First Winner of the SPECTROstar Nano Promotion: Dr. Gary Spedding!

A Research Proposal for use of a SPECTROstar Nano
in the Brewing Analytical Laboratory.

Gary Spedding,
Managing Owner, BDAS, LLC.

 

Abstract and goals:

The brewing industry has a requirement and expectancy for it to perform multiple routine analytical tests to ensure quality of product and raw materials. Unfortunately the industry has lagged behind other industries and academic laboratories in implementing new micro-methods and modern approaches in fulfilling its quality control needs. This proposal provides an outline of research topics that could benefit the industry through the use of the SPECTROstar Nano technology in developing new and improved methods for beer analysis such as: 1) free amino nitrogen and protein analysis and 2) beer bitterness evaluation and prompting further impetus for the American Society of Brewing Chemists as a whole to push further ahead in providing much needed newer and more modern techniques. Moreover, such methods (and the possibilities for newly developed approaches) would also be applicable to a wider range of beverages and process raw materials.

Methods and Materials:

1. A SPECTROstar Nano with uv/visible scanning and full spectrum output capabilities obtained from BMG LABTECH by us providing a winning proposal! Beer samples will be procured and routine chemicals and solvents will be obtained from recognized scientific supply houses.
   Cuvettes and microplates will also obtained from recommended suppliers. Additional equipment and instrumentation is readily available in our small laboratory.
2. Standard methods from the ASBC (American Society of Brewing Chemists) Methods of Analysis (2010 edition) and the Analytical EBC (5th Edition or later) will be used and modified. For the main research here the methods outlined by us (Abernathy et al, 2009) will be used.

Introduction

The Brewing industry worldwide relies on a number of methods for the analysis of finished product and process raw materials. Such methods are rather antiquated for the most part and have not kept up with modern food process quality control methods. In fact many methods for most craft brewers rely on methods that are decades old. The American Society of Brewing Chemists and the European Brewery Convention provide manuals for the industry but only a handful of laboratories can afford sophisticated equipment such as GC or HPLC systems for routine analysis on many analytes of interest.

Our laboratory is a firm believer in spectroscopic techniques (along with ELISA and microplate approaches and the use of enzymatic assays) which do provide for some application in the brewing laboratory. Many methods could, however, be modernized and especially scaled down. A spectrophotometer with extensive plate reading sample capabilities and providing full uv and visible spectra output would dramatically improve methods and throughput.

Proposed Experiments:

A brief listing of potential methods for use with a SPECTROstar Nano is followed by a specific research topic of interest to our laboratory and some discussion of methods already scaled down and amenable to mass multi-sample analysis. Brewing laboratories routinely measure color (absorbance at 430 nm), polyphenols (600 nm), flavonoids and tannins, bitterness (following organic extraction - at 275 nm), sulfite content (550 nm), free amino nitrogen or FAN (discussed further below – at 570 nm) and numerous other analyses, via spectroscopy, on a less widely used basis.

The proposal to use the SPECTROstar is multifaceted. With its capabilities to measure small volumes in a vast array and its incubation and shaking capabilities it will enable the reduction in assay size for numerous methods requiring spectroscopy. Polyphenol assays are readily performed by rapid mixing of several components and immediate measurement at an absorbance of 600 nm and multiplication of the result by the factor 820. The instrument will facilitate the further reduction in volume for this assay. Bitterness assays in beer involve an organic extraction and spectral measurement at 275 nm. Hop alpha acids can be extracted and tested over a spectrum of absorbencies. The scanning capabilities of the SPECTROstar Nano will facilitate this type of analysis and reduce the volume needs for solvents for extraction and analysis. These are just three assays that we will research to see if they can be scaled down and adapted to the SPECTROstar Nano. Other enzymatic assays for a wide range of metabolites and species can also be tested using routine or ELISA-based methods.

The main area of research for immediate application of this new unit will be a continuation of our earlier studies into the measurement of protein content in beer (much needed in order to eliminate the unpleasant and dangerous Kjeldahl assay), ammonia and free amino nitrogen (beer and wine). In collaboration with a laboratory in Texas we thoroughly reviewed and revised a standard ninhydrin-based assay to measure free amino nitrogen in beer (Abernathy et al, 2009). The method was adapted for use in a microwell format (tested in Texas) but thus far we have not been able to apply that approach to the assay. We believe the SPECTROstar Nano will provide the full capabilities to develop this critically important assay into a reliable and rapid throughput method. Furthermore, the basic approach was applied to hydrolyzed and dialyzed samples of beer for protein analysis providing a true indication of beer protein. The Kjeldahl assay, in addition to its many drawbacks (dangerous boiling of acids, general difficulty in measuring accurately low amounts of protein), does not in fact measure only protein. A few approaches to measure protein (in beer samples) spectrally have been investigated and collaboratively tested by the American Society of Brewing Chemists but have proven unreliable. Using our initially developed methods and the SPECTROstar Nano will allow us to generate useful assays for ammonia, protein and free amino nitrogen determination in beer, wine and other beverages.

Expected outcomes and alternate experiments:

These topics were both discussed briefly above. With our main thrust of research we expect to develop a useful set of methods to more accurately and rapidly determine protein in beer (for nutritional labeling purposes) and to solidly define new and better methods for measuring free amino nitrogen (a key to determining efficiency of fermentation and stability of beer and wine). It is unlikely that the Kjeldahl method (or Combustion methods) will ever be abandoned outright but we could generate more accurate protein quantitation values. Other routine assays will be modified to reduce assay size and waste of precious reagents (some having waste disposal issues) thus promoting a greener environment. Such will include; the measurement of polyphenols in beer, possibly extended to tannins and flavonoids; to beer bitterness and alpha acid determination; to sulfites (an allergen) in beer and wine and some soft drinks; to mycotoxins analysis via ELISA methods and to numerous enzymatic assays relying on spectral measurement of the cofactor NAD. There are numerous possibilities and potentials for the use of a SPECTROstar Nano and ancillary equipment in our laboratory.

Conclusion:
We are mainly a small but dedicated analytical testing facility for the beverage industry. We do, however, try to do some basic research along with scaling down routine assays to promote a safer environment, to speed up return of results, and reduce cost and use of reagents, solvents and other chemicals. Using standard spectroscopy methods and ELISA technology, a SPECTROstar Nano with its incubation, shaking capabilities, its scanning across the uv and visible wavelengths and reference corrections to our existing assays would benefit our laboratory and limited research efforts enormously.

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

Proposal respectfully submitted,
Gary Spedding,
Managing Owner, BREWING & DISTILLING ANALYTICAL SERVICES, LLC
1141 RED MILE ROAD, SUITE 202, LEXINGTON, KY 40504

See the proposals of the other winners here.