Victoria and Alana demonstrate the Vintessential™ 4A165 Malic Acid Test Kit with the Absorbance One portable photometer. The 4A165 format provides 100 tests for monitoring L-malic acid in wine, grape juice and fermenting samples.
The demonstration covers kit setup, reagent activation, sample dilution, incubation, measurement, quality-control checks and result export. Before testing, use the current 4A165 Malic Acid Kit 100 Instructions and the relevant Absorbance One data sheet.
Learn more about the Malic Acid test kit, or browse the Vintessential Video Library.
The 4A165 kit measures L-malic acid using an absorbance-based enzymatic method. Reagent 2 contains the co-enzyme NAD, and the absorbance change at 340 nm is used by the Absorbance One software to calculate the result.
Vintessential Malic Acid Test Kits are available in 30-test and 100-test formats. The 4A160 is the 30-test format and the 4A165 is the 100-test format. The correct instructions and reagent preparation volumes must be used for the kit format being tested.
The demonstration uses the Vintessential Absorbance One, a portable 340 nm cuvette photometer, with the customised software workflow for measurement, calculation and result handling.
A sample should be diluted when its expected concentration is outside the kit's working range. In the demonstration, a sample concentration below 0.4 g/L is recommended for the Malic Acid Test Kit. Suggested dilutions vary with the stage of production, and the correct dilution factor must be entered into the Absorbance One software.
A quality-control standard provides a known reference value that helps verify the performance of the reagents, instrument and analytical process. It should be assessed according to the current method before sample results are accepted.
The demonstration explains that the method can be used with samples from grapes through to ferments and finished wines. Sample preparation may include dilution, filtration or centrifugation depending on the concentration and the amount of solids in the sample.
The video explains that most wine samples do not need to be decolourised. If a red wine is already being diluted sufficiently to bring the malic acid concentration into range, additional decolourisation is generally not required. Users should follow the current Vintessential sample preparation guidance for their sample.
Yes. Results remain available in the software's results list and can be reviewed later. The demonstration also shows the results file being named and exported in CSV or another format supported by the software.
This transcript has been lightly edited for readability and to correct obvious automatic-caption errors. Refer to the current Vintessential instructions and data sheets before performing a test.
Today we have a Malic 100 Test Kit and we will run through it. The kit has a batch number and an expiry date. If anything goes wrong with the kit, those are the two pieces of information Vintessential will often ask the customer for. They help with troubleshooting and traceability.
Each reagent also has an individual batch number. This supports traceability and helps maintain the integrity of the kit. The 100-test kit includes reagent 1, two bottles of buffer and co-enzyme, a standard and reagent 4. Vintessential test kits are available in 100-test and 30-test versions to suit larger and smaller testing requirements.
The first step in the malic acid protocol is to set up the workspace. Prepare the pipettes, cuvettes and cuvette rack, and work in a clean, clear space. This protocol uses the 2 to 20, 10 to 100 and 100 to 1,000 microlitre pipettes. Keeping the workspace organised is particularly important while becoming familiar with the kit.
The first reagent preparation step is to activate reagent 2. This contains NAD, the co-enzyme in the kit. It is the compound that changes the absorbance when it reacts with malic acid. The reaction is measured at 340 nm and provides the result.
For the 100-test protocol, add 22 mL of distilled water to reagent 2 and invert the bottle several times until the powder has dissolved. One advantage of using Absorbance One is that its workflow helps prevent confusion between the preparation requirements for the 30-test and 100-test kits.
Sample preparation depends on the stage of production and the expected concentration. During harvest, malic acid and other analytes may be present at concentrations above the range the kit can measure.
The kits use absorbance spectroscopy, which is linear over a defined concentration range. At higher concentrations the response becomes nonlinear. When the Absorbance One reading approaches 1.8 absorbance units, the sample needs to be diluted because the signal can be lost in noise. Vintessential provides a fact sheet with suggested dilutions and workable ranges for each kit.
The appropriate dilution depends on the time of year and the expected sample concentration. For the Malic Acid Test Kit, the demonstration recommends a sample concentration below 0.4 g/L. During vintage, a 1 in 20 or 1 in 10 dilution may be appropriate, while a finished wine may require only a 1 in 2 dilution.
