3

= moles of N in the sample [5]

A reagent blank should be run to subtract reagentnitrogen from the sample nitrogen.

% N = N HCl×Corrected acid volumeg of sample

×14 g Nmol

×100

[6]

9-2table

Nitrogen to Protein Conversion Factors forVarious Foods

Percent N in Protein Factor

Egg or meat 16.0 6.25Milk 15.7 6.38Wheat 18.76 5.33Corn 17.70 5.65Oat 18.66 5.36Soybean 18.12 5.52Rice 19.34 5.17

Data from (1,8).

where:

NHCl = normality of HCl,in mol/1000 ml

Corrected acid vol. = (ml std. acid for sample) –(ml std. acid for blank)

14 = atomic weight of nitrogen

A factor is used to convert percent N to percent crudeprotein. Most proteins contain 16% N, so the conver-sion factor is 6.25 (100/16 = 6.25).

% N/0.16 = % protein [7]

or% N ×6.25 = % protein

Conversion factors for various foods are given inTable 9-2.

9.2.1.3.6 Alternate Procedures In place of distilla-tion and titration with acid, ammonia or nitrogen canbe quantitated by:

1. Nesslerization

4NH4OH + 2HgI2 + 4KI + 3KOHmercuric iodide

→ NH2Hg2IO + 7KI + 2H2Oammonium dimercuric iodide,red-orange, 440 nm

[8]

This method is rapid and sensitive, but theammonium dimercuric iodide is colloidal andcolor is not stable.

2. NH3 + phenol + hypochloride−→OH−

indophenol (blue, 630 nm) [9]

3. pH measurement after distillation into knownvolume of boric acid

4. Direct measurement of ammonia, using ionchromatographic method

Protein(-N)+H2SO4 (NH4)2SO4+CO2+H2O(Sample) Catalyst

Heating

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3) Neutralization (Distillation) • Sampel diencerkan dengan air, ditambahkan alkali

(NaOH), lalu dilakukan destilasi, dan detilate langsung masuk ke larutan asam (H3BO3) yang sudah diketahui volumenya.

• NaOH mengubah ion ammonium menjadi amonia (NH3), NH3 bereaksi dengan H3BO3 berubah menjadi ion borat (H2BO3⁻)

A. METODE KJELDAHL ………………………

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

NH3 + H3BO3 NH4⁺+ H2BO3⁻

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Kjeldahl Method Apparatus

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3) Titration (Analysis) • Ion Borat (= N) di titrasi dengan HCl yang sudah

distandarisasi • HCl mengubah ion borat menjadi asam borat.


H2BO3⁻ + HCl H2BO3 + Cl⁻

4) Perhitungan

Chapter 9 • Protein Analysis 137






(NH4)2SO2 [1]




[2]


(borate ion)


H2BO3− + H+ → H3BO3 [4]






×14 g Nmol

×100

[6]

9-2table




Data from (1,8).

where:





% N/0.16 = % protein [7]




1. Nesslerization



[8]






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A. METODE KJELDAHL (Perhitungan) ………………………

• Sebelum melakukan, perlu dilakukan titrasi blanko. • Volume titrasi blanko menjadi faktor koreksi volume

titrasi sampel.







(NH4)2SO2 [1]




[2]


(borate ion)


H2BO3− + H+ → H3BO3 [4]






×14 g Nmol

×100

[6]

9-2table




Data from (1,8).

where:





% N/0.16 = % protein [7]




1. Nesslerization



[8]






Corrected acid volume (ml) = Sample HCl volume (ml) - Blank HCl volume (ml)







(NH4)2SO2 [1]




[2]


(borate ion)


H2BO3− + H+ → H3BO3 [4]






×14 g Nmol

×100

[6]

9-2table




Data from (1,8).

where:





% N/0.16 = % protein [7]




1. Nesslerization



[8]






Or

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A. METODE KJELDAHL ………………………Chapter 9 • Protein Analysis 137






(NH4)2SO2 [1]




[2]


(borate ion)


H2BO3− + H+ → H3BO3 [4]






×14 g Nmol

×100

[6]

9-2table




Data from (1,8).

