Lactose / D - Galactose

Transcription

1 Lactose / D - Galactose Microtiter plate test for the quantitative determination of lactose / D - galactose 140 determinations Article No. E1001 In - vitro test store at 2 8 C ifp Institut für Produktqualität GmbH Berlin, GERMANY

2 EnzymeFast Lactose / D - Galactose Brief information Easy to use enzymatic test in a microtiter plate format for the quantitative determination of lactose and D - galactose in foods, pharmaceutical products and other sample material. The test kit contains all the required reagents and one standard. It has sufficient material for 140 determinations (e.g. 116 lactose determinations plus 4 standard series with 4 calibration points, blank and test control each). For evaluation a microtiter plate photometer (340 nm) is required. Sample preparation Liquid, clear samples: Solid samples: dilute homogenise and extract sample, clarify with Carrez I and II, filtrate and dilute Time required for five samples Sample preparation: approx. 30 min Conducting the test: approx. 80 min Evaluation: approx. 5 min Test characteristics Test volume: 330 µl Measurement in: microtiter plate, 96 wells Wavelength: 340 nm Temperature: C Weighed sample: 1-5 g (ml) Sample volume: µl Blank measurement: water Standard range: Limit of detection: g / 100 g (ml) D - galactose with 5 g (ml) weighed sample, 100 µl sample volume g / 100 g (ml) lactose g / 100 g (ml) lactose monohydrate Limit of quantification: with 1 g (ml) weighed sample, 20 µl sample volume 0.30 g / 100 g (ml) lactose 0.31 g / 100 g (ml) lactose monohydrate Recovery rate: % with 5 g (ml) weighed sample, 100 µl sample volume g / 100 g (ml) lactose g / 100 g (ml) lactose monohydrate EnzymeFast Lactose / D - Galactose E

3 1. Intended use EnzymeFast Lactose / D - Galactose is a UV test in a microtiter plate format for the quantitative determination of lactose and D - galactose in foods, pharmaceutical products and other sample material. The test kit complies with the regulations of the German Offical Collection of Test Methods (ASU) as specified in 64 of the German Food and Feed Code (LFGB). The ASU is published by the German Federal Office of Consumer Protection and Food Safety (BVL). Information on lactose: Lactose (also referred to as milk sugar) is derived from the Latin word lac, lactis for milk, and the suffix -ose for sugar. It is a sugar contained in milk and dairy products in amounts of %. Lactose belongs to the group of disaccharides and consists of the two molecules D - galactose and D - glucose, which form a β-1,4 glycosidic linkage. IUPAC defines lactose as 4 - (β - D - galactopyranosyl) - D - glucopyranose. 2. Principle of the test Lactose is cleaved into D - galactose and D - glucose in the presence of the enzyme β - galactosidase and water at ph 6.6 (Equation 1). β - galactosidase (1) Lactose + H 2 O D - galactose + D - glucose D - galactose is oxidized from β - galactose dehydrogenase (Gal - DH) into D - galactonic acid by nicotinamide adenine dinucleotide (NAD) at ph 8.6 (Equation 2). Gal - DH (2) D - galactose + NAD + D - galactonic acid + NADH + H + The quantity of NADH formed according to Equation (2) is equal to the D - galactose quantity and can be used to back - calculate the lactose content. D - galactose is used to calibrate the test system. The absorbance of the NADH formed is measured in a microtiter plate photometer at 340 nm. EnzymeFast Lactose / D - Galactose E

