ANALISIS PROTEIN --------------------------------------------- Disampaikan oleh Sri Naruki Fakultas Teknologi Pertanian UGM.

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1 ANALISIS PROTEIN --------------------------------------------- Disampaikan oleh Sri Naruki Fakultas Teknologi Pertanian UGM

2 PROTEIN Protein are an abundant component in all cells (Nielsen, 2004) Food protein : very complex; MW ranging from 5000 to more than a million Daltons Function : growth & maintenance of tissue, formation of essential body compounds, transportation of nutrients, etc. (Guthrie, 1982)

3 Protein consist of polypeptides, extremely long chains of many amino acid units All protein in all species, regardless of their function or biological activity, are built from the same basic set of 20 standard amino acids, which by themselves HAVE NO intrincsic biological activity

4 General structure of amino acids : COOH H 2 N  C  H R R = side chain or R group The simples amino acid : glycine  R = H In all amino acids, except glycine, the  carbon atom has 4 different substituent groups and is thus asymetric or chiral carbon (Lehninger, p. 96)

5 The Peptide Bond Peptide bond : how the amino acids are linked together to make a protein H H H 2 NCCOOH + H 2 NCCOOH R R’ H O H H 2 N-C-C-N-C-COOH + H 2 O R H R’

6 Proteins are composed of H, C, N, O, and S (Nielsen, 2004) Composition of elements in proteins : -C : 50 – 55 % -O : 20 – 25 % -N : 13.4 – 19.1 % -H : 5 – 7 % -S : 0.4 – 2.5 % -P, Fe, Cu : trace

7 C (karbon) : merupakan penyusun terbesar Bila C digunakan sebagai dasar analisis protein Analisis protein berdasarkan C mempunyai beberapa keuntungan : a.Digesti lebih mudah bila dibandingkan dengan digesti pada metoda analisis berdasarkan N b.Kadar C protein = tinggi sehingga - mengurangi error - faktor konversi relatif konstan

8 TAPI INGAT : ada beberapa senyawa lain yang juga mengandung C  karbohidrat dan lipida Oleh karena itu, C nonprotein tersebut harus dihilangkan terlebih dahulu Pemisahan C nonprotein tersebut  sulit Oleh karena itu, yang lazim dilakukan adalah : penentuan kadar protein berda- sarkan kadar nitrogen

9 Nitrogen is the most distinguishing element present in proteins N content in various food protein ranges from 13.4% to 19.1%  an average = 16.0% Therefore, protein content (%) =  100/16  x % N = 6.25 x % N conversion factor/CF (faktor konversi/FK)

10 N content in various food protein ranges from 13.4% to 19.1%  conversion factors for various food are varied : -Egg or meat  CF = 6.25 -Dairy products 6.38 -Wheat 5.70 -Other cereal grains & oilseeds 6.25 -Almonds 5.18 -Peanuts & Brazil nuts 5.46 -Other tree nuts & coconut 5.30 Conversion Factors

11 Analysis of protein is very complicated since some food components posses similar physicochemical properties Nonprotein nitrogen could come from : -Free amino acids - Some vitamins -Small peptide - Alkaloids -Nucleic acids - Uric acid -Porphyrin - Urea -Ammonium ions - Etc ANALYSIS OF PROTEIN

12 Aspartame is a small peptide composed of Asp, Phe, and methanol

13 Nucleic acids are polymer formed from linking of various nucleotides

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15

16 Struktur vitamin B 1 (tiamin)  juga mengandung N

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19 Caffeine alkaloid

20 Chemical structure of uric acid

21 Chemical structure of urea

22 The total organic nitrogen in foods would represent nitrogen : -Primarily from protein -A lesser extent from all organic nitrogen- containing nonprotein substances Therefore, % Protein = CF x % N is called : crude protein (protein kasar) Other major food components, including carbohydrate and lipid, may interfere physically with analysis of food proteins

23 1.Nutrition labeling 2.Functional property investigation Proteins of food have unique functional properties. Example : gliadin & glutenin in wheat flour for bread making 3.Biological activity determination, including enzymes & enzyme inhibitor. Enzyme activity is expressed in terms of specific activity : unit of enzyme/mg of protein Importance of Analysis

