Abstract:
Diastase activity is an inherent property of honey and an excellent indicator of the quality of honey.A very low diastase activity indicates that the honey has been subjected to unfavourable temperature conditions. As per FSSAI Regulation limit of diastase activity for honey is minimum 3. A standard solution of starch, capable of developing, with iodine, a colour in a defined range of intensity, is acted upon by the enzyme in the sample under standard conditions. The intensity of the blue colour is measured at intervals by a UV-Visible spectrophotometer. A plot of absorbance against time, or a regression equation, is used to determine the time (tx) required to reach the specified absorbance, i.e., 0.235. The present study is designed to estimate diastase activity of different brands of honey by spectrophotometric (Schade’s) Method. It was observed that, the method was successfully carried out in our laboratory and found that different brands were different diastase activity ranging from 2.5 to 20.69 as diastase number.
Keyword:
Diastase Activity, Diastase number, Honey, Spectrophotometry, Schade’s unit.
Introduction:
Diastase activity is a function of alpha, beta and gamma amylase, that degrade carbohydrate or starch to a short chain sugar like glucose and/or maltose. Diastase activity is expressed as the diastase number in Schades’s unit and is defined as one diastase unit corresponds to the enzyme activity of 1 g honey ,which can hydrolyse 0.01 g of starch in 1 hr at 40 deg C, the activity of this enzyme decreases with the time of storage and that of heating. Enzyme activity is very specific at 37 to 40 deg C.
Main function of honey is to supply energy to our body and reduce muscle fatigue. Honey has known antibacterial action. it is used in medicine for over 5000 years. Only proper function of all amylase can give that benefit to human beings. During processing time of honey, heat is required before storage. Heat processing of honey eliminates the micro organisms responsible for spoilage and reduces the moisture content to a level that inhibits fermentation process. But heating of honey above 40 deg C destroys enzyme activity. So, if honey is mixed with hot milk or water, it is of no use. Main reasons for decrease diastase number are overheating of honey during processing time, another reason for degradation is poor storage condition.
Current regulation as per Regulation No 2.8.1of Food Safety and standard Authority of India (FSSAI), limit of diastase number is not less than 3.
As this parameter is one of the important quality parameter and also has a regulatory requirement, after standardization the method, it can be used for routine analysis of honey samples, and, thereby has a huge potential for business growth.
Materials and Method:
Samples, chemicals and reagents:
Honey samples from 5 different brands were purchased from local market of Kolkata, west Bengal. Solid starch, solid Iodine, Potassium Iodide, Sodium acetate, Sodium chloride were of AR grade and were purchased from Merck, India.
Instrument /equipment:
Shimadzu UV1900 spectrophotometer, thermostatic digital Water Bath and analytical balance were used for this analysis.
Procedure:
10.0 g of honey was taken into a beaker and dissolved completely in approximately 15 ml of water and 5 ml of acetate buffer (pH 5.3) without heating. The solution was transferred quantitatively to a 50 ml volumetric flask containing 3 ml of sodium chloride solution (2.9% w/v) and the volume has been adjusted to the mark with water (sample solution).
Calibration of the Starch Solution / Adjustment of Blue Value:
This procedure was carried out to determine the amount of water that has to be added to the reaction mixture so that the absorbance range of the iodine starch solution is 0.745 to 0.770. 20, 21, 22, 23, 24 and 25 ml of water was pipetted out into 6 suitable glass flasks and 5 ml of dilute iodine solution was added. Starting with the first test tube, 0.5 ml of a mixture containing 10 ml of water and 5 ml of starch solution was added, mixed well by agitating and immediately the absorbance was read at 660 nm against water blank in a 1 cm cell. The other test tubes were proceeded in the same way until an absorbance in the range 0.770 to 0.745 is obtained. The amount of water determined in this way is the standard dilution for every determination carried out with the starch solution.
Determination in the honey sample solution:
10 ml of honey solution was pipetted into a 50 ml flask and placed in water bath at 40°C with a second flask containing about 10 ml of starch solution. After 15 minutes, 5 ml starch solution was pipetted into the honey solution, mixed and the timer was started. At periodic intervals, for the first time after 5 minutes, 0.5 ml aliquots was taken and added rapidly to 5 ml of diluted iodine solution. the amount of water was added (as determined in “Calibration of the starch solution”), mixed well and immediately read the absorbance of each separate solution at 660 nm against a water blank in a 1 cm cell.
