Table 2 Troubleshooting advice.
Step | Problem | Possible Reason | Suggested solution |
|---|---|---|---|
4.5.3 DNSA colour reagent | Precipitation | Low storage temperature | Heat the DNSA solution (maintaining the temperature ~ 45 °C) under constant stirring for 10–15 min |
4.5.4 starch solution | Cloudy solution | Wrong potato starch reference | Make sure that the potato starch reference used is the same as that recommended in the text |
Starch not fully gelatinized | Make sure that the starch solution is thoroughly heated during preparation | ||
4.4.5 α-amylase solutions | Enzyme preparation not properly dispersed | Solubility issues | Make sure that the solution is mixed homogenously for 20 min in an ice bath. Certain solutions (e.g. pancreatin tested here), can sometimes be cloudy. In the present study, the recommended dispersion step was effective as reflected by the similar repeatability and reproducibility as compared with the other references tested |
If poor solubility is a suspected cause of inconsistent results: Sonication may enhance solubility and subsequent centrifugation may reduce cloudiness37. However, depending on the pre-treatment followed, removal of insoluble material may reduce enzymatic activity by as much as 10%38. It is therefore recommended to investigate the impact of changing the enzyme preparation method by comparing results obtained when using different pre-treatments. Regardless of the method selected, the procedure followed when determining enzyme activity should be adopted for subsequent experiments | |||
4.6 enzymatic assay | Inconsistent results and difficulty maintaining target temperature during incubations | Different instruments may require different temperature settings to ensure that the correct temperature is reached within the microtubes | Check the temperature reached within the microtubes: |
1- Set the water bath or heating block to 37 °C | |||
2- Place a microtube containing 1 mL of water in it | |||
3- Monitor internal temperature using a thermocouple until equilibrium is reached | |||
4- If the temperature inside the microtube is not 37 °C, adjust the settings as appropriate and continue monitoring until an internal temperature of 37 °C is reached and maintained stable over at least 15 min | |||
Substrate solution not at 37 °C | The substrate solution should be pre-heated to 37 °C to ensure that the temperature equilibrium is reached quickly | ||
High variability between repetitions | Inconsistent sampling times | It is important to be accurate to the secondSignificant (> 5 s) deviations from the sampling times and failing to immediately inactivate α-amylase straight after by dispensing the sample into a microtube pre-filled with DNSA are likely to have a significant impact on the results | |
Inconsistent absorbance values | Dilution with water performed after boiling | The addition of water to the sample/calibrator and DNSA mixtures should only be carried out after these are heated to 100 °C for 15 min | |
Flat or nonlinear reaction curve1 | Enzyme solution too concentrated | Re-test the product at lower concentrations | |
Absorbance close to zero and (almost) flat reaction curve | Enzyme solution too diluted | Re-test the product at higher concentrations |