4 3. Contents of the test kit 1) 1 x UV microtiter plate, 96 wells (contained in each test kit) 2) Solution 1: Lyophilisate with citrate buffer, ph approx. 6.6; approx. 15 mg NAD; approx. 37 mg magnesium sulphate Solubilize contents of bottle 1 with 2.7 ml of sterile deionized or distilled water. The solution will remain stable for three months if kept at 2-8 C. 3) Solution 2 (red screw cap): 0.78 ml β-galactosidase suspension, approx. 550 U 4) Solution 3: 2 x 24 ml potassium diphosphate buffer, ph approx ) Solution 4 (green screw cap): 3.1 ml Gal - DH suspension, approx. 5 U 6) Solution 5: 2 ml D - galactose standard solution: 10.0 g / 100 g (ml) for calibration 7) Solution 6: 2 ml lactose monohydrate control solution: 5.0 g / 100 g (ml) Bring all solutions to C prior to use. Mix by carefully shaking or swirling immediately before pipetting. 4. Required devices and reagents 4.1 Devices and accessories 1) Analytical scale 2) Microtiter plate photometer (340 nm wavelength) 3) Water bath, heatable to 60 C 4) 50 ml centrifuge tubes 5) 100 ml volumetric flask 6) Erlenmeyer flask 7) Funnel and pleated filter 8) 1.5 or 2.0 ml reaction tubes 9) Pipette tips for micropipettes µl; µl 4.2 Reagents 1) Water, deionized or distilled (= water) 2) Carrez I solution (potassium ferrocyanide [II]): Dissolve 3.60 g of K 4 [Fe(CN) 6 ] 3 H 2 O in 100 ml water (stable for six months if stored at C) 3) Carrez II solution (zinc sulphate): Dissolve 7.20 g of ZnSO 4 7 H 2 O in 100 ml water (stable for six months if stored at C) 4) NaOH 0.1 mol / l: Dissolve 0.4 g NaOH pellets in 100 ml water (stable for six months if stored at C) EnzymeFast Lactose / D - Galactose E

5 5. Storage instructions Store the test kit / the reagents at 2-8 C. 6. Sample preparation - Clarify samples, emulsions and solutions containing protein with Carrez reagents (see below). - Clarify turbid solutions by means of filtration. - Degas solutions containing carbonic acid e. g. by means of filtration. - Treat strongly coloured solutions with polyamide or polyvinylpolypyrrolidone (PVPP, e. g. 1 g / 100 ml sample). - Extract samples containing fat with hot water in a 100 ml volumetric flask (extraction temperature above the fat melting point); leave to cool to room temperature, make up to 100 ml, place into an ice bath for 15 min and then filtrate; alternatively, clarify with Carrez reagents after extraction with hot water. Increasing sensitivity - Increase the amount of weighed sample to max. 5 g (ml) (to be taken into account in the evaluation). - Increase the sample volume to max. 100 µl (Sections 7.3 and 7.4, Step 4): In Sections 7.3 and 7.4, Step 5, the volume of Solution 3 must be reduced accordingly in that case (example: a sample volume of 100 µl requires reducing Solution 3 by 80 µl). 6.1 Foods without Carrez clarification (e. g. samples low in protein) 1) Weigh 1 g (ml) of homogenised sample into a 50 ml centrifuge tube. 2) Add approx. 30 ml water (60 C), seal the tube and extract at 60 C for 15 min; shake well at least five times during extraction. 3) Leave to cool to below 30 C. 4) Adjust ph to ) Transfer quantitatively to a 100 ml volumetric flask and rinse centrifuge tubes. 6) After cooling to room temperature, make up to 100 ml with water, mix and filtrate into an Erlenmeyer flask through a pleated filter; discard the first millilitres. 7) If necessary, dilute the sample extract with water in 1.5 or 2.0 ml reaction tubes, depending on the concentration of lactose (= dilution factor F). Use prepared samples on the same day. Note: Steps 2 to 4 can be omitted for liquid, clear, colourless and close to neutral samples. EnzymeFast Lactose / D - Galactose E

6 6.2 Foods with Carrez clarification 1) Weigh 1 g (ml) of homogenized sample into a 50 ml centrifuge tube. 2) Add approx. 30 ml of water (60 C), seal tube and extract at 60 C for 15 min; shake well at least five times during extraction. 3) Add 5 ml Carrez I solution and mix. 4) Add 5 ml Carrez II solution and mix. 5) Leave to cool below 30 C. 6) Adjust ph to (in the case of chocolate, e. g., by adding 7-10 ml 0.1 mol / NaOH). 7) Transfer quantitatively to a 100 ml volumetric flask and rinse centrifuge tube. 8) After cooling to room temperature, make up to 100 ml with water, mix and filtrate into an Erlenmayer flask through a pleated filter; discard the first millilitres. 9) Use clear, also slightly opalescent solution for the test. 10) If necessary, dilute the sample extract with water in 1.5 or 2.0 ml reaction tubes, depending on the lactose concentration (= dilution factor F). Use prepared samples on the same day. 6.3 Examples for sample preparation a) Milk chocolate with an expected lactose content of: 10.0 g / 100 g (equals a D - galactose content of 5.0 g / 100 g) Weighed sample: 1 g Dilution factor F of the sample extract: dilute 1 : 2 (F = 2) in order to reach the middle range of the standard curve (dispense 200 µl of sample into a 1.5 or 2.0 ml reaction vial, add 200 µl of water, mix). Sample volume in the test: 20 µl b) Dark chocolate with an expected lactose content of: < 0.1 g / 100 g (equals a D - galactose content of < 0.05 g / 100 g) Weighed sample: 5 g Dilution factor F of the sample extract: undiluted (F = 1) Sample volume in the test: 100 µl c) Pastry with an expected lactose content of: 5.0 g / 100 g (equals a D - galactose content of 2.5 g / 100 g) Weighed sample: 1 g Dilution factor F of the sample extract: undiluted (F = 1) Sample volume in the test: 20 µl EnzymeFast Lactose / D - Galactose E

