COEI-1-ACTGLU Determination of beta-glucanase (β 1-3, β 1-6) Activity in enzyme preparations

General specifications

These enzymatic activities are usually present within a complex enzymatic preparation. In the degradation of ß-glucans from Botrytis cinerea, endo- β-glucanase activities of the type endo- β-1,3 and of the type exo- β-1,6 glucosidase, as well as exo- β-1,3 type activities are involved. They are summarized here under the commonly used term, “β-glucanases”. These enzymatic preparations are also capable of degrading β-glucans in the cell walls of dying Saccharomyces yeast cells which supports the process called “élevage de vin sur lie“ (aging of wine laying on lees). Endo- β-1,3 activities, endo- β-1,6 activities as well as exo- β-1,3 and exo- β-1,6 activities are involved in this process. Unless otherwise stipulated, the specifications must comply with resolution OIV/OENO 365/2009 concerning the general specifications for enzymatic preparations included in the International Oenological Codex.

1. Origin

Reference should be made to paragraph 5, “Sources of enzymes and fermentation environment”, of the general monograph on Enzymatic preparations.

The enzyme preparations containing β-glucanase activities are produced by direct fermentations, for example, of Trichoderma harzianum, Trichoderma longibrachiatum (T. reesei) and Penicillium funiculosum.

2. Scope of application

Reference should be made to the International Code of Oenological Practices, OENO 11/2004; OENO 12/2004; OENO 15/20 04 and AG 3/85- OEN.

The enzymatic preparations containing β 1-3 and β 1-6 glucanase activities are able to hydrolyse the glucan produced by Botrytis cinerea (noble rot and gray rot). This polysaccharide causes great difficulties during wine clarification and filtration. Such β-glucanases are therefore specifically used for clarification and filtration of wines made from botrytised grapes.

The glucans contained in the yeast cell walls are also hydrolysed by these β-glucanases. They may be used to improve the process of maturing on lees as well as the filterability.

3. Principle

The method of analysis is based on measuring the glucose released by the enzyme, using a standardised solution of Schizophyllum sp. glucan as substrate.

3.1.         Definition of units

A unit of β-glucanase (β-Glu-U) is defined as the quantity of reducing sugars, expressed as glucose, released in test conditions by 1 g (or 1 mL) of enzyme per minute.

3.2.         Role of the enzyme

As it grows on infected grapes (as noble or grey rot), Botrytis cinerea excretes a β-1,3-glucan which, at every third unit of glucose, possesses a β-1,6 glycosylated residue of glucose (Fig. 1). This glucan is very similar to glucan synthetised by Schizophyllum sp.

3.3.         Principle of measurement

The enzymatic activity releases glucose which, in an alkaline salt solution, reduces 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. The addition of phenol increases the sensitivity of the reaction. Sodium bisulphite serves to stabilise colour.

4. Apparatus

4.1.         Spectrophotometer and cuvettes with an optical path length of 1 cm

4.2.         40°C, 100°C water bath

4.3.         Standard magnetic stirrer

4.4.         Submersible multi-point magnetic stirrer set at 300 rpm

4.5.         Measuring containers (volumetric flasks, beakers, conical flasks, etc.)

4.6.         Beaker

4.7.         Micro-pipettes

4.8.         Timer

4.9.         Ultrasonic bath

4.10.     pH meter

5. Reagents and products

5.1.         Substrate

Glucan stock solution supplied by the University of Braunschweig^[1]; the glucan content of which has been determined by the University of Braunschweig.

5.2.         Pure products

5.2.1.  Citric acid monohydrate (CAS No.  5949-29-1)

5.2.2.  Sodium hydroxide (CAS No.  1310-73-2)

5.2.3.  Potassium sodium tartrate (CAS No.  304-59-6)

5.2.4.  Sodium metabisulphite Na2S2O5 (CAS No.  7681-57-4)

5.2.5.  Phenol (CAS No. 108-95-2)

5.2.6.  Anhydrous glucose

5.2.7.  3,5-dinitro-2-hydroxybenzoic (3,5-dinitrosalicylic) acid (CAS No. 609-99-4)

5.2.8.  Distilled water

5.3.         Solutions

5.3.1.  1M sodium hydroxide solution

In a 100-mL volumetric flask, dissolve 4.0 g of sodium hydroxide (5.2.2) in distilled water (5.2.8) and make up to the required volume.

5.3.2.  Citrate buffer solution (pH 4.0) - 0.2 mol/L

In a 500-mL volumetric flask, dissolve 21.0 g of citric acid monohydrate (5.2.1) in 400 mL of distilled water, then adjust the pH to 4.0 with a molar solution of sodium hydroxide (5.3.1) and make up to the required volume with distilled water (5.2.8).

5.3.3.  Citrate buffer solution (pH 4.0) - 0.1 mol/L

In a 1,000-mL volumetric flask, dissolve 21.0 g of citric acid monohydrate (5.2.1) in 900 mL of distilled water (5.2.8), then adjust the pH to 4.0 with a molar solution of sodium hydroxide (5.3.1) and make up to the required volume with distilled water (5.2.8).

5.3.4.  Titrating solution: DNS (dinitrosalicylic) acid colour reagent with phenol

This is prepared from solutions A, B and C below:

5.3.4.1.                  Solution A:

Weigh out 154.2 g of potassium sodium tartrate (5.2.3) in an 800-mL beaker and dissolve completely in 500 mL of distilled water (5.2.8). Add 9.7 g of sodium hydroxide (5.2.2).

5.3.4.2.                  Solution B:

In a 2,000-mL beaker, completely dissolve 5.3 g of 3,5-dinitrosalicylic acid (5.2.7) in 500 mL of distilled water (5.2.8). The best results are obtained using an ultrasonic bath.

5.3.4.3.                  Solution C:

In a 100-mL beaker, dissolve 4.2 g of phenol (5.2.5) in 50 mL of distilled water (5.2.8). Then add 1g of sodium hydroxide (5.2.2) and, when completely dissolved, 4.2 g of sodium metabisulphite (5.2.4) and dissolve again.

5.3.4.4.                  0.3% glucose solution

In a 100-mL volumetric flask, put exactly 300 mg of glucose (5.2.6), dissolve in distilled water (5.2.8) and make up to the required volume with distilled water.

5.3.4.5.                  DNS acid colour reagent with phenol

Solutions A and C are mixed with solution B in a 2,000-mL beaker, which is then covered with aluminium foil.

Before using, keep in the dark for at least 3 days.

Transfer the reagent to a brown glass container.

If stored in a dark place at 15-20° C, this solution can be kept for a month.

For each newly-prepared reagent and before each measurement, a new calibration is carried out prior to each enzyme analysis.

Before each use, 3 mL of 0.3% glucose solution (5.3.3.4) should be added to 200 mL of the DNS acid colour reagent with phenol.

5.3.5.  Glucan in solution at 0.1%, pH 4.0

Weigh out the exact quantity of glucan stock solution (5.1) to obtain a final concentration of 1 g /L.

The final substrate solution should contain 50% of the citrate buffer solution (pH 4.0) - 0.2 mol/L (5.3.2).

To obtain 100 mL of substrate solution from the glucan stock solution (5.1) (actually containing 5.2 g/L), weigh out 19.2 g in a 100-mL beaker. Add 50 mL of the citrate buffer solution (pH 4.0) - 0.2 mol/L (5.3.2). Homogenize the glucan mixture by stirring for at least 15 minutes. When well-mixed, adjust the pH to 4.0 with a sodium hydroxide molar solution (5.3.1). Then transfer the solution to a 100- mL volumetric flask and make up to the required volume later with distilled water (5.2.8).

