Enzymes

Codified File

Enzymatic preparations

COEI-1-PRENZY Enzymatic preparations

The prescriptions described below concern all enzymatic preparations susceptible of being used during various operations that can be applied to grapes and their derivatives.

The prescriptions are based on the recommendations from the “General Specifications and Considerations for Enzymes used in Food Processing” drafted by the “Joint FAO/WHO Expert Committee on Food Additives (JECFA), 67th Session, Rome 20 -29 June 2006 published in 2006 in the FAO JECFA monographs.

 

  1. General considerations

Enzymatic preparations can be made from any safe biological sources. When looking for synergies between various enzymatic activities including pectinase, cellulase and hemicellulase, mixtures of preparations made from different strains can be carried out. These preparations can contain one or more active compounds, in addition to supports, diluents, preservatives, antioxidants and other substances compatible with the good manufacturing practices and in accordance with local regulations. In certain cases, preparations can contain cells or cell fragments. Furthermore they can be in either liquid or solid form. The active substances can also be immobilised on a support admitted for food use.

  1. Labelling

The labelling of enzymatic preparations must at least specify the enzyme name according to IUBMB rules (ex. polygalacturonase), the activity (in units by g or mL), the batch number storage condition for maintaining stability and the expiry date. Enzymatic preparations with multiple technological activities (cf. 4.1) should bear the name of each enzyme on which the preparation is standardized.

If there is available space, it is desirable that the label has the additional information: recommended dose and implementation conditions, the nature of additives and carriers used, the nature of enzymatic activities. If there is not enough space, this information shall be indicated on the technical data sheet of the preparation.

The indication that enzymatic preparations were obtained by genetically modified organisms must be mentioned. If it is not mentioned in the labelling, the fact that genetic engineering was used to improve the microorganism that produces the enzyme has to be mentioned in related documentation.

  1. Admitted enzymatic preparations

All enzymatic preparations with activities presenting a technological interest duly proven in practice and meeting the conditions and criteria mentioned above, are accepted for the treatment of grapes and their by-products.

Enzymatic preparations used must not contain any substance, microorganism, nor enzymatic activity that:

  • is harmful to health,
  • is harmful to the quality of the products manufactured, particularly concerning the colour, the aroma and the taste of the wines,
  • can lead to the formation of undesirable products,
  • or that will give rise or facilitate fraud.
  1. Enzymatic activities

 

4.1.    General considerations

[Enzymatic preparations contain many enzymatic activities. Other than the main enzymatic activities, (activities for which, respectively, the enzymatic preparation has been standardised) whose technological interest has been duly proven, secondary enzymatic activities are only tolerated if they are set within the technological constraint limits for manufacturing of enzymatic preparations.]

Generally speaking, the secondary activities present in a given preparation must not become the main reason to use the said preparation unless this preparation is declared as multiple technological effects. Referring to the International Code of Oenological Practices, OENO 11/2004 – OENO 18/2004 and AG 3/85-OEN, on a technological level, a distinction is made between the following  types of preparations

  • Maceration preparations: facilitate extraction of compounds such as colour, tannins,...
  • Clarification / filtration preparations: facilitate clarification and filtration of musts and wine
  • Aroma enhancers: reinforces and/or modifies aromatic profile of musts and wine
  • Stabilisation preparations: facilitates extraction of macromolecules or other substances with a stabilising effect on wine (yeast mannans).

When an enzymatic preparation generates multiple technological effects, duly noted in a practice, (ex. Clarification and aroma enhancer enzymes), whether they are the result of a main and/or secondary activity, they must be declared as such on the label. Different enzymatic activities responsible for these effects must be measured and indicated in the technical preparation data sheet.

 

4.2.    Activity measurement

The enzymatic activities presented are measured under the conditions corresponding to their biochemical characteristics. (pH, temperature) and if possible, the closest to activities encountered in the practice (grape juice, must or wine). The methods implemented must correspond to state of the art in analytical terms and, if possible, be validated in accordance with appropriate international standards (for example: ISO 78-2; ISO 5725).

Results are expressed in nanokatal/g or nanokatal/mL or in viscosity units in the case of enzymes with endo-type of activities. (nkat = 1 nmole of transformed substrate or product formed per second by g or mL of the preparation). Results should be given with reference to the method used.

When the sought out technological effect results from the action of different enzymes within the same preparation, it is necessary to measure each enzymatic activity. Each of these activities will require special Codex monograph, with the details of the analytical method.

  1. Sources of enzymes and fermentation environment

The sources of enzymes must be non-pathogenic, non-toxic and genetically stable, and the fermentation broth should not leave harmful residues in enzymatic preparations. In the case of microorganisms, a safety study must be conducted in order to ensure that enzymatic preparation produced by a microorganism species (e.g. Aspergillus niger) does not present any health risk. This study can be based on principles brought forth on food enzyme guidelines published by the European Food Safety Authority (EFSA), or other equivalent organisations.

The techniques implemented must be compatible with good manufacturing practices and the prescriptions of the International Oenological Codex if yeast and/or lactic bacteria are used.

  1. Carriers, diluents, preservatives and other additives

Substances used as carriers, diluents, preservatives or other additives must not, with a “carry over” effect, disseminate compounds in the grapes and derivative products, which are not compatible with regulations in force in different countries. Moreover, these compounds must not have a negative effect on the organoleptic properties of wine. In the case of immobilised enzymes, the carriers used must comply to standards on material in contact with foodstuffs. For this type of preparation, the content of compounds of the carriers used, susceptible to enter the musts and wine, should be determined and indicated on the label of the enzymatic preparation.

Preservatives such as KCl are added in the liquid enzyme concentrate during manufacturing. These substances prevent the development of micro-organisms during the different formulation operations of products. These substances can be found not only in liquid preparations but also in solid preparations. Given the inevitable “carry over” effect, only preservatives which are compatible to regulations in force in the different countries are authorised.

These substances must be clearly identified and indicated on the label or on the technical data sheet of the commercial product.

  1. Hygiene and maximal level of contaminants

 

Enzymatic preparations must be produced in accordance with good manufacturing practices:

 

7.1.    Lead

Proceed with the determination according to the method described in chapter II of the International Oenological Codex.

Content less than 5 mg/kg.

 

7.2.    Mercury

Proceed with the determination according to the method described in chapter II of the International Oenological Codex.

Content less than 0.5 mg/kg.

7.3.    Arsenic

Proceed with the determination according to the method described in chapter II of the International Oenological Codex.

Content less than 3 mg/kg.

7.4.    Cadmium

Proceed with the determination according to the method described in chapter II of the International Oenological Codex.

Content less than 0.5 mg/kg.

 

7.5.    Salmonella sp

Proceed with counting according to the method described in chapter II of the International Oenological Codex.

Absence checked on a 25 g sample.

 

7.6.    Total coliforms

Proceed with counting according to the method described in chapter II of the International Oenological Codex.

Content less than 30/per gram of preparation.

 

7.7.    Escherichia coli

Proceed with counting according to the method described in chapter II of the International Oenological Codex.

Absence checked on a 25 g sample.

7.8.    Antimicrobial activity

Non-detectable 

 

7.9.    Specific mycotoxins of different production strains

Non-detectable

  1. Technical data sheet to be supplied by manufacturer

Each type of enzymatic preparation must be defined using a technical data sheet.

It must contain at least the following information:

  • Name of enzyme and biological origin (e.g. pectolytic enzymes of Aspergillus niger or pectolytic enzyme of A. oryzae expressed as A. niger),
  • Declared activity (in nKat/g or nKat/ml of preparation)
  • Fields and application mode (technological effects and useful details for the implementation of the preparation),
  • Stability of the preparation and expiration date period based on production date guaranteeing the maintaining of activity, under the given storage conditions (temperature),
  • Types of reactions catalysed by the main enzymatic activities,
  • Main enzymatic activities with IUB number (for example Tannase 3.1.1.20),
  • Secondary enzymatic activities with, if possible, the IUB number
  • Types of carriers, diluents, preservatives and additives used and their respective contents,
  • If deemed useful, further information can be added to this technical data sheet.

Beta-glucanase activity (β 1-3, β 1-6)

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.

  1. 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.

  1. 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.

  1. 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

  1. 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).

  1. 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.

  1. 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

  1. 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

Cellulase

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.

  1. 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.

  1. 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).

  1. 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

  1. 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

  1. 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)
    1.   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.

6.4.         Stock glucose solution at 250 μg/mL

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.

  1. 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

  1. 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

  1. 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).

  1. 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

  1. 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%.

  1. 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

Cinnamoyl esterase activity

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.

  1. 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.

  1. 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.
    1.   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
  1. 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)

4.6.2.  A sample of 200 μL is taken with a precision syringe (4.2.5) after 3 hours, 24 hours, 72 hours.

4.6.3.  The reaction is stopped by adding the sample (4.6.2) of 200 μL in a 10-mL flask containing 0.5 mL of methanol (4.3.1) and 1 mL of ether (4.3.8)

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.

