RESOLUTION OENO 10/2008

CODEX  POLYGALACTURONASE

THE GENERAL ASSEMBLY

IN VIEW of article 2, paragraph 2 IV of the Agreement of 3 April 2001, by which the International Organisation of Vine and Wine was founded,

Following a proposal made by the Sub-Commission of Methods of Analysis and Appraisal of Wine and the Specifications of Oenological Procedures expert group,

HAS HEREBY DECIDED to add the following monograph to the international Oenological Codex :

POLYGALACTURONASE

endo- and exo-polygalacturonase activities (PG)

(EC. 3.2.1.15 – CAS N° 9032-75-1)

GENERAL SPECIFICATIONS

These enzymes are not found in a pure state but they are present within an enzyme complex. Unless otherwise stipulated, the specifications must comply with Oeno resolution 14/2003 concerning the general specifications for enzyme preparations included in the International Oenological Codex.

1.      ORIGIN AND OENOLOGICAL SCOPE

These enzyme activities are used to support grape maceration, to clarify musts and wines, to improve the filterability of musts and wines and grape pressing.

The enzyme preparations containing such activity come from directed fermentations of Aspergillus niger.

Principle enzyme activities accompanying polygalacturonase activity include:

• Pectin lyase
• Pectin methyl esterase

Secondary activities: Various hemicellulases (see “hemicellulase” sheet) as well as cellulases can be considered as secondary enzyme activities, but are also extremely useful for the hydrolysis of pectic substances. In this case, given their usefulness, it is not appropriate to apply the clause of resolution 14/2003 requiring that the sum of secondary enzyme activities should not be higher than 50% of the sum of enzyme activities needed by the designated function since they usefully contribute to reaching that objective.

On the contrary, the 50% clause can be applied for the following secondary enzyme activities: protease, beta-glucosidase.

2.      SCOPE

The method of determination was developed using a commercially available polygalacturonase. The conditions and the method were developed for application to the commercial enzyme preparations such as those found on the oenological market.

3.      PRINCIPLE

Polygalacturonases cut pectin chains with a low degree of methylation and thus release the galacturonic acids forming the pectin located at the ends of the chain. Once released, the galacturonic acids are determined by the Nelson method (1944). In an alkaline medium, the pseudo aldehyde group of sugars reduces the cupric ions . The latter react with the arsenomolybdate reagent to produce a blue colour, whose absorbance, measured at 520 nm, varies linearly with the concentration in monosaccharides (between 0 and 250 g/mL).

4.      EQUIPMENT

4.1. magnetic stirrer with hot-plate

4.2. water bath at 40°C

4.3. water bath at 100°C

4.4. 100-ml beaker

4.5. centrifuge capable of housing 15-mL glass test tubes

4.6. stop-watch

4.7. 100-ml graduated flask

4.7.1. 500-ml graduated flask

4.8. 200-µl precision syringe

4.8.1. 1-ml precision syringe

4.9. 10-ml straight pipette graduated to 1/10 mL

4.10. spectrophotometer

4.11. 15-mL glass test tubes

4.12. Vortex-type mixer

4.13. 500-mL amber glass bottle

4.14. room at 4°C

4.15. drying oven at 37°C

4.16. cotton-wool

4.17. brown paper

4.18. pH-meter

4.19. metal rack for 15-mL test tubes

4.20. disposable spectrophotometer cuvettes with a 1-cm optical path length, for measurement in the visible spectrum.

5.      REAGENTS

5.1. sodium acetate (COONa 99% pure - MW = 82g/mole)

5.2. acetic acid (COOH 96% pure - MW = 60 g/mole, density = 1.058)

5.3. polygalacturonic acid 85% pure. "Polygalacturonic acid sodium salt" from citrus fruit (Sigma, P3 850) is an example.