Store all glucan stock solutions at ambient temperature. If a new glucan stock solution is used, a glucan substrate factor (Gf = glucan factor) should be determined by means of the standard enzyme. The "Gf" is essential for comparing the results from previous glucan stock solutions with the new ones. The “Gf” is calculated with the values measured considering that standard enzymatic activity is 10,000 β-Glu U/g in the formula (See: Calculation of enzymatic activity).

5.4.         Enzyme preparations

5.4.1.  Glucanase standard enzyme solution:

Dissolve 0.5 g of glucanase standard enzyme preparation in 25 mL of the citrate buffer solution (pH 4.0; 0.1 mol/L) (5.3.3) and make up to 100 mL with distilled water (5.2.8).

5.4.2.  For all other enzyme preparations: Dissolve 1 mL of enzyme preparation or 0.5 g of solid powdered or granulated enzyme preparation in 25 mL of the citrate buffer solution (pH 4.0; 0.1 mol/L) (5.3.3) and make up to 100 mL with distilled water (5.2.8). If the absorption values are too high or too low (absorbance range 0.1-0.6), appropriate dilution is necessary. The enzyme dilution should contain 25% of citrate buffer solution (5.3.3).

6. Procedure

6.1.         Reagent “blank” test

Add 7 mL of DNS acid colour reagent with phenol (5.3.4) to 3 mL of distilled water (5.2.8) in a 50-mL volumetric flask and heat for exactly 10 minutes over a bath of boiling water. Cool for 5 minutes in an ice bath, then transfer the flask into a water bath at 20° C and add distilled water (5.2.8) to just below the mark. After 10 minutes at 20°C, make up to the required volume.

6.2.         Glucose calibration curve with DNS acid colour reagent with phenol

Dissolve 2.00 g of glucose (5.2.6) in a 200-mL volumetric flask and make up to volume with distilled water (5.2.8). Using this solution, prepare the following dilutions:

No.	V standard solution / 100 mL	glucose/100 mL	glucose (μg) in the trial (= 0.5 mL)
1	2 mL	20 mg	100 μg
2	5 mL	50 mg	250 μg
3	10 mL	100 mg	500 μg
4	15 mL	150 mg	750 μg
5	20 mL	200 mg	1,000 μg
6	30 mL	300 mg	1,500 μg
7	40 mL	400 mg	2,000 μg

Use a pipette to put 0.5 mL of each glucose dilution into a 50-mL volumetric flask and add 7 mL of DNS colour reagent with phenol (5.3.4) and 2.5 mL of distilled water (5.2.8). Heat the measuring containers for exactly 10 minutes in a bath of boiling water. Cool for 5 minutes in a bath of ice, then transfer the flask to a water bath at 20°C and add distilled water (5.2.8) to just below the mark. After 10 minutes at 20°C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

On a diagram, plot the quantity of glucose released in the test against the absorbance at 515 nm (Fig. 2).

The calibration curve is produced the same day before every enzyme analysis.

6.3.         “Blank” testing of enzymes

Use a pipette to put 0.5 mL of each enzyme solution (5.4.1 or 5.4.2) into a 50-mL volumetric flask and add 7 mL of DNS acid colour reagent with phenol (5.3.4). Mix carefully and add 2.5 mL of substrate solution (5.3.5). Stir well by hand. Then heat all samples over a bath of boiling water for exactly 10 minutes, cool for 5 minutes in a bath of ice and transfer the flask to a water bath at 20° C, adding distilled water (5.2.8) to just below the mark. After 10 minutes at 20° C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

6.4.         Measuring the activity of enzyme preparations

For each sample of enzymes, put 10 mL of substrate (5.3.5) into a conical flask in a water bath at 40° C for 5 minutes. Samples should be homogenized using a submersible multi-point magnetic stirrer set at 300 rpm. After 5 minutes, 2 mL of the enzyme solution (5.4.1 or 5.4.2) are added to the first sample and a timer started just after adding the first enzyme solution.

Then add the following enzyme solutions to all the other samples with an interval of 30 seconds between samples.

Samples should then be stirred at 300 rpm throughout the entire reaction time.

After exactly 15 minutes, remove 3 mL of the first mixture, followed by all the other samples, at intervals of 30 seconds.

Using a pipette, put each 3-mL mixture into as many 50-mL volumetric flasks as required, each of which contains 7 mL of DNS acid colour reagent with phenol (5.3.4).

Then heat all the samples, at 30-second intervals, for exactly 10 minutes over a bath of boiling water.

Cool for 5 minutes in a bath of ice, transfer the flask to a water bath at 20° C and add distilled water (5.2.8) to just below the mark.

After 10 minutes at 20° C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

The difference in the absorbance between the “blank” reading of enzymes and the value after reaction should be between 0.1 and 0.6 absorbance units.

If the values are over the measuring range of the calibration curve, repeat the experiment with dilutions adapted to the enzymes.

For all enzymes, always prepare 1 “blank” enzyme reading and 2 values after reaction. The two values after reaction should be similar.

7. Calculations

To calculate the enzyme activity, use the mean value of the two readings.

The enzymatic activity of an enzyme preparation is calculated according to the following formula:

β-Glu-Unit activity/ g or mL=

Nkat/g or mL = (Actiβ-Glu-Unit/g or mL)

Where:

• G = Quantity of reducing sugars released during the test (reducing sugars released by Δ = the mean of 2 repetitions of the absorbance after reaction minus the absorbance of the “blank” enzyme, calculated in glucose from the glucose calibration curve in µg).
• E = Quantity of enzyme diluted to 100 mL in g or mL
• 200 = Dilution factor
• 15 = Reaction time in min
• Gf = Glucan factor (to be calculated)

Example of a calculation:

Enzyme	Measured value 1 2		“Blank” enzyme	E	μg glucose	β-Glu units /g or mL
Enzyme used	0.621	0.618	0.415	0.503	662	10325
Penicillium funiculosum ß-Glucanase	0.417	0.416	0.023	1	1249	9799

Gf calculation:

• 1 Measure using old substrate and standard enzyme (Value 1)
• 2 Measure using new substrate and standard enzyme (Value 2)

Calculation: Value 1 / Value 2

8. Bibliography

• Bertrand A. Détermination de l'activité β-glucanase de Botrytis des préparations enzymatiques, OIV FV 1263

^[1] Prof. Dr Udo Rau, Technical University Braunschweig, Department of Biochemistry and Biotechnology Spielmannstr. 7, 38106 Braunschweig - Germany

COEI-1-ACTCEL Determination of cellulase activity in enzymatic preparations (endo-(1 4)- β-D- glucanase)

EC 3.2.1.4 – CAS N° 9012-54-8

General specifications

These enzymes are generally present among other activities, within an enzyme complex. Unless otherwise stipulated, the specifications must comply with the resolution OIV-OENO 385–2012 concerning the general specifications for enzyme preparations included in the International Oenological Codex.

1. Origin

Reference is made to paragraph 5 “Source of enzyme and fermentation environment” of the general monography on enzymatic preparation

The enzyme preparations containing this activity are produced by directed fermentations, as exemple, of Aspergillus Niger Trichoderma longibrachiatum (T. reesei), Penicillium sp., Talaromyces emersonii or Rhizopus oryzae.

2. Scope / Applications

Reference is made to the International Code of Oenological Practices, OENO

11/2004; OENO 12/2004; OENO 13/2004; OENO 14/2004 and OENO 15/2004.

