Enzytec Glucose Remover

Transcription

1 R-Biopharm AG Enzytec Remover Product Information Ready-to-use All reagents ready to use and stable for handling 32 samples Up-to-date Corresponds to the German law 64 for testing tose-free samples

2 Intended use The Remover kit is especially helpful when testing lactose-free samples in conjunction with the tose/ kit ( ). In Germany, this combination is mandatory by law ( 64-Methode L ). In addition, the Remover kit can be used in every case where a glucose excess must be eliminated. This can be the case when using for example the Sucrose/ ( ) or the Sucrose//Fructose ( ) assays for the determination of sucrose in honey. The procedure can also be applied for the Maltose/ Sucrose/ ( ) and the Starch ( ) assays. Test principle Several enzymatic sugar tests from the Roche product line use the glucose pathway with glucose dehydrogenation and production of NADH: tose/ ( ), Sucrose/ ( ), Sucrose//Fructose ( ), Maltose/ Sucrose/ ( ) and Starch ( ). All these tests do not work properly when the sample contains a large excess of glucose, because the difference between the two cuvettes will be low and not reproducible (ΔA < 0.100). For this reason, the instructions for use of these assays include a special procedure to remove the glucose excess from the sample, by using the enzymes Oxidase and catalase: D- + H 2 O + O 2 GOD D-Gluconate + H 2 O 2 2H 2 O 2 Catalase 2H 2 O + O 2 Since glucose has been removed, it is possible to perform the enzymatic tests listed above with an increased sample volume, and thus reach a high sensitivity and precision. The test-kit (E3400) contains the reagents that are needed to apply this procedure on 32 samples: buffer glucose oxidase catalase control sample (1 g/l lactose, 1 g/l sucrose, 25 g/l glucose) The control sample is a liquid solution. It contains 25 g/l glucose and 1 g/l lactose in order to simulate the concentrations in lactose-free milk (galactose has been omitted). It also contains 1 g/l sucrose in order to simulate a sample with low sucrose and high glucose concentrations. The control sample is handled like a fluid sample where the glucose excess must be removed via the Remover procedure (simplified procedure). 2

3 The application for the Remover is different depending if the sample is liquid or solid: for liquid sample, the application starts with glucose oxidation and continues with Carrez clarification; the whole procedure is performed in the same tube and is faster, so it is called simplified procedure in the insert; it has also the advantage that only one dilution step takes place, thus increasing the sensitivity of the test for solid samples, extraction and Carrez clarification are performed first; an aliquot of the sample is used for glucose oxidation, leading to an additional dilution of the sample (factor 2) and thus reducing the sensitivity of the test Measurement of lactose-free products Milk contains around 50 g/l lactose, which must be eliminated by adding the enzyme tase. This leads to the hydrolysis of lactose into 25 g/l galactose + 25 g/l glucose. The efficiency of the lactose elimination is > 99 %, so the remaining lactose concentration in the milk is usually below 0.5 g/l (0.05 %) while the accepted limit is 0.1 %. The lactose content of food is differentiated into three levels: low-lactose (< 1 %) very low lactose (< 0.1 %) lactose-free (< 0.01 %) Please note that the limit of 0.01 % is only for lactose-free products where no milk has been used as raw material (for example soya drinks). It is extremely difficult to reach for products based on milk, because this means that the lactose hydrolysis should be higher than 99.9 %. For milk-containing products, the limit of 0.1 % is used in almost every country. These products cannot be described as lactose-free, because lactose has been present in the raw material and is still present at very low concentrations (< 0.1 %). Measurement with the tose-galactose test The measurement is very different depending on the two situations A and B described here. A) The tose-free product is not based on milk When there is no milk included in the raw materials, lactose has never been present in the product. For example, lactose can be tested when products are manufactured with soya (e.g. soya drink or soya yoghurt), but in the same manufacturing plant than milkcontaining products. In this case, using the tose/galactose kit will allow proving that the lactose concentration is indeed at zero (no contamination of the product). 3