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

Glycosidase

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.

  1. 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

  1. 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.

  1. 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

  1. 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

5.4.    β-D-glucosidase (Fluka; 250 mg; 6.3 U/mg, ref. 49290) as an example. One unit corresponds to the quantity of enzyme required to release 1 µmole of glucose per minute with pH 5 and 35°C.

5.5.    ρ-nitrophenol - Np) (C6H5NO3 99.5% pure - PM: 139.11 g/mole)

5.6.    Distilled water

5.7.    Commercial enzymatic preparation for analysis

  1. 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

6.2.    Solution of p -nitrophenyl - β-D-Glucopyranoside 4mM

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).

6.4.    Stock solution of ρ -nitrophenol (p-Np) at 125 μg/ml

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

6.5.    Preparation of the standard range of p-nitrophenol (p-Np) from 0 to 50 μg/ml

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

  1. 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).

  1. 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).

  1. 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:

  1. 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.

  1. 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.

  1. 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.

  1. 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
  1. 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
  1. 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.).

6.3.2.  Solution of p-nitrophenyl β-D-galactopyranoside 4 mM

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.).&

6.3.4.  Solution of p-n i t rop hen yl β-D-xylopyranoside 4 mM

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.

  1. 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

  1. 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).

  1. 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).

  1. 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:

  1. 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).

  1. 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.

Pectinlyase activity

COEI-1-ACTPLY Determination of pectinlyase activity in enzymatic preparations (Pectinlyase activity)

EC. 4.2.2.10. – CAS no. 9033-35-6)

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 356/2009 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 monography on Enzymatic preparation

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

  1. 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.

These enzyme activities are used to support grape maceration and grape juice extraction as well as to help the clarification of musts and wines and finally to improve their filterability.

  1. Principle

This enzymatic activity results in the decomposition of highly methylated pectins by the β-elimination of methylated galacturonic acids. In so doing, a system of highly delocalised conjugated double bonds is created, absorbing in the ultraviolet range.

  1. Apparatus

 

4.1.     magnetic stirrer

4.2.     water bath at 25 °C

4.3.     water bath at 100 °C

4.4.     1000-mL graduated flask

4.4.1.  100-mL graduated flask

4.5.     Chronometer

4.6.     quartz cuvets with a 1-cm optical path length, for spectrophotometer, for measurement in the UV spectrum

4.7.     pH-meter

4.8.     100- μL precision syringes

4.9.     1000- μL precision syringes

4.10. spectrophotometer

4.11. 15-mL test tubes

4.12. shaker of the vortex type

4.13. metal rack for 15-mL test tubes

4.14. chamber at 4 °C

4.15. carded cotton

4.16. Kraft paper

  1. Products

5.1.     Citrus fruit pectin with a 63-66 % degree of esterification

(Pectin from citrus peel, Fluka, Ref. 76280), as an example.

5.2.     Sodium hydroxide (NaOH, 99 % pure - PM = 40 g/mole)

5.3.     Citric acid (C6H8O7·H2O, 99.5 % pure - PM = 210.14 g/mole)

5.4.     Sodium dihydrogenophosphate (NaH2PO4·2H2O, 99 % pure PM = 156.01 g/mole) 

5.5.     Distilled water

5.6.     Commercial enzymatic preparation for analysis

  1. Solutions

6.1.     Solution of sodium hydroxide 1M

Introduce 40 g of sodium hydroxide (5.2) into a 1000-mL graduated flask (4.4) and make up with distilled water (5.5).

6.2.     Mc Ilvaine buffer (Devries et al).

It consists of solutions A and B.

6.2.1.  Solution A:  acid citric at 100 mM: dissolve 4.596 g of citric acid (5.3) in 200 mL of distilled water (5.5)

6.2.2.  Solution B: sodium dihydrogenophosphate at 200 mM: dissolve 6.25 g of sodium dihydrogenophosphate (5.4) in 200 mL of distilled water (5.5).

6.2.3.  Preparation of the Mac Ilvaine buffer

Mix 50% of solution A (6.2.1) + 50 % of solution B (6.2.2) and adjust pH to 6 using the solution of sodium hydroxide (6.1).

The solution must be maintained at 4 °C (4.13). Check the pH of the buffer using a pH-meter (4.7)

6.3.     Solution of citrus fruit pectin at 1 % (p/v)

Dissolve 0.5 g of pectin (5.1) in 50 mL of Mc Ilvaine buffer (6.2).

  1. Preparation of the sample

It is important to homogenise the enzymatic preparation before taking a sample by turning over the recipient, for example. The enzymatic solutions and blanks should be prepared at time of use.

7.1.     Enzymatic solution at 10 g/L to be prepared just before use.

Place 1g of commercial preparation (5.6) in a 100-mL graduated flask (4.4.1), make up with distilled water (5.5), stir (4.1) in order to obtain a homogeneous mixture.

7.2.     Blank denatured by heating to be prepared just before use

Place 10 mL of the enzymatic solution at 10 g/L (7.1) in a 15-mL test tube (4.10), plug with carded cotton (4.14) covered with Kraft paper (4.15) and immerse the tube for 5 minutes in the water bath at 100°C (4.3). Then chill and centrifuge 5 min at 6500 g.

  1. Procedure

 

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

In 5 x 15-mL test tubes (4.10) numbered from 1 to 5, placed in a rack (4.12) in a water bath at 25°C, introduce

  • 400 μL of Mc Ilvaine buffer (6.2) using a 1000-µL precision syringe (4.8.1)
  • 100 μL of the enzymatic solution at 10 g/L (7.1) using a 100- μL precision syringe (4.8)
  • 500 μL of citrus fruit pectin solution (6.3) beforehand warmed at 25°C in water bath; start the chronometer (4.5)

After stirring (4.11), the tubes plugged with carded cotton (4.14) and Kraft paper (4.15), are placed in the water bath at 25 °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 rapid (30 seconds max) heating by placing each tube numbered from 1 to 5 in the water bath at 100 °C (4.3) and adding acid or basic concentrated solutions as stop reagent. The tubes are then cooled under running cold water.

8.2.     Determination of released substances

The reactional medium (8.1) is diluted to one tenth with distilled water (5.5). The dilution is placed in a cuvet (4.6) with an optical path of 1 cm.

Zero spectrophotometer using distilled water.

Immediately measure the absorbance at 235 nm, using a spectrophometer (4.9).

8.3.     Blank

Proceed as described in 8.1, replacing the enzymatic solution by the blank denatured by heating (7.2). For each kinetic point, the enzymatic reaction of each blank is carried out at the same time as that of the enzymatic solution.

  1. 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 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 DO/T slope (1) of the straight regression line, taking into account only the points of the ascending phase (see figure 1).

9.2.     Calculating the enzymatic activity

The enzymatic activity of the pectinlyase is calculated using the molar extinction coefficient of the molecule formed (ε= 5500 M-1cm-1). The formula to be applied is as follows:

Activity in

Where

  • DOT: absorbance value at time T (min)
  • V: quantity of enzymatic solution introduced
  • (mL): in this case, 0.1 mL
  • C: concentration of the enzymatic solution
  • (g/L): in this case 10 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

  1. Characteristics of the method

r=

0,028

R=

0,112

Sr=

0,01

SR=

0,04

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 the pectinlyase the mean standard deviation of the values is 0.01 with a percentage error of 4.66, 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 standard deviation of reproductibility given by this variance analysis is 0,04.
  • 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 1.

Determination

Variance analysis hypotheses

Probability

Power of

test (α= 5 %)

Newman-

Keuls test (*)

Bonferroni test

(**)

PL

Adhered to

0.00725

87 %

Significant

Significant

Table 1: 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 determination method involves a probability of occurrence of a discrepancy in activity (between samplings) lower than 5 %.

  1. Bibliography
  • DE VRIES J.A., F. M. ROMBOUTS F.M., VORAGEN A.g.J., PILNIK W. Enzymic degradation of apple pectins. Carbohydrate Polymers, 2, 1982, 25-33.

Pectinmethylesterase activity

COEI-1-ACTPME Determination of pectin methylesterase activity in enzymatic preparations ((Pectin Methyl-Esterase Activity (PME)
(EC. 3.1.1.11 – CAS N° 9025-98-3)

General specifications

These enzymes are usually present within an complex enzymatic preparation. 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 monography on Enzymatic preparation

The enzyme preparations containing such activity are produced by directed fermentations such as Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus Tubigensis, Aspergillus Awamori, Rhizopus oryzae and Trichoderma longibrachiatum (T.reesei)

  1. 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.

These enzyme activities are used to support grape maceration and grape juice extraction as well as to help the clarification of musts and wines and finally to improve their filterability.

Determination of Pectine methylesterase activity using methanol dosage

  1. Principle

The enzyme activity of demethylation of the pectin results in the appearance of free carboxyl groups associated with the galacturonic acids making up the chains.