24 Protein analysis is required if you want to know : 1.Total protein content 2.Content of particular protein in mixture 3.Protein content during isolation and purification of protein 4.Nonprotein nitrogen (ex. TMA content) 5.Amino acid composition (profil asam amino) 6.Nutritive value of protein (ex. PER, BV) 7.Activity (of enzyme)

25 Protein Content of Selected Foods Food item% Protein (wet weight basis) CEREALS & PASTA Rice, brown, raw Wheat flour Corn flour, yellow Spaghetti, dry, enriched Cornstarch 7.9 13.7 6.9 12.8 0.3 DAIRY PRODUCTS Milk, whole, fluid Cheese, cheddar 3.3 24.9 FRUITS AND VEGETABLE Apple, raw, with skin Potato, whole, flesh and skin 0.2 2.0 LEGUMES Soybean, mature seeds, raw36.5 MEAT, POULTRY. FISH Egg, raw, whole Ham, sliced, regular 12.5 17.6

26 Determination of nitrogen, peptide bonds, aromatic amino acids Based on the dye-binding capacity of protein Based on the UV absorptivity of protein Based on the light scattering properties of protein The Basic Principles of Some Methods to Measure Protein Content

27 I. Determination of nitrogen, peptide bonds, aromatic amino acids A.Kjeldahl method D. Titrasi formol B.Biuret method E. Dumas method C.Lowry method F. BCA method

28 A. KJELDAHL METHOD

29 On March 7, 1883, Johann Kjeldahl presented his method of N analysis Today, the Kjeldahl method for the determination of organic nitrogen is the worldwide standard for the purpose of calculating the protein content in both human food and animal feed The Kjeldahl method has been adapted as a standard method of N analysis in fertilizer, fossil fuel, waste water, etc.

30 Proteins & other organic food components in a sample are digested with sulfuric acid in the presence of catalysts The total organic nitrogen is converted to ammonium sulfate The digest is neutralized with alkali and distilled into a boric acid solution The borate anions formed are titrated with standardized acid, which is converted to N in the sample PRINCIPLE

31 The result of analysis represents the crude protein content of the food since N also comes from nonprotein components The Kjeldahl method for nitrogen analysis is composed of three distinct steps : 1.Digestion (destruksi) 2.Neutralization and Distillation 3.Titration

32 1.Katalis logam seperti Hg, Cu, dan Se ditam- bahkan ke asam sulfat untuk menyempurna- kan oksidasi. - Yang terbaik : Hg - Selenium dioksida dan Cu-sulfat (3:1) juga efektif untuk digesti - Copper and titanium dioxide also have been used as a mixed catalyst for digestion  the use of them poses less safety concern than Hg in the post-analysis disposal of the waste IMPROVEMENTS

33 Bila digunakan Hg, selama digesti akan terbentuk senyawa kompleks Hg-amonia Hg harus diendapkan (sebagai HgS) dengan penambahan Na-tiosulfat (Na 2 S 2 O 3 )  presipitan Na 2 S 2 O 3 dicampur dengan NaOH yang digunakan untuk membebas- kan NH 3 (saat tahap netralisasi sebelum distilasi) Na-tiosulfat juga ditambahkan untuk membantu membebaskan N dari ikatan- nya dengan Hg (lihat butir 3)

34 Di lapangan, campuran (NaOH + Na 2 S 2 O 3 ) dikenal dengan istilah NaOH-thio Katalis Se (selenium) : -efek lebih hebat daripada Hg -Tidak perlu step tambahan (yaitu penam- bahan Na-tiosulfat /Na 2 S 2 O 3 ) -TAPI : bila jumlah Se berlebihan maka suhu destruksi/digesti tidak terkontrol sehingga ada kemungkinan kehilangan N (karena menguap)

35 2. Penambahan potasium sulfat dapat dila- kukan untuk menaikkan titik didih asam sulfat sehingga meningkatkan digesti Suhu digesti yang ideal : 370 – 410 o C. TAPI bila K-sulfat berlebihan maka akan terjadi kehilangan N 3. Sulfide or sodium thiosulfate are added to the diluted digest to help release nitrogen from mercury, which tends to bind to ammonium

36 4. The ammonia is distilled directly into a boric acid solution, followed by titration with standard acid 5. Colorimetry Nesslerization, or ion chroma- tography to measure ammonia, is used to determine nitrogen content after digestion