Reaction blank:
10 ml of sample solution prepared according to “Preparation of Test Samples” was added to 5 ml of water and mixed thoroughly. 0.5 ml of this solution was removed and added to 5 ml of dilute iodine solution. the amount of water determined in “Calibration of the Starch Solution” was added, mixed well and read the absorbance at 660 nm against a water blank in a 1 cm cell. If there is an absorbance this blank value must be subtracted from the values obtained under “Determination of Test Solution”.
Calculation and Expression of Results:
The diastase activity is calculated as diastase number (DN) as follows:
DN=60*0.10*1.0/tx*0.01*2.0 =300/tx
tx = reaction time in minutes obtained as follows:
The absorbance values of the test sample solutions were plotted against the corresponding reaction times in minutes after subtracting the absorbance of the blank value (see “Blank Value Control”). The regression line is drawn through the measuring points in the range of absorbance 0.155 to 0.456 in order to determine the time tx for absorbance 0.235. There should be at least three points in the absorbance range 0.155 to 0.456. The time for A = 0.235 can also be calculated from the regression equation and this is generally considered preferable to graphical interpolation.
Results and Discussions:
Table 1-5 and Figure 1-5 denotes graphical representation of Absorbance Vs Time and finding (tx) from each graph for each sample.
Table 1, Figure 1: Absorbance Vs Time for Brand 1
| Brand 1 | |
| Time | Absorbance |
| 5 | 0.4942 |
| 10 | 0.3149 |
| 15 | 0.2241 |
| 20 | 0.1554 |
| 30 | 0.0788 |
| 40 | 0.058 |
| 50 | 0.052 |
| 60 | 0.049 |
Table 2, Figure 2: Absorbance Vs Time for Brand 2
| Brand 2 | |
| Time | Absorbance |
| 5 | 0.7198 |
| 10 | 0.7172 |
| 20 | 0.651 |
| 30 | 0.5082 |
| 40 | 0.4572 |
| 50 | 0.3564 |
| 60 | 0.2198 |
Table 3, Figure 3: Absorbance Vs Time for Brand 3
| Brand 3 | |
| Time | Absorbance |
| 5 | 0.6322 |
| 10 | 0.5784 |
| 20 | 0.4513 |
| 30 | 0.3683 |
| 40 | 0.3019 |
| 50 | 0.2029 |
| 60 | 0.1866 |
Table 4, Figure 4: Absorbance Vs Time for Brand 4
| Brand 4 | |
| Time | Absorbance |
| 5 | 0.4946 |
| 10 | 0.4879 |
| 20 | 0.3624 |
| 30 | 0.2592 |
| 40 | 0.1778 |
| 50 | 0.1287 |
| 60 | 0.0936 |
Table 5, Figure 5: Absorbance Vs Time for Brand 5
| Brand 5 | |
| Time | Absorbance |
| 5 | 0.7163 |
| 10 | 0.7065 |
| 20 | 0.7008 |
| 30 | 0.6424 |
| 40 | 0.6251 |
| 50 | 0.5894 |
| 60 | 0.55541 |
From the derived tx values, Diastase number for each brands of honey was estimated and the results are incorporated in Table 6 and Figure 6.
Table 6: Estimation of Diastase number in honey samples
| Diastase Number | |
| Brand-1 | 20.69 |
| Brand-2 | 5.08 |
| Brand-3 | 6.38 |
| Brand-4 | 8.82 |
| Brand-5 | 2.5 |
Figure 6: Estimation of Diastase number in honey samples
Conclusion:
Thus, the method is successfully adopted and diastase number of different brands of honey samples were analysed. The samples showed various diastase numbers ranging from 2.5 to 20.69. This variation may be attributed to heat generated during blending and processing of different brands of honey samples as well as the age and storage condition of honey samples. The adopted method was simple, quick, accurate, and precise, do not require expensive instrumentation, and could be easily incorporated in routine analysis of Honey to meet the requirement of regulatory authorities.