The pectin methyl-esterase activity is estimated by determination of the methanol according to the Klavons & Bennet method (1986). The alcohol oxydase of Pichia pastoris is specific to primary alcohols with a low molecular weight and catalyses the oxidation of the methanol into formaldehyde. 2,4-Pentanedione condenses exclusively with aldehydes of low molecular weight such as formaldehyde, forming a chromophore absorbing at 412 nm.

  1. Equipment

2.1.    water bath at 25°C

2.2.    water bath at 30°C

2.3.    water bath at 60°C

2.4.    water bath at 100°C

2.5.    100-ml cylindrical flask

2.6.    stop-watch

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

2.8.    1-L graduated flask

2.9.    100-ml graduated flask

2.10. pH-meter

2.11. 500-5000 μl precision syringe

2.12. 100-1000 μl precision syringe

2.13. 0-200 μl precision syringe

2.14. 0-20 μl precision syringe

2.15. spectrophotometer

2.16. 15-ml sealed glass screw-top test tubes

2.17. metal rack for 15 ml test tubes

2.18. Vortex-type mixer

2.19. magnetic stirrer

  1. Reagents

3.1.    citrus fruit pectin with a degree of esterification of 63-66%. (Pectins ex-citrus: Fluka, ref: 76280 as an example).

3.2.    orange peel pectin esterase (Fluka; 20 U/mg, ref: 76286 as an example).

3.3.    sodium acetate (CH3COONa 99% pure - MW = 82g/mole)

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

3.5.    alcohol oxydase of Pichia Pastoris (Sigma, 250 U; 0.2 ml, ref: A2404 as an example). One unit of alcohol oxydase oxidizes one µmole of methanol into formaldehyde per minute at pH 7.5 and at 25°C.

3.6.    ammonium acetate (CH3COONH4, 99.5% pure - MW = 77.08g/mole)

3.7.    pentane-2.4-dione (C5H8O2 - MW = 100.12g/mole)

3.8.    methanol (CH20H, Analytical Reagent grade - MW = 32g/mole)

3.9.    potassium dihydrogen phosphate (KH2PO4, 99% pure - MW = 136.06 g/mole)

3.10. disodium hydrogen phosphate (Na2HPO4.2H2O 98.5% pure - MW = 178.05 g/mole)

3.11. distilled water

3.12. commercial enzyme preparation to be analysed

  1. Solutions

4.1.    Sodium acetate buffer 50 mM, pH 4.5

This consists of 2 solutions, A and B.

4.1.1.  Solution A: introduce 4.10 g of sodium acetate (5.3) into 1 liter of distilled water (5.11).

4.1.2.  Solution B: introduce 2.8 ml of acetic acid (5.4) into 1 liter of distilled water (5.11). 6.1.3 Preparation of the sodium acetate buffer: mix 39.2% of solution A (6.1.1) + 60.8% of solution B (6.1.2),. Check that the pH equals 4.5 using a pH-meter (4.10). Maintain at 4°C

4.2.    Citrus fruit pectin solution at 0.5% (p/v)

Introduce 0.5 g of citrus fruit pectin (5.1) into 100 ml of sodium acetate buffer (6.1) in a 100-ml graduated flask (4.9).

The solution must be prepared as needed.

4.3.    Acetic acid solution 0.05 M

Introduce 0.283 5 ml of acetic acid (5.4) into 100 ml of distilled water (5.11), in a 100-ml graduated flask (4.8).

4.4.    Ammonium acetate solution 2 M

Dissolve 15.4 g of ammonium acetate (5.6) in 100 ml of acetic acid (6.3), in a 100-ml graduated flask (4.9).

4.5.    2,4-Pentanedione 0.02 M

Introduce 40.8 µl 2,4-pentanedione (5.7) into 20 ml of ammonium acetate solution (6.4). The solution must be prepared as needed.

4.6.    Sodium phosphate buffer (0.25 M; pH 7.5)

This consists of solutions A and B.

4.6.1.  Solution A: introduce 34.015 g of potassium dihydrogen phosphate (5.9) into 1 liter of distilled water (5.11).

4.6.2.  Solution B: introduce 44.5125 g of disodium hydrogen phosphate (5.10) into 1 liter of distilled water (5.11).

4.6.3.  Preparation of the sodium phosphate buffer: mix 16.25 % of solution A (6.6.1) + 83.75% of solution B (6.6.2) to obtain a pH of 7.5.

Check the pH using a pH-meter (4.10).

Maintain at 4°C, for a maximum of one week

4.7.    Stock solution of methanol at 40 µg/ml

Introduce 5 µl of methanol (5.8) using a precision syringe (4.14) into 100 ml of sodium phosphate buffer (6.6) in a 100-ml graduated flask (4.9).

4.8.    Alcohol oxydase at 1U/ml

Dilute alcohol oxydase of Pichia pastoris (5.5) in a phosphate buffer (6.6) in order to obtain a solution at 1U/ml. The solution must be prepared as needed.

Preparation of the standard solutions of methanol

The standard solutions are produced from 0 to 20 μg methanol as indicated in Table 1. They are made up from the stock solution of methanol (6.7.)

Quantity of Methanol (μg)

0

5

10

15

20

Quantity of Methanol (μmole)

0

0.1563

0.3125

0.4688

0.625

Vol. stock solution (6.7.) (μl)

0

75

150

225

300

Vol. buffer (6.6.) (μl)

600

525

450

375

300

Table 1: standard solutions of methanol

  1. 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 prepared at the time of use.

5.1.    Enzyme solution with 1 g/l to be prepared just before use

Place 100 mg of commercial preparation (5.12) in a 100-ml graduated flask (4.9), make up to the mark with distilled water (5.11), and stir (4.19) in order to obtain a homogeneous mixture.

5.2.    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 screw-top test tube (4.16), and immerse the test tube for 5 minutes in the water bath at 100°C (4.4). Cool and centrifuge for 5 min at 6500 g.

  1. Procedure
    1.     Enzyme kinetics: The test tubes are prepared at least in duplicate.

In 5 x 15-ml test tubes (4.16) numbered from 1 to 5, placed in a rack (4.17) in a water bath at 30°C introduce:

  • 100 μl of the enzyme solution at 1 g/l (8.1), using the precision syringe (4.13),
  • 500 μl of the citrus fruit pectin solution (6.2) warmed beforehand at 30°C in a water bath, start the stop-watch (4.6).

After shaking (4.18), the test tubes are replaced in the water bath at 30°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 30°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

6.2.    Determination of methanol released

In a 15-ml screw-top test tube (4.16)

Add 1 ml of the alcohol oxydase solution (6.8) to the reaction medium (9.1), using the precision syringe (4.12), start the stop-watch (4.6).

After shaking (4.18), the test tube is placed in the water bath at 25°C (4.1) for 15 min.

Then add 2 ml of 0.02 M 2,4-pentanedione (6.5) using the precision syringe (4.11), start the stop-watch (4.6).

After shaking (4.18), the test tube is placed in the water bath at 60°C (4.3) for 15 min.

The test tube is then cooled under running cold water.

Place the supernatant liquid in a cuvette (4.7).

Zero the spectrophotometer using distilled water.

Immediately measure the absorbance at 412 nm (4.15).

6.3.    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.

6.4.    Standard solutions

Proceed as described in 9.2, replacing the reaction mixture (9.1) by the various mixtures of the standard solutions of methanol from 0 to 20 μg (7).

  1. Calculations

 

7.1.    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).

7.2.    Producing the calibration line

The calibration line corresponds to plotting a graph whose X-coordinates are the various concentrations of the standard solutions of methanol (from 0 to 0.625 μmole) and whose Y-coordinates are the corresponding values of optical densities, obtained in 9.4. Then calculate the straight regression line (2) resulting from the linearity of the data of the graph.

7.3.    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 methanol 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 methanol released in µmoles during time T (min)
  • V: quantity of enzyme solution introduced (ml), in this case 0.1 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

  1. Characteristics of the method

 

r

0.14

R

0.112

Sr

0.05

SR

0.04

The intralaboratory repeatability of the method is estimated using the mean standard deviation of the absorbance values resulting from the same sampling of the enzymatic preparation, determined 5 times. In this way, for the pectin-methyl-esterase determination the mean standard deviation of the values is 0.05 with a percentage error of 5.46, in which the % error corresponds to:

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 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 0.04.
  • the power of the test for the first type of risk α (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

(α= 5%)

Newman- Keuls test (*)

Bonferroni test

(**)


PME

Adhered to

0.00001

99%

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 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  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%.

  1. Bibliographical references

 

  • KLAVONS J.A., BENNET R.D., Determination of methanol using alcohol oxydase and its application to methyl ester content of pectins. J. Agr. Food. Chem, 1986. Vol 34, p 597-599.
  • Enzyme activities and their measurement – OIV Document, FV 1226, 2005

Determination of Pectinmethylesterase activity using acid based titration

  1. Principle

The demethylation activity of the pectinmethylesterase results in the appearance of free carboxylic groups at the level of the galacturonic acids forming the chains. To determine the activity of pectinmethylesterase, the carboxyl groups can be titrated during the enzymatic hydrolysis with sodium hydroxide solution at constant temperature and constant pH-value.