4 The Lambert-Beer law allows to calculate the sensitivity of the test, as stated in the insert under point 4: with ΔA = and v = 500 µl, the detection limit is 7 mg/l (7 ppm). But the samples must be pre-treated for clarification. If we take as example the Carrez clarification with 5 g sample weight into 100 ml (= 50 g/l), the calculation will be: lowest detection limit = 7 mg/l (v = ml and ΔA = 0.020) content = [7 (mg/l) / 50 (g/l)] x 100 = 14 mg/100 g = % (140 ppm). This detection limit is sufficient if the limit for lactose has been set at 0.1 % (1 g/l). It is even possible to work with ΔA = 0.050, which is our recommendation for the LOD: lowest detection limit in the test c = 18 mg/l (v = ml and ΔA = 0.050) content = [18 (mg/l) / 50 (g/l)] x 100 = 36 mg/100 g = % (360 ppm). But since the product never contained lactose, the limit as lactose-free product is usually set to 0.01 %. In this case, it is necessary to improve the detection limit. For this purpose, we recommend extracting 18 g sample into 100 ml during Carrez clarification: C = 3.3 x x = g/l (v = 500 µl and ΔA = 0.050) 6.3 x 1 x 0.5 x 1000 Content = g/l lactose x 100 = 0.01 % 180 g/l sample extract If the sample result is below ΔA = 0.050, this means that the lactose concentration is below 0.01 %. The Carrez reaction with a high sample weight (18 g/100 ml) is difficult but possible when done properly: prepare fresh solutions of Carrez-I and Carrez-II mix thoroughly after each addition (5 ml of Carrez-I and mix, add 5 ml Carrez II and mix) adjust ph between The mixture obtained is very turbid and will hardly pass through a filter. For this reason we recommend: either to make a first filtering with a 150 µm paper and then a second filtering with paper around 5-10 µm (or syringe filter 0.45 µm) or to make first a centrifugation step, remove the fatty layer on the top with a spatula, and filter the liquid part with a filter of 5-10 µm (or syringe filter 0.45 µm) 4

5 If the normal Carrez procedure does not succeed to clarify the sample, because the protein concentration is too high for the specified weight (18 g/100ml), two possibilities exist: double the amount of the Carrez solutions (10 ml of each Carrez solution) or use concentrated Carrez solutions (5 ml of Carrez I solution = 15 g K 4 [Fe(CN) 6 ] x 3H 2 O per 100 ml and 5 ml of Carrez II solution = 30 g ZnSO 4 x 7 H 2 O per 100 ml) B) The lactose-free product is based on milk If the product contains milk as raw material, the measurement of the low lactose level (< 0.1 %) is very difficult with the tose/galactose test, because of the high galactose concentration in the sample (25 g/l). Indeed, when the very-low lactose sample (usually < 0.5 g/l) is tested, the dilution during Carrez is 1:50 or 1:100, so the lactose concentration will be 5 to 10 mg/l, which is below the LOD of the test (18 mg/l with v= and ΔA = 0.050). But it is not possible to increase the sample weight during Carrez precipitation, because the 25 g/l galactose will also react in the test. So a compromise must be found: the dilution must be low enough to detect the low concentration of lactose the dilution must be high enough to measure the high concentration of galactose This question was investigated at R-Biopharm, and the result was that a 1:25 dilution is a good compromise, as shown in the results below. The first table shows that it is possible to measure precisely a sample containing 1 g/l lactose (0.1 %). The ΔA is low since it is around 0,020 (ΔA), but the 4 measurements are not too far from the target. Test Sample Dilution A1 A2 A2-A1 ΔA ΔA g/l Recovery LAC Blank ACS 1 g/l % ACS 1 g/l % ACS 1 g/l % ACS 1 g/l % GALAC Blank ACS 1 g/l ACS 1 g/l ACS 1 g/l ACS 1 g/l

