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                            Comparison of Russian and international methods for determination of diastase                                                                      activity in natural honey

                                                      E.U. Balashova, E.V. Alexandrova, I.V.Gadalina
                                     Apis Analytical Centre LLC Report on the international conference
                                                                      Beekeeping-XXI century

                                                                          Moscow, May of 2010

   All natural honey that is stored in compliance with necessary conditions contains enzymes. One of the most important enzymes is amylase. Amylase (or diastase) catalyzes the cleavage reactions of starch and other polysaccharides. Amylase is an important enzyme not so much for the qualities attributed to it, but for its implication in quality control of honey. Amylase is usually present in all kinds of honey. From a quantitative point of view, it is directly associated with other enzymes contained in honey. Since methods for determining the activity of amylase in honey are much more accessible than methods for determining the content of other enzymes, amylase activity is used to determine the relative amounts of the latter. Besides that, diastase is the most stable of all the honey enzymes, therefore its absence or presence in small quantities points out to a violation of honey processing and storage conditions.
   The activity of diastase in honey is determined by diastatic index, which is measured in units of Gothe and is expressed in cubic centimeters of a 1% starch solution, which decomposes at a temperature of 40 in 1 hour in presence of amylolytic enzymes contained in 1 gram of dehydrated honey substance.
   Determination of diastatic index abroad refers to 1g of honey without taking in account the water content. 1 cm3 of starch solution corresponds to 1 unit of Gothe.
   The magnitude of diastatic index depends on several factors, different combination of which results in a dissimilar content of diastase enzyme in different types of honey. Diastatic index in natural high-quality honey is placed within the range of 3 to 50.
   In Russian Federation the quality indicator of honey diastatic index is regulated by GOST 197922001 Natural honey. Technical conditions, GOST R 52451-2005 Monofloral honey. Technical condition, in European Union , USA, Canada and other countries by Codex Alimentarius [1] and Directive of European Council 2001/110/ [2].
   Standard values of diastatic index in Russia, European Union and other countries are shown in table 1.

      Table1. Standard values of diastatic index in Gothe units for Russia and European Union
   
GOST 19792-2001* GOST 52451-2005* Directive
2001/110/**
Codex Alimentarius**
All
types
Acacian Linden Sunflower Buckwheat All Types*** Honey
containing
little of
enzymes
and HMF
<=
15 mg/kg
All types Honey
containing
little of
enzymes
>= 7 >= 5 >= 11 >= 15 >= 18 >= 8 >= 3 >= 8 >= 3


*      taking into account the anhydrous honey substance
**    without accounting for anhydrous honey substance
***  Except for honey used in baking and confectionary industry

   Purpose of the study: assessment of methods for diastatic index determination in honey and use of the data obtained to create GOST pertaining to methods for diastatic index determination. This is a relevant issue at the present, because there are studies being done on standardization of methods for honey quality analysis.

   The cause of the study: : In 2009, with the aim to confirm the competency of the staff, Apis Analytical Centre LLC participated in international comparison trials, which are carried out by Food Environment Research Agency from Great Britain. A control sample of honey FAPAS 2818F was obtained, in which it was necessary to determine the content of water, fructose, glucose, sucrose, 5-hydroxymethilfurfural (by spectrophotometric method and high performance liquid chromatography), as well as the diastatic index. The results were sent to Great Brittan. After a statistic processing of the results obtained in all laboratories participating in the comparison trials, the report produced by Fera agency was published. The results of Apis Analytical Centre LLC and FERA agency for diastatic index are listed in Table 2.

      Table 2. The results of the analysis of honey sample FAPAS 2816F, obtained in
                   Apis Analytical Centre LLC, and those obtained by the FERA agency.

   
Name of the indicator Results of Center Apis
Results of FERA agency
    Data obtained Acceptable range
   Water content, % 18,00 18,21 17,69 - 18,74
   Glucose content, % 30,97 31,85 30,58 33,13
   Fructose content, % 35,92 36,52 35,06 37,98
   Sucrose content, % 1,44 1,70 1,31 2,09
   5-hydroxymethylfurfural    content, mg/kg    (spectrophotometric method) 8,90* 8,76 4,97 12,54
   5-hydroxymethylfurfural    content, mg/kg
   (HPLC method)
9,22 9,08 7,09 11,08
   Diastatic index, in Gothe 5,10**
   4,18***
10,55 7,06 - 14,04


*      according to method of White (GOST R 52834-2007)
**    according to GOST 19792-2001 taking in account the anhydrous honey substance
***  according to GOST 19792-2001 without taking in account the anhydrous honey substance

