The Secrets of Beers
Standard Methods for Beer Analysis
- Teaser (Copyright: Liana.salanta)
- Teaser (Copyright:_Bene16)
- Teasern (Copyright: Tinelot Wittermans)
- Teaser (Copyright_Lilly_M.)
- Figure 1: Copper calibration (standard addition method).
- Figure 2a: Calibration curves for 24 elements.
- Figure 2b: Radial view for high element concentrations.
- Figure 2c: Axial view for low element concentrations.
- Figure 3: SIM chromatograms of 38 derivatized amino acids in beer.
- Table 1: Results of the determination of amino acids in beer samples in µmol/L.
It is the most popular alcoholic drink in Europe, and next to drinking water, it is one of the most researched food products with the highest standards regarding quality, freshness, appearance and flavor: it is beer.
These standards have to do with the German Beer Purity Law of 1516 (the “Reinheitsgebot”), which uniquely defines the ingredients of beer to be hops, malt, yeast and water (Teasers) Today, consumer interest in the diversity and variety of beers available has never been higher - of the 6,500 breweries in Europe, 700 have been established in 2014. Statistically, per capita beer consumption in European countries is around 68 liters a year. The highest consumption rate is in the Czech Republic (144 L) followed by Germany (107 L) and Austria (106 L) . The popularity of beer and its high per capita consumption leads to the questions: how healthy is beer, and what ingredients does it contain?
To ensure that beer meets the highest expectations of European consumers, the so called “classical four elements” hops, malt, yeast and water should be tested for characteristics, quality and contamination.
Beer contains all major B vitamins. Vitamins B2 and B6, important for metabolism, are particularly abundant. Furthermore, bitter substances and essential oils are indisputably effective against loss of appetite, gastric disorders and states of anxiety.
Last but not least, beer contains a variety of minerals and trace elements (e.g. Ca, Na, Mg and Zn) that are important for human nutrition. However, there is some potential contamination risk through undesirable substances such as heavy metals (for instance Cd, Pb, Hg and As). There are also other contaminants of organic origin, the so-called mycotoxins such as ochratoxin A (OTA), that can be formed on grains/brewers barley by fungi of the Aspergillus and Penicillium genera.
Brewing beer may appear simple with only four “elements”, but the chemistry is quite complex, requiring many types of analytical instruments for that perfect appearance, aroma, flavor, mouthfeel and overall impression. This article gives an overview of important measureable parameters.
Contaminants in Hops and Water
Hops and hop products provide the bitterness in beer.
They originate from compounds such as isohumulones, which are part of a class of compounds referred to as iso-alpha acids.
The bitter substances, aroma substances and polyphenols make a considerable contribution to the character of the beer. Along with these, hops contain components which are not necessarily desirable. As hops are a natural product and consequently exposed to environmental influences during the vegetation phase, contaminants such as metals and mycotoxins are to be expected. Other substances are nitrate and zinc which enter the plant when the soil is fertilized. Also, pesticide residues in hops need to be analyzed regularly.
Determination of Elements
The quality standards for beer analysis are described in the European Brewery Convention (EBC), which includes the determination of elements like copper, zinc, sodium, potassium, calcium and more, anions such as nitrate and sulfite, as well as organic components, e.g. ethanol, glycerin and others .
A meticulous quality control procedure is essential, and during each stage of the production process analytical methods such as spectroscopy, chromatography and mass spectrometry are applied for quality assurance or for product characterization. For the quantitative determination of metals like copper and zinc, atomic absorption spectrometry is the method of choice, since the sensitivity in flame atomization enables a fast and precise analytical procedure.
Even though the most abundant constituent of beer is water, it is important to control all other constituents which are dissolved in it. Determination of copper is important as high concentrations affect the colloidal stability and the taste of the beer. The same applies with zinc, an essential trace element for yeast influencing metabolic processes such as protein synthesis and nucleic acid metabolism. Typical concentration levels of copper and zinc in beer are 0.2 mg/L .
A typical measuring procedure according to EBC is the method of standard addition. This requires the preparation of standard solutions containing the same amount of beer, but different amounts of copper standard, for example, five standard solutions with 0.0, 0.1, 0.2, 0.4 and 0.6 mg/L. The flasks are filled up to the mark and mixed well. The procedure intends that total analyte concentration is the combination of the unknown (beer) and the standard, and that the total concentration varies linearly. If the signal response is linear in this concentration range, a calibration curve similar to Figure 1 is generated.
For low levels of toxic ingredients such as lead, cadmium and arsenic, electrothermal atomization combined with a high sensitivity graphite furnace is needed in order to achieve calibration ranges as low as 1 to 10 µg/L.
Simultaneous Multi-Element Analysis
Water, the universal solvent, is required to be free of contaminants and odors that may affect the beer’s flavor, color and taste. Heavy metal or inorganic contaminants such as Cd, Pb, Hg and As can be harmful to human health. These and other metals can be analyzed in a sequential way by atomic absorption spectrometry as described earlier.