  1. Equipment and materials
  • titration equipment (burette)
  • temperature controlled heat plate and magnetic stirrer/magnetic stir bar
  • pH meter
  • glass cup, filled with water
  • chronometer
  • graduated flasks (different volume)
  • beakers ( preferably 50 mL)
  • precision pipettes (different volume)
  1. Chemicals and reagents
  • Pectin; highly esterified; p.a. quality (Sigma P9135-100G); CAS 9000-69-5
  • 0,01 M NaOH solution (Titrisol) p.a. quality; CAS 1310-73-2
  • NaOH pellets p.a. quality ; CAS 1310-73-2
  1. Preparation of soultions

4.1.    1 M NaOH

Dissolve 4 g NaOH in 100 mL H2O

4.2.    substrate solution

As substrate solution 1 % Pectin in H2O, is used by solving 2.0 g Pectin very slowly in 150 ml H2O. Subsequently the pH value is adjusted at pH 4.0 and at 40 °C with 1 M NaOH. The solution must be filled up to 200 mL exactly. Just before measuring, the pH-value should be controlled and adjusted again at pH 4,0, if necessary

4.3.    enzymatic solution

The enzymatic solution consists of approximately 30 to 50 mg/L commercial enzyme preparation diluted in cold water. This solution should be prepared directly before using.

4.4.    0.01M NaOH

This precast solution should be diluted according to the description of the producer.

4.5.    Performance of enzyme activity determination

20 ml of substrate solution are put in a beaker (magnetic stirrer is added) on the temperature controlled heat plate in a glass cup, which is filled with water heated up to 40 °C. The pH electrode is put in substrate solution. It is necessary to have a control and maybe a new setting up of the pH-value at 40 °C before starting the analysis. Then 0.1 ml of the enzymatic solution is added. Exactly at this time the chronometer is started. During the analysis the pH value must be measured and the sample has to be titrated up to pH 4.0 with 0.01 M NaOH for 10 minutes at 40 °C. After 10 min the analysis is stopped and the consumption of 0.01 M NaOH is read off.

The consumption of 0,01 M NaOH should amount to values between 3,5 mL and 8,5 mL. Otherwise it is recommended to dilute or concentrate the enzymatic solution.

  1. Calculation of the enzymatic activity

Enzymatic activity is calculated by using following formula:

Activity

Activity

  • n = consumption of 0.01 M NaOH in µmol
  • t = time in min (in this case 10 min)
  • v = quantity of enzymatic solution introduced in ml (=0.1 ml)
  • c = concentration of the enzymatic solution in g/L

Validation of the acid based titration to determine the activity of Pectin methylesterase

The mean value of the standard deviation was determined of 8 different enzymes.

Each enzyme was analysed 6 times.

Mean value of the standard deviations of the different enzymes = 3.91 %

Validation of the acid based titration to determine the activity

of PME

Proteases (aspergillopepsine I)

COEI-1-PROTEA Comparative evaluation of protease activity (Aspergillopepsin I) in enzyme preparations (EC 3.4.23.18)

  1. Origin

Enzyme preparations that have an Aspergillopepsin I activity are formed by controlled fermentation of Aspergillus spp., in particular of Aspergillus niger.

This enzyme is usually referred to as Aspergillopepsin I or Aspergillus acid protease (EC 3.4.23.18). Proteases are usually present as an enzyme complex. Unless otherwise stated, the specifications of resolution OIV-OENO 365-2009 must comply with "general specifications of enzyme preparations" set out in the International Oenological Code.

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

  1. Scope/Applications

Reference is made to the International Code of Oenological Practices, OIV-OENO 541A-2021 and OIV-OENO 541B-2021.

Enzymatic preparations containing protease activities (Aspergillopepsin I) are able to degrade the native must or wine protein under specific conditions of heat treatment. These proteins are causing great difficulties during must and wine clarification and stabilization steps. Such Proteases are therefore specifically used for the stabilization of protein rich must and wine.

In order to verify that the treatment has led to the removal of proteases (Aspergillopepsin I) and to the reduction of the native level of proteins, the proteins can be assayed in finished wines using the SDS-PAGE method described in the Appendix I of this monograph.

  1. Principle

This procedure is only for the determination of proteolytic activity in enzyme preparations, expressed in spectrophotometric acid protease units (SAPU), of preparations derived from, e.g., Aspergillus niger, and Aspergillus oryzae. The test is based on a 30-min enzymatic hydrolysis of a Hammarsten Casein Substrate at pH 3.0 and 37 °C. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. The quantity of solubilized casein in the filtrate is determined spectrophotometrically (reference: Food Chemical Codex).

  1. Reagents and solutions

4.1.    Casein: Use Hammarsten-grade casein, (CAS 9000-71-9, e.g. Merck article number 102242)

4.2.    Glycine-Hydrochloric Acid Buffer (0.05 M): Dissolve 3.75 g of glycine in about 800 mL of water. Add 1 M hydrochloric acid until the solution is pH 3.0, determined with a pH meter. Quantitatively transfer the solution to a 1000-mL volumetric flask, dilute to volume with water, and mix.

4.3.    TCA Solution: Dissolve 18.0 g of trichloroacetic acid and 11.45 g of anhydrous sodium acetate in about 800 mL of water and add 21.0 mL of glacial acetic acid. Quantitatively transfer the solution to a 1000-mL volumetric flask, dilute to volume with water, and mix.

4.4.    Substrate Solution: Pipet 8 mL of 1 M hydrochloric acid into about 500 mL of water and disperse 7.0 g (moisture-free basis) of Casein (4.1) into this solution, using continuous agitation. Heat for 30 min in a boiling water bath, stirring occasionally, and cool to room temperature. Dissolve 3.75 g of glycine in the solution and adjust to pH 3.0 with 0.1 M hydrochloric acid, using a pH meter. Quantitatively transfer the solution to a 1000-mL volumetric flask, dilute to volume with water, and mix.

  1. Sample preparation

Weigh the enzyme preparation, quantitatively transfer it to a glass mortar, and triturate with Glycine-Hydrochloric Acid Buffer (4.2).

Quantitatively transfer the mixture to an appropriately sized volumetric flask, dilute to volume with Glycine-Hydrochloric Acid Buffer (4.2), and mix.

The solution of the sample enzyme preparation must be prepared so that 2 mL of the final dilution gives a corrected absorbance of enzyme incubation filtrate at 275 nm (A, as defined in the Procedure) between 0.200 and 0.500.

  1. Procedure

Pipet 10.0 mL of Substrate Solution (4.4) into each of a series of 25 x 150 mm test tubes, allowing at least two tubes for each sample, one for each enzyme blank, and one for a substrate blank.

Stopper the tubes, and equilibrate them for 15 min in a water bath maintained at 37°C 0.1°C.

At zero time, start the stopwatch, and rapidly pipet 2.0 mL of the Sample Preparation into the equilibrated substrate.

Mix by swirling and replace the tubes in the water bath. (Note: The tubes must be stoppered during incubation).

Add 2 mL of Glycine-Hydrochloric Acid Buffer (instead of the Sample Preparation) to the substrate blank.

After exactly 30 min, add 10 mL of TCA Solution (4.3) to each enzyme incubation and to the substrate blank to stop the reaction. (Caution: Do not use mouth suction for the TCA Solution).

In the following order, prepare an enzyme blank containing 10 mL of Substrate Solution, 10 mL of TCA Solution, and 2 mL of the Sample Preparation.

Heat all tubes in the water bath for 30 min, allowing the precipitated protein to coagulate completely.

At the end of the second heating period, cool the tubes in an ice bath for 5 min, and filter through Whatman No. 42 filter paper, or equivalent. The filtrates must be perfectly clear.

Determine the absorbance of each filtrate in a 1-cm cell at 275 nm with a suitable spectrophotometer, against the substrate blank. Correct each absorbance by subtracting the absorbance of the respective enzyme blank.

6.1.    Standard Curve

Transfer 181.2 mg of L-tyrosine, chromatographic-grade or equivalent (CAS 60-18-4, e.g. Merck article number 108371), previously dried to constant weight, to a 1,000-mL volumetric flask.

 Dissolve in 60 mL of 0.1 M hydrochloric acid.

When completely dissolved, dilute the solution to volume with water, and mix thoroughly. This solution contains 1.00 μmol of tyrosine in 1.0 mL.

Prepare dilutions from this stock solution to contain 0.10, 0.20, 0.30, 0.40, and 0.50 μmol per mL.

Determine the absorbance of each dilution in 1-cm cell at 275 nm, against a water blank.

Prepare a plot of absorbance versus μmol of tyrosine per mL. A straight line must be obtained.