5.4. anhydrous sodium sulphate ( 99.5% pure - MW = 142 g/mole)

5.5. anhydrous sodium carbonate ( 99.5% pure - MW = 105.99 g/mole)

5.6. sodium potassium tartrate (99% pure - MW = 282.2 g/mole)

5.7. anhydrous sodium bicarbonate (NaH 98% pure - MW = 84.0 1 g/mole)

5.8. copper sulfate penta-hydrated (Cu. 99% pure - MW = 249.68 g/mole)

5.9. concentrated sulphuric acid (98% pure)

5.10. ammonium heptamolybdate ((99% pure - MW = 1235.86 g/mole)

5.11. sodium hydrogenoarsenate (98.5% pure - MW = 3 12.02 g/mole). Given the toxicity of this product, special attention must be paid during manipulation. Waste material must be treated in an appropriate manner.

5.12. D-galacturonic acid (- MW: 2 12.16 g/mole)

5.13. distilled water

5.14. commercial enzyme preparation to be analysed

6.      SOLUTIONS

6.1.      Reagents of the oxidizing solution

These reagents have to be prepared first, taking into account the 24-hour lead-time for solution D.

6.1.1.     Solution A:

Place successively in a 100-mL beaker (4.4):

• 20 g of anhydrous sodium sulphate (5.4)
• 2.5 g of anhydrous sodium carbonate (5.5)
• 2.5 g of sodium potassium tartrate (5.6)
• 2 g of anhydrous sodium bicarbonate (5.7)

Dissolve in 80 ml of distilled water (5.13). Heat (4.1) until dissolution and transfer into a 100-ml graduated flask (4.7). Make up to the mark with distilled water (5.13). Maintain at 37°C (4.15); if a deposit forms, filter on a folded filter.

6.1.2.     Solution B:

Dissolve 15 g of copper sulfate pentahydrate (5.8) in 100 mL of distilled water (5.13) and add a drop of concentrated sulphuric acid (5.9). Maintain at 4°C.

6.1.3.     Solution C:

This solution is prepared just before use in order to have a satisfactory proportionality between the depth of colour and the quantity of glucose by mixing 1 mL of solution B (6.1.2) with 24 mL of solution A (6.1.1).

6.1.4.     Solution D:

In a 500-mL graduated flask (4.7.1), dissolve 25 g of ammonium molybdate (5.10) in 400 mL of water (5.13). Add 25 ml of concentrated sulphuric acid (5.9) (cooled under running cold water).

In a 100-mL beaker (4.4) dissolve 3 g of sodium arsenate (5.11) in 25 mL of water (5.13) and transfer quantitatively into the 500-mL graduated flask (4.7.1) containing the ammonium molybdate (5.10).

Make up to the mark with water (5.13) to have a final volume of 500 mL.

Place at 37°C (4.15) for 24 hours then maintain at 4°C (4.14) in a 500 mL amber glass bottle (4.13).

6.2.      Sodium acetate buffer (pH 4.2, 100 mM)

This consists of solutions A and B.

6.2.1.     Solution A

Sodium acetate 0.1 M: dissolve 0.5 g of sodium acetate (5.1) in 60 mL of distilled water (5.13)

6.2.2.     Solution B

Acetic acid 0.1 M: dilute 1 mL of acetic acid (5.2) with 175 mL of distilled water (5.13)

6.2.3.     Preparation of the sodium acetate buffer

Mix 23.9 ml of solution A (6.2.1) + 76.1 ml of solution B (6.2.2).

Check the pH of the buffer using a pH-meter (4.18).

The solution must be maintained at 4°C (4.14).

6.3.      Polygalacturonic acid solution at 0.4 % (p/v)

In a 100 mL graduated flask (4.7) dissolve 0.4 g of polygalacturonic acid (5.3) in 100 mL of sodium acetate buffer (6.2).

The solution must be prepared just before use.

6.4.      Stock solution of D-galacturonic acid at 250 µg/ml

In a 100 mL graduated flask (4.7), dissolve 0.0250 g of D-galacturonic acid (5.12) in distilled water (5.13) and make up to 100 mL.

7.      PREPARATION OF THE STANDARD SOLUTIONS OF D-GALACTURONIC ACID

The standard range is produced from 0 to 250 µg/mL, according to table 1.