Enzymes catalysing the degradation of cellulose-type of grape cell walls polysaccharides, mainly endo-(1 4)- β-D-glucanases, are useful to speed up and fulfill the maceration process of the grapes.  They also have a positive effect on filtration and clarification in allowing a more complete enzymatic degradation of polysaccharides.

3. Principle

 

The endo-(14)- β-D-glucanase catalyses the hydrolysis of the oside bonds within cellulose in a random way. Its activity can therefore be assessed by determination of the reducing sugars (expressed in glucose), released during incubation, by the NELSON method (1944).

Only the activities of the "endo-" type are measured because of the presence of carboxymethyl groups that block the action of the exo-glucanases. The endo-glucanases act inside the chains in non-carboxymethylated regions. In an alkaline environment, the pseudo-aldehydic group of sugars reduces the cupric ions Cu²⁺. 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 μg/ml).

4. Apparatus

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.     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

4.21.     ultrasonic probe

5. Reagents

5.1.         Sodium acetate (CH₃COONa 99% pure - MW = 82g/mole)

5.2.         Acetic acid (CH₃COOH 96% pure - MW = 60 g/mole, density = 1.058)

5.3.         Carboxy-methyl-cellulose (CMC) with a degree of substitution from 65 to 95%.

5.4.         Cellulase of Trichoderma reesei (Fluka, 4U/mg, ref: 22173 as an example). One unit releases 1 µmole of glucose from carboxy-methyl-cellulose per minute.

5.5.         Anhydrous sodium sulphate (Na₂SO₄ 99.5% pure - MW = 142 g/mole)

5.6.         Anhydrous sodium carbonate (Na₂CO₃ 99.5% pure - MW = 105.99 g/mole)

5.7.         Sodium potassium tartrate (KNaC₄H₄O6.4H₂O 99% pure - MW = 282.2 g/mole)

5.8.         Anhydrous sodium bicarbonate (NaHCO₃ 98% pure - MW = 84.0 1 g/mole)

5.9.         Copper sulfate penta-hydrate (CuSO₄.5H₂O 99% pure - MW = 249.68 g/mole)

5.10.     Concentrated sulphuric acid (H₂SO₄ 98% pure)

5.11.     Ammonium heptamolybdate ((NH₄)₆Mo₇O₂₄.4H₂O 99% pure - MW = 1235.86 g/mole)

5.12.     Sodium hydrogenoarsenate (Na₂HAsO₄.7H₂O 98.5% pure - MW = 312.02 g/mole). Given the toxicity of this product, special attention must be paid during manipulation. Waste material must be treated in an appropriate manner.

5.13.     Anhydrous D-glucose (C₆H₁₂O₆ 99% pure - MW = 180.16 g/mole)

5.14.     Distilled water

5.15.     Commercial enzyme preparation for analysis

6. Solutions

6.1.         Reagents of the oxidizing solution

These reagents must 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 (5.5)
• 2.5 g of anhydrous sodium carbonate (5.6)
• 2.5 g of sodium potassium tartrate (5.7)
• 2 g of anhydrous sodium bicarbonate (5.8)

Dissolve in 80 mL of distilled water (5.14). Heat with stirring (4.1) until dissolution and decant into a 100-mL graduated flask (4.7). Make up to the mark with distilled water (5.14). Maintain at 37°C (4.15); if a deposit is formed, filter using a folded filter.

6.1.2.  Solution B:

Dissolve 15 g of copper sulfate pentahydrate (5.9) in 100 mL of distilled water (5.14) and add a drop of concentrated sulphuric acid (5.10). 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 (5.11) in 400 mL of water (5.14). Add 25 mL of concentrated sulphuric acid (5.10) (cooled under cold running water).

In a 100-mL beaker (4.4), dissolve 3 g of sodium arsenate (5.12) in 25 mL of water (5.14) and quantitatively transfer into the 500-mL graduated flask (4.7.1) containing ammonium molybdate (5.11).

Make up to the mark with water (5.14).

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 below.

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 (5.14)
  2.   Solution B: acetic acid 0.1 M: dilute 1 mL of acetic acid (5.2) with 175 mL of distilled water (5.14)

6.2.3.  Preparing 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.         Carboxy-methyl-cellulose solution (CMC) at 2% (p/v) to be prepared just before use

Into a 100-mL graduated flask (4.7) introduce 2 g of CMC (5.3) and 100 mL of distilled water (5.14)

Given the high viscosity and in order to have a homogeneous solution, it must be subject to ultrasonic treatment (4.21), stirred without heating (4.1) and kept in suspension while constantly stirring.

In a 100-mL graduated flask (4.7), dissolve 0.0250g of glucose (5.13) in distilled water (5.14), and make up to 100 ml.

7. Preparing the standard solutions of glucose

This is produced using the stock solution of glucose at 250 µg/mL (6.4.), as indicated in Table 1.

Glucose (μg/ml) 0	25	50	100	150	200	250
Glucose (µmole/ml)0	0.139	0.278	0.555	0.833	1.110	1.388
Vol. (μl) stock solution (6.4)0	100	200	400	600	800	1000
Vol. (μl) distilled water (5.14) 1000	900	800	600	400	200	0
Table 1: standard solutions of glucose based on the stock solution

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 have to be preparedat the time of use.

8.1.         Enzyme solution at 2 g/l to be prepared just before use

Place 200 mg of commercial preparation (5.15) in a 100-mL graduated flask (4.7), make up to the mark with distilled water (5.14), and shake 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 2 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). Then chill 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 2 g/l (8.1), using the precision syringe (4.8),
• 400 μl of sodium acetate buffer (6.2), using the precision syringe (4.8.1),
• 600 μl of the carboxy-methyl-cellulose solution (6.3) previously warmed at 40°C in a water bath, start the stop-watch (4.6).

After mixing (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 pemits the evaluation of the enzymatic activity

9.2.         Determination of the reducing substances released

In a 15-mL test tube (4.11):

Place 1 mL of the reaction mixture (9.1)

Add 1 mL of solution C (6.1.3)

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 (5.14) using the graduated 10-mL pipette (4.9)

Wait 10 min. for the colour to stabilise.

Centrifuge (4.5) each test tube at 2340 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 (4.10).

9.3.         Blanks

Proceed as described in 9.1, replacing the enzymatic solution at 2 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 enzymatic 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 glucose 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 enzymatic 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 range of glucose (from 0 to 0.693 µmole/ml) and whose Y-coordinates are the corresponding values of optical densities, obtained in 9.4. Then calculate the (Q/T) slope of the straight regression 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 glucose 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

Where

• Q: quantity of glucose 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 2 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

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, for the determination with carboxy-methyl-cellulose the mean standard deviation of the values is 0.03 with a percentage error of 13.56, in which the % error corresponds to:

• (mean standard deviation of values x 100) mean test value

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

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

2 tests were used in order to determine good 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 α (5%) – first type of risk α 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	Variance analysis hypotheses	Probability	Power of Test (α= 5%)	Newman- Keuls test (*)	Bonferroni test (**)
Endo-(1 4)- β-D-glucanase	Adhered to	0.00011	95%	Significant	Significant
Table 2: Variance analysis – 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

• 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.
• Enzymatic activities and their measurement – OIV Document, FV 1226, 2005

COEI-1-CINEST Measurement of cinnamoyl esterase activity in enzymatic preparations

Two different methods are proposed to measure the cinnamyl esterase activity since we have no principal precursor, para-coumaroyltartric acid; the first method uses the chlorogenase activity of Aspergillus niger i.e. the hydrolysis of chlorogenic acid (caffeoylquinic); this requires the implementation of conventional enzymatic measuring apparatus.