Determine the slope and intercept for use in the Calculation below. A value close to 1.38 should be obtained for the slope. The slope and intercept may be calculated by the least squares method as follows:

in which

  • n is the number of points on the standard curve
  • M is the μmoL of tyrosine per ml for each point on the standard curve,
  • A is the absorbance of the sample.

6.2.    Calculation

One spectrophotometric acid protease unit is that activity that will liberate 1 μmoL of tyrosine per min under the conditions specified. The activity is expressed as follows:

in which

  • A is the corrected absorbance of the enzyme incubation filtrate;
  • I is the intercept of the Standard Curve;
  • 22 is the final volume of the incubation mixture, in mL;
  • S is the slope of Standard Curve;
  • 30 is the incubation time, in min; and
  • W is the weight, in g, of the enzyme sample contained in the 2.0-mL aliquot of sample preparation added to the incubation mixture in the Procedure.

Appendix I: SDS-PAGE protein assay

  1. Principle

This test is based on a modified Bradford method (Marchal et al., 1997; Marchal et al., 1996) combined with SDS-PAGE electrophoresis.

The quantification of proteins is realized with a Bradford test using an ultrafiltration at 3kDa to reduce the interferences due to ethanol and phenolic compounds (Marchal et al., 1996), and SDS-PAGE (Sodium Dodecyl Sulfate - PolyAcrylamide Gel) electrophoresis to separate the proteins according to their molecular weight (Laemmli, 1970).

  1. Protocol

The samples (wines before treatment, wines with Aspergillopepsin I just added, wines after treatment) are ultrafiltrated with centrifuge filters 3 kDa (for example: Amicon® Ultra-4, Merck Millipore, Irlande) at 4500 g during 20 minutes at 18 °C, and the ultrafiltrate is collected.

400 µL ultrapure water are added to 400 µL of sample (wine or ultrafiltrate) and 200 µL Bradford reagent (Bio-Rad, USA) in a semi micro-cuvette (path length 10mm).

The solution is mixed twice, and the absorbance is measured at 595 nm after 30 minutes, compared to ultrapure water.

To obtain the absorbance of proteins (AP), the absorbance of the ultrafiltrate (AUF) has to be deduced from the absorbance of wine (AW):

  • A standard curve with 5 concentrations (from 0 to 20 mg/L) is made with BSA (Bovin Serum Albumin) with 10-minutes reaction.
  • The total protein content is calculated in mg/L eq. BSA, with the average value of 3 different measures.

Polyacrylamide gels are used, at 4% for stacking and 13% for resolving (composition in Table 1).

Samples are mixed with Laemmli buffer 4X (3 volumes of samples + 1 volume of buffer; Bio-Rad, USA) and analyzed by SDS-PAGE. Markers from 10 to 250 kDa are used as standards: Precision Plus Protein TM Unstained Standards, Bio-Rad, USA. The analyses are made by triplicate.

Table 1. Composition of resolving and stacking gels (for 4 gels).

 

Composition

Resolving gel

(13%)

Stacking gel

(4%)

Ultrapure water

6.2 mL

4.88 mL

Bis-Acrylamide (30%)

8.6 mL

1.04 mL

Buffer Tris-HCl 1,5M pH 8.8

5.0 mL

-

Buffer Tris-HCl 0,5M pH 6,8

-

2.0 mL

Sodium dodecyl sulfate (SDS) 10%

0.2 mL

80 μL

Ammonium Persulfate (APS) 10%

100 μL

40 μL

Tetramethylethylenediamine (TEMED)

20 μL

8 μL

Gels are run on a vertical electrophoresis apparatus (for example: Mini-PROTEAN III; Bio-Rad, USA) at room temperature and stained with Coomasie blue R250.

After migration, gels are stained with silver nitrate at room temperature according Rabilloud (1994): see Table 2.

Table 2. Protocol for silver staining SDS-PAGE gels.

 

Step

Solution: final concentration

Time

Fixing

Ethanol 99%: 30% (v/v)

Acetic acid: 10% (v/v)

Over night

Sensitization

Ethanol 99%: 20% (v/v)

Potassium acetate: 0.5M

Potassium tetrathionate: 3 g/L

Glutaraldehyde 50%: 1% (v/v)

2 h 30 min

(in the dark)

Washing

Ultrapure water

3 x 20 min

Staining

Silver nitrate: 2 g/L

Formaldehyde 37%: 0.7 mL/L

30 min

Washing

Ultrapure water

15 sec

Development

Potassium carbonate: 30 g/L

Formaldehyde 37%: 0.5 mL/L

Sodium thiosulfate. 5H2O 2.48 g/L: 3.75 mL/L

5 min

Stop

Tris: 50 g/L

Acetic acid: 25 mL/L

5 min

  1. Results

The molecular weight of chitinases and TLP (Thaumatin Like Proteins) is below 15 kDa and the proteases’ one is close to 40 kDa. A visual analysis of the gels allows an initial observation of the residual proteins.

Precise results are obtained after digitalization of the SDS-PAGE gels and analysis with a specific software.

  1. References
  • Marchal R., Seguin V. et Maujean A. Quantification of interferences in the direct measurement of proteins in wines from the Champagne region using the Bradford method. American Journal of Enology and Viticulture, 1997, 48, 303-309.
  • Marchal R., Bouquelet S. et Maujean A. Purification and partial biochemical characterization of glycoproteins in a Champenois Chardonnay wine. Journal of Agricultural and Food Chemistry, 1996, 44, 1716-1722.

Polygalacturonase activity

COEI-1-ACTPGA Determination of polygalacturonase activity in enzymatic preparations (endo- and exo-polygalacturonase activities (PG)

(EC. 3.2.1.15 – CAS N° 9032-75-1)

 

General specifications

These enzymes are generally present among other activities, within an enzyme complex, but may also be available in purified form, either by purification from complex pectinases or directly produced with Genetically Modified Microorganisms. 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 “Sources of enzymes and fermentation environment” of the general monograph on enzymatic preparations.

The enzyme preparations containing such activity are produced by directed fermentations such as Aspergillus niger, Rhizopus oryzae and Trichoderma reesei or longibrachiatum

 

  1. 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.

These enzyme activities are used to contribute to the effectiveness of grape maceration and grape juice extraction as well as to help the clarification of musts and wines and finally to improve their filterability.

Methods

  1. Methods 1
  1. 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.

  1. 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 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).

  1. 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.

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

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

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

5.6.    sodium potassium tartrate (KNaC4H2O6.4H2O 99% pure - MW = 282.2 g/mole)

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

5.8.    copper sulfate penta-hydrated (CuSO4.5H2O 99% pure - MW = 249.68 g/mole)

5.9.    concentrated sulphuric acid (H2SO4 98% pure)

5.10. ammonium heptamolybdate ((NH4)6MO7O24.4H2O 99% pure - MW = 1235.86 g/mole)

5.11. sodium hydrogenoarsenate (Na2HASO4.7H2O 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 (C5H10O7.H2O - MW: 2 12.16 g/mole)

5.13. distilled water

5.14. commercial enzyme preparation to be analysed

  1. Solutions
    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.

  1. 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

  1. 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.

  1. Procedure
    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).

  1. Calculation

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

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

  1. 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 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

Analysis of variance hypotheses

Probability

Power of

Test

(α= 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%.

  1. 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.
  • Enzyme activities and their measurement – OIV Document, FV 1226, 2005

Methods 2: Determination of Polygalacturonase activity with cyanoacetamide

  1. Principle

Polygalacturonases cut the principal pectin chains (homogalacturonan domain) with a low degree of methylation. This enzyme activity leads to the release of galacturonic acids along with the homogalacturonan oligomers.  Therefore the reducing ends are released. This ultraviolet method with cyanoacetamide, based on Knoevenagel reaction, which means the condensation between an active methylen group and a carbonyl group in a strongly alkaline medium, is existing to find out the activity of various enzymes amongst others of polygalacturonase. It has been developed for the determination of the enzymatic degradation of polysaccharides through an endo- and exo- mechanism that generates reducing monosaccharides.

  1. Equipment and materials
  • Spectrophotometer
  • quartz cuvette ( λ=274 nm, optical path length 1 cm)
  • analytical scale
  • magnetic stirrer and stir bar
  • water-bath (40°C; 100°C)
  • chronometer
  • graduated flasks (different volume)
  • beakers (different volume)
  • precision pipettes (different volume)
  • spectrophotometer
  • glass tubes (closable)
  • vortex mixer
  1. Chemicals and reagents
  • polygalacturonic acid, ~95 % enzymatic (CAS 25990-10-7)
  • pH 4.0 Na-citrate/HCl buffer, 1.06 g/cm3 (Titrisol), p.a. quality
  • pH 9.0 H3BO3/KCl/NaOH buffer ≈0.05 M/≈0.05 M/≈0.022M (Titrisol), p.a. quality
  • cyanoacetamide,  ≥ 98 %, purum (CAS 107-91-5)
  • D-galacturonic acid monohydrate ≥ 97 % (CAS 91510-62-2)
  1. Preparation of solutions

4.1.    Stock solution of D-galacturonic acid (250 µg/mL)

Dissolve 0,025 g of D-galacturonic acid in 100 mL H2O.

