Beer Reference Sample Correlation Between Free Amino Nitrogren

75th ASBC Annual Meeting
53
2013 ASBC Annual Meeting
May 18-21, 2013
Hilton Tucson El Conquistador
Tucson, Arizona
Beer Reference Sample Correlation Between Free Amino Nitrogen (FAN) and NOPA
(Nitrogen by OPA)
Otama, L.1,3, Tikanoja, S1, Kane, H.2, Hartikainen, S.1, Kaski, L.1 and Suoniemi-Kähärä, A.1
1
2
Thermo Fisher Scientific, Ratastie 2, 01620 Vantaa Finland.
Thermo Fisher Scientific, Stafford House, Boundary Way, Hemel Hempstead, HP2 7GE UK.
2
Corresponding author: [email protected]
Introduction
In the fermentation process to produce beer yeast, normally Saccharomyces, converts sugars to ethanol and carbon dioxide.
The yeast synthesises proteins required for healthy growth from amino acids, which may be created by the yeast from
ammonia or by removal of the amino group from other alpha amino acids. The alpha amino acids available to the yeast in the
fermentation are therefore known as the Free Amino Nitrogen (FAN). The ninhydrin method measures the ammonia content
in addition to the FAN so measures the total assimilable nitrogen. Beer industry EBC (European Brewery Convention)
Methods of Analysis collection, MEBAK and ASBC describe the Free Amino Nitrogen (FAN) measurement by the ninhydrin
based assay.
In this paper we show the correlation between the BAPS beer samples measured according to EBC FAN protocol and the
Thermo Scientific Gallery system NOPA (alpha-amino nitrogen by OPA) method. Rapid 2-reagent method was developed for
a multipurpose discrete analyzer. Method is easily adopted to a manual spectrophotometer as well. The use of blank buffer
eliminates possible sample color interference. Total analysis time for six samples and 60 requests was approximately 45
minutes.
Materials and Methods
Instrument
NOPA method was tested using a Thermo Scientific Gallery discrete photometric analyzer. We also obtained similar results
with Thermo Scientific Gallery Plus, Arena and Gallery Plus Beermaster analyzers.
Reagents
Alpha-Amino Nitrogen (NOPA) test kit from Thermo Fisher Scientific (Cat. No. 984342) was used. Kit provides ready-to-use
non-hazardous reagents for 350 tests when performed by the automated analyzer.
Method principle
Primary Amino groups are derivated by OPA (o-Phthaldialdehyde) and NAC (N-acetyl cysteine) to form isoindoles. In
optimized conditions isoindoles form a chromogenic complex proportional to the concentration of alpha-amino nitrogen in the
sample. Reaction is measured photometrically at 340 nm wavelength, side wavelength being at 700 nm.
Samples
Six commercial reference beer samples (BAPS) were purchased from LGC Standards. Assigned values originate from the
proficiency test average from different laboratories analyzing the samples according to the official EBC method. Beer and
wort samples were analyzed for method comparison and performance studies.
In this paper, in addition to the BAPS samples, 16 commercial beer samples and two wort samples were analyzed. Values
used as a reference were assigned by the ninhydrin method. For the NOPA analysis, beer samples were degassed by
shaking 10 minutes. Wort samples were additionally centrifuged.
33 malt or malt extract samples were analyzed in an industry setting. In-house ninhydrin results were assigned as reference
values. Malt samples were prepared as either an IOB or an EBC congress mash extract. Malt extract samples were diluted
1:4.
Method calibration and quality control
Method was calibrated with NOPA standard from Thermo Fisher Scientific (Cat. No. 984394). NOPA std has alpha-amino
nitrogen concentration of 150 mg/L.
Glycine standard in water was used as a quality control sample (Sigma-Aldrich, Cat # G7403). Standard stock was prepared
by weighting 0.1085 g of pure glycine standard (C2H5NO2, MW = 75.07 g/mol, purity 99 %) into a 100 ml volumetric flask
and filled up to mark with deionized water. The solution has an alpha-amino nitrogen concentration of 200 mg/l. QC samples
20, 50 and 125 mg/l were diluted from stock with deionized water.
Application for automation
Automatic Gallery NOPA application consists of two reagents, end-point measurement with sample blank and linear
calibration curve used for result calculation. Tested measuring range is from 20 to 300 mg/L with secondary dilution 1+2 at
150 mg/L.First 200 µl of NOPA R1 reagent and 2 µL of sample is incubated for 120 seconds and reaction is blanked. After
addition of 20 µL R2 reagent and 300 s incubation, the reaction is measured at 340 nm. Additional side wavelength of 700 nm
or 750 nm can be used. Method is performed at 37 °C.
Method calibration
The results were calculated automatically by the analyzer using a linear calibration curve. Calibrator solution with 150 mg/L of
alpha-amino nitrogen and deionized water were used for calibrating the test. Example of the calibration curve is shown in
Figure 2.
Results and discussion
Repeatability and reproducibility
Repeatability was performed by analyzing BAPS samples with the Gallery analyzer in three batches. Six different beer
samples were analyzed with the number of measurements being n=30. Test was calibrated at the beginning of each batch.
