an overview of uv-vis spectrophotometry applications in the...methods of analysis –color analysis...

An Overview of UV-Vis Spectrophotometry Applications in the

Craft Brewing Industry

Rick Blankemeier

Quality Assurance Manager

Stone Brewing Co.

Goals

• Brief overview of the brewing process

• Identify key aspects of brewing chemistry in the process

• How we utilize the UV-Spec for key analyses

Brewing Process Overview

• Ingredients

– Malted Barley -> Sugar source for fermentation

– Hops -> Adds flavor and bitterness to beer

– Water -> Solvent for extracting sugars and flavors from the malt

– Yeast -> Ferments

Brewing Process Overview

• Terminology

– “Mash” is the mixture of the crushed malted barley and 150F water to convert starches to sugar

– “Wort” is the term for the sugary water that is produced after lautering

– “Lauter” is the term for separating the spent grain husks from the liquid wort using a giant colander-like vessel called a lauter tun.

Brewing Process Overview

Analytical Testing

• American Society of Brewing Chemists (ASBC) Methods of Analysis

– Color Analysis (ASBC Method Beer 10A)

– Bitterness (ASBC Method Beer 23)

– FAN (ASBC Method Beer 31)

Color Analysis

• One of the simplest assays using a spectrophotometer

• Measures in units of SRM (Standard Reference Method) or EBC (European Brewing Convention)

• Maps consistency of raw materials and brewing process

Color• Read absorbency at

430nm

• Measures absorbency in the blue part of the visible spectrum of light

• Helps determine issues with the boiling phase or grain handling process

Bitterness (IBUs)

• Measures the approximate* amount of iso-alpha acids in a sample of beer

• Uses liquid-liquid extraction to separate the iso-alpha acids* from acidified wort/beer into 2,2,4-trimethylpentane (iso-octane)

• Vital for determining the correct hopping rate and bitterness level of beer

• Good measure for consistent boiling temperature

*1 IBU ≠ 1 ppm iso-alpha acid

Bitterness• Measures

absorbency at 275nm

• Uses 6M HCl and iso-octane

• Need to protonate IAA in order for it to dissolve into iso-octane

• Abs x 50 = IBU

Free Amino Nitrogen (FAN)

• FAN represents the concentration of individual amino acids, ammonia, and end-group α-amino nitrogen in small peptide chains and proteins

• Basically, it measures the bioavailability of nitrogen to yeast as nutrients.

• Too much FAN = potential micro issues and fusel oil formation in high gravity beers. Also denotes a bad fermentation

• High FAN can also lead to haze issues and some amino acids can also lead to higher diacetyl formation rates in fermenting beer

• Also good to measure FAN post-fermentation for the rate of FAN uptake

Free Amino Nitrogen

• Ninhydrin-based dying method

• Ninhydrin color reagent keeps for about 2 weeks

• Measures against a glycine standard curve

• Absorbency at 575nm

• Good range is 50-150 mg/L after fermentation

Iron Testing

• Iron acts as a nutrient in some enzymatic activities within the yeast’s metabolic pathway

• Too much iron can lead to metallic flavor in beer or premature oxidation issues with beer

• Excessive iron comes with some specialty malts and filter media (diatomaceous earth most often)

• Iron helps with free radical formations with certain fatty acids in beer that cause the off-flavor trans-2-nonenal to form, which makes the beer taste like cardboard.

Iron• Ferric iron in beer is

reduced to the ferrous form by ascorbic acid.

• Ferrous iron then reacts to Ferrozine to form a colored complex.

• Absorbency at 562nm

• Compared with a series of standards

• You have to nitric acid wash lots of glassware for this method to be accurate.

• Looking for values less than 0.05 mg/L

Thank You!

• If you have any questions about the procedures or anything else, you can reach me via e-mail

– rick.blankemeier@stonebrewing.com

References

• Briggs, D., Boulton, C., Brookes, P., Stevens, R., 2004. Brewing: Science and Practice. Cambridge: Woodhead Publishing.

• Caballero, I., Blanco, C., Porras, M., Iso-alpha-acids, bitterness and loss of beer quality during storage. Trends in Food Science & Technology, in press

• Intelmann, D., Batram, C., Kuhn, C., Heseleu, G., Meyerhof, W., Hofmann, T., Three TAS2R Bitter Taste Receptors Mediate the Psychophysical Responses to Bitter Compounds of Hops (Humulus lupulus L.) and Beer. Chemosensory Perception 2 (2009) 118-132

• Kappler, S., Krahl, M., Geissinger, C., Becker, T., Krottenthaler, M., Degradation of Iso-Alpha-Acids During Wort Boiling. Journal of the Institute of Brewing 116 (2010) 332-338

• Khatib, A., 2006, Studies of iso-alpha-acids: analysis, purification, and stability. Doctoral Dissertation, Leiden University.

• Kishimoto, T., 2008. Hop-Derived Odorants Contributing to the Aroma Characteristics of Beer. Doctoral Dissertation, Kyoto University.

• Malowicki, M., Hop Bitter Acid Isomerization and Degradation Kinetics in a Model Wort-Boiling System, M. Sc. Thesis, 2004, Oregon State University

• Malowicki, M., Shellhammer, T., Isomerization and Degradation Kinetics of Hop (Humulus lupulus) Acids in a Model Wort-Boiling System. Journal of Agricultural and Food Chemistry 53 (2005) 4434-4439

• Schönberger, C., Kostelecky, T., 125th Anniversary Review: The Role of hops in Brewing. Journal of the Institute of Brewing 117 (2011) 259-267

• Techakriengkrai, I., Paterson, A., Taidi, B., Piggott, J., Relationships of Sensory Bitterness in Lager Beers to Iso-Alpha-Acid Contents. Journal of the Institute of Brewing 110 (2004) 51-56

• Research Newsletter of the International Centre for Brewing and Distilling, Winter 2003.

• AMERICAN SOCIETY OF BREWING CHEMISTS, Report of Subcommittee on Free Amino Nitrogen. Proc. 1974, p. 34; Proc. 1975 (Vol. 33, No.3), p.88

• EUROPEAN BREWERY CONVENTION. Analytica-EBC (4th ed.). Schweizer Brauerei-Rundschau, CH-8047, Zurich, Switzerland (1987)

• LIE, S. J Inst. Brew. 79.37 (1973)

• WYLIE, E. B., and JOHNSON, M.J> Biochim. Biophys, Acta 59:450(1962)

• Abernathy, D.G., Spedding, G. and Starcher, B. (2009) Analysis of Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell NinhydrinAssay. J. Inst. Brew. 115(2), 122-127.

• Grigsby, J.H. and Palamand, S.R. Studies on the Staling of beer: the use of 2-thiobarbituric acid in the measurement of beer oxidation. ASBC J. 1975, 34 (2), 49-55.

• Speers, R.A.1976. The Effect of Copper, Iron and EDTA on Beer Oxidation. B.Sc. (Agr.) Thesis, Dept. of Food Science, Univ. British Columbia, Vancouver, BC.

• American Society of Brewing Chemists. Report of Subcommittee on Polyphenols in Beer. Journal 36:129, 1978.

• European Brewery Convention. Analytica-EBC, 3rd ed. Method 7.7, p. E64.