4.2.    1 % cyanoacetamide solution

Dissolve 1 g of cyanoacetamide in 100 mL H2O

4.3.    Borate buffer (pH 9.0)

This precast solution should be diluted according to the description of the producer.

4.4.    Na-citrate/HCl buffer (pH 4.0)

This precast solution should be diluted according to the description of the producer.

4.5.    Polygalacturonic acid solution

Stirring constantly dissolve polygalacturonic acid very slowly in the concentration of 5 g/l in Na-citrat/HCl buffer (pH 4.0)

  1. Performance of enzyme activity determination

 

5.1.    Calibration curve and procedure

The standard range is produced from 0 μg/mL to 250 μg/mL of D-galacturonic acid. Use stock solution for dilution.

D-galacturonic acid monohydrate μg/mL

0

25

50

100

150

200

250

D-galacturonic acid monohydrate μmol/mL

0

0.118

0.236

0.471

0.707

0.943

1.178

Stock solution μL

0

100

200

400

600

800

1000

H2O μL

1000

900

800

600

400

200

0

Cyanoacetamide assay: 1mL of D-galacturonic acid and 2 mL borate buffer (pH 9) and.1 mL of 1 % cyanoacetamide solution are mixed. After incubation in a test tube at 100°C for 10 min, the solution is cooled down in a cold water bath. Then the absorbance must be measured at 274 nm immediately. The photometer must be set to zero with water.

For calculation the intersection point of the regression line must be set to zero.

5.2.    Enzymatic hydrolysis and procedure of the sample

For the enzymatic hydrolysis of polygalacturonic acid 10 mL of polygalacturonic acid solution must be heated at 40°C in a closable glass tube. Then 0,01 g of the sample is added and the mixture must be incubated at 40°C. After exactly 5 min and exactly 10 min, 500 μL are removed from the reaction mixture and directly heated up to 100°C in preheated test tubes for 10 min. Afterwards this 500 μL are diluted with water to a total volume of 25 mL.

For analysing the blank the same concentration of enzyme in polygalacturonic acid is heated up to 100 °C for 10 min (the polygalacturonic acid solution must be heated at 100°C before adding the enzyme!). In case of cloudiness the solution should be centrifuged at 5000 rpm for 5 min. Then the blank must also be incubated at 40°C. 500 μL of the blank solution are removed after 5 min and also placed in the water bath at 100°C for 10 min. Afterwards this 500 μL are diluted with water to a total volume of 25 mL.

Cyanoacetamide assay: 1 mL of the diluted solution and 1 mL of 1 % cyanoacetamide solution are added to 2 mL borate buffer (4.3.). After incubation in a test tube at 100°C for 10 min, the solution must be cooled down in a cold water bath. Then the absorbance must be measured at 274 nm immediately.

  1. Calculation of the enzymatic activity

Enzymatic activity is calculated by relating the absorbance value and the quantity of product formed using a standard range with the formula:

Activity (U/g) = q/ (t*c*F)

Activity (nkat/g) = q/ (t*c*F) *(1000/60)

  • q = quantity of galacturonic acid in µmol/mL
  • t = time in min
  • c = concentration of the enzymatic solution in g/L (= 0.01 g/L) pro 10 mL substrat
  • F = correction factor of the volume (=2)
  1. Literature

Bach E. and Schollmeyer E. (1992): An Ultraviolett-Spectrophotometric Method with 2-Cyanoacetamide for the Determination of the Enzymatic Degradation of Reducing Polysaccharides. Anal. Biochem. 203, 335-339.

  1. Intra-laboratory validation of the determination of the activity of Polygalacturonase with 2- Cyanoacetamide

The mean value of the standard deviation was determined of 6 different enzymes.

Each enzyme was analysed 6 times.

Mean value of the standard deviations of the different enzymes = 6,93 %

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Intra-laboratory validation of the determination of the activity of PG with 2-Cyanoacetamide

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Urease

COEI-1-UREASE Urease

E.C.3.5.1.5

CAS N°: 9002-13-5

General specifications

The specifications must be in compliance with general specifications for enzymatic preparations as provided for in the International Oenological Codex.

  1. Object, origin and field of application

The purpose of an enzyme is to break down urea into ammonia and carbon dioxide. Urease is produced from Lactobacillus fermentum. It belongs to the urease group collectively called “urease acids”. They are activated at low pH levels.

L. fermentum is grown in a synthetic environment. After fermentation, the culture is filtered, washed in water and the cells are killed in 50% vol alcohol. The suspension is freeze dried or dried by pulverisation.

The preparation consists of a powder made up of whole dead cells containing enzymes.

Urease contains no substances, nor micro-organisms nor collateral enzymatic activities which are:

  • harmful to health,
  • harmful to the products treated,
  • lead to the formation of undesirable products
  • produces or facilitates fraud
  1. Labelling

The concentration of the product must be indicated on the label in addition to security and storage conditions and the to the expiration date.

  1. Enzymatic activity

The claimed enzymatic specific activity is posted at 3.5 U/mg. Note that one unit is defined as the quantity of enzymes which release one micromole molecule of ammonia hydroxide from 5 g/l dose of urea, per minute at pH level 4 in a citrate buffer 0.1 M medium, at 37 0C.

This activity is the only isolation.

  1. Characteristics

Urease can be found in the crystal powder form, white, odourless, with a mild taste

 

  1. Supports, diluents, preservation agent

The only substance added for conditioning is dextrin.

  1. Trials

6.1.    Sulphuric ashes

Determine sulphuric ashes according to the method in Chapter II in the International Oenological Codex. The rate of sulphuric ashes in urease must not be over 8%.

6.2.    Solution for trials:

Dissolve 5 g of urease in 100 ml of water.

6.3.    Heavy metals

A 10 ml of solution for trials (6.2), add 2 ml of buffer solution pH 3.5 (R), 1.2 ml of thioacetamide (R) reagent. There should be no precipitation. If brown colouring occurs, it should be less than demonstrated in the trial prepared as indicated in Chapter II of the International Oenological Codex.

The contents of heavy metals expressed in lead, must be less than 30 mg/kg.

6.4.    Arsenic

Measure arsenic according to the method which appears in Chapter II of the International Oenological Codex from the trial solution (6.2).

The contents of arsenic must be less than 2 mg/kg.

6.5.    Lead

Measure lead according to the method which appears in Chapter II of the International Oenological Codex from the trial solution (6.2).

The contents of lead must be less than 5 mg/kg.

6.6.    Mercury

Measure mercury according to the method which appears in Chapter II of the International Oenological Codex from the trial solution (6.2).

The contents of mercury must be less than 0.5 mg/kg.

6.7.    Cadmium

Measure cadmium according to the method which appears in Chapter II of the International Oenological Codex from the trial solution (6.2).

The contents of cadmium must be less than 0.5 mg/kg.

  1. Biological contaminants

Carry out a counting according the method described in Chapter II of the International Oenological Codex

7.1.    Total bacteria under 5 x 104 CFU/g

7.2.    Coliformesteneur under 30 CFU/g of preparation

7.3.    Escherichia coli absence checked on 25 g sample

7.4.    St. aureus absence checked on 1 g sample

7.5.    Salmonella absence checked on 25 g sample.

No mutagenic or bacterial activity should be detectable

It is also admitted that no Lactobacillus strain should produce antibiotics.

  1. Application to wine

Urease must be carefully incorporated and mixed in wine to be aged more than 1 year if it contains more than 3 mg/l of urea. The dose to be used will be 25 mg/l to 75 mg/l, according to tests carried out beforehand. This procedure is carried out in less than 4 weeks at a temperature above 15°C and when there is less than 1 mg/l fluoride ions.

After a noticeable decrease in urea, for example less than 1 mg/l, all enzymatic activity is eliminated by filtering the wine. (diameter of pores under 1 μm).

  1. Storage conditions

Urease can be stored for several months at a low temperature (+ 5 °C). There is a 50% loss in activity annually.

Arabinanase activity

COEI-1-ACTARA Determination of endo- α(1,5) arabinanase activity in pectolytic enzyme preparations

 

General specifications

These enzymes are usually present among other activities, within a complex enzymatic preparation. 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 fermentation of microorganisms such as Aspergillus niger, Aspergillus Tubigensis, Aspergillus Awamori Trichoderma reesei, Penicillium funiculosum or Arabinanases belong to the family glycohydrolases.

  1. Scope / Applications

Reference is made to the International Code of Oenological Practices, OENO 11/2004; OEN 12/2004; OENO 13/2004; OENO 14/2004 and OENO 15/2004.