The NOPA method showed excellent repeatability both within run and between run. Within run typical CV’s below 1.5 %. The
total repeatability of the measurement for the samples was between 2.2 and 3.2 %. Repeatability results are shown in Table
1.
Calculating Z-score
Instructions from the BAPS were followed.
Interpretation of the Z-score calculation results are seen below:
Linearity
Method linearity has tested with pure chemicals dissolved in deionized water. Primary measurement was designed from 20150 mg/L and it was extended with a automatic secondary dilution (1+2) to up to 300 mg/L. All linearity samples were
measured as triplicates. Example of the method linearity is shown in Figure 3.
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Method comparison to FAN
For method comparison study, 16 commercial beer samples and two wort samples were analyzed. Values used as a
reference were assigned by the FAN ninhydrin method. For the NOPA analysis, beer samples were degassed by shaking 10
minutes. Wort samples were additionally centrifugated. Recovery rates varied between 88-114 %. For most samples the
recovery rates were 96-109 % which shows excellent correlation between the two methods. Based on this preliminary study,
our method shows good correlation to FAN (R2=0,99). Method comparison curve is shown in Figure 4.
Manual method
Thermo Scientific Alpha-Amino Nitrogen (NOPA) method can also be performed with manual spectrophotometer at 340 nm
with 1 cm cuvette path length. Baseline is done against air or deionized water at 37 °C. Method was designed as an endpoint method, reaction time being 5 minutes. Sample/R1/R2 ratio is 1/100/10. Manual method linearity was determined up to
200 mg/L.
Maltings Industry Comparison to FAN
For an in-industry comparison 2 malt extract samples and 31 malt samples (either IOB or congress mash extracts) were
analyzed. NOPA results obtained on the Gallery were compared to in-house FAN ninhydrin results. Calibration was carried
out using 200 mg/L glycine standard. The results show good correlation to FAN (R2=0.95). Comparison results are shown in
Figure 5.
Table 1. Method repeatability (n=30)
Sample_206-2L
Sample_207-2L
Sample_209-2L
Assign value
92,50
Assign value
73,00
Assign value
30,00
N
30
N
30
N
30
Mean
98,63
Mean
74,07
Mean
29,83
z score
1,23
z score
0,21
z score
-0,03
SD
CV %
SD
CV %
SD
CV %
Within run
1,14
1,2
0,79
1,1
0,75
2,5
Between run
1,86
1,9
1,85
2,5
0,21
0,7
Total
2,19
2,2
2,02
2,7
0,78
2,6
Results
Sample 206-2B
Sample 209-2B
Sample 210-2L
Assign value
52,74
Assign value
29,00
Assign value
114,17
N
30
N
30
N
30
Mean
54,36
Mean
29,10
Mean
126,49
z score
0,32
z score
0,02
z score
2,46
SD
CV %
SD
CV %
SD
CV %
Within run
0,44
0,8
0,32
1,1
1,52
1,2
Between run
1,66
3,1
0,62
2,1
3,24
2,6
Total
1,72
3,2
0,69
2,4
3,58
2,8
Results
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Figure 1. Thermo Scientific Gallery Plus Beermaster
discrete photometric analyzer
Figure 2. Calibration curve example. Calibration was
performed with water and NOPA standard (150 mg/L),
both run as duplicates. Responses (Abs) were from 0.098
to 1.866.
Figure 3. Method linearity
Linearity 20 ‐ 300 mg/l 350
1,5
Result mg/l
Response (A)
2,0
1,0
0,5
0,0
250
150
50
‐50 0
0
50
100
150
y = 1,0078x ‐ 2,5038
R² = 0,9998
50
100
200
150
200
250
Theoretical mg/l
300
350
Concentration (mg/l)
Figure 4. Method comparison. The two wort samples are
the ones with highest NOPA concentration.
Figure 5. Industry comparison
400
y = 1,0836x ‐ 4,6005
R² = 0,9922
300
200
100
0
0
100
200
FAN concentration (mg/L)
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300
NOPA concentration (mg/l)
NOPA concentration (mg/L)
250
200
150
y = 1,231x ‐ 6,8424
R² = 0,9507
100
50
0
0
50
100
150
200
FAN concentration (mg/l)
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Conclusion
According to this study, all samples tested by Thermo Scientific Alpha-Amino Nitrogen (NOPA) method show similar result
levels as samples tested according to EBC FAN protocol for beer (9.10). Furthermore we see that no additional Ammonia
measurements are needed. Gallery method is easy to use and a robust method for beer and wort FAN measurement. The
advantage of using automated analyzer is the usability to measure many analytes like beta-glucan and SO2 in addition of
NOPA measurement from the same sample. The results of all measured analytes can be printed to the same report.
Acknowledgements
Thanks to Muntons plc for the provision of malt and malt extract samples & analysis results. References
1. Daniel G. Abernathy, Gary Spedding and Barry Starcher. Analysis of Protein and Total Usable Nitrogen in Beer and
Wine Using a Microwell Ninhydrin Assay. J. Inst. Brew. 115(2), 122–127, 2009
2. Analytica-EBC, Method collection from European Brewery convention (2008)
3. ASBC (The American society of brewing chemists) methods of analysis, 2009 edition
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