6 The second table shows the same, but for a sample containing also 25 g/l galactose (so similar to lactose-free milk). The galactose signal (ΔA) is therefore high, and generates big differences when it must be subtracted from the lactose result. For this reason, the results vary from 0.9 g/l down to 0.48 g/l (48 % from the expected 1 g/l). Test Sample Dilution A1 A2 A2-A1 ΔA ΔA g/l Recovery LAC Blank g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % GALAC Blank g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % 1.0 g/l % Conclusion The results in the tables above are below the expected target value, because the specific lactose signal is "covered" by the unspecific galactose signal that is subtracted in the calculation. In order to be really precise in the determination of the lactose concentration, it would be necessary to repeat the measurement between 5 and 10 times. Therefore, we do not recommend this method and we have developed the Enzytec remover kit shown in the next pages. 6

7 Measurement with glucose oxidation and tose/ testing Principle As stated above, the principle is to remove the glucose excesss (25 g/l) from the sample, which allows performing the tose/ test with 500 µl sample volume and thus increasing the sensitivity and precision. We compare here: the classical method without glucose oxidation and with tose/galactose testing the new method with glucose oxidation and tose/ testing The tables below shows the results obtained with following samples: control (C) = 1 g/l lactose (0.1 %), 25 g/l galactose (2.5 %), 25 g/l glucose (2.5 %) milk (M) = lactose-reduced milk from a food shop Test tose/galactose testing tose/ testing Control 1 Milk 1 Control 2 (+GOD) Milk 2 (+GOD) Oxidation No Yes (simplified method E3400) Carrez Clarification Yes Yes In all cases, the sample volume was 0.5 ml and ended in 10 ml during Carrez clarification (dilution 1:20) tose/galactose results D-Galactose tose / Galactose Cuvette Sample Dist. water Sol. 1 Susp. 2 Sol. 3 Dist. water Susp. 4 - ΔA sample ΔA g/l % Rec Blank ACS Control Control Mix. Incubate for 20 min at room temp. Mix. Wait approx. 2 min and read Control Milk Milk Milk Blank ACS Control Control Control Milk Milk Milk Mix. Wait approx. 20 min and read 7

8 tose/ results tose / Galactose D-Galactose Cuvette Sample Dist. water Sol. 1 Susp. 2 Blank Mix. Incubate for 20 min at room temp. Sol. 3 Dist. water Mix. Wait approx. 2 min and read Susp Mix. Wait approx. 20 min and read ACS Control 2 Control 2 Control 2 Milk 2 Milk 2 Milk Blank ACS Control 2 Control 2 Control 2 Milk 2 Milk 2 Milk ΔA sample ΔA g/l % Rec These results show that: with the tose/galactose test, the control and the milk have not been pre-treated so there is an excess of galactose. The resulting ΔA lac is very low because the difference between the 2 cuvettes is very low, so the result is not reproducible (from 64 to 145 % recovery) and it is necessary to perform the test several times. with the tose/ test, the control and the milk have been pre-treated with GOD, and the sample volume in the test has been increased to 500 µl. The resulting ΔA is above and is reproducible (from 99 to 107 % recovery). Therefore, one test is enough. For this reason, we recommend using the combination of oxidation and tose/ testing (with increased sample volume). In Germany, this combination is mandatory by law ( 64-Methode L ). 8