The second method relates to the hydrolysis of ethyl cinnamate, the content of which is measured by gas chromatography.

Both methods were compared, their give similar results.

 

General specifications

Unless otherwise stipulated, the specifications must comply with the resolution OIV/OENO 365/2009 concerning the general specifications for enzymatic preparations included in the International Oenological Codex.

1. Origin

 

These enzymatic activities are often present in preparations like pectolytic enzymes by directed fermentations of Aspergillus sp.

2. Scope of application

This activity is responsible for the production of volatile phenols which impacts negatively the sensory properties of wines, especially white wines. On the other hand, only limited studies have demonstrated that this activity seems to have a positive effect in stabilising the colour of red wines.

3. Method A. Chlorogenate hydrolase or chlorogenase (EC. 3.1.1.42 – CAS no. 74082-59-0)

 

3.1.    Principle

Cinnamyl esterase degrades chlorogenic acid releasing caffeic acid. The reduction in measured absorbance at 350 nm linked to the disappearance of this substrate can be used to quantify the cinnamyl esterase activity.

An enzymatic unit is defined as being the quantity of enzyme enabling a drop in the absorbance of 1 unit at pH 6.5 and 30°C.

3.2.    Apparatus

3.2.1.  water bath at 30°C

3.2.2.  water bath at 100°C

3.2.3.  litre graduated flask

3.2.4.  125-mL Erlenmeyer flask

3.2.5.  100-mL graduated flask

3.2.6.  1000-mL graduated flask

3.2.7.  Chronometer

3.2.8.  100- μL precision syringe

3.2.9.  1000- μL precision syringe

3.2.10.     5000- μL precision syringe

3.2.11.     graduated 5-mL straight pipette

3.2.12.     pH-meter

3.2.13.     spectrophotometer

3.2.14.     15 mL glass screw-top test tubes

3.2.15.     metal rack for 15-mL test tubes

3.2.16.     cuvets with a 1-cm optical path length, for single use, for spectrophotometer, for measurement in the UV spectrum

3.2.17.     stirrer of the Vortex type

3.3.    Products

3.3.1.  methanol (Analytical Reagent Rank – CH3OH - PM = 32.04 g/mole)

3.3.2.  sodium dihydrogenophosphate (NaH2PO4.2H2O 99% pure - PM = 156.01 g/mole)

3.3.3.  sodium hydroxide (NaOH 99% pure - PM = 40 g/mole)

3.3.4.  chlorogenic acid (95% pure - PM = 354.30 g/mole)

3.3.5.  distilled water

3.3.6.  commercial enzymatic preparation for analysis

 

3.4.    Solutions

3.4.1.  Methanol at 80% (v/v)

Introduce 100 mL of methanol (3.3.1) into a 125-mL Erlenmeyer flask (3.2.4) to which 25 mL of distilled water (3.3.5) have been added.

3.4.2.  Sodium hydroxide solution at 9M:

Introduce 360g of sodium hydroxide (3.3.3) into a 1000-mL graduated flask (3.2.6) and make up with distilled water (3.3.5).

3.4.3.  Phosphate buffer 0.1M (pH 6.5)

Introduce 31.5 g of sodium dihydrogenophosphate (3.3.2) into a 2-litres graduated flask (3.2.3) to which 1.8 litres distilled water (3.3.5) have been added.

Adjust the pH to 6.5 using the sodium hydroxide solution (3.4.2) and a pH-meter (3.2.11). Then adjust the volume with 2 litres with distilled water (3.3.5).

3.4.4.  Chlorogenic acid solution at 0.06% (p/v)

Dissolve 0.06 g of chlorogenic acid (3.3.4) in a 100-mL graduated flask (3.2.5) to which the phosphate buffer (3.4.3) has been added up to the gauge line.

 

3.5.    Preparation of the sample

It is important to homogenise the enzymatic preparation before sampling, by upturning the container for example. The enzymatic solution and the blanks will have to be prepared extemporaneously.

3.5.1.  Enzymatic solution at 10 g/L

• Place 1g of commercial preparation (3.3.6) in a 100-mL graduated flask (3.2.5), make up with the phosphate buffer (3.4.3), and stir (3.2.17) in order to obtain a homogeneous mixture.
  2.   White denatured by heating
• Place 10 mL of the enzymatic solution at 10 g/L (3.5.1) in a 15-mL test tube (3.2.14) and immerse the tube for 5 minutes in the water bath at 100°C (3.2.2).

3.6.    Procedure

3.6.1.  Enzymatic reaction: The test tubes are produced at least in duplicate.

In 4 x 15-mL test tubes (3.2.14) numbered from 1 to 4, placed in a rack (3.2.15)

Introduce 100 µL of the enzymatic solution at 10 g/L (3.5.1), using the precision syringe (3.2.8),

500 µL of the chlorogenic acid solution (3.4.4), start the chronometer (3.2.7).

After shaking (3.2.17), the test tubes are placed in the water bath at 30°C (3.2.1)

• for 120 min. for test tube no.1
• for 240 min. for test tube no.2
• for 330 min. for test tube no.3
• for 400 min. for test tube no.4

The reaction is stopped by adding 5 mL of methanol at 80% (3.4.1) using a straight pipette (3.2.11) in each of the numbered tube 1 to 4, immediately after they have been removed from the water bath at 30°C. The tubes are then shaken.

3.6.2.  Proportioning of the released substances (caffeic acid)

The reactional medium (3.6.1) is placed in a cuvet with a 1-cm optical path length (3.2.16). Immediately measure the absorbance at 350 Nm, using a spectrophotometer (3.2.13). The measurement is to be compared with a blank of methanol 80% pure (3.4.1).

3.7.    Calculations

3.7.1.  Determining the 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 representation: the enzymatic activity is linear in time. Otherwise, the activity would be underestimated (Figure 1)

Figure 1: Kinetics of an enzymatic reaction

The kinetics is determined over 400 minutes. The activity concerned is measured at T=120 min T=180 min, T=240 min, T=300 min T=360 min T=400 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 enzymatic 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).

3.7.2.  Calculation of the enzymatic activity

The cinnamyl esterase activity is calculated based on the reduction in absorbance per hour since this activity is very weak in the preparations. The calculation formula is as follows

• DO0: Value of the absorbance of the blank
• DOT: Value of the absorbance at time T (hour)
• V: quantity of enzymatic solution introduced (μL), in this case 100 µL
• C: concentration of the enzymatic solution (g/L), in this case 10 g/L

4. Method B. Hydrolysis of ethyl cinnamate

4.1.    Principle

Cinnamyl esterase hydrolyses ethyl cinnamate. The reduction in this ester measured by gas chromatography can be used to quantify the cinnamyl esterase activity.

4.2.    Apparatus

4.2.1.  Gas phase chromatograph with a flame ionisation detector or mass spectrometry equipped with a capillary tube of the Carbowax 20 M type 50 m x 0.2 mm x 0.2 μm phase thickness

4.2.2.  Magnetic stirrer and stirrer bars

4.2.3.  Laboratory glassware (5-mL precision pipettes, conical flasks, 50-mL and 100- mL graduated flasks, 10-mL, 60-mL, 150-mL laboratory glass bottles etc.)