where:





% N/0.16 = % protein [7]




1. Nesslerization



[8]






10



5) Kelebihan dan Kekurangan ➡ Kelebihan:

• Untuk semua jenis bahan pangan • Relatif murah • Akurat (high accuracy) • Dapat dimodifikasi (micro Kjeldahl)

➡ Kekurangan: • Menentukan TON, bukan hanya protein • Waktu analisis lama • Low precision (reproducible) • Corrosive reagent

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1) Prinsip • Menggunakan visible (Vis) Spoktrofometer. • Reagent (dye) Coomassie Brilliant Blue G-250

ditambahkan dalam sample. • Warna reagent kan berubah dari merah menjadi biru,

akibat dari pembentukan komplek antara reagent dengan ikatan polipeptida.

• Dianalisis dengan Spektofometer pada panjang gelombang 595nm. Besarnya absorbant menunjukan konsentrasi protein.

B. METODE BRADFORD DYE-BINDING

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B. METODE BRADFORD DYE-BINDING …………….2) Kelebihan dan Kekurangan

➡ Kelebihan: • Cepat (2 menit). • Sensitif (dapat menganalisis kadar protein

yang rendah) • Menganalisis melokul protein kecil (≥

4000Da) ➡ Kekurangan:

• Complex reagent (dye)-protein dapat berikatan dengan kuvet quart, gunakan kuvet gelas atau plastik

• Warna komplek reagent (dye)-protein bervariasi, tergantung jenis dan asal protein. (pemilihan standar yang sesuai)

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1) Prinsip • Menggunakan Visible (Vis) Spektrofometer. • Sampel direaksikan dengan reagent Biuret,

menghasilkan komplek yang berwarna ungu (ion Cu⁺⁺ bereaksi dengan ikatan peptida).

• Dilakukan filtrasi. Dan filtrat dianalisis dengan Spektofometer pada panjang gelombang 540nm. Besarnya absorbant menunjukan konsentrasi protein.

C. METODE BIURET

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C. METODE BIURET …………….

2) Kelebihan dan Kekurangan ➡ Kelebihan:

• Metode paling sederhana. • Lebih murah dan lebih cepat dibandingkan

metode Kjeldahl (30 menit). • Tidak menganalisis nitrogen dari sumber

nonpeptida atau protein. ➡ Kekurangan:

• Kurang sensitif. • Ada varaiasi warna hasil reaksi, tergantung jenis

proteinnya (warna harus distandarisasi). • Warna juga tergantung pada kadar

karbohididrat dan lipid dalam sampel.

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1) Prinsip • Menggunakan Ultraviolet (UV) Spektofometer. • Sampel direaksikan dengan Buffer atau Alkali,

Residu asam amino Triptofan dan Tirosin terbentuk. • Dilakukan filtrasi. Dan filtrat dianalisis dengan

Spektofometer pada panjang gelombang 280nm. • Konsetrasi protein dihitung berdasarkan rumus:

‣ C = A/(a x b) ‣ Dimana: C = Konsentrasi protein, A =

Absorbance, a = Molar absorptivity, dan b = lebar kuvet.

D. METODE ABSORPSI ULTRAVIOLET

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C. METODE BIURET …………….

2) Kelebihan dan Kekurangan

➡ Kelebihan: • Cepat dan sangat sensitive. • Bebas gangguan komponen lainnya.

➡ Kekurangan: • Kalau ada asam amino aromatis lainnya, harus

di hitung molar absorptivity. • Larutan (filtrat) yang dihasilkan harus tidak

berwarna dan bebas partikulat.

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1) Prinsip • Menggunakan Gas Chromatography. • Sampel dibakar dengan cepat (700-1000°) untuk

menghasilkan N2 dan NOx, kemudian direduksi dengan Cu.

• Hasil reduksi selanjutnya di lewatkan ke kolom GC dan dianalisis dengan Thermal Conductivity Detector (TCD).