37 Beberapa Modifikasi 1.Cara makro Kjeldahl -Berat sampel : 1 g -Katalis : K 2 SO 4 -HgO -Distilasi : memapak lempeng Zn dengan tujuan agar supaya * tidak terjadi superheating * tidak terjadi percikan cairan * tidak terbentuk gelembung gas yang besar

38 2. Cara mikro Kjeldahl -Berat sampel : 10 – 30 mg -Katalis : Na 2 SO 4 –HgO 3. Cara Gunning -Berat sampel : 0,7 – 5,5 g -Katalis : K 2 S atau Na 2 SO 4 dan CuSO 4

39 GENERAL PROCEDURE & REACTIONS As stated before, the Kjeldahl method for nitrogen analysis is composed of three distinct steps. Sample preparation should be done prior to these three steps. 1.Sample preparation 2.Digestion (destruksi) 3.Neutralization and Distillation 4.Titration

40 Solid food are ground to pass a 20 mesh screen Samples for analysis should be homogeneous No other special preparation are required 1. Sample Preparation

41 2. Digestion Step The purpose of digestion : to break the intricate structure and chemical bonds that hold a chemical substances (piece of meat, cup of flour, or quart of oil) down into simple chemicals and ionic structures Specifically, proteins and other forms of nitrogen are broken down and converted to ammonia

42 To digest the sample, 1 – 2 g of the sample are placed on a digestion tube with 12 – 15 mL of concentrated H 2 SO 4. Seven grams of K 2 SO 4 and a metallic catalyst, usually Cu, are the added. The digestion tube is heated to the boiling temperature of the mixture Digestion was done to complete oxidation and conversion of N to ammonium sulfate The digestion is usually completed after one hour at 370 – 400 o C

43 a. Procedure of Digestion Place sample (accurately weighed) in a Kjeldahl flask Add sulfuric acid and catalyst Digest until CLEAR to get complete breakdown of all organic matter Digesti diakhiri bila larutan menjadi jernih dan tak berwarna UNTUK MENGOKSIDASIMEMERLUKAN H 2 SO 4 1 g Protein+ 9,0 g 1 g Lemak+ 17,8 g 1 g Karbohidrat + 7,3 g

44 Nonvolatile ammonium sulfate is formed from nitrogen and sulfuric acid : Sulfuric acid Protein (NH 4 ) 2 SO 4 Heat, catalyst During digestion : -protein nitrogen is liberated to form ammonium ions b. Reactions During Digestion

45 -Sulfuric acid oxidized organic matter & combines with ammonium formed -C is converted to CO 2 (volatile/menguap) -H is converted to H 2 O (volatile/menguap) Lama digesti bervariasi, tergantung dari jenis sampel yang dianalisis -Protein kaya akan asam amino histidin (His) dan triptofan (Trp) : digesti lama (  perlu katalis lebih banyak) -Sampel kaya lemak : digesti lama (  perlu H 2 SO 4 lebih banyak)

46 HgO + H 2 SO 4  HgSO 4 + H 2 O HgSO 4  Hg 2 SO 4 + SO 2 + O n (CHON) + O n + H 2 SO 4  CO 2 + H 2 O + (NH 4 ) 2 SO 4 protein c. Reaksi lengkap

47 Pada tahap ini (NH 4 ) 2 SO 4 dipecah menjadi NH 3 dengan penambahan NaOH (sampai dica-pai kondisi alkalis) dan pemanasan (distilasi) Selama distilasi dapat ditambahkan lempeng Zn NH 3 yang dibebaskan ditangkap oleh larutan asam standar berlebihan. Asam dapat berupa -Asam borat 4% -HCl 0,1N 3. Neutralization & Distillation Step

48 Ujung alat distilasi harus tercelup larutan asam Distilasi diakhiri bila destilat yang dihasil- kan tidak lagi bereaksi basa. Caranya ???? Reaksi yang terjadi saat netralisasi dan distilasi : (NH 4 ) 2 SO 4 + 2NaOH  2NH 3 + Na 2 SO 4 + 2H 2 O Selanjutnya NH 3 yang dibebaskan akan bereaksi dengan larutan penampung sebagai berikut :