   The analysis of obtained data showed that all the results of Apis Analytical Centre LLC, with the exception of the diastatic index value, fall within the acceptable range of values for the indicators determined.
   All the methods for analysis that were used by the participants were listed in an attachment to the report of FERA agency. One of the methods was the harmonized method of Schade, recommended by International Honey Comission (IHC) [3].
   Laboratory employees have mastered this technique and compared it with the method given in GOST 19792-2001 Natural honey. Technical conditions.
   Results and discussion. The main procedure stages, equipment and reagents in both methods are similar:
      -  Spectrophotometric method;
      -  Iodimetry;
      - The reaction is carried out in the combined reagent mixture, consisting of a starch solution, acetate          buffer (pH 5,0 - 5,3) and sodium chloride solution.
    The main differences:
       Wavelength, which is used to determine the magnitude of optical density, according GOST 19792 - 580 nm,           for Shade - 660 nm;
      - Calculation of diastatic index according to GOST - based on the optical density of the control and
          trial solutions on the anhydrous substance of honey; according to Shade time tx, at which the           optical density reaches 0.235 (fig.1), is determined from the graph and the calculation is carried out without           taking into account the mass fraction of water.

         

                            

            Fig. 1 Variation of optical density of honey solution during the determination of diastatic index
                      by method of Shade

      Absorbance spectra obtained by both of the methods look similar. (Fig.2)

         

                            

               Fig. 2: The absorbance spectrum of the solution during the determination of the diastatic index of honey

      As it is known from the theory of spectroscopy, for accurate spectroscopic measurements, it is essential to choose the correct wavelength to work with. In most cases it's best to take measurements in the region of maximal absorption, at a wavelength of 540-580 nm (close to the values specified in GOST 19792). It is not recommended to work with the descending region of spectral curve (660 nm according to Sheyd method), because in this case minor deviations in the wavelength value bring to a noticeable inaccuracy in determination of optical density, i.e. to wrong results.
      It should be noted that during the process of starch decomposition by amylase, the region of maximal absorption can be shifted from 580 to 540 nm. It can be seen from the graph that in this wavelength range the optical density of the solution practically remains unchanged, thus to reduce the time it takes for the analysis without significant errors, it is likely to fix the wavelength at 580 nm, as indicated in GOST 19792. In the region of wavelength range 660 - 620 nm, a minor variation in ? results in a significant change in optical density, and hence it brings to major dissimilarities in product
     In our center, a study was performed to test the dependency of diastatic index values with the type of honey, the research method, and the wavelength used in each method. In total 9 samples of honey were selected: 3 of each kind (flower, buckwheat, linden), in order to get the full range of possible values of diastatic index for each type of honey. Calculation of diastatic index according to GOST was carried out with and without taking into consideration the anhydrous honey substance. The results of the analysis are represented in Table 3.

             Table 3. Diastatic index values for various types of honey

   
Type of honey Wavelength used 580 nm
Wavelength used 660 nm
  According
to GOST
According
to GOST without
taking in account the humidity
According
to Sheyd
According
to GOST
According
to GOST without
taking in account the humidity
According
to Sheyd
flower 4
12
28
3
10
23
5
10
24
6
17
36
5
14
30
7
14
35
buckwheat 6
18
37
5
15
30
6
14
28
10
25
47
9
20
38
8
22
48
linden 4
8
12
3
7
10
5
9
12
6
12
18
5
10
15
6
14
17


   From data given in Table 3, it can be seen that for all types of honey an analogous trend of variation is observed:
      -  regardless of research method and type of honey, the values of diastatic index are close when measured at          the same wavelength.
      - the values of diastatic index, while measuring the optical density in the range of maximal absorbance at          wavelength values of 540-580 nm, are about 1.5 times smaller that the diastatic index values obtained at a          wavelength of 660 nm.
      - taking into account water content in honey decreases the deviation in the results obtained, when          calculating the diastatic index according to GOST.
   Data indicated above confirmed the negative result of Apis Analytical Centre LLC, obtained in international comparison test.
   To make sure that the method of Shade is mastered correctly, the staff of Apis Analytical Centre LLC once again took part in the international round. The results are presented in table 4.

      Table 4: Results of analysis of honey sample FAPAS 2818F, obtained in Apis
                    Analytical Centre LLC and compared to data of FERA agency.

   
Name of the indicator Results of
LLC Center Apis
Results of FERA agency
    Data obtained Acceptable range
   Water content, % 16,80 116,38 15,90 16,85
   Glucose content, % 32,21 31,54 30,27 32,80
   Fructose content, % 37,73 39,15 37,58 40,71
   Sucrose content, % 1,60 1,74 1,34 2,15
   5-hydroxymethylfurfural    content, mg/kg    (spectrophotometric method) 10,48 9,98 5,67 14,30
   5-hydroxymethylfurfural    content, mg/kg
   (HPLC method)
9,93 8,59 6,70 10,48
   Diastatic index in Gothe units 9,27 9,24 6,00 12,47


   The results obtained were excellent; the determined diastatic index value was accurate.
   Using the same sample, the diastatic index value was determined according to GOST 19792. The results are shown in table 5.