Table 1: standard solutions of D-galacturonic acid

Galacturonic acid (µg/mL) 0	25	50	100	150	200	250
Galacturonic acid (µmole/mL) 0	0.118	0.236	0.471	0.707	0.943	1.178
Vol. (µl) stock solution (6.4) 0	100	200	400	600	800	1000
Vol. (µl) distilled water (5.13) 1000	900	800	600	400	200	0

8.      PREPARATION OF THE SAMPLE

It is important to homogenise the enzyme preparation before sampling, by upturning the container for example. The enzyme solution and the blanks will have to be prepared at the time of use.

8.1. Enzyme solution at 1 g/l to be prepared just before use

Place 100 mg of commercial preparation (5.14) in a 100-ml graduated flask (4.7), make up with distilled water (5.13), and stir in order to obtain a homogeneous mixture.

8.2. Blank denatured by heating, to be prepared just before use

Place 10 mL of the enzyme solution at 1 g/l (8.1) in a 15-ml test tube (4.11), plug with cotton wool (4.16) covered with brown paper (4.17) and immerse the test tube for 5 minutes in the water bath at 100°C (4.3). Cool and centrifuge 5 min at 6500 g.

9.      PROCEDURE

9.1.      Enzyme kinetics:

The test tubes are prepared at least in duplicate.

In 5 x 15-ml test tubes (4.11) numbered from 1 to 5, placed in a rack (4.19) in a water bath at 40°C, introduce

200 µl of the enzyme solution at 1 g/l (8.1), using the precision syringe (4.8),

400 µl of distilled water (5.13), using the precision syringe (4.8.1),

600 µl of the polygalacturonic acid (6.3) warmed beforehand at 40°C in a water bath, start the stop-watch (4.6).

After shaking (4.12), the test tubes plugged with cotton-wool (4.16) and brown paper (4.17) are replaced in the water bath at 40°C (4.2)

• for 1 min. for test tube N°1
• for 2 min. for test tube N°2
• for 5 min. for test tube N°3
• for 10 min. for test tube N°4
• for 15 min. for test tube N°5

The reaction is stopped by placing each of the test tubes numbered from 1 to 5, immediately after they have been removed from the water bath at 40°C, in the water bath at 100°C (4.3) for 10 min.

The test tubes are then cooled under running cold water.

Note: the kinetic point at 10 min is used for the evaluation of the enzyme activity

9.2.      Determination of reducing substances released

In a 15-mL test tube (4.11)

Place 1 mL of the reaction medium (9.1) using the precision syringe (4.8.3)

Add 1 mL of solution C (6.1.3) using the precision syringe (4.8.3)

After shaking (4.12), the test tube is placed in the water bath at 100°C (4.3) for 10 min. The test tube is then cooled under running cold water.

Add 1 mL of solution D (6.1.4)

Add 9.5 ml of water (5.13) using the straight 10-mL pipette (4.9)

Wait 10 min. for the colour to stabilise.

Centrifuge (4.5) each test tube at 2430 g for 10 min.

Place the supernatant liquid in a cuvette (4.20).

Zero the spectrophotometer using distilled water

Immediately measure the absorbance at 520 nm, using a spectrophotometer (4.10).

9.3.      Blanks

Proceed as described in 9.1, replacing the enzyme solution at 1 g/l (8.1) by the blank denatured by heat (8.2). For each kinetic point, the enzymatic reaction of each blank is carried out at the same time as that of the enzyme solution. .

9.4.      Standard solutions

Proceed as described in 9.2, replacing the reaction mixture (9.1) by the various mixtures of the standard solutions of D-galacturonic acid from 0 to 250 µg/mL (7).

10.    CALCULATIONS

10.1. Determining the reaction kinetics

In general, calculating the enzymatic activity can only be done when the substrate and the enzyme are not in limiting quantities. This therefore refers to the ascending phase of the kinetic curve: the enzymatic activity is linear in time. Otherwise, the activity would be underestimated (Figure 1).

Figure 1: Kinetics of an enzymatic reaction

The kinetics are determined over 15 minutes. The activity concerned is measured at T=1 min T=2 min, T=5 min, T=10 min, T=15 min.