49 Bila penampungnya asam borat : NH 3 + H 3 BO 3 (boric acid)  NH 4 + H 2 BO 3 – (borate ion) Bila penampungnya HCl : 2NH 3 + 2HCl (berlebihan)  2NH 4 Cl + HCl (sisa)

50 The digest is diluted with water Alkali-containing sodium thiosulfate is added to neutralize the sulfuric acid The ammonia formed is distilled into a boric acid solution containing the indicator methylene blue and methyl red Procedure of Netralization & Distillation

51 a.Bila digunakan penampung asam borat (METODA TITRASI LANGSUNG) -Borate anion (proportional to the amount of nitrogen) is titrated with standardized HCl (anion borat dalam penampung dititrasi dengan HCl standar 0,1N ) -Reaction : H 2 BO 3 – + H +  H 3 BO 3 -Calculation Moles of HCl = moles of NH 3 = moles N in the sample 4. Titration

52 A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen % N = N HCl x (corrected acid volume/g of sample) x (14g N/mol) x 100 Where : -N HCl = normality of HCl, in moles/1000ml -Corrected acid volume = (ml std acid for sample) – (ml std acid for blank)

53 Atau : % N =  (ml HCl sampel – ml HCl blanko ) : berat sampel (g) x 1000  x N HCl x 14,008 x 100% Untuk menghitung kadar protein, maka % N dikalikan faktor konversi (CF atau FK)  CF beberapa jenis bahan makanan : lihat slide sebelumnya

54 Bila digunakan penampung HCl, maka HCl sisa dititrasi dengan NaOH standar (0,1N) Reaksi - Saat distilasi : 2NH 3 + 2HCl (berlebihan)  2NH 4 Cl + HCl (sisa) -Saat titrasi : HCl (sisa) + NaOH  NaCl + H 2 O b. Bila digunakan penampung HCl (METODA KONVENSIONAL / BACK TITRATION / TITRASI BALIK)

55 Perhitungan Pada metoda konvensional, perlu dua (2) macam larutan standar yaitu HCl standar dan NaOH standar meq NH 3 hasil distilasi = (meq asam mula- mula – meq asam setelah distilasi) meq NH 3 = (ml asam x N asam) – (ml NaOH x N NaOH) Berat N (g) = meq N x 0,014007 Kadar N (%) =  (ml HCl x N HCl) – (ml NaOH x N NaOH)  x 1,4 g sampel

56 Atau : %N =  (ml NaOH blanko – ml NaOH sampel ) : berat sampel (g) x 1000  x N NaOH x 14,008 x 100% A factor is used to convert % N to % crude protein Most proteins contain 16% N, so the conversion factor (CF) is 6.25 % Protein = % N x 6.25

57 PROSEDUR LENGKAP METODA KJELDAHL 0,7 – 2.2 g sampel (dalam labu Kjeldahl) 0,7g HgO (atau 0,65g logam Hg) 15g K 2 SO 4 (atau Na 2 SO 4 anhidrat) 25ml H 2 SO 4 pekat DESTRUKSI/DIGESTI (didihkan) pelan-pelan, tambahkan sedikit parafin Setelah jernih, didihkan lagi + 30 menit PENDINGINAN (next slide)

58 PENDINGINAN + 200 ml aquades dingin Butiran Zn 25ml larutan Na 2 S 2 O 3 8% + 15g NaOH PEMASANGAN RANGKAIAN DISTILASI PEMASANGAN ERLENMEYER PENAMPUNG (15ml HCl 0,02N + 6 tetes MR-BCG) DISTILASI (Diakhiri bila distilat mencapai > 150ml ) (Cuci ujung kondenser) TITRASI HCl sisa dalam Erlenmeyer dengan NaOH LAKUKAN PENENTUAN BLANKO !!

59 + 30mg Sampel (dalam labu Kjeldahl) 2g K 2 SO 4 ; 40mg HgO 2ml H 2 SO 4 pekat Batu didih DESTRUKSI/DIGESTI Bila larutan sudah jernih, tambahkan aquades dan didihkan lagi + 1,5 jam PENDINGINAN & PEMINDAHAN KE LABU DISTILASI PROSEDUR LENGKAP METODA MIKRO KJELDAHL

60 PENDINGINAN 8 – 10ml NaOH-Na 2 S 2 O 3 DISTILASI - Tampung distilat dalam Erlenmeyer berisi : 5ml H 3 BO 3 4% + 4 tetes indikator (0,2% MR + 0,2% BCG) - Distilasi diakhiri saat distilat sudah tidak lagi alkalis TITRASI - Distilat dititrasi dengan HCl 0,02N LAKUKAN PENENTUAN BLANKO !!