Most wines do not need to be decolourised. For wines with a very strong red colour, PVP can be used to reduce the colour without affecting the analyte concentration. If a red wine is already being diluted sufficiently to bring it below 0.4 g/L, it generally does not also need to be decolourised.
During vintage, samples can be centrifuged or filtered when necessary. Because the sample volume is small compared with the buffers and other reagents, this is not always required. Very thick grape samples are more likely to need additional preparation.
The Absorbance One and Vintessential test kits can be used with samples ranging from grapes and grape juice through to ferments and finished wines. Folding filter paper can be a practical option when testing in a vineyard or at a site without a centrifuge. Information about dilution and sample preparation is included in the protocol workflow, with additional downloads available from the Vintessential website.
Each kit includes a quality-control standard with a certificate of analysis. The standard has been tested in Vintessential laboratories and has a known value. The malic acid kits include a standard with a value of 0.2 g/L.
A standard is run with every batch of samples. The result should normally be within 10 per cent of the value shown on the bottle or certificate of analysis. A result substantially outside the expected range can indicate a pipetting or sample-handling error. The sample can be reread and the setup checked before continuing.
Vintessential enzymes are tested for stability at 37 degrees Celsius for five days. Running the standard provides an indication of both kit performance and analyst performance. If a kit has been exposed for a prolonged period, particulate or aggregated material may be visible. In that situation, contact a local Winechek laboratory or Vintessential supplier for advice.
If the standard produces the correct result, that indicates the kit is performing correctly. Standards are included with the benchtop spectroscopy kits used with Absorbance One, and additional standards are also available separately.
Open the software and proceed to the reagent setup step. The workflow shows what must be added to each cuvette. Cuvette 1 is the blank. Add 500 microlitres of buffer using the 100 to 1,000 microlitre pipette and use a clean pipette tip for each addition.
After reagent 1 has been added to the cuvettes, add distilled water. The blank receives 500 microlitres. The standard and samples receive 450 microlitres because a further 50 microlitres of standard or sample will be added. The blank is used in the software calculations that determine the final result.
Add 100 microlitres of activated reagent 2, the NAD solution prepared earlier. Then add 5 microlitres of GOT. Add 50 microlitres of the standard to cuvette 2. Cuvettes 3 and 4 are used for samples, with 50 microlitres of sample added to each.
A dilution can be prepared directly in the cuvette when appropriate. For example, 25 microlitres of water and 25 microlitres of wine creates a 1 in 2 dilution. The dilution used must be entered correctly in the software at the sample setup step.
Cover the cuvettes and mix them well by inversion. The first incubation period for this kit is three minutes. Absorbance One includes a countdown timer within the protocol workflow.
After the first incubation period, the software prompts for the blank. Insert cuvette 1 into Absorbance One in the correct orientation. The software then requests the standard and each sample in sequence.
Sample names can be entered in the software. If a sample was diluted, enter its dilution factor. A 1 in 5 dilution made from 10 microlitres of wine and 40 microlitres of water has a dilution factor of five. A dilution fact sheet is available on the Vintessential website to assist with these calculations. When all samples have been read, select Finish Readout.
The next protocol step is to add 5 microlitres of reagent 4 to each cuvette. Some enzyme reagents may look slightly cloudy because the kits contain a small excess of enzyme. This supports complete reaction and shelf life.
Use a clean pipette tip for each enzyme and co-enzyme addition. Returning liquid from a cuvette to a reagent vial can contaminate the reagent and cause it to react prematurely. Mix the cuvettes by inversion, then incubate for 10 minutes using the software timer.
After the second incubation, repeat the readings in the same order, beginning with the blank and then the standard and samples. The demonstration produces a standard result of 0.21 g/L, which is within 10 per cent of the standard's expected value.
The results file can be given a name and exported in CSV or another format supported by the software. Exporting is optional because completed tests remain in the software's results list and can be reviewed later.
The Absorbance One software guides the user through each step of the process. Compared with conventional spectroscopy, users do not need to record calculations on worksheets or prepare a calibration curve and calculate results manually. The method uses small volumes and requires only a compact workspace, making enzymatic testing practical within a winery.