7 7. Test procedure / assay setup 7.1 Dilution of the standard Dilute Solution 5 (D - galactose standard 10.0 g / 100 g [ml]). To do so, dispense water into 1.5 or 2.0 ml reaction tubes (A - D) as shown in Table 1. Add 100 µl of Solution 5 to A, mix (= g / 100 g (ml)). Transfer 100 µl from A to B, mix (= g / 100 g (ml)). Continue according to Table 1. Table 1: Dilution of the D - galactose standard *) Standard series* Water Standard g / 100 g (ml) A 100 µl µl of Solution B 100 µl µl of A C 300 µl µl of B D 300 µl µl of C The standard series already contains the extraction dilution factor of 1 : 100 for a sample amount of exactly 1 g. 7.2 Determination of lactose and free D - galactose (D - galactose total ) Preparation of solutions The lactose must be cleaved enzymatically. For each required well of the microtiter plate, mix 15 µl of Solution 1 and 5 µl of Solution 2 in a 1.5 or 2.0 ml reaction vial (see Table 2 for examples). Table 2: Premix Solutions for the required number of samples (standards, blank, control and pipetting reserve are included in the calculation below) Number of Solution 1 Solution 2 Total samples µl + 40 µl 160 µl µl + 45 µl 180 µl µl + 50 µl 200 µl µl + 60 µl 240 µl µl + 65 µl 260 µl µl + 70 µl 280 µl µl + 75 µl 300 µl µl + 85 µl 340 µl µl + 90 µl 360 µl µl + 95 µl 380 µl µl µl 480 µl µl µl 600 µl Note: After pipetting Solutions 1 and 2 the mixture can be stored up to four weeks at 2-8 C. EnzymeFast Lactose / D - Galactose E

8 a) Assay setup The enzyme solution (Solution 1 + 2) is dispensed into the separate wells of the microtiter plate and blank, standards, assay control and sample solutions are added. For the individual steps, see Table 3. Note: - Always rinse the pipette tip with the solution to be pipetted and make sure it contains no air bubbles. - After the measurements, rinse the microtiter plate thoroughly with tap water followed by deionized or distilled water, and then leave to air dry. The plate can then be used for further measurements. - You do not need to additionally determine free D - galactose if the sample does not contain any free D - galactose. Table 3 shows the pipetting scheme for the determination of lactose and free D - galactose. Table 3: Pipetting scheme for the determination of lactose + free D - galactose Lactose + free D - galactose Blank well The reaction is completed when the measured absorbance remains constant; this is usually the case after 30 minutes. Standard/ Control well Sample well Notes 1. Solution µl 20 µl 20 µl Dispense into wells. 2. Water 20 µl - - Add and mix by pipetting 3. Standard/Control - 20 µl - up and down three 4. Sample times µl (= sample volume) 5. Incubate for 20 min at C. Add and mix by pipetting 6. Solution µl 270 µl 270 µl up and down three times. 7. Incubate for 3 min at C. 8. Measurement 1 A1 blank A1 std. / contr. A1 sample Start reaction by adding Solution 4. Measure absorbance at 340 nm Add and mix by pipetting up and down three 9. Solution 4 20 µl 20 µl 20 µl times; additionally use (green cap) pipette tip to thoroughly stir assay in well. 10. Incubate at C (wait until reaction is completed - approx. 30 min) a). Measure absorbance at 11. Measurement 2 A2 blank A2 std. / contr. A2 sample 340 nm. EnzymeFast Lactose / D - Galactose E