61 In place of distillation & titration with acid, ammonia or nitrogen can be quantitated by : 1.NESSLERIZATION : 4NH 4 OH + 2HgI 2 + 4KI + 3KOH  mercuric iodide NH 2 Hg 2 IO + 7KI + 2H 2 O ammonium dimercuric iodide ALTERNATE PROCEDURES

62 NH 2 Hg 2 IO (red-orange) can be determined spectrophotometrically at 440nm This method is rapid & sensitive, but the ammonium dimercuric iodide is colloidal and color is not stable OH – 2. NH 3 + phenol + hypochlorite indophenol (blue, 630nm) 3. pH measurement after distillation into known volume of boric acid 4. Direct measurement of ammonia, using ion chromatographic method

63 1.Applicable to all types of foods 2.Inexpensive(if not usingan automated system) 3.Accurate; an official method for crude protein content) 4.Has been modified (micro Kjeldahl method) to measure microgram quantities of protein) ADVANTAGES

64 1.Measures total organic nitrogen, not just protein nitrogen 2.Time consuming (at least 2 hr to complete) 3.Poorer precision than the Biuret method 4.Corrosive reagent DISADVANTAGES

65 CONTOH SOAL Pada analisa kadar protein dengan metoda Kjeldahl, diperoleh hasil kadar protein susu bubuk skim adalah 40%. Penampung distilat yang digunakan adalah HCl berlebihan. Bila berat sampel = 0,25g dan titrasi blanko membutuhkan NaOH 0,1N sebanyak 45ml, hitunglah volume NaOH untuk titrasi sampel. Faktor konversi N ke protein skim = 6,38.

66 B. BIURET METHOD

67 A violet-purple color is produced when cupric ions are complexes with > 2 pep- tide bonds, under alkaline conditions) The absorbance of the color produced is read at 540 nm The color intensity (absorbance) is proportional to the protein content of the sample 1. PRINCIPLE

68 H O H HOO–C–N–C –C –NH 2 + CuSO 4 + NaOH  R H R R H H R 2 HN–C–C–N–C–COOH H O H Cu + Na 2 SO 4 + H 2 O H O HOO–C–N–C–C–NH 2 R H H R (senyawa kompleks ungu) 2. REACTIONS

69

70 Hasil yang diperoleh dapat dipengaruhi oleh adanya lipida dan komponen lain yang dapat mengubah warna ataupun memberikan respons yang sama dengan ikatan peptida. Contoh gugus yang memberi respons yang sama dengan ikatan peptida : –CSNH 2 –CHNH 2 CH 2 OH –C(NH)NH2 –CHNH 2 CHOH – CH 2 NH 2 –CHOHCH 2 NH 2 –CRHNH 2

71 1.A 5-ml biuret reagent is mixed with a 1-ml portion of protein solution (1 to 10mg protein/ml). The reagent include copper sulfate, NaOH, and K-Na-tartrate, which is used to stabilize the cupric ion in the alkaline solution 2.After the reaction mix is allowed to stand at room temperature for 15 or 30 min, the absorbance is read at 540 nm against a reagent blank 3. PROCEDURE

72 Filtration or centrifugation before reading absorbance is required if the reaction mixture is not clear A standard curve of concentration versus absorbance is constructed using bovine serum albumin (BSA) BSA : 0, 20, 40, 60, 80, 100, 120  g/mL

73 Lebih murah daripada metoda Kjeldahl, cepat (dapat selesai dalam < 30 menit), paling sederhana Deviasi warna jarang terjadi dibanding dengan metoda Lowry, absorpsi UV, atau turbidimetri Senyawa yang dapat mengganggu analisis hanya sedikit sekali Tidak mendeteksi nitrogen dari sumbernonprotein 4. ADVANTAGES