      Table 5.Diastatic index in FAPAS 2818F honey sample, obtained
                   According to GOST 19792 and the method of Shade

   
Wavelength used: 580 nm
Wavelength used: 660 nm
According
to GOST
According
to GOST without
taking in account the humidity
According
to Sheyd
According
to GOST
According
to GOST without
taking in account the humidity
According
to Sheyd
5,9 4,8 5,6 9,5 7,9 9,3


   From the results presented in table 5, it can be seen that the values of diastatic index obtained by both methods at a wavelength of 660nm, fall into acceptable range of values, conversely the use of 580 nm wavelength does not allow to obtain the wanted results.
   To confirm the validity of using any of these methods, we decided to test the linearity of both of these methods for the whole range of amylase concentrations which can be found in honey. To do this, when preparing honey solutions, we took samples that were 2 times greater and 2 times smaller than those indicated in corresponding methods. Measurements were performed at a maximal wavelength peak and at 660 nm. The results are shown in Tables 6 and 7, rounded up to whole units.

         Table 6. Linearity of diastatic index determination by method of Shade.

   
Type of honey Wavelength used: 540- 580 nm
Wavelength used: 660 nm
  Concentration of honey solution, g/cm3
Concentration of honey solution, g/cm3
  0,1 0,2 0,4 0,1 0,2 0,4
Flower 6
13
10
24
14
30
8
18
14
35
21
47
Buckwheat 8
17
14
28
19
41
11
24
22
48
30
65
Linden 5
6
9
12
13
15
7
9
14
17
21
23


         Table 7. Linearity of diastatic index determination according to GOST 19792-2001.

   
Type of honey Wavelength used: 540- 580 nm
Wavelength used: 660 nm
  Concentration of honey solution, g/cm3
Concentration of honey solution, g/cm3
  0,05 0,1 0,2 0,05 0,1 0,2
Flower 6
13
12
28
25
54
9
19
17
36
34
71
Buckwheat 8
18
18
37
33
72
12
23
25
47
49
88
Linden 4
6
8
12
17
25
6
9
12
18
24
35


   Based on the data obtained, the variation of diastatic index with honey concentration in the studied sample was plotted. The plotted graphs demonstrate the linear dependency of diastatic index on the enzyme concentration in honey at any wavelength, given that the study is carried out in accordance with GOST 19792 (Fig 3). The confirmation of validity of these results lies in the fact that all the graphs come out from the origin of coordinates.

         

                            

               Fig 3. Variation of diastatic index with enzyme concentration in flower honey,
                                        using the method of analysis according to GOST 19792

        

                            

               Fig 4. Variation of diastatic index with enzyme concentration in flower honey,
                                        using the method of Shade

   The results obtained by method of Shade, at all fixed wavelengths, are poorly aligned with the straight line, more over, these straight lines do not begin from the origin of the coordinates.Only the results obtained from samples with concentrations of not more that 0.2 g/m3 satisfy the given requirement.

   These graphs allow to conclude that the determination of diastatic index according to GOST 19792 is more preferable for the entire range of determination of this indicator in honey.

   Conclusion.According to the results obtained, the method described in GOST 19792 is more preferable.

   Advantages of the method for determination of diastatic index by method of GOST compared with the method of Shade:
       - Universal procedure for every sample of honey (in method of Shade - specific);
       - Does not require calibration of starch solution ;
       - Shorter duration of analysis (in method of Shade long process);
       - Possibility of performing 10 analyzes simultaneously, while taking into account
         parallel results (in method of Shade no more than 1)
       - quick processing of data obtained using the formula (in method of Shade-plotting
         of the graph for each determination, finding of time tx, calculation of results).
   The only disadvantage of method of GOST is instability of diastase inside of the studied sample upon contact of the latter with air. To eliminate that disadvantage, a research was conducted on the stability of the studied samples in both of the methods. In method of Sheid, the samples are stable, probably because they are prepared in presence of acetate buffer and solution of sodium chloride. Analogous procedure, in our method, lead to the distortion of the analysis results. To obtain reliable results, it is sufficient to limit the access of air to the sample or to use a freshly prepared sample.
   The study of these methods is imperative at the present, because there are studies being done on standardization of methods for honey quality analysis.



[1] (Codex Alimentarius) Revised Codex Standard for Honey, CODEX STAN 12-1981, Rev.1 (1987), Rev.2 (2001)
[2] 2001/110/ 20.12.2001 (Council Directive 2001/110/EC 20.12.2001 )
[3] Harmonised Methods of the International Honey Commission (2002)