4.2.4.  Pasteur pipettes

4.2.5.  200- μL, 50- μL and 10- μL precision syringes

4.2.6.  Drying oven at 25°C

4.2.7.  Precision balance to within 0.1 mg/L

4.2.8.  pH-meter

4.3.    Products

4.3.1.  Methanol (Analytical Reagent Rank – CH3OH - PM = 32.04 g/mole)

4.3.2.  Citric acid 99% pure

4.3.3.  Sodium hydroxide (NacOH 99% pure - PM = 40 g/mole)

4.3.4.  Ethyl cinnamate (99% pure - PM = 176 g/mole)

4.3.5.  Distilled or permuted water

4.3.6.  Commercial enzymatic preparation for analysis

4.3.7.  Pure ethanol 99% vol.

4.3.8.  Diethylic ether 99% pure.

4.3.9.  Pure Dodecanol

4.4.    Solutions

4.4.1.  Ethanol at 12% (v/v)

Introduce 12 mL of ethanol (4.3.7) into a 100-mL graduated flask (4.2.3) make up to volume with distilled water (3.3.5).

4.4.2.  Sodium hydroxide solution 4 M

Introduce 16 g of pure sodium hydroxide into a 100-mL graduated flask; make up with distilled water; stir until dissolution.

4.4.3.  Citrate buffer at pH 6.5

Introduce 0.05 g of citric acid (4.3.2) into a 150 mL bottle (4.2.3), add 100 mL of ethanol to 12% vol. (4.4.1) dissolve using a magnetic stirrer. Place under magnetic stirring in the presence of the electrode of the pH-meter (4.2.8) bring to pH 6.5 byadding the sodium hydroxide 4 M drop by drop (4.4.2).

4.4.4.  Stock solution of ethyl cinnamate at 500 mg/L

Using a precision syringe (4.2.5) place 50 μL of ethyl cinnamate (4.3.4) in a 100-mL graduated flask containing a little pure ethanol (4.3.7) make up to the gauge line with pure ethanol (4.3.7); homogenise

4.4.5.  Ethyl cinnamate solution at 25 mg/L in the citrate buffer

In a 100-mL graduated flask, place 5 mL of stock solution of ethyl cinnamate at 500 mg/L (4.4.4) measured with a precision pipette (4.2.3); make up to 100 mL with the citrate buffer at pH 6.5 vol. (4.4.3). Homogenise.

Note: a more concentrated ethyl cinnamate solution must not be prepared because the ester is liable to be partially insoluble.

4.4.6.  Dodecanol solution at 0.5 g/L(internal standard)

Using a precision syringe (4.2.5) place 50 μL of pure dodecanol (4.3..9) in a 100-mL graduated flask containing a little pure ethanol (4.3.7); make up the gauge line with pure ethanol (4.3.7); homogenise.

4.5.    Preparation of the sample

It is important to homogenise the enzymatic preparation before sampling, by upturning the container for example.

4.6.    Procedure

4.6.1.  Enzymatic reaction: In a 60-mL laboratory flask, place 50 mL of ethyl cinnamate solution at 25 mg/L (4.4.5) add approximately 100 mg of the commercial enzymatic preparation to be analysed (4.3.6) weighed with precision (4.2.7), i.e. weight P.

After stirring (4.2.2), the bottle is plugged and left on the laboratory bench or if possible in a drying oven at 25°C (4.2.6)

4.6.4.  Addition of the internal standard

In the preparation (4.6.3), using a precision syringe (4.2.5) add 50 μL of dodecanol to 500 mg/L (4.4.6); homogenise.

4.6.5.  Blank

Proceed as in 4.6.3 and 4.6.4 without adding the 200 μL of the sample from the enzymatic reaction (4.6.2)

4.6.6.  Reference solution

Proceed as in 4.6.3 and 4.6.4 by placing in the bottle (4.2.3) 200 μL of ethyl cinnamate solution at 25 mg/L (4.4.5) instead of the sample of enzymatic reaction (4.6.2)

4.6.7.  Chromatography

4.6.7.1. Inject 2 μL of the blank (4.6.5) into the chromatograph to locate the internal standard. Start the temperature programmer and the data acquisition.

4.6.7.2. Inject 2 μL of reference solution to locate the ethyl cinnamate (ec) and the internal standard (is); measure their respective surface areas Sec0 Sis0

4.6.7.3. Under the same conditions as 4.6.7.2 inject the samples (4.6.4) after 3 hours, after 24 hours and after 72 hours, i.e. the respective surface areas of residual ethyl cinnamate and internal standard S3 and Sis3; S24 Sis24, S72 Sis72.

Determine the quantity of residual ethyl cinnamate for each sample; for example for 72 hours.

Cinnamyl esterase activity in mg of hydrolysed ethyl cinnamate per hour and g of enzymatic preparation

EC activity in EC mg/g enzyme/hour =

• P = weight of enzyme added in the preparation (6.1) in mg/L.

4.7.    Comments: The method has been freely adapted from Barbe (1995).

The reaction taking place at pH 6.5 is much more complete than with the pH in the wine where it is approximately 10 times slower; therefore, if after 72 Hours, only a few mg of ethyl cinnamate have been degraded, the EC activity in the wine can be considered negligible.

Examples of cinnamoyl esterase activities measured at pH 6.5, of commercial enzymatic preparations.

5. Bibliography

• Barbe CH, 1995: On the contaminating esterase activities of pectolytic preparations. PhD Thesis, Univer

COEI-1-GLYCOS Determination of glycosidase activity in enzymatic preparations

Introduction

Enzymes of the glycosidase type are used to reveal the flavours of wines based on their glycosylated precursors.

Aromatic molecules are partially in the form of heterosides; they are for the main part associated with glucose; the measurement of enzymatic activity sufficient to break this specific bond has been described under " β-D-glycosidase activity". However, this activity is not really functional if the glucose is itself bound to another type of sugar (which is the case for most aromatic precursors). These are essentially apiose, arabinose, rhamnose and yxlose.

In order to measure the true efficiency of an enzymatic preparation so as to obtain the aromatic potential of the grape or wine, the measurement concerning β-D-glucosidase activity should include the measurement of apiofuranosidase, arabinofuranosidase, β-D-galactosidase, rhamnosidase, and xylosidase activities.

Determination of glucosidase activity in enzymatic preparations

(activity β-D-glucosidase)

(EC 3.2.1.21 – CAS no. 9001-22-3)

(OENO 5/2007; OIV-OENO 489-2012)

General specifications

These enzymes are usually present among other activities, within an enzymatic complex. Unless otherwise stipulated, the specifications must comply with the resolution OIV/OENO 365/2009 concerning the general specifications for enzymatic preparations included in the International Oenological Codex.

1. Origin

Reference is made to paragraph 5 “Source of enzyme and fermentation environment” of the general monograph on Enzymatic preparation.

The enzymatic preparations containing these activities are produced by directed fermentations of Aspergillus niger.

2. Scope/Applications

Reference is made to the International Code of Oenological Practices, OENO 16/2004 and OENO 17/2004.

Enzymes belonging to the glycosidase type are used to reveal and enhance the flavours of wines. This is realized through the hydrolysis of the glycosylated aroma precursors. The enzymes can also be added to the wine before the end of alcoholic fermentation but they will become active only after completion of the alcoholic fermentation

3. Principle

The enzymatic hydrolysis of p-nitrophenyl- β-D-Glucopyranoside, which is colourless, releases glucose and para-Nitrophenol (ρ-Np); the latter turns yellow in the presence of sodium carbonate, the absorbance of which is measured at 400 nm.

