enzymatic determinations of kecap Page 1 of 13

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1 enzymatic determinations of kecap Page 1 of 13 Contains enzymatic determinations of: - citrate - glycerol - glucose, fructose, starch - galactose - L-glutamic acid - formic acid - malate - ethanol - acetate

2 enzymatic determinations of kecap Page 2 of 13 Determination of Citrate Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, Principe: Citrate Lyase (CL) catalyzes the reaction: Citrate Oxalacetate + Acetate Malate Dehydrogenase (MDH) catalyzes the reaction: Oxalacetate + NADH + H + L-Malate + NAD +. and Lactate Dehydrogenase (LDH) catalyzes the reaction: Pyruvate + NADH + H + L-lactate + NAD + The decrease in NADH can be measured at 340 nm. Reagensia - Buffer: Solve 6.37 g Glycylglycin in ca. 200 ml bidest, adjust to ph 7.8 with ca. 11 ml 5 M NaOH, add 8.9 ml of ZnCl-solution (80mg/100ml). Adjust to 250 ml. The buffer can be kept for four weeks at 4 o C. - NADH-solution: solve 30 mg NADH-Na 2 and 60 mg NaHCO 3 with 6 ml bidest. This buffer can be kept for 4 weeks at 4 o C - MDH/LDH: 0.1 ml MDH, 0.4 ml (NH 4 ) 2 SO 4 (3.2M) ml LDH. This solution can be kept for 1 year at 4 o C. - CL: Solve168 mg lyophilisate with 1 ml cold bidest. Stable for 1 week at 4 o C and 4 when frozen. Add to a cuvette: ml buffer ml NADH ml sample (less than 0.4 g citrate/l) or blanc (water) ml MDH/LDH wait for 3-5 minutes, and measure E340 before against a blanc. Start reaction by the addition of 0.02 ml CL. Measure after 5-10 minutes the E340 after. Calculation c= 3.30 x (E340 after - E340 before )/6.3 correct for the reaction of the blanc!

3 enzymatic determinations of kecap Page 3 of 13

4 enzymatic determinations of kecap Page 4 of 13 Determination of galactose Principe Galactose is oxidized to galacton acid in the presence of galactose-dehydrogenase (Gal- DH). During this reaction NAD is simultanously converted into NADH. The NADH is measured at 340 nm. Methode - Buffer: 4.80 g Na 2 HPO 4, 0.86 g NaH 2 PO 4.H 2 O and 0.10 g MgSO 4.7H 2 O are dissolved in 290 ml water, the ph should be 7.5. This solution can be kept at 4 C for 1 year. - NAD-solution: 60 mg NAD is dissolved in 6 ml bidest - Gal-DH (5mg/ml) Reaction Add to a cuvette: ml buffer ml sample ml NAD-solution Mix and measure E 340 before - add: 0.02 ml Gal-DH Mix and wait until E 340 after is stable, which takes about 30 minutes. Calculation: mm = dilution factor x 3.12 x (E 340 after - E 340 before )/ (0.1 x 6.3)

5 enzymatic determinations of kecap Page 5 of 13 Determination of Glycerol Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, Principe Glycerol kinase (GK) catalyzes the reaction: Glycerol + ATP Glycerol-3-P + ADP ADP is removed by PEP and Pyruvate Kinase (PK): ADP + PEP ATP + Pyruvate The pyruvate is removed by NADH and Lactate Dehydrogenase (LDH): Pyruvate + NADH + H + Lactate + NAD The NADH can be measured at 340 nm Reagensia - Buffer:8.6 gr Glycylglycine and 0.22 g MgSO 4.7H 2 0 are solved in about 240 ml bidest, the ph is set at 7.4 by the addition of ca. 2.2 ml NaOH (5 mol/l). Bidest is added until the volume reaches 250 ml. The buffer can be kept for 3 months at 4 o C - NADH/ATP/PEP solution: 42 mg NADH-Na 2, 120 mg ATP-Na 2 H 2, 60 mg PEP-Na and 300 mg NaHCO 3 are dissolved in 6 ml bidest. This solution can be kept for 2 weeks at 4 o C. - PK/LDH (3 resp. 1 mg/ml) - GK (1 mg/ml) Reaction Add to a macro-cuvette: ml Buffer ml NADH/PEP/ATP ml sample or blanc ml PK/LDH mix and wait 3 min., measure E340 before, start the reaction by the addition of: ml GK Measure after 10 minutes the E340 after calculations