74 1.Kurang sensitif bila dibandingkan dengan metoda Lowry; perlu minimal 2 – 4 mg protein untuk analisa 2.Absorbansi dapat berasal dari pigmen empedu, bila pigemen tersebut ada di dalam sampel 3.Garam amonium dengan konsentrasi tinggi dapat mengganggu reaksi 5. DISADVANTAGES

75 4. Warna yang dihasilkan bervariasi dengan perbedaan jenis protein. Misal : gelatin menghasilkan warna pinkish-purple 5. Kondisi “tak tembus cahaya” dapat terjadi pada larutan final apabila terdapat lipida atau karbohidrat dalam jumlah banyak 6. Bukan merupakan metoda yang absolut : warna harus distandardisasi dengan protein yang diketahui (misal BSA) atau dicocokkan dengan metoda Kjeldahl

76 Hubungan antara absorbansi pada 540 nm pada Biuret method dengan kadar protein menurut metoda Kjeldahl A at 540 nm % Protein (Kjeldalh)

77 C. LOWRY METHOD

78 The Lowry method combines the biuret reaction (see point B) with the reduction of the Folin-Ciocalteau phenol reagent (phosphomolybdic-phosphotungstic acid) by tyrosin & tryptophan residues in the protein The bluish color developed is read at 750 nm (high sensitivity for low protein concentration) or 500 nm (low sensitivity for high protein concentration) 1. PRINCIPLE

79 Protein reaction with cupric ion under alkaline condition

80 Terdapat 2 macam reagen Lowry, yaitu -Lowry A (mengandung fosfotungstat-fosfo molibdat 1 : 1) -Lowry B (mengandung Na-karbonat 2% dalam NaOH 0,1N serta Cu-sulfat dan Na- K-tartrat 2%) 2a. PROSEDUR

81 Cara : -1 mL larutan protein sampel + 5mL reagen Lowry B, gojog dan biarkan 10 menit -Kemudian tambahkan 0,5 mL reagen Lowry A dan biarkan 20 menit -Baca nilai absorbansi pada 600 nm

82 1.Proteins to be analyzed are diluted to an appropriate range (20 – 100  g) 2.K Na Tartrate-Na 2 CO 3 solution is added after cooling and incubated at room temperature for 10 min 3.CuSO 4 -K Na Tartrate-NaOH solution is added after cooling and incubated at room temperature for 10 min 2b. PROCEDURE

83 4. Freshly prepared Folin reagent is added, then the reaction mixture is mixedand incubated at 50 o C for 10 min 5. Absorbance is read at 650 nm 6. A standard curve of bovine serum albumin (BSA) is carefully constructed for estimating protein concentration of the unknown

84 Cu ++ in alkaline solution to form complexity with protein. Cu ++ catalyses oxidation of phenol group of tyrosine with phosphomolybdic-phosphotungstic acid. A at 750 nm g of protein (Kjeldahl) 

85 1.Very sensitive : a.50 – 100 times more sensitive than biuret method b.10 – 20 times more sensitive than 280nm UV absorption method c.Similar sensitivity as Nesslerization; however, more convenient than Nesslerization 2. Less affected by turbidity of the sample 3. More specific than most other methods 4. Relatively simple, can be done in 1 – 1.5 hr 3. ADVANTAGES

86 1.Color varies with different proteins to a greater extent than biuret method 2.Color is not strictly proportional to protein concentration 3.The reaction is interfered with to varying degrees by sucrose, lipids, phosphate buffers, monosaccharides, and hexoamines 4.High concentration of reducing sugars, ammonium sulfate, and sulfhydryl compounds interfere with the reaction 4. DISADVANTAGES

87 D. TITRASI FORMOL

88 Larutan protein dinetralkan dengan basa (NaOH), kemudian ditambah formalin sehingga terbentuk dimetilol Dengan terbentuknya dimetilol maka gugus amino protein sudah terikat dan tidak mempengaruhi reaksi antara asam (gugus karboksil) dengan basa NaOH sehingga akhir titrasi dapat diakhiri dengan tepat 1. PRINSIP

89 Indikator yang dipakai adalah PP Akhir titrasi ditandai saat terjadinya perubahan warna menjadi merah muda yang tidak hilang dalam 30 detik Titrasi formol baik untuk digunakan untuk evaluasi proses terjadinya pemecahan protein (misal : pada fermentasi protein pada tempe, kecap, tauco, dzsb.) Proses hidrolisis protein ditandai dengan meningkatnya titrasi formol