Arabinanases are useful  for  the maceration  of the grapes,  the clarification of musts and wines, the filterability of musts and wines since they are facilitating. the action of other enzyme activities hydrolysing the constituents of the cell wall of grape.

  1. Principle

 

The substrate employed is Azurine-crosslinked debranched arabinan (AZCL-Arabinan). Highly purified arabinan from sugar-beet pulp is treated with α-L-arabinofuranosidase to remove 1,3- and 1,2- α -linked arabinofuranosyl residues, leaving linear 1,5- α -arabinan. This polysaccharide still contains a small percentage of galacturonic acid, galactose and rhamnose (6, 4 and 2 % respectively), but is resistant to attack by polygalacturonase and endo-1,4- β-D-galactanase. The polysaccharide is then dyed and crosslinked. Treatment of this substrate with a large excess of α-L-arabinofuranosidase results in a limited release of arabinose but no release of dye labelled fragments.

AZCL-Arabinan is a highly sensitive and very specific substrate for the assay of endo arabinanase, when you measure the supernatant after the reaction at 590 nm.

  1. Apparatus

 

4.1.         Glass test tubes (15 ml )

4.2.         Water bath set 40 °C

4.3.         Vortex tube mixer

4.4.         Qualitative Filter circle, retented particle diameter : 11 μm (in liquid)

4.5.         1 cm light path cuvettes

4.6.         Spectrophotometer set 590 nm

4.7.         Chronometer

4.8.         Pipet (500 μl, 10 ml)

4.9.         pH meter

4.10.     15 ml glass test tubes

4.11.     Metal rack for 15 ml test tubes

4.12.     Funnel

4.13.     100 ml graduated flask

  1. Reagents and products:

5.1.         Arabinazyme Tablets (Megazyme, batch 60701 as an example)

5.2.         Trizma base (CAS no. 77-86-1)

5.3.         Glacial acetic acid (CAS No. 64-19-7)

5.4.         Sodium hydroxid solution (CAS No. 1310-73-2)

  1. Solutions
    1.          Dilution Buffer
  • (Sodium Acetat buffer, 50 mM, pH 4.0)
    1.          Glacial acetic acid is added to 900 ml of distilled water. This solution is adjusted to pH 4,0 by the addition of 1 M sodium hydroxide solution. The volume was adjusted to 1 L with distilled water. 2 % Trizma Base Solution
  • Dilute 2 g Trizma Base in 100 ml distilled water.
  1. Preparation of the sample

7.1.          Enzyme dilution

For most commercial pectinase enzyme preparations, a dilution of 500-fold is required. Place 200 mg of commercial preparation in a 100 ml graduated flask, make up with dilution buffer (6.1), and stir in order to obtain a homogeneous mixture.

  1. Procedure

8.1.         Enzymatic reaction

The test tubes are prepared at least in duplicate.

500 μl of diluted enzyme in dilution buffer (7.1) are pre-equilibrated to 40 °C for 5 min.

The reaction is initiated by the addition of an Arabinazyme tablet. Start the chronometer.

The tablet hydrates rapidly. The suspension should not be stirred.

After exactly 10 min at 40 °C the reaction is terminated by the addition of 10 ml Trizma Base solution (6.2) and stir.

After about 5 min standing at room temperature, the slurry is stirred again and filtered through a qualitative filter circle.

The absorbance of the reactions solutions are then measures at 590 nm against the reaction blank

8.2.         Reaction blank

A reaction blank is prepared by adding 10 ml Trizma base solution (6.2) to 500 μl enzyme solution and stir before the addition of the Arabinazyme tablet.

  1. Calculations

 

Endo-Arabinanase activity being assayed is determined by reference to the calibration curve of the test kit (i.e. Lot.No. 60701)

Where:

  • Y endo-arabinase activity (in milliUnits/assay)
  • M slope of the calibration graph
  • X absorbance of the reaction at 590 nm (minus the reaction blank, or read against the reaction blank)
  • C intersection on the Y-axis (intercept point)
  • 2 conversion from 0,5 ml enzyme dilution to 1 ml in the test
  • FV Dilution factor of the original enzyme preparation (i.e. 500-fold)
  • 1000 conversion from milliUnits to Units

 

  1. References

Xylanase activity

COEI-1-XYLANA Determination of endo-1,4- β-xylanase activity in enzymatic preparations

General specifications

Hemicellulases are generally present in enzymatic preparations among other activities within an enzymatic complex. Unless otherwise stated, the specifications must be compliant with Resolution OIV-OENO 365-2009 on the general specifications of enzymatic preparations that appear in the International Oenological Codex.

  1. Origin and application

 

Hemicellulases catalyse the degradation of hemicelluloses. The hemicelluloses of the cell walls of grape berries are principally composed of xyloglucans and arabinoxylans; these two polysaccharides constitute almost 90% of grape hemicelluloses.

The hemicellulase activity of enzymatic preparations is evaluated by measuring the 1,4- β-xylanase activity.

Enzymatic preparations containing hemicellulase activities are used during grape maceration, and in the clarification and improvement of the filterability of musts and wines.

Enzymatic preparations containing these activities are derived from the managed fermentation of, for example, Aspergillus sp. or Trichoderma sp., or mixtures of enzymes thus obtained.

  1. Scope of application

The method of determination was developed using commercial xylanase. The conditions and the method were developed for use with commercial enzymatic preparations such as those available on the market of oenological products.

  1. Principle

 

Xylanases hydrolyse xylan chains and thus liberate the constitutive monosaccharides at the reducing ends. The measurement of the xylanase activity is estimated by measuring the reducing monosaccharides (xylose) liberated during the incubation period, according to the Nelson method (1944). In the alkaline environment the pseudo aldehyde groups of the sugars reduce the cupric Cu2+ ions. These ions react with the arsenomolybdate reagent, giving it a blue colouring, for which the absorbance – measured at 520 nm – varyies in a linear manner with the monosaccharide concentration (between 0 and 400 μg/mL).

  1. Apparatus

4.1.    Magnetic-stirrer hotplate

4.2.    Water bath at 40°C

4.3.    Water bath at 100°C

4.4.    100-mL Cylindrical flask

4.5.    Centrifuge compatible with 15-mL glass tubes

4.6.    Stopwatch

4.7.    100-mL Calibrated flasks

4.7.1.  500-mL Calibrated flask

4.8.    200-µL Precision syringe

4.8.1.  1-mL Precision syringe

4.9.    10-mL Straight pipette calibrated with graduations at 0.1-mL intervals

4.10. Spectrophotometer

4.11. 15-mL Glass tubes

4.12. Vortex-type stirrer

4.13. 500-mL Brown-glass flask

4.14. Chamber at 4°C

4.15. Oven at 37°C

4.16. Cotton wool

4.17. Kraft paper

4.18. pH Meter

4.19. Metal tray for 15-mL tubes

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

  1. Products

5.1.    Sodium acetate (pure CH3COONa at 99% - PM = 82 g/mol

5.2.    Acetic acid (pure CH3COOH at 96% - PM = 60 g/mol, density = 1.058)

5.3.    Xylan (beechwood) P-XYLNBE-10G, Lot No. 171004a, Megazyme

5.4.    Sodium sulphate anhydrous (pure Na2SO4 at 99.5% - PM = 142 g/mol)

5.5.    Sodium carbonate anhydrous (pure Na2CO3 at 99,5% - PM = 105.99 g/mol)

5.6.    Potassium sodium tartrate (pure KNaC4H4O6·4H2O at 99% - PM = 282.2 g/mol)

5.7.    Sodium hydrogen carbonate anhydrous (pure NaHCO3 at 98% - PM = 84.01 g/mol)

5.8.    Copper sulphate pentahydrate (pure CuSO4·5H2O at 99% - PM = 249.68 g/mol)

5.9.    Concentrated sulphuric acid (pure H2SO4 at 98%)

5.10. Ammonium heptamolybdate (pure (NH4) 6Mo7O24·4H2O at 99% - PM = 1235.86 g/mol)

5.11. Sodium hydrogen arsenate (pure Na2HAsO4·7H2O at 98.5% - PM = 312.02 g/mol)

5.12. D-xylose (pure C5H10O5 at 99% - PM = 150 g/mol)

5.13. Distilled water

5.14. Commercial enzymatic preparation for analysis

  1. Solutions

 

6.1.    Reagents for the oxidising solution

These reagents should be prepared first, considering the 24-hour time limit for solution D.

6.1.1.  Solution A

Successively place in a 100-mL cylindrical flask (4.4):

  • 20 g sodium sulphate anhydrous (5.4),
  • 2.5 g sodium carbonate anhydrous (5.5),
  • 2.5 g potassium sodium tartrate (5.6),
  • 2 g sodium hydrogen carbonate anhydrous (5.7).

Dissolve in 80-mL distilled water (5.13). Heat and mix (4.1) until dissolution and transfer to a 100-mL flask (4.7). Make up to the calibration mark with distilled water (5.13).