9 Calculation of the sensitivity The sensitivity of tose/ testing is calculated with the Lambert-Beer formula: c = x Δ A (g/l) for lactose With the tose/ procedure described above, the sample volume is increased to ml and the lowest ΔA that we recommend is (A), so the lowest detection limit of the tose/ test is 18 mg/l. Depending on the sample dilution, the sensitivity of the application will be: with the protocol described above (0.5 ml sample in 10 ml), the dilution is 1:20 so the method is able to detect 360 mg/l (0.036 %) with the simplified protocol in the Remover kit (1 ml sample in 10 ml), the dilution is 1:10 so the limit is 180 mg/l (0.018 %) for the solid sample application in the Remover kit (20 g in 100 ml for Carrez, plus dilution 1:2 for glucose oxidation), the sensitivity is [(0.018 / 200)*100] x 2 = % in all cases, the sensitivity is sufficient for milk-based products (< 0.1 %) For lactose-free products having no milk as raw material, the limit is 0.01 % (0.1 g/l) and we recommend using the simple tose/galactose test as described previously. But if the same limit of 0.01 % should be applied to milk-based products, it will be necessary to apply the combination of Remover and tose/ testing, while increasing the weight of sample. We recommend to take ΔA = as measurement limit, so the calculation shows that it is necessary to weight 1.8 ml of milk into 10 ml during sample preparation: C = x x = g/l (with v = 500 µl and ΔA = 0.050) 6.3 x 1 x 0.5 x 1000 Content = g/l lactose x 100 = 0.01 % 180 g/l sample extract With this protocol, every sample below A = is below 0.01 % and is therefore lactose-free. With this high sample amount (1.8 ml in 10 ml), it will take more time to achieve the oxidation of, so we recommend to incubate overnight. For solid sample, the protocol leads to an additional dilution of factor 2, so the limit will be achieved only with ΔA = or by doubling the sample amount to 36 g in 100 ml (which will be difficult to clarify). 9

10 So overall, we don t recommend applying the limit of 0.01 % (0.1 g/l) to milk-based products: during the manufacturing process, the efficiency of lactose hydrolysis with tase enzyme must be > 99.9 % (from 50 g/l to 0.05 g/l = factor 10000) during the testing procedure, the sample weight must be increased to 18 g/100 ml for liquid samples and the oxidation from 3 h to overnight; for solid samples, the sample weight must be increased to 36 g/100 ml or the ΔA must be reduced to (A). Performance data of the kit As seen above, the measurement of lactose-free products is not possible when lactose has been enzymatically removed, because there is a large excess of galactose and glucose (25 g/l each). For this reason it is necessary to remove the glucose excess with the kit (E3400). Once the glucose has been removed, the sample is tested via the tose/ kit with an increased sample volume. This procedure is mandatory by law in Germany ( 64). In the instructions of the kit (E3400), two protocols are described: the general protocol according to the German law ( 64) that is suited for solid or liquid samples; sample extraction starts by Carrez clarification with g sample; then 5 ml of this sample are transferred into a separate tube for the glucose oxidation the simplified protocol that is applicable only to liquid samples; the whole procedure takes place in the same tube with glucose oxidation first, followed by Carrez clarification The samples were tested as described in these two procedures, and some relevant results are shown below. 10

11 Incubation time for GOD The glucose oxidation was measured via the Roche assay ( ), with following results: Repeats Samples Recovery (%) Blank assay control solution Remover control sample without oxidation Same after 1/2 h oxidation c (g/l) c (g/l) c (g/l) Oxidation [%] h oxidation c (g/l) Oxidation [%] h oxidation c (g/l) Oxidation [%] h oxidation c (g/l) Oxidation [%] Overnight oxidation c (g/l) Oxidation [%] The conclusion is that the minimum oxidation time is 3 h, and it can be extended to overnight incubation. Attention: When sucrose is tested, the incubation time is limited to 3 h. If the incubation is performed overnight, part of the sucrose is lost because a side-activity of the GOD causes a partial hydrolysis of sucrose (data not shown). 11

12 Robustness with decreased amount of GOD Two samples were tested: the control sample from the Remover kit a lactose free milk from a food shop Both samples were tested twice (A and B), with a decreasing amount of GOD (100 µl and 75 µl) in order to test the robustness of the kit. Both samples were prepared according to the simplified method, meaning that the calculations below are taking into account a pre-dilution of the samples of 1:10 and a sample volume of 500 µl in the test. The glucose concentration of the control sample is set at 25 g/l. The glucose concentration of the milk sample before oxidation was measured at 27.3 g/l via the Roche kit. Results obtained with the tose/ assay ( ): tose/ cuvette cuvette Sample Concentration (g/l) Target value (g/l) tose calculation Concentration (g/l) Recovery (%) oxidation (%) Blank Control Sample A µl GOD Control Sample B µl GOD Milk 00 µl GOD Milk B 100 µl GOD Control Sample A µl GOD Control Sample B µl GOD Milk A 75 µl GOD Milk B 75 µl GOD These results indicate that, even if the GOD amount is reduced to 75 µl, oxidation takes place with the same efficiency. 12