9 7.3 Determination of free D - galactose Assay setup The enzyme solution (Solution 1) is dispensed into the separate wells of the microtiter plate and blank, standards and sample solutions are added. For the individual steps, see Table 4. Note: - For the determination of free D - galactose, Solution 2 is not needed. Hence, it is not necessary to mix Solutions 1 and 2. - Always rinse the pipette tip with the solution to be pipetted and make sure it contains no air bubbles. Table 4 shows the pipetting scheme for the determination of free D - galactose. Table 4: Pipetting scheme for the determination of free D - galactose Free D - galactose Blank Standard b) Sample well well well Notes 1. Solution 1 20 µl 20 µl 20 µl Dispense into wells. 2. Water 20 µl Standard - 20 µl - Add and mix by pipetting up 4. Sample and down three times µl (= sample volume) 5. Incubate for 20 min at C. 6. Solution µl 270 µl 270 µl Add and mix by pipetting up and down three times. 7. Incubate for 3 min at C. 8. Measurement 1 A1 blank A1 standard A1 sample Measure absorbance at 340 nm. Start reaction by adding Solution 4. Add and mix by pipetting up and down three times; 9. Solution 4 20 µl 20 µl 20 µl additionally use pipette tip (green cap) to thoroughly stir assay in well. 10. Incubate at C (wait until reaction is completed - approx. 30 min) c). 11. Measurement 2 A2 blank A2 standard A2 sample Measure absorbance at 340 nm. b) c) Not required if already included in The reaction is completed when the measured absorbance remains constant; this is usually the case after 30 minutes. EnzymeFast Lactose / D - Galactose E

10 8. Evaluation Determine the absorbance differences A (A2 - A1) for blanks, standards, controls, and samples. Subtract A of the blank from A of the corresponding standard / control / sample. Table 5 shows the assay evaluation for Sections 7.2 and 7.3. Note: The raw data can be transferred to the EnzymeFast Excel evaluation sheet. The results are then calculated automatically. The evaluation sheet is available on request. Table 5: Assay evaluation scheme A1 340 nm A2 340 nm A 340 nm Blank A blank 0 g / 100 g (ml) A1 blank A2 blank (A2 blank - A1 blank ) Standards A standard g / 100 g (ml) A A (A A ) A blank g / 100 g (ml) A A (A A ) A blank g / 100 g (ml) A A (A A ) A blank g / 100 g (ml) A A (A A ) A blank Control solution A control solution g / 100g (ml) A1 control A2 control (A2 control - A1 control ) A blank Sample A sample unknown conc. A1 sample A2 sample (A2 sample - A1 sample ) A blank Calibration and evaluation are carried out with D - galactose. For the assay control and samples, the result has to be converted to lactose or lactose monohydrate. 8.1 Calibration The determined absorbance values A standard are plotted against the concentration of the standards in a diagram. Linear regression is used to determine the linear equation (Equation 3), which is then solved for D - galactose [g / 100 g (ml)] (Equation 4). The values of A sample or A control are then substituted into the equation. (3) A = slope conc. D - galactose + y-intercept (4) Conc. D - galactose * = ( A sample y-intercept) F 1 g (ml) 1 20 µl 1 Slope weighed sample [g (ml)] sample volume [µl] * in g / 100 g (ml) F = Dilution factor of the sample extract; 1 based on calibration EnzymeFast Lactose / D - Galactose E

11 Note: The result is expressed as D - galactose. For conversion to lactose or lactose monohydrate, the factors shown in Table 6 should be used: Molecular weight D - galactose g x mol -1 Lactose g x mol -1 Lactose monohydrate g x mol -1 Table 6: Conversion factors for lactose and lactose monohydrate Conversions Conversion to lactose Conversion to lactose monohydrate D - galactose Factor of Factor of Lactose - Factor of Lactose monohydrate Factor of Evaluation examples Determination of lactose content in the absence of free D - galactose If there is no free D - galactose present in the sample, the assay for the determination of the lactose content is prepared as described in Section 7.2. Milk chocolate with an expected lactose content of: 12 g / 100 g Weighed sample: g Dilution factor F of the sample extract: 1 : 3 (F = 3) Sample volume: 20 µl Table 7: Evaluation example A1 340 nm A2 340 nm A 340 nm Blank 0 g / 100 g A1 blank Standards g / 100 g A g / 100 g A g / 100 g A g / 100 g A A2 blank A A ,184 A A A blank (A2 blank - A1 blank ) A standard (A A ) A blank (A A ) A blank (A A ) A blank (A A ) A blank EnzymeFast Lactose / D - Galactose E