90 O H O R–CH–C–OH + NaOH  R–C–C–O – NH 2 NH 3 + pada pH netral H O H R–C–C–O – + CH 2 O  R–C–COOH NH 3 + (formalin) HOH 2 C–N–CH 2 OH (dimetilol) H R–C–COOH H O HOH 2 C–N–CH 2 OH + NaOH  R–C–C–ONa (dimetilol) HOH 2 C–N–CH 2 OH 2. REAKSI PADA TITRASI FORMOL

91 10 ml larutan protein sampel + 20 ml aqu- ades + 0,4 ml larutan K-oksalat jenuh (K- oksalat : air = 1 : 3) dan 1 ml indikator PP 1%. Diamkan selama 2 menit Titrasi larutan tersebut dengan 0,1N NaOH sampai terbentuk warna pink atau warna standar (10 ml susu + 10 ml aquades + 0,4 ml K-oksalat jenuh + 1 tetes indikator rosanilin-khlorida 0,01%) 3. PROSEDUR

92 Setelah warna tercapai, tambahkan 2 ml formaldehid 40% dan titrasilah kembali dengan larutan NaOH sampai warna seperti warna standar tercapai lagi. Catatlah nilai titrasi kedua ini. Dibuat titrasi blanko yang terdiri dari : 20 ml aquades + 0,4 ml larutan K-oksalat jenuh + 1 ml indikator PP + 2 ml formal- dehid, dan titrasilah dengan larutan NaOH Titrasi formol = titrasi terkoreksi = titrasi kedua – titrasi blanko

93 Bila nilai titrasi formol akan digunakan untuk menentukan kadar protein, maka harus dibuat percobaan serupa dengan menggunakan larutan yang telah diketahui kadar proteinnya (misalnya dengan metoda Kjeldahl) Selanjutnya ditentukan hubungan antara titrasi formol dengan % protein. Misal : -% protein susu = 1,83 x ml titrasi formol -% kasein = 1,63 x ml titrasi formol

94 E. DUMAS (NITROGEN COMBUSTION) METHOD

95 Sample are combusted at high tempera- ture (700 – 1.000 o C) The nitrogen released is quantitated by gas chromatography using thermal conductivity detector (TCD) The nitrogen determined is converted to protein content in the sample 1. PRINCIPLE

96 PROCEDURE Sample (100 – 500 mg) are weighed into a tin capsule and introduced to a combust- on reactor in automated equipment The nitrogen released is measured by a built-in gas chromatograph APLICATION It is an alternative to the Kjeldahl method It is suitable for all type of foods 2. PROCEDURE & APLICATION

97 ADVANTAGES Requires no hazardous chemicals Can be accomplished in 3 minutes Recent automated instrument can analyze up to 150 samples without attention DISADVANTAGES Expensive equipment is required Measures total organic nitrogen, not just protein nitrogen 3. ADVANTAGES & DISADVANTAGES

98 F. BICINCHONINIC ACID (BCA) METHOD

99 Protein mampu mereduksi ion kupri (Cu 2+ ) menjadi ion kupro dalam suasana alkalis Dengan reagen BCA (berwarna apple- greenish), ion kupro tersebut membentuk kompleks berwarna purplish, yang intensitasnya dapat ditera pada 562 nm Intensitas warna purplish tersebut propor- sional dengan kadar protein 1. PRINSIP

100

101

102 Mix (one step) the protein solution with the BCA reagent, which contain BCA sodium salt, Na-carbonate, NaOH, and Cu- sulfate pH 11.25 Incubate at room temperature for 2 hr, or 60 o C for 30 min. A higher temperature gives a greater color respon Read the solution at 562 nm against a reagent blank Construct a standard curve using BSA 2. PROSEDUR

103 BCA method had been used in protein isolation and purification The suitability of BCA method for measuring protein in complex food has not been reported 3. APPLICATION

104 1.Sensitivity of the micro BCA method (0.5 – 10  g) is better than Lowry method 2.One-step mixing is easier than in the Lowry method 3.The reagent is more stable than for the Lowry method 4.Nonionic detergent and buffer salts do not interfere with the reaction 4. ADVANTAGES

