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
enzymatic determinations of kecap Page 2 of 13 Determination of Citrate Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, 1983. 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) + 0.5 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: - 2.80 ml buffer - 0.10 ml NADH - 0.20 ml sample (less than 0.4 g citrate/l) or blanc (water) - 0.02 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!
enzymatic determinations of kecap Page 3 of 13
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: - 2.90 ml buffer - 0.10 ml sample - 0.10 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)
enzymatic determinations of kecap Page 5 of 13 Determination of Glycerol Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, 1983. 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: - 2.90 ml Buffer - 0.10 ml NADH/PEP/ATP - 0.10 ml sample or blanc - 0.01 ml PK/LDH mix and wait 3 min., measure E340 before, start the reaction by the addition of: - 0.01 ml GK Measure after 10 minutes the E340 after calculations
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
enzymatic determinations of kecap Page 7 of 13 Determination of glucose, fructose and starch Method: Boehringer Mannheim, Methoden der enzymatischen Lebensmittel-analytik mit Einzelreagentien, 1983. 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: - 0.20 ml citrate-buffer - 0.10 ml sample
enzymatic determinations of kecap Page 8 of 13-0.02 ml AGS (or 0.02 ml bidest incase you only want to measure the free glucose) Mix and incubate for 15 min. at 55-60 o C, close the cuvettes during this. (2) The glucose can then be determined by the addition of: - 2.50 ml tri-ethanolamine-buffer - 0.10 ml NADP - 0.10 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: - 0.02 ml HK/G6P-DH Measure after 15 minutes the E340 after (3) For fructose measurement: Add after the measurement of glucose: - 0.01 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 100-1000 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.
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: 0.075 M Na-pyrophosphate, 0.075 M semicarbazide, 0.021 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. - 0.024 M NAD (17mg/ml) - Alcoholdehydrogenase Add to a tube: - 4.8 ml buffer - 0.1 ml NAD - 0.1 ml sample - 0.02 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
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. - 40 mm NAD: 30 mg/ml - MDH Add to a cuvette: - 2.5 ml Buffer - 0.2 ml NAD - 0.2 ml sample - 0.02 ml MDH Incubate for 60 minutes at 25 o C and measure E340
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 - 2.00 ml Buffer - 1.00 ml sample - 0.20 ml NAD - 0.05 ml GlDH Mix and incubate at room temperature for 45 minutes. Measure E340.
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 100.0 ml H 2 O (20 mm) a. Pipet into test tubes (in duplo) - 0.5 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) - 0.04 ml 30% NaAc - 1.0 ml reagens solution - 3.5 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.
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 7.2-7.5, 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: - 0.05 ml Tris - 0.05 ml MgCl2-0.125 ml Hydroxylamine - 3 mg ATP - 0.015 ml acetate kinase Add to a 4 ml cuvette (do every determination in duplo) - 0.25 ml sample or standaard (0, 4, 6 and 8 mm NaAc). Also include a sample of uninoculated sample. - 0.24 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.