After determining the kinetics of the enzymatic reaction, plot the curve for the variation in absorbance in relation to reaction time. The absorbance corresponds to the difference between the absorbance at time T of the enzyme preparation and that of the corresponding blank. Then calculate the equation (1) of the straight regression line, taking into account only the points of the ascending phase (see figure 1).

10.2. Producing the calibration line

The calibration line corresponds to plotting a graph whose X-coordinates are the various concentrations of the standard solutions of D-galacturonic acid (from 0 to 0.589 µmole/mL) and whose Y-coordinates are the corresponding values of optical densities, obtained in 9.4. Then calculate the straight regression slope line (2) resulting from the linearity of the data of the graph.

10.3. Calculating the enzymatic activity

Based on the straight regression line (1) calculate the absorbance for an average time T (for example 4 min. in the case of figure 1) deduct from it the quantity Q of D-galacturonic acid released (in µmoles) for this intermediate time using equation (2).

The formula used to calculate the enzymatic activity in U/g of the preparation is as follows

• Activity in U/g = 1000 x (Q/T)/(VxC)

Where Q: quantity of D-galacturonic acid released in µmoles during time T (min)

V: quantity of enzyme solution introduced (mL), in this case 0.2 mL

C: concentration of the enzyme solution (g/l), in this case 1 g/l

It is then possible to express the enzymatic activity in nanokatals. This unit corresponds to the number of nanomoles of product formed per second under the conditions defined by the determination protocols and therefore:

• Activity in nkat/g = (activity in U/g) x (1000/60)

11.    CHARACTERISTICS OF THE METHOD

r	0.084
R	0.056
Sr	0.03
SR	0.02

The intralaboratory repeatability of the method is estimated using the mean standard deviation of the absorbance values resulting from the same sampling of the enzyme preparation, determined 5 times. In this way, to analyse the polygalacturonase the mean standard deviation of the values is 0.03 with a percentage error of 3.78, in which the % error corresponds to:

In this way, the determination method as presented is considered repeatable.

The intralaboratory reproducibility tests were carried out using 2 enzyme preparations with 5 samplings for each.

2 tests were used in order to determine the satisfactory reproducibility of the method:

• analysis of variance (the study of the probability of the occurrence of differences between samplings). Analysis of variance is a statistical method used to test the homogeneity hypothesis of a series of K averages. Performing the analysis of variance consists in determining if the "treatment" effect is "significant or not". The standard deviation of reproducibility given by this analysis of variance is 0.02.
• the power of the test for the first type of risk (5%) – first type of risk  is the risk of deciding that identical treatments are in fact different.

If the power is low ( 20%), this means that no difference has been detected between treatments, but there is little chance of seeing a difference if one did in fact exist.

If the power is high ( 80%), this means that no difference has been detected between the treatments, but, if there was one, we have the means of seeing it.

The results are given in table 2.

Determination	Analysis of variance hypotheses	Probability	Power of test (= 5%)	Newman- Keuls test (*)	Bonferroni test (**)
PG	Treatment*block interaction	0.0256	77%	Significant	Significant

Table 2: analysis of variance– study of the sampling effect

* Newmann-Keuls test: this comparison test of means is used to constitute homogeneous groups of treatments: those belonging to the same group are regarded as not being different to risk  of the first species selected

** Bonferroni test: also referred to as the "corrected T test", the Bonferroni test is used to carry out all the comparisons of pairs of means, i.e., (t (t-1))/2 comparisons before treatments, respecting the risk  of the first species selected.

In this way, the tests set up are used to see a difference if there really is one (high power test); in addition, the method of determination involves a probability of occurrence of a discrepancy in activity (between samplings) lower than 5%.

12.    BIBLIOGRAPHY

1. NELSON N, A photometric adaptation of the SOMOGYI method for the determination of glucose. The may Institute for medical research of the Jewish hospital, 1944. p 375-380.
2. Enzyme activities and their measurement – OIV Document, FV 1226, 2005