105 1.Color is not stable with time. The analyst needs to carefully control the time for reading absorbance 2.Any compound capable of reducing Cu 2+ to Cu + will lead to color formation 3.Reducing sugar interfere to a greater extent than in Lowry method 4.Color variation among proteins are similar to those in the Lowry method 5. DISADVANTAGES

106 II. Based on the UV absorptivity of protein The Basic Principles of Some Methods to Measure Protein Content

107 ULTRAVIOLET (UV) 280 nm METHOD

108 Protein show strong absorption at 280 nm, primarily due to tyrosine (Tyr) and tryptophan (Trp) residues in the protein Because the content of Tyr & Trp in protein from each food source is fairly constant, the A 280 could be used to estimate the concentration of protein 1. PRINCIPLE

109 Protein are solubilized in buffer or alkali Absorbance of protein solution is read at 280 nm against a reagent blank Protein concentration is calculated according Beer’s law : A = abc A = absorbance, a = konstanta (molar absorp- tivity for individual protein), b = cuvette path length, c = concentration Bisa juga memakai standar BSA 2. PROCEDURE

110 It has been used to determine the protein content of milk and meat products It has not been used widely in food system It is better applied in a purified protein system It is also better applied in proteins that have been extracted in alkali or denaturing agents such as 8M urea 3. APPLICATION

111 1.Rapid and relatively sensitive (at 280 nm, + 100  g protein is required, several times more sensitive than the Biuret method) 2.Nondestructive : sample can be used for other analysis after protein determination 3.No interference from ammonium sulfate and other buffer salts 4. ADVANTAGES

112 1.Nucleic acid also absorb at 280 nm. 2.The solution must be clear and colorless. Turbidity due to particulates in the solution will increase absorbance falsely 3.A relatively pure system is required to use this method 4. DISADVANTAGES

113 III. Based on the dye-binding capacity of protein A. Dye : acid orange-12 B. Dye : coamassive brillian blue G-250 The Basic Principles of Some Methods to Measure Protein Content

114 A. DYE BINDING METHOD with acid orange-12 as a dye

115 Principle: at low pH, basic groups of protein are (+) charged. These will quantitatively bind a (-) charged dye. What are these basic groups?

116 Acid Orange 12: Procedure: 1.Mix protein, dye, buffer pH = 2.  protein + dye : membentuk komplex tidak larut 2.Filter or centrifuge. 3.Measure optical density (OD) or absorbancy of filtrate (filtrat mengandung dye sisa yang tak bereaksi dengan protein)

117 Absorbance of dye bound by protein = Absorbance of initial dye – Absorbance of filtrate A. at 470 nm % Protein (Kjeldahl) 681012 14 16 Skim milk Kadar protein  maka intensitas warna filtrat 

118 Factors Influencing Dye Binding determination: 1.Temperature 2.Non-proteins. 3.Buffers systems 4.Protein quality

119 B. DYE BINDING METHOD with coamassie brillian blue G-250 as a dye

120

121 PRINSIP : dye + protein  kompleks larut Yang ditera : absorbansi senyawa kompleks yang larut tersebut Oleh karena itu : bila kadar protein  maka absorbansi juga  Dengan tabel konversi yang menunjukkan hubungan antara cat yang terikat protein dengan kadar protein  kadar protein sampel dapat diketahui Dapat pula dibuat garis regresi yang yang menunjukkan hubungan antara cat yang terikat protein dengan kadar protein

122 1. SAMPLE PREPARATION Kjeldahl & Dumas methods require little preparation  sample particle size of 20 mesh is satisfactory Other methods require fine particles for extraction of protein from the complex food systems COMPARISON OF METHODS

123 2. PRINCIPLE Kjeldahl & Dumas methods measure directly the total amount of organic N in the foods Other method measure the various properties of protein. For examples : -The Biuret method measures peptide bonds -The Lowry methos measures a combination of peptide bonds and the amino acids Tyr & Trp

124 3. SENSITIVITY Kjeldahl, Dumas, and Biuret method are less sensitive than Lowry, BCA, or UV method 4. SPEED Speed of determination in spectrophoto- metric method and the Dumas method are faster than with the Kjeldahl method

125 AKHIR DARI KULIAH DENGAN POKOK BAHASAN PROTEIN TERIMA KASIH