6 enzymatic determinations of kecap Page 6 of 13 E sample =E before - E after E corrected = E sample - E blanc c = 3.12 x E/630

7 enzymatic determinations of kecap Page 7 of 13 Determination of glucose, fructose and starch Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, Principe Before starch can be measured it must be dissolved and converted to glucose. The glucose is measured. Starch is converted to glucose by Amyloglucosidase (AGS): Starch + (n-1)h 2 0 n Glucose The glucose can be determined by the action of hexokinase (HK) and glucose 6-P Dehydrogenase (G6P-DH): glucose + ATP G-6-P + ADP G-6-P + NADP Gluconate-6-phosphate + NADPH + H + Fructose can be determined after the determination of glucose, by the addition of phospho glucose isomerase (PGI). This turns fructose-phospate into glucose phosphate (G-6-P). Fructose is converted into fructose 6-P bij hexokinase. The NADPH can be measured at 340 nm. Reagensia - Citrate-buffer: 88 mg citrate.h 2 O and 170 mg Na 3 -Citrate.2H 2 O are solved in 20 ml bidest. - AGS, use undiluted - Tri-ethanolamine: solve 14.0 g Tri-ethanolamine and 0.25 g MgSO 4.7H 2 O in ca. 200 ml bidest. Adjust ph to 7.6 with ca. 5 ml NaOH (5 M) and fill up to 250 ml with bidest. This solution can be kept at 4 o C for 4 weeks. - NADP-solution: solve 60 mg NADP in 6 ml bidest, this solution can be kept for 4 weeks at 4 o C. - ATP-solution: 300 mg ATP and 300 mg NaHCO 3 are dissolved in 6 ml bidest, this solution can be kept for 4 weeks at 4 o C. - HK/G6P-DH (resp. 2 and 1 mg/ml), use undiluted. (1) Add to a cuvette: ml citrate-buffer ml sample

8 enzymatic determinations of kecap Page 8 of ml AGS (or 0.02 ml bidest incase you only want to measure the free glucose) Mix and incubate for 15 min. at o C, close the cuvettes during this. (2) The glucose can then be determined by the addition of: ml tri-ethanolamine-buffer ml NADP ml ATP (In case you want to measure only glucose the first part (1) can be skipped and simple replaced bij 0.10 ml sample) Measure E340 before after 3 minutes and start reaction: ml HK/G6P-DH Measure after 15 minutes the E340 after (3) For fructose measurement: Add after the measurement of glucose: ml PGI Measure after 30 minutes the E340after(fructose) (=fructose + glucose) Calculation Esample = E after - E before E corrected = E sample - E blanc C = 3.04 x E/630 (or incase of direct glucose determination: 2.82 x de/630 real amount of starch can be calculated from C starch + glucose - C glucose. fructose can be calculated from C(fruc + gluc) - C(gluc) The solvation of starch Add mg sample to a100 ml erlenmeyer, add 20 ml Dimethylsulfoxide and 5 ml HCl (8M). For samples containing fat first add the DMSO. Close erlenmeyer with parafilm and incubate for 30 minutes at 60 o C, cool quickly, add 50 ml of bidest and adjust ph to 4-5 with 5 M NaOH. Adjust the total volume to 100 ml and filtrate if necessarly.

9 enzymatic determinations of kecap Page 9 of 13 Determination of ethanol Principe Alcoholdehydrogenase (ADH) calalyses the reaction: Ethanol + NAD+ Aceetaldehyde + NADH Semicarbazide and a high ph stimulate the quantitative conversion of ethanol, by the reaction of semicarbazide with aceetaldehyde to a semicarbazon. Reagentia - Buffer: M Na-pyrophosphate, M semicarbazide, M glycine. Dissolve 10 g Na 4 P 2 O 7.10H 2 O, 2.5 g Semicarbazide.HCl and 0.5 g glycine in ca. 250 ml bidest, add ca. 5 ml 4N NaOH until a ph of 8.7 is reached, then fill up to 300 ml. The buffer can be kept for 4 weeks at 4oC M NAD (17mg/ml) - Alcoholdehydrogenase Add to a tube: ml buffer ml NAD ml sample ml ADH Close the tubes and incubate in a waterbath of 25 o C for 70 minutes. Measure at 340 nm. Calculations mm ethanol = E x 5.02/6.2