12 Table 7 continued: Evaluation example A1 340 nm A2 340 nm A 340 nm Control solution 5.0 g / 100 ml A1 control 0.07 A2 control A Control solution (A2 control - A1 control ) A blank 0.43 Sample unknown conc. A1 sample A2 sample A Sample (A2 sample - A1 sample ) A blank Fig. 1: Standard curve Control solution: D - galactose in g / 100 ml = ( ) D - galactose in g / 100 ml = 2.50 Lactose monohydrate in g / 100 ml = = 5.00 (target: 5.0 g / 100 ml) Sample: D - galactose in g / 100 g = ( ) 3 x x 20 D - galactose in g / 100 g = 5.99 Lactose in g / 100 g = = 11.4 Lactose monohydrate in g / 100 g = = 12.0 EnzymeFast Lactose / D - Galactose E

13 8.2.2 Determination of the lactose content if the sample contains free D - galactose If the sample contains free D - galactose along with lactose, the assays for total D - galactose (or lactose + free D - galactose) according to Section 7.2 and for free D - galactose according to Section 7.3 are pipetted in parallel in one microtiter plate. The lactose content is then calculated according to Equation (5): (5) Lactose in g / 100 (ml) = (content D - galactose total - content free D - galactose ) x Test properties 9.1 Lactose monohydrate control solution 5.00 g / 100 ml The content of the control solution should be between 4.75 and 5.24 g / 100 ml. 9.2 Specificity The test is specific for D - galactose in the absence of L - arabinose. 9.3 Limit of detection and limit of determination Limit of detection The limit of detection was calculated based on the six-fold standard deviation of the blank. Weighed sample: 5 g (ml) Dilution factor F of the sample extract: undiluted Sample volume: 100 µl Detection limit g / 100 g (ml) D - galactose Lactose Lactose monohydrate EnzymeFast Lactose / D - Galactose E

14 9.3.2 Limit of quantification The quantification limit is defined as the concentration of the lowest standard. Weighed sample: 1 g (ml) Dilution factor F of the sample extract: undiluted Sample volume: 20 µl Quantification limit g / 100 g (ml) D - galactose Lactose Lactose monohydrate Weighed sample: 5 g (ml) Dilution factor F of the sample extract: undiluted Sample volume: 100 µl Quantification limit g / 100 g (ml) D - galactose Lactose Lactose monohydrate Recovery rates The average recovery rates are %. 10. Bibliography The test kit complies with the regulations of the German Offical Collection of Test Methods (ASU) as specified in 64 of the German Foodstuffs and Animal Feed Code (LFGB). The ASU is published by the German Federal Office of Consumer Protection and Food Safety (BVL). BVL L : Bestimmung des Laktose- und Galaktosegehaltes von Milch und Milchprodukten BVL L : Determination of the lactose content in meat products BVL L : Determination of lactose in bread including rolls of bread dough BVL L : Determination of the lactose content of chocolate BVL L : Determination of the lactose content in partially adapted milkbased baby food BVL L : Determination of the lactose content in zwieback for children and zwieback flour Beutler, H. O. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) 3rd ed., Vol. VI, pp , Verlag Chemie, Weinheim, Deerfield Beach / Florida, Basel EnzymeFast Lactose / D - Galactose E

15 11. Abbreviations ASU German Offical Collection of Examination Procedures as specified in 64 of the German Foodstuffs and Animal Feed Code BVL German Federal Office of Consumer Protection and Food Safety A absorbance A absorbance difference Gal - DH β - galactose dehydrogenase LFGB German Foodstuffs and Animal Feed Code NAD nicotinamide adenine dinucleotide NaOH sodium hydroxide EnzymeFast is a registered trademark of ifp Institut für Produktqualität GmbH. ifp also offers analyses as a service. Manufacturer: ifp Institut für Produktqualität GmbH Teltowkanalstr. 2, Berlin, GERMANY Contact Phone +49 (0)30 / Fax +49 (0) E - mail info@produktqualitaet.com Ordering E - mail order@produktqualitaet.com EnzymeFast Lactose / D - Galactose E