As soon as the number of elements increases, the use of a simultaneous inductively coupled plasma optical emission spectrometer is required. In the described experiments a ICPE-9820 (Shimadzu) with vertical minitorch position and ‘dual view’ (axial and radial) plasma observation was used. This method enables the analysis of samples with low concentrations of just a few µg/L (axial) such as Pb, Cd, Mn, Hg, As and Sb and high concentrations of approximately 0.5 mg/L up to 150 mg/L (radial) such as Na, K, Ca and Mg within a single analysis sequence. Implementation of this method is carried out in accordance with DIN EN ISO 11885 (2009). The beer samples were degassed for 5 minutes in an ultrasonic water bath and subsequently diluted with water to 1:1 and 1:5 respectively. Calibration was performed against aqueous standard solutions. Figure 2 shows the calibration curves for 24 elements.
Screening Hop for Pesticides
Contamination through organic compounds such as pesticides is a major concern due to the potential for causing illnesses. 34 pesticides from five classes of compounds including organonitrogen, synthetic pyrethroids, organochlorines, organophosphorus and carbamates were analyzed in hops. Samples were prepared by the QuEChERS method before introduction into a triple quadrupole mass spectrometer (GCMS-TQ8040, Shimadzu). Determination of pesticides at 1 ppb level has been performed. The pesticides include the toxic herbicide Terbuthylazine and the insecticide Lindane, a known carcinogen and neurotoxin.
Determination of Mycotoxins
Mycotoxins often exist as contaminants in grains. To ensure consumer food safety, manufactures of food and beverages have to strictly manage risks from such contaminants. To maintain high-quality food standards, it is essential to determine rapidly the concentrations of hazardous mycotoxins in foods or beverages.
UHPLC-MS/MS offers the best combination of selectivity, sensitivity and speed for detection of these compounds in complex matrices such as beer. A high-throughput method for the quantification of 14 mycotoxins in beer was developed and the 14 mycotoxins patulin (PAT), nivalenol (NIV), deoxynivalenol (DON), aflatoxin (AF) B1, B2, G1, G2, T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZON), fumonisin (FM) B1, B2, B3 and ochratoxin A (OTA) were analyzed in beer by LC-MS/MS using a UHPLC system coupled to a triple quadrupole mass spectrometer with fast polarity switching and high-speed scanning .
Amino Acids in Beer
Generally, there are two methods to color beer, not including additives. One is by the Maillard Reaction in which an amino acid and a reducing sugar go through a chemical reaction. This is the same reaction as browning meat or toast. The other reaction is a caramelization process where sugars decompose through pyrolysis. These reactions not only add color, but also add flavor to the beer. Thus, when brewing beer, it is important to know the free amino acids available. There are a number of methods for analyzing amino acids such as GC-MS, UHPLC and LC-MS/MS. Figure 3 shows the SIM chromatograms of the determination of 38 derivatized amino acids in beer by LC-MS/MS .
Analysis of Vicinal Diketones (VDK)
In the caramelization process coloring beer, the sugars undergo a thermal decomposition, forming compounds such as VDK, including diacetyl (2,3-butanedione) as well as 2,3-pentanedione. Vicinal diketones contain two C=O ketone groups on adjacent carbons. These compounds occur naturally in alcohol beverages and produce a buttery flavor. According to the European Brewery Convention (EBC), analysis is performed with a headspace GC with an ECD detector at typically low concentrations around 0 - 100 ppb range in beers .
Determination of CO2
Quality assurance is only one of the aspects of branded products such as beer. And quality of the final product is dependent on the starting materials, strict production processes and specialists overseeing the entire production.
An important player in the production process and a condition for quality is invisible: carbon dioxide (CO2), formed during the fermenting process. After filling the beer into barrels and bottles, carbon dioxide ensures that the necessary pressure is maintained. This is an important factor for guaranteeing shelf life and fresh and steady taste, and consistent quality of the beer. Beer contains 4 - 6 g/L CO2.
Carbon dioxide in the world of Total Organic Carbon (TOC) analyzers is referred to as Inorganic Carbon (IC). The IC method is a suitable alternative to the classical Corning reference method for measuring CO2 in beer.
Beer is made from natural grain and vegetable base products which are exposed to environmental impacts as well as agricultural treatment. Beer may therefore contain a variety of heavy metals such as arsenic, lead and cadmium and additional undesirable substances such as mycotoxins and pesticides. To track and analyze these elements and compounds various analytical technologies and sensitive analytical systems are required, such as UV-Vis spectrophotometers, atomic absorption-, ICP-OES spectrometers, liquid- and gas chromatography as well as mass spectrometry, and TOC analyzers to permanently guarantee the quality of beer. Hardware and software systems from a single source ensure compatibility and seamless processes.
1Market Manager Food, Shimadzu Europa GmbH
2Market Manager Food, Shimadzu Scientific Instruments
Shimadzu Europa GmbH
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 Schad, G.J. et al., The column, Vol. 10, Issue 7 (2015)
 Analysis Guidebook: Food Product Analyses, C180-E060A, P. 61
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