Store at 37 °C (4.15); if a deposit forms, filter through fluted filter.

6.1.2.  Solution B

Dissolve 15 g copper sulphate pentahydrate (5.8) in 100 mL distilled water (5.13) and add a drop of concentrated sulphuric acid (5.9).

6.1.3.  Solution C

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

6.1.4.  Solution D

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

In a 100-mL cylindrical flask (4.4), dissolve 3 g sodium hydrogen arsenate (5.11) in 25 mL water (5.13) and quantitatively transfer to a 500-mL calibrated flask (4.7.1) containing ammonium molbydate (5.10).

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

Place at 37 °C (4.15) for 24 hours then store at 4°C (4.14) in a 500-mL brown-glass flask (4.13).

6.2.    Sodium acetate buffer (pH 4.2, 100 mmol/L) 

It is made up of solutions A and B.

6.2.1.  Solution A (0.1 M sodium acetate): dissolve 0.5 g sodium acetate (5.1) in 60 mL distilled water (5.13).

6.2.2.  Solution B (0.1 M acetic acid): dilute 1 mL acetic acid (5.2) with 175 mL distilled water (5.13)

6.2.3.  Preparation of sodium acetate buffer: mix 23.9 mL solution A (6.2.1) + 76.1 mL solution B (6.2.2).

Verify the pH o the buffer using a pH meter (4.18).

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

6.3.    2% Oat-spelt xylan solution (p/v)

In a 100-mL calibrated flask (4.7), dissolve 1 g oat-spelt xylan (5.3) in 100 mL sodium acetate buffer (6.2).

6.4.    Xylose stock solution at 400 μg/mL

Dissolve 0.040 g D-xylose (5.12) in 100 mL distilled water (5.13).

  1. Preparation of xylose calibration range

Prepare the calibration range (from 0 to 400 μg/mL) based on the xylose stock solution (6.4) as presented in Table 1.

Table 1: Xylose calibration range

 

Xylose (μg/mL)

0

50

100

150

200

250

300

400

Xylose (μmol/mL)

0

0.232

0.462

0.694

0.925

1.156

1.387

1.890

Vol. stock solution (μL) (6.4.)

0

125

250

375

500

625

750

1000

Vol. distilled water (μL) (5.13)

1000

875

750

625

500

375

250

0

  1. Sample preparation

It is important to mix the enzymatic preparation before sampling, by inverting the container, for example. The enzymatic solution and the blanks should be prepared just before use.

8.1.    Enzymatic solution 2 g/L

Place 200 mg of enzymatic preparation (5.14) in a 100-mL calibrated flask (4.7), make up to the mark with distilled water (5.13) and stir in order to obtain a homogenous mixture.

8.2.    Heat-denatured blank

Place 10 mL enzymatic solution at 2 g/L (8.1) in a 15-mL tube (4.11) stoppered with cotton wool (4.16) covered with Kraft paper (4.17) and immerse the tube in the water bath at 100 °C for 5 min (4.3).

  1. Procedure

9.1.    Enzymatic reaction

Prepare the tubes in duplicate at the minimum.

In 5 x 15-mL tubes (4.11) numbered from 1 to 5 and placed in a tray (4.19),

use the 200- μL precision syringe (4.8) to add 200 μL enzymatic solution at 2 g/L (8.1), then

use the 1-mL precision syringe (4.8.1) to add 400 μL sodium acetate buffer (6.2) and

600 μL 2% oat-spelt xylan (6.3), and start the stopwatch (4.6)

After stirring (4.12), place the tubes stoppered with cotton wool (4.16) and Kraft paper (4.17) in the water bath at 40 °C (4.2):

  • for 1 min for tube 1,
  • for 2 min for tube 2,
  • for 5 min for tube 3,
  • for 10 min for tube 4,
  • for 20 min for tube 5.

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

Cool the tubes under a current of cold water.

9.2.    Determination of liberated reducing substances (xylose in this case)

In a 15-mL tube (4.11),

place 1 mL reaction medium (9.1),

add 1 mL solution C (6.1.3),

after stirring (4.12), place the tube in a water bath at 100°C (4.3) for 10 min.

Then cool the tube under a current of cold water.

Add 1 mL solution D (6.1.4),

add 9.5 mL water (5.14) using the10-mL straight pipette (4.9)

wait 10 min for colour stabilisation.

Centrifuge (4.5) each of the tubes at 5000 rpm for 10 min.

Place the supernatant in a cuvette (4.20).

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

9.3.    Blanks

Proceed as described in 9.1, replacing the enzymatic solution at 2 g/L (8.1) by the heat-denatured blank (8.2). Ideally perform the enzymatic reaction of the blanks at the same time as that of the enzymatic solution.

9.4.    Calibration range

Proceed as described in 9.2, replacing the reaction medium (9.1) by the different media of the xylose calibration range from 0 to 400 μg/mL (7).

  1. Calculation

10.1. Kinetics

Generally, calculation of enzymatic activity may only be carried out when the substrate and the enzyme are not in limited quantities. This therefore refers to the ascending phase of the representation of kinetics: the enzymatic activity is linear over time. Otherwise, the activity would be underestimated (Figure 1).

Figure 1: Enzmatic reaction kinetics

Determine the kinetics over 15 min. Measure the activity concerned at T=1 min T=2 min, T=5 min, T=10 min and T=15 min.

After determining the enzymatic reaction kinetics, plot the curve for the absorbance variation in relation to the reaction time. The absorbance corresponds to the difference between the absorbance at time T of the enzymatic preparation and of the corresponding blank.

Then calculate the equation (1) of the regression line, considering the points of the ascending phase (see Figure 1).

10.2. Calibration line

For the calibration line, plot a graph showing the different concentrations of the xylose calibration range (0-1,89 µmol/mL) as the abscissa and the corresponding optical density values as the ordinates, obtained in 9.4. Then calculate the slope (Q/T) of the regression line (2) resulting from the linearity of the graph data.

10.3. Calculation of enzymatic activity

Based on the regression line (1), calculate the absorbance for a mean time, T (e.g. 4 min in the case of Figure 1), by deducing from it quantity Q of xylose released (in micromoles) for this intermediary time using equation (2).

The enzymatic activity in U/g of preparation is calculated as follows:

Where

  • Q: quantity of xylose released in µmols during time T (min),
  • V: quantity of enzymatic solution introduced (mL) – 0.2 mL in this case,
  • C: concentration of enzymatic solution (g/L) – 2 g/L in this case.

The enzymatic activity in nanokatals:

This unit corresponds to the number of nanomoles of product formed per second.

  1. Method characteristics

 

r= 0.056

R= 0.056

Sr= 0.02

SR= 0.02

The repeatability of the method is estimated using the mean standard deviation of the absorbance values derived from the same sampling of the enzymatic preparation, determined 5 times. Therefore, for the determination of xylanase the mean standard deviation of the values is 0.02 with a percentage of error of 9.7%. The % error corresponds to the following:

As such, the method of determination as presented is deemed repeatable.

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

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

Variance analysis (study of the probability of the occurrence of differences between samplings). Variance analysis is a statistical method that makes it possible to test the hypothesis of homogenity of a series of k-means. Carrying out variance analysis consists of determining whether the ‘treatment’ effect is ‘significant or not’.

The power of the test for type I -risk (5%). The type I α risk is the risk of concluding that the identical treatments are in fact different.

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

If the power is high ( 80%), this means that no difference has been detected between treatments, however, if one does exist, the means are available to see it.

The results are given in Table 2.

 

Table 2: Variance analysis – study of the sampling effect

Determination

Variance analysis hypotheses

Probability

Test power

α

Newman-Keuls test (*)

Bonferroni test (**)

 Xylanase

Respected

0.00087

93%

Significant

Significant

* Newman-Keuls test: this comparison test of means makes it possible to constitute homogenous treatment groups: those belonging to the same group are considered as not being different to the given type I -risk.

** Bonferroni test: also called the ‘corrected t-test’, the Bonferroni test makes it possible to carry out all comparisons of pairs of means, i.e. (t (t-1) )/2 comparisons before treatments, respecting the given type I -risk.

Therefore, the tests put in place make it possible to see a difference if there really is one (high test power); in addition, the method of determination shows a probability of occurrence of differences in activity (between samplings) of less than 5%, strengthened by membership of the same group (Newman-Keuls test non-significant) and considered as not being different to the given type I -risk (Bonferroni test non-significant).

  1. Bibliographic references
  • Nelson, N., ‘A photometric adaptation of the SOMOGYI method for the determination of glucose’, Journal of Biological Chemistry, May Institute for Medical Research of the Jewish Hospital, vol. 153, 1944, pp. 375-380.
  • Doco, T., et al., ‘Polysaccharides from grape berry cell walls. Part II. Structural characterization of the xyloglucan polysaccharides’, Carbohydrate Polymers, vol. 53, Issue 3, 15 August 2003, pp. 253-261.