10 enzymatic determinations of kecap Page 10 of 13 Determination of Malate Principe Malate-dehydrogenase (MDH) catalyses the reaction: L-malate + NAD+ Oxalacetate + NADH Oxalacetate is removed by hydrazine. Reagentia - Buffer: 0.5 M Glycine, 0.4 M Hydrazine. Dissolve 9.4 g glycine and 13.0 hydrazine sulphate in about 150 ml water, adjust the ph to 9.0 with 4 N KOH (about 54 ml) and adjust the volume to 250 ml. Keep the solution at 4 o C mm NAD: 30 mg/ml - MDH Add to a cuvette: ml Buffer ml NAD ml sample ml MDH Incubate for 60 minutes at 25 o C and measure E340

11 enzymatic determinations of kecap Page 11 of 13 Determination of L-glutamate Principe Glutamate dehydrogenase (GlDH) catalyses the reaction: L-glutamate + NAD + H 2 O α-ketoglutarate + NADH + NH4 + α-ketoglutarate is removed by the action of hydrazine Reagentia - Buffer: 0.5 M Glycine, 0.4 M Hydrazine. Dissolve 9.4 g glycine and 13.0 hydrazine sulphate in about 150 ml water, adjust the ph to 9.0 with 4 N KOH (about 54 ml) and adjust the volume to 250 ml. Keep the solution at 4 o C. - GlDH - NAD (17 mg/ml) Add to a cuvette ml Buffer ml sample ml NAD ml GlDH Mix and incubate at room temperature for 45 minutes. Measure E340.

12 enzymatic determinations of kecap Page 12 of 13 Determination of Formic Acid (Lang, E and Lang, H.: Spezifische farbreaction zum direkten Nachweis der Ameisensäre. Z. Anal. Chem. 260, 8-10, 1972) Principle Formiate/formic acid reacts with citric acid in the presence of a acetamide-i-propanol solution and a small amount of alkali, yielding a red colour which can be measured at 515 nm. Reagents a. Reagens solution: 0.5 g citric acid and 10 g Acetamide dissolved in 100 ml isopropanol b. 30% (w/v) sodium acetate solution c. Acetic acid anhydride d. standaard solution: 136 mg sodium formiate in ml H 2 O (20 mm) a. Pipet into test tubes (in duplo) ml sample or standaard (Include a standaard with 0, 5, 10, 15 and 20 mm Na-formiate (make appropriate dilutions of the standaard solution), and a sample of uninoculated medium) ml 30% NaAc ml reagens solution ml HAc b. Incubate for 30 min at 50 o C c. Cool the tube and transfer its content to a cuvette, measure the extinction at 515 nm.

13 Determination of acetate Principle enzymatic determinations of kecap Page 13 of 13 Acetate kinase catalyses the reaction of acetate and ATP to acetyl-p. Hydroxylamine reacts with acetyl-p. FeCl 3 reacts with this complex, yielding a brown colour which can be measured at 540 nm. Reagents a. 0.5 M Tris-HCl (ph 7.4) b. 1.0 M MgCl 2 c. 10% TCA d. 1.25% in 1 M HCl e. 3 M Hydroxylamine (prepare freshly: add to 2.08 g, 4 M KOH till ph , fill up to 10 ml) f. acetate kinase g. ATP h. standaard solution of sodium acetate: 10 mm Prepare a reaction mixture, which contains for every single determination: ml Tris ml MgCl ml Hydroxylamine - 3 mg ATP ml acetate kinase Add to a 4 ml cuvette (do every determination in duplo) ml sample or standaard (0, 4, 6 and 8 mm NaAc). Also include a sample of uninoculated sample ml reaction mixture Incubate 1-2 h at 30 o C Add 0.5 ml TCA-solution Add 2 ml FeCl 3 -solution and mix After 5-30 min, measure E 540.