Quality analysis for beer includes testing the free amino nitrogen (FAN), which provides an estimate of the protein content. Generally, protein content is measured using the Kjeldahl method, which is expensive and complex. Another standard assay used for several decades by the brewing industry is the ninhydrin-based FAN assay using cuvettes. Ninhydrin can be used to detect primary amino acids, resulting in a blue color with an absorbance maximum at 570 nm. The standard used here is the amino acid glycine.
Subsequently, Starcher and staff and our lab led the reduction of the volume of the standard cuvette assay and use of a multi-well format. This decrease in the volume reduces the amount of costly reagents used, limits waste, and makes for a simpler and less expensive waste disposal.
BMG LABTECH’s SPECTROstar Nano integrates cuvette and microplate measurements in one instrument. Spectra are recorded by the integrated spectrometer over time and the MARS software displays all the plots.
Materials and Methods
- Clear 96-well plates from Greiner
- SPECTROstar Nano, BMG LABTECH
Standard FAN Assay at pH 6.8
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Ninhydrin color reagent: Anhydrous Na2HPO4 (4 g), KH2PO4 (6 g), ninhydrin (0,5 g), and fructose (0.3 g) were dissolved in 100 mL distilled water. This stock solution can be stored in a refrigerator in an amber bottle for up to 2 weeks.
Glycine standard stock: Glycine (107.2 mg) was dissolved in distilled water and made up to 100 mL. This can be stored at 0–4 °C to prevent mold growth.
Glycine standard solution: One milliliter of glycine standard stock is diluted to 100 mL final volume with distilled water. The standard contains 2 mg amino-nitrogen/L. It should be freshly made for each day’s assay.
Dilute solution: Potassium iodate (2 g) was dissolved in 600 mL of distilled water and then 400 mL of 96% ethanol was added. It is stable for a long time at 4 °C, but should be used at room temperature to prevent condensation on the cuvettes.
Assay: One milliliter of beer is diluted to 50 mL using distilled water. Two milliliters of this are placed in 16 test tubes of size 150 mm. Ninhydrin color reagent (1 mL) is added and the loosely covered tubes are heated in a boiling water bath for 16 min. The tubes are then transferred to a cold water bath to bring the temperature down to 20 °C within 20 minutes. Then, 5 mL of dilution reagent is added, mixed, and the absorbance at 575 nm is measured immediately against a blank with 2 mL water.
Microplate FAN Assay at pH 5.5
Ninhydrin stock solution: Ninhydrin (8 g) was dissolved in 300 mL ethylene glycol and 5 N sodium acetate buffer at pH 5.5 was added (544 g sodium acetate tetrahydrate + 400 mL glacial acetic acid in 1 L water). This is stable at room temperature for at least 6 months.
Stannous chloride solution: SnCl2 (500 mg) was dissolved in 5 mL ethylene glycol and the solution is stable at room temperature for at least 6 months.
Ninhydrin reagent/working solution: Before performing the assay, 25 µL of SnCl2 solution is added to every 1 mL of ninhydrin stock solution and mixed well.
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Assay: 2 µL (0.4 µg N) glycine standard or 2 µL of beer or grape juice is added to a 96-well micro-well plate. Then, 100 µL of the pH 5.5 acetate buffered ninhydrin reagent is added. The plate is heated for 10 min at 104 °C. Using the SPECTROstar Nano microplate reader, the absorbance at 575 nm is measured simultaneously for several samples.
Reduced Volume Cuvette FAN Assay at pH 5.5
Assay: To 200 µL of the pH 5.5 acetate buffered ninhydrin reagent, 30 µL beer or grape juice or 20 µL (4 µg N) glycine standard is added and placed in a boiling water bath for 10 min. Then, the samples are removed and 2.8 mL cold water is added. These are vortexed and the absorbance at 575 nm is recorded in the SPECTROstar Nano against a blank sample with 30 µL water.
Controls and Calculations
Tests for all the samples, water blanks, and glycine standard should be performed two or three times. The average blank value is subtracted from the samples and glycine standard. The FAN content in ppm is obtained by dividing the average absorbance of the blank corrected sample by the average absorbance of the standard glycine and multiplied by 2 and the dilution factor (50 for beer diluted 1 mL into 50 mL or 100 for beer wort diluted 1 mL into 100 mL). All samples, including those that are mashed, should be free of haze/debris by filtering or centrifugation before dilution).
Results and Discussion
Wavelength Optimization
Using the scanning capabilities of the SPECTROstar Nano the optimal wavelength for this assay can be determined. The absorbance spectra of ninhydrin reaction with either glycine or a beer sample were recorded, and the instrument showed a clear absorbance maximum at 570–575 nm (Fig. 1).
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Figure 1. Spectra captured for a glycine standard (red line) and a beer sample (green line).
Reaction Times Required for Completion of FAN Assays
Apart from the three assays explained before, another reduced cuvette FAN assay at pH 6.8 was investigated (Fig. 2). The reaction rates are different for the different assays. The standard FAN and the rFAN pH 6.8 reactions required at least 16 min to reach maximum absorbance, whereas the rFAN 5.5 and the microplate FAN assay attained the maximum within 8 min. There was no absorbance loss even when the samples were incubated for up to 20 min.
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Figure 2. Reaction times required for completion of 4 different FAN assays).
Conclusions
The SPECTROstar Nano is well suited for the FAN microplate and cuvette assays for beer samples and has several advantages over the old standard FAN cuvette assay. The reduced volume cuvette and use of a microplate based FAN uses a concentrated ninhydrin solution, reducing the cooling time and doing away with dilution or quenching of the solution. In addition, no further dilution of the beer/wort samples is required.
The pH and buffers for the assay were optimized and the developed method should be useful for several brewing, enology, distilling, and food laboratories for the routine determination of free or total useable nitrogen.
This information has been sourced, reviewed and adapted from materials provided by BMG LABTECH Ltd.
For more information on this source, please visit BMG LABTECH Ltd.