4. Apparatus
   1.     magnetic stirrer
   2.     water bath at 30°C 4.3 water bath at 100°C
   3.     cuvets with a 1-cm optical path length, for single use, for spectrophotometer, for measurement in the visible spectrum
   4.     crushed ice

precision syringe 500 – 5000 μl

precision syringe 100 μl

precision syringe 1000 μl

4.5.    spectrophotometer

4.6.    Eppendorf tubes

4.7.    100-mL graduated flask

4.8.    pH-meter

4.9.    cold room at 4°C

4.10. metal rack for Eppendorf tubes

4.11. carded cotton

4.12. Kraft paper

4.13. agitator of the vortex 4.18 chronometer

4.14. 15-mL glass tubes

5. Products
   1.     Sodium carbonate (Na₂CO₃ 99.5% pure - PM:105.99 g/mole)
   2.     Sodium acetate (CH₃COONa 99% pure - PM: 82g/mole)
   3.     Acetic acid (CH₃COOH 96% pure - PM: 60g/mole)

ρ-nitrophenyl- β-D-Glucopyranoside (Fluka, ref. 73676) as an example

5.6.    Distilled water

5.7.    Commercial enzymatic preparation for analysis

6. Solutions
   1.     Sodium acetate buffer (100 mM, pH 4.2)

It consists of solutions A and B.

6.1.1.  Solution A: introduce 0.5 g of sodium acetate (5.2) into 60 ml of distilled water (5.7)

6.1.2.  Solution B: introduce 1 ml of acetic acid (5.3) into 175 mL of distilled water (5.7) 6.1.3

Preparation of the sodium acetate buffer: mix 47.8 ml of solution A (6.1.1) + 152 ml of solution B (6.1.2).

Check the pH of the buffer using a pH-meter (4.12).

Maintain at 4°C

Place 0.096 g of p -nitrophenyl - β-D-Glucopyranoside (5.4) in 80 mL of sodium acetate buffer (6.1.).

6.3.    Sodium carbonate solution 1M

Dissolve 10.6 g of sodium carbonate (5.1) in 100 mL of water distilled (5.7) in a 100-ml graduated flask (4.11). The solution can be maintained at 4°C (4.13).

Dissolve 0.01 g of p-Np (5.6) in 80 mL of distilled water (5.7). The stock solution must be prepared extemporaneously.

It is made up using the stock solution of p-nitrophenol (p-Np) (6.4.) as indicated in table 1.

 

Table 1: Standard range of para-Nitrophenol
Quantity of p-Np (μg)	0	2	4	6	8	10
P-Np concentration (μg/mL)	0	10	20	30	40	50
P-Np concentration (μmol/mL)	0	.07222	0.14	0.22	0.29	0.36
Volume of stock solution (6.4) (μl)	0	16	32	48	64	80
Distilled water (5.7) (μl)	200	184	168	152	136	120

7. Preparation of the sample

 

It is important to homogenise the enzymatic preparation before sampling, by upturning the container for example. The enzymatic solution and the blanks will have to be prepared extemporaneously.

7.1.    Place 1 g of commercial preparation (5.8) in a 100-mL graduated flask (4.11), make up with distilled water (5.7), and stir (4.1) in order to obtain a homogeneous mixture.

7.2.    Blank denatured by heating

Place 10 mL of the enzymatic solution at 10 g/l (8.1) in a 15 mL tube (4.19), plug with carded cotton (4.15) covered with Kraft paper (4.16) and immerse the tube for 5 minutes in the water bath to 100°C (4.3).

8. Procedure

 

8.1.    Enzymatic reaction: The tubes are produced at least in duplicate..

In 5 Eppendorf tubes (4.10) numbered 1 to 5, placed in a rack (4.14) in ice crushed (4.5) introduce

100 µl of the solution of p-nitrophenyl- β-D-Glucopyranoside (6.2), using a precision syringe (4.7),

• 100 µl of the enzymatic solution with 2 g/l (8.1), start the chronometer (4.18)

After stirring (4.17), the Eppendorf tubes are placed in the water bath at 30°C (4.2)

• for 1 min. for tube no.1
• for 2 min. for tube no.2
• for 5 min. for tube no.3
• for 10 min. for tube no.4
• for 15 min. for tube no.5

The reaction is stopped by placing each of the tubes numbered from 1 to 5 immediately after they have been removed from the water bath at 30°C, in a bath of crushed ice (4.5)

8.2.    Determination of p-nitrophenol released

From the Eppendorf tubes containing the various reactional mediums (9.1)

Add 600 μl of sodium carbonate solution (6.3), using a precision syringe (4.8),

1.7 ml of distilled water (5.7), using a precision syringe (4.6),

Place the resulting mixture in a tank (4.4).

Immediately measure the absorbance at 400 nm, using a spectrophotometer (4.9)

8.3.    Blanks

Proceed as described in 9.1 by replacing the enzymatic solution with 2 g/l (8.1) by the blank denatured by heat (8.2). The ideal situation is to carry out the enzymatic reaction of the blank at the same time as that of the enzymatic solution.

8.4.    Standard range

Proceed as described in 9.2 by replacing the reactional medium (9.1) by the various mediums of the standard range of p -nitrophenol from 0 to 50 μg/mL (7).

9. Calculations

9.1.    Determining the 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 representation: 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 12 minutes. The activity concerned is measured at

• T=1 min
• T=2 min
• T=4 min
• T=6 min
• T=8 min
• T=10 min
• T=12 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 enzymatic 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).

9.2.    Producing the calibration line

The calibration line corresponds to plotting a graph whose X-coordinates are the various concentrations of the standard range of p.nitrophenol (from 0 to 0.36 μmole/ml) and whose Y-coordinates are the corresponding values of optical densities, obtained in 9.4. Then calculate the Q/T slope of the straight regression line (2) resulting from the linearity of the data of the graph.

9.3.    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 p.nitrophenol 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

Where

• Q: quantity of p.nitrophenol formed in µmoles during time T (min)

• V: quantity of enzymatic solution introduced (ml) here 0.1 ml
• C: concentration of the enzymatic solution (g/l) here 2 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:

10. Characteristics

The repeatability of the method is estimated using the mean standard deviation of the absorbance values resulting from the same sampling of the enzymatic preparation, proportioned 5 times. In this way, to proportion β-D-glucosidase the mean standard deviation of the values is 0.01 with a percentage error of 8.43, in which the % error corresponds to:

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

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

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

• variance analysis (the study of the probability of the occurrence of differences between samplings). Variance analysis is a statistical method used to test the homogeneity hypothesis of a series of K averages. Performing the variance analysis consists in determining if the "treatment" effect is "significant or not"
• the power of the test for the first species of risk α (5%) – first species of risk α 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.

Table 2: Variance analysis – study of the sampling effect
Determination	Variance analysis hypotheses	Probability	Power of test (α= 5%)	Newman- Keuls test(*)	Bonferroni test (**)
β-D-glucosidase	Adhered to	0.0285	42%	Non Significant	Non Significant

* 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 α 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 α 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 determination method involves a probability of occurrence of a discrepancy in activity (between samplings) lower than 5%, reinforced by belonging to the same group (Newmann-Keuls test not significant) and considered not to be different to the first species of risk α (Bonferroni test not significant).

Determination of various glycosidase activities in enzyme preparations

β-D-galactosidase (EC 3.2.1.23 – CAS n° 9031-11-2)

α-L-arabinofuranosidase (EC 3.2.1.55 – CAS n° 9067-74-7)

α-L-rhamnosidase (EC 3.2.1.40 – CAS n° 37288-35-0)

β-D-xylosidase (EC 3.2.1.34 – CAS n° 9025-53-0)

(OIV-OENO 451-2012)

General specifications

These enzymatic activities are usually present among other activities within an enzymatic complex. Unless otherwise stipulated, the specifications must comply with the resolution OIV/OENO 365/2009 concerning the general specifications for enzymatic preparations included in the International Oenological Codex.