E. METODE DUMAS (PEMBAKARAN N)

138 Part II • Compositional Analysis of Foods

9.2.1.4 Applications

Advantages:

1. Applicable to all types of foods2. Inexpensive (if not using an automated system)3. Accurate; an official method for crude protein

content4. Has been modified (micro Kjeldahl method) to

measure microgram quantities of proteins

Disadvantages:

1. Measures total organic nitrogen, not just pro-tein nitrogen

2. Time consuming (at least 2 h to complete)3. Poorer precision than the biuret method4. Corrosive reagent

9.2.2 Dumas (Nitrogen Combustion) Method

9.2.2.1 Principle

The combustion method was introduced in 1831 byJean-Baptiste Dumas. It has been modified and auto-mated to improve accuracy since that time. Samplesare combusted at high temperatures (700–1000◦C)with a flow of pure oxygen. All carbon in the sampleis converted to carbon dioxide during the flash com-bustion. Nitrogen-containing components producedinclude N2 and nitrogen oxides. The nitrogen oxidesare reduced to nitrogen in a copper reduction col-umn at a high temperature (600◦C). The total nitrogen(including inorganic fraction, i.e., including nitrateand nitrite) released is carried by pure helium andquantitated by gas chromatography using a thermalconductivity detector (TCD) (9). Ultra-high purityacetanilide and EDTA (ethylenediamine tetraacetate)may be used as the standards for the calibration ofthe nitrogen analyzer. The nitrogen determined is con-verted to protein content in the sample using a proteinconversion factor.

9.2.2.2 Procedure

Samples (approximately 100–500 mg) are weighed intoa tin capsule and introduced to a combustion reac-tor in automated equipment. The nitrogen released ismeasured by a built-in gas chromatograph. Figure 9-1

9-1f igure

General components of a Dumas nitrogen ana-lyzer. A, the incinerator; B, copper reductionunit for converting nitrogen oxides to nitrogen;and GC, gas chromatography column.

shows the flow diagram of the components of a Dumasnitrogen analyzer.


The combustion method is an alternative to the Kjel-dahl method (10) and is suitable for all types of foods.AOAC Method 992.15 and Method 992.23 are for meatand cereal grains, respectively.

Advantages:

1. Requires no hazardous chemicals.2. Can be accomplished in 3 min.3. Recent automated instruments can analyze up

to 150 samples without attention.

Disadvantages:

1. Expensive equipment is required.2. Measures total organic nitrogen, not just pro-

tein nitrogen.

9.2.3 Infrared Spectroscopy

9.2.3.1 Principle

Infrared spectroscopy measures the absorption ofradiation (near- or mid-infrared regions) by moleculesin food or other substances. Different functionalgroups in a food absorb different frequencies of radia-tion. For proteins and peptides, various mid-infraredbands (6.47 µm) and near-infrared (NIR) bands (e.g.,3300–3500 nm; 2080–2220 nm; 1560–1670 nm) charac-teristic of the peptide bond can be used to estimatethe protein content of a food. By irradiating a sam-ple with a wavelength of infrared light specific forthe constituent to be measured, it is possible to pre-dict the concentration of that constituent by measuringthe energy that is reflected or transmitted by the sam-ple (which is inversely proportional to the energyabsorbed) (11).

9.2.3.2 Procedure

See Chap. 23 for a detailed description of instrumen-tation, sample handling, and calibration and quantita-tion methodology.


Mid-infrared spectroscopy is used in Infrared MilkAnalyzers to determine milk protein content, whilenear-infrared spectroscopy is applicable to a widerange of food products (e.g., grains; cereal, meat,and dairy products) (3,12,13) (AOAC Method 997.06).Instruments are expensive and they must be calibratedproperly. However, samples can be analyzed rapidly(30 s to 2 min) by analysts with minimal training.

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C. METODE DUMAS (PEMBAKARAN N) …………….

2) Kelebihan dan Kekurangan

➡ Kelebihan: • Tidak memerlukan bahan kimia berbahaya. • Cepat, 3 menit • Dengan sistem otomatis, dapat melalkukan

analisis 150 sampel sekaligus. ➡ Kekurangan:

• Alat/instrument yang diperlukan sangat mahal. • Menentukan TON, bukan hanya dari protein.

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analisis protein - unila

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