13 Example of results with the first application method (equivalent to 64) The method equivalent to 64 was applied as described in the kit instructions: cheese and milk are weighed into 100 ml for Carrez clarification; then 5 ml are oxidated in 10 ml total volume (dilution = 1:2), and 0.5 ml are used for the test the control sample from the kit E3400 does not need Carrez clarification; it is treated for oxidation by adding 1 ml sample (instead of 5 ml), 2 ml tri-ethanolamine buffer (reagent 1), 100 µl GOD solution (reagent 2), 10 µl Catalase (reagent 3) and ml water (instead of ml); the dilution factor for result calculation is 10 (instead of 2). the target value for glucose was measured with the Roche assay before glucose removal; the glucose oxidation was calculated by comparing glucose concentration before and after glucose removal. The first table contains results with milk samples and shows that: the recovery of the control is excellent (99.5 %) the lactose content of the samples is below the limit of 0.1 % for milk-based products (~25 g/l) is removed by more than 98 % in 3 h oxidation time. Extraction as solid samples tose/ cuvette cuvette tose calculation Weight (g) in Volume (ml) Concentration (g/l) Target value (g/l) Content (g/100g or %) Recovery (%) oxidation (%) Sample Water Control E Milk 1 (A) Milk 1 (B) Milk 2 (A) Milk 2 (B)

14 The second table contains results with cheese samples and shows that: the recovery of the control is excellent (103 %) the lactose content of all sample is very low; although these are all milk-based products, they are not far from the limit for non-milk products (0.01 %) from cheese is removed by more than 98 % in 3 h oxidation time. For the goat cheese, the glucose concentration was already very low with 17 mg/kg before removal. After glucose oxidation it was below the measuring range of the test and therefore not reliable, and this led to variable results from 77 % to 128 %. Extraction as solid samples tose/ cuvette cuvette tose calculation Weight (g) in Volume (ml) Concentration (g/l) Target value (g/l) Content (g/100gor %) Recovery (%) oxidation (%) Sample Water Control E Mozzarella Mozzarella B Goat cheese Goat cheese B Fresh cheese Fresch cheese B Example of results with the second application (simplified procedure for liquid samples only) The simplified method for liquid samples was applied as described in the kit instructions: milk is processed by pipeting 1 ml sample directly into the 50 ml tube were oxidation and Carrez clarification take place; yogurt is distributed in several tubes for centrifugation and the supernatant is taken as liquid sample after ph adjustment all samples are calculated with a dilution factor of 1:10 and a sample volume of 0.5 ml, and the results are given as liquid samples in g/l the target value for was measured with the Roche assay before glucose removal; the glucose oxidation was calculated by comparing glucose concentration before and after glucose removal. 14

15 The results in the table below show that: the recovery of the control is excellent (> %) the lactose content of the samples is below the limit of 0.1 % (1 g/l) for milk-based products (~ 25 g/l) is removed by more than 95% in 3 h oxidation time, even with a reduced amount of GOD the reproducibility of the method is good (each samples was tested twice) tose/ cuvette cuvette Sample Concentration (g/l) Target value (g/l) tose calculation Concentration (g/l) Recovery (%) oxidation (%) Blank Control Sample A µl GOD Control Sample B µl GOD Milk 00 µl GOD Milk B 100 µl GOD Control Sample A µl GOD Control Sample B µl GOD Milk A 75 µl GOD Milk B 75 µl GOD For further information or orders please contact R-Biopharm AG: Product Management: Dr. FranÇois Guillot Phone: xxxxxxx - xxxxxxxx Fax: +49 (0) f.guillot@r-biopharm.fr International Sales: Phone: +49 (0) Fax: +49 (0) sales@r-biopharm.de Order Department: Phone: +49 (0) Fax: +49 (0) orders@r-biopharm.de 15