1. Origin

Reference is made to paragraph 5 “Source of enzyme and fermentation environment” of the general monograph on Enzymatic preparation

The enzymatic preparations containing these activities are produced by directed fermentations of Aspergillus niger for example.

2. Scope/ Applications

Reference is made to the International Code of Oenological Practices, OENO 16/2004 and OENO 17/2004.

The glycosidase activities are used to reveal and enhance the flavours of wines based on hydrolysis of the sugar part of their glycosylated precursors. The enzymes can also be added to the must but their technological efficiencies will become active only after completion of the alcoholic fermentation.

3. Principle

Available enzymatic preparations with glycosidase activity contain enzymes that are able to hydrolyse the glycosidic bonds between glucose and other types of sugar, and in particular: apiose, galactose, arabinose, rhamnose and xylose- which then liberate the aromatic compounds contained in glucose by means of glycosidase activity. Similarly, the enzymes are capable of hydrolysing the bond of synthetic compounds that includes these various types of osidic compounds and p-nitrophenol. This enables to measure

these different activities.

Determination of β-D-galactosidase activity

The enzymatic hydrolysis of β-D- galactopyranoside of p-nitrophenyl, which is colourless, liberates galactose and para-nitrophenol (p-Np); the latter takes on a yellow colour when mixed with sodium carbonate, the absorbance of which is measured at 400nm.

Determination of α-L-arabinofuranosidase activity

The enzymatic hydrolysis of α-L-arabinofuranoside of p-nitrophenyl, which is colourless, liberates arabinose and p-nitrophenol (p-Np); the latter takes on a yellow colour when mixed with sodium carbonate, the absorbance of which is measured at 400nm.

Determination of α-L-rhamnosidase activity

The enzymatic hydrolysis of α-L-rhamnopyranoside of p-nitrophenyl, which is colourless, liberates rhamnose and p-nitrophenol (p-Np); the latter takes on a yellow colour when mixed with sodium carbonate, the absorbance of which is measured at 400nm.

Determination of β-D-xylosidase activity

The enzymatic hydrolysis of β-D-xylopyranoside of p-nitrophenyl, which is colourless, liberates xylose and p-nitrophenol (p-Np); the latter takes on a yellow colour when mixed with sodium carbonate, the absorbance of which is measured at 400nm.

4. Apparatus
   1.      magnetic stirrer
   2.      40°C water bath
   3.      100°C water bath
   4.      single-use 1 cm optical path vats for spectrophotometer measurement in the visible range
   5.      crushed ice
   6.      precision syringes 500 – 5000 μl
   7.      precision syringe 100 μl
   8.      precision syringe 1000 μl
   9.      spectrophotometer
   10. eppendorf tube
   11. 100 ml volumetric flask
   12. pH meter
   13. 4°C cold room
   14. metal tray for eppendorf tubes
   15. absorbent cotton
   16. Kraft paper
   17. vortex type stirrer
   18. timer
   19. 15 ml glass tubes

5. Products
   1.      Sodium carbonate (pure Na2CO3 at 99.5% - PM: 105.99 g/mole)
   2.      Sodium acetate (pure NaCH3COO at 99% - PM: 82g/mole)
   3.      Acetic acid (pure CH3COOH at 96% - PM: 60g/mole)
   4.      p- nitrophenol (p-Np) (pure C6H5NO3 at 99.5% - PM: 139.11 g/mole)
   5.      Distilled water
   6.      Commercial enzymatic preparation to be analysed, and depending on the measurement of the considered activity:
   7.      a β -D-galactopyranoside de p-nitrophenyl (Sigma ref. N1252, 250 mg) as an example
   8.      b α -L-arabinofuranoside de p-nitrophenyl (Sigma ref. N3641, 10 mg) as an example
   9.      α -L-rhamnopyranoside de p-nitrophenyl (Sigma ref. N7763, 100 mg) as an example
   10. β -D-xylopyranoside de p-nitrophenyl (Sigma ref. N2132, 500 mg) as an example

6. Solutions

For the determination of α-L-arabinofuranosidase or α-L- rhamnosidase

6.1.     Sodium acetate buffer (100 mM, pH 4.4) It is made of solutions A and B.

6.1.1.  Solution A: add 0.984 g of sodium acetate (5.2) in 60 ml of distilled water (5.6)

6.1.2.  Solution B: add 2 ml of acetic acid (5.3) in 175 ml of distilled water (5.6)

6.1.3.  Preparation of the sodium acetate buffer: Add 78 ml of solution A (6.1.1) + 122 ml of solution B (6.1.2).

Control the pH of the buffer with the pH meter (4.12).

Keep at 4°C

For the determination of β-D-galactosidase or β-D-xylosidase activity

6.2.     Sodium acetate buffer (100 mM, pH 4.0) It is made of solutions A and B.

6.2.1.  Solution A: add 0.984 g of sodium acetate (5.2) in 60 ml of distilled water (5.6)

6.2.2.  Solution B: add 2 ml of acetic acid (5.3) in 175 ml of distilled water (5.6)

6.2.3.  Preparation of the sodium acetate buffer: Add 36 ml of solution A (6.1.1) + 164 ml of solution B (6.1.2).

Control the pH of the buffer with the pH meter (4.12).

Keep at 4°C

6.3.     Reagent solution (depending on the measurement of the considered enzymatic activity)

6.3.1.  Solution of p-nitrophenyl α -L-arabinofuranoside 4 mM

Add 0.086 g of p-nitrophenyl α -L-arabinofuranoside (5.4) in 80 ml of sodium acetate buffer (6.1.).

Add 0.096 g of p-nitrophenyl β-D-galactopyranoside (5.4) in 80 ml of sodium acetate buffer (6.1).

6.3.3.  Solution of p-nitrophenyl α -L-rhamnopyranoside 4 mM

Add 0.091 g of p-nitrophenyl α -L-rhamnopyranoside (5.4) in 80 ml of sodium acetate buffer (6.1.).&

Add 0.0868 g of p-nitrophenyl β-D-xylopyranoside (5.4) in 80 ml of sodium acetate buffer (6.1).

6.4.     Solution of sodium carbonate 1M

Dissolve 10.6 g of sodium carbonate (5.1) in 100 ml of distilled water (5.6) in a 100 ml volumetric flask (4.11). The solution may be kept at 4°C (4.13).

6.5.     Stock solution of p-nitrophenol at 125 µg/ml

Dissolve 0.01 g of p-nitrophenol (5.5) in 80 ml of distilled water (5.6). The stock solution must be prepared extemporaneously.

7. Preparation of the standard range of p-nitrophenol from 0 to 100 µg/ml

It is made of the stock solution of p-nitrophenol (6.4.) as indicated in table 1.

Table 1: Standard range of p-nitrophenol (p.Np)
Quantity of p-Np (μg)	0	4	8	12	16	20
Concentration of p-Np (μg/ml)	0	20	40	60	80	100
Concentration of p-Np (μmol/ml)	0	0.14	0.2	0.43	0.5	0.72
		9		8
Volume of stock solution (6.4) (μl)	0	16	32	48	64	80
Distilled water (5.5) (μl)	200	184	168	152	136	120

8. Preparation of the sample

It is important that the enzymatic preparation be homogeneous before sampling, by shaking it for example. The enzymatic solution and whites are to be prepared extemporaneously.

8.1.     Enzymatic solutions

 

For the determination of α-L-rhamnosidase or β-D- xylosidase activity

10 g/l enzymatic solution

Put 1 g of commercially available preparation (5.6) in a 100 ml volumetric flask (4.11), add distilled water (5.5), and stir (4.1) in order to achieve a homogeneous solution.

For the determination of α-L-arabinofuranosidase activity

1 g/l enzymatic solution

Put 100 mg of commercially available preparation (5.6) in a 100 ml volumetric flask (4.11), add distilled water (5.5), and stir (4.1) in order to achieve a homogeneous solution.

For the determination of β-D-galactosidase activity

2 g/l enzymatic solution

Put 100 mg of commercially available preparation (5.6) in a 100 ml volumetric flask

(4.11), add distilled water (5.5), and stir (4.1) in order to achieve a homogeneous solution.

8.2.     Denatured white through heating

Put 10 ml of the enzymatic solution (8.1) in a 15 ml tube (4.19), plug with absorbent cotton (4.15) covered with Kraft paper (4.16) and immerse the tube for 5 minutes in the

100°C water bath (4.3).

9. Procedure
   1.      Enzymatic reaction: The tubes must be at least doubled.

In 6 eppendorf tubes (4.10) numbered from 1 to 6 and placed in a tray (4.14) of crushed ice (4.5), introduce

• 100 μl of the considered reagent solution (6.2), with a precision syringe (4.7),
• 100 μl of the corresponding enzymatic solution (8.1), start the timer (4.18)

After stirring (4.17), the eppendorf tubes are placed in the 40°C water bath (4.2)

• for 2 mn in tube n° 1 for 5 mn in tube n° 2
• for 10 mn in tube n° 3 for 15 mn in tube n° 4 for 20 mn in tube n° 5 for 30 mn in tube n° 6

The reaction is stopped by placing each numbered (1-6) tube immediately after extraction from the 40°C water bath in the tray of crushed ice (4.5).

9.2.     Determination of liberated p-nitrophenol

With the eppendorf tubes containing the various reactive media (9.1)

add 600 μl of the considered reagent solution (6.3), with a precision syringe (4.8), and

1.7 ml of distilled water (5.5) with a precision syringe (4.6), Place the resulting mixture in a vat (4.4).

Immediately measure the absorbance at 400 nm with a spectrophotometer (4.9)

(This can also be simplified by indicating: See point 8.2 pertaining to the measurement of

β-D-glycosidase activity)

9.3.     Blank

Proceed as per indications given in point 9.1 by replacing the enzymatic solution (8.1) with whites denatured by heating (8.2). Ideally, the enzymatic reaction of whites should be carried out at the same time as the reaction of the enzymatic solution.

9.4.     Standard range

Proceed as described for point 9.2 by replacing the reactive medium (9.1) with various media of the standard range of p-nitrophenol from 0 to 100 μg/ml (7).

10. Calculations
    1. Chemical kinetics

Generally, the calculation of the enzymatic activity can only be carried out when the substrate and the enzyme are not in limiting quantities. This corresponds to the ascending phase of the kinetic representation: the enzymatic activity is linear in time. If this were not to be the case, the activity would be underestimated (Illustration 1).

A kinetic calculation is performed for 30 minutes. The activity under consideration is measured at T=2 min, T=5 min, T=10 min, T=15 min, T=20 min, T=30 min.

After having calculated the kinetic rate of the enzymatic reaction, establish the variation curve of absorbance according to reaction times. Absorbance is the difference between absorbance at time T of the enzymatic preparation and the corresponding white.

Then calculate the equation (1) of the regression curve by considering only the points of the ascending phase (see illustration 1).

10.2. Establishing the standard line

The standard calibration line is established in a graph where the x-axis represents the various concentrations of the standard range of the p-nitrophenol (0 to 0.72 µmole/ml) and the y-axis represents the various corresponding optical densities established in 8.4. Then calculate the regression curve (2) that results from the linearity of the graph’s data.

10.3. Calculation of enzymatic activities

Based on the regression curve (1), calculate the absorbance for an average time of T (for example 4 mn in the case of illustration 1) and deduce the Q quantity of liberated p- nitrophenol (in μmoles) for this intermediate time with equation (2).

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

Where

• Q: quantity of p-nitrophenol formed in µmoles during time T (min)
• V: quantity of introduced enzymatic solution (ml), in this instance 0.1 ml
• C: concentration of the enzymatic solution (g/l), in this instance 10 g/l

It then becomes possible to represent the enzymatic activity in nanokatal. This unit corresponds to the number of nanomoles of the amount of product created per second in the conditions defined in determination protocols, and therefore:

11. Reproducibility

The reproducibility of the method is estimated with the average of standard deviations of absorbance values resulting from a sample taken from the same enzymatic preparation, determined five times.

The table below summarises the results:

Activity	Average of values’ standard deviations	Error percentage (%)
α-L-arabinofuranosidase	0	5
β-D-galactosidase	0.03	3.78
α-L-rhamnosidase	0.001	4.66
β-D-xylosidase	0.03	3.78

The % of error corresponds to:

Hence, the determination method as presented herein is deemed to be reproducible.

The reproducibility trails were carried out with 2 enzymatic preparations and 5 samplings for each.

Two tests were used to determine the proper reproducibility of the method:

• the analysis of variance (the study of the probability of deviations between samples). The variance analysis is a statistical method that enables to test the homogeneity hypothesis of a set of average k values. The variance analysis consists in determining whether the "treatment" effect is "significant or not"
• the strength of the trial with type I error (5%) – type I error is the risk of deciding that identical treatments are different

If the strength is feeble ( 20%), this means that no difference has been detected between treatments, but there is little chance of seeing a difference if there actually were one.

If the strength is high ( 80%), this means that no difference has been detected between treatments, but we would have the means of seeing it if such a difference

were present.

The results are given in table 2.

Table 2: Variance analysis – stuffy of the sampling effect
Determinations	Hypothses of variance analysis	Probability	Strength of the trial (α	Newman-Keuls test (*)	Bonferroni test (**)
α-L-arabinofuranosidase	Satisfied	0.0125	45%	Not significant	Not significant
β-D-galactosidase	Satisfied	0.01	75%	Not significant	Not significant
α-L-rhamnosidase	Satisfied	0.006	65%	Not significant	Not significant
β-D-xylosidase	Satisfied	0.0253	73%	Not significant	Not significant

* Newman-Keuls test: this test is used to compare averages and enables to establish homogeneous treatment groups: those that belong to a same group are considered as not different to the chosen type I error

** Bonferroni test: also known as the “Bonferroni correction” the Bonferroni test enables to carry

out all 2 on 2 average comparisons. i.e. (t(t-1))/2 comparisons before treatments. respecting the chosen type I error.

Therefore. the tests conducted enable to identify a difference if such a difference exists (high trial strength); furthermore the determination method presents the probability of activity deviations (from one sampling to the next) of less than 5% reinforced by belonging to the same group (non-significant Newmann-Keuls test) and considered to be not different from type I error (non-significant Bonferroni test).

12. Bibliography

• M. Lecas. Thèse de Doctorat. 1994. Composition et structure des polymères constitutifs des parois cellulaires de la pellicule de la baie de raisin. Application d’enzymes fongiques liquéfiant les parois à fins d’extraction des substances odorantes.
• I. Dugelay. Thèse de Doctorat. 1993. L’arôme du raisin : étude des précurseurs hétérosidiques et des activités enzymatiques exogènes impliquées dans leur hydrolyse – Applications technologiques.