Beer Archives - NMR Testing Laboratory

The Chemical Fingerprint of Beer from a Single Experiment with Minimum Sample Preparation – A Rapid Quantitive Analysis by 1H NMR Spectroscopy

September 7, 2016 by process nmr Beer, Benchtop NMR, Chemistry, Cider, Craft Beverage, NMR, NMR Test Methods, NMR Validation, qNMR Tagged: Beer, Ethanol, Fingerprint, Malt, NMR, Organic Acid, Quality

NMR Spectroscopy is the premier tool utilized by chemists to obtain detailed chemical information on molecular structure and is used extensively in molecular structure verification, chemical purity analysis, and complex mixture analysis. We have developed a quantitative NMR analysis that yields a chemical fingerprint that brewers can utilize to follow detailed variations in the chemistry observed in the various stages of the brewing process (malting, mashing, boiling, fermentation, ageing, and blending). The analysis observes all molecules in the beer at the same time and each molecular component (acids, alcohols, amino acids, malt-oligosaccharides) yields a unique spectral fingerprint pattern that is related to the structure of the molecule. Though the spectrum consists of a large number of overlapped individual fingerprints it is possible to identify and quantify individual components because many components have signals that appear at unique and specific points in the spectrum. The quantitative analysis is performed by comparing the area under the individual molecule signals to that of an internal standard (Maleic Acid 99%). Molecular components are quantified on a weight/volume basis in mg/L (parts-per million). Ethanol is also quantified on a %volume/volume basis.

The technique is not only applicable to the brewing process but is also being utilized to gain detailed chemical understanding of cider-making process, as well as the production of wine, mead, sake, spirits, and kombucha. Our laboratory has been developing this method with the help of a number of breweries following changes in batches of standard beers (Kolsch, Stout, Scots Ale, Barley Wine) as the brewing process is tweaked and changed over the course of 2 years. We have looked not just at finished beers but have studied dextrin solubility and chemistry of wort made from different malts, the effect of temperature on sour mashing, the effect of wild yeast and bacteria on various aspects of beer chemistry, as well as troubleshooting of “out of sensory target range” beers. The analysis requires very little sample preparation, has a large (orders of magnitude) linear concentration range of applicability and observes a large number of components in a single test that does not require constant re-calibration with expensive standards.

A poster was presented on this topic at the World Brewing Congress held August 1-17 in Denver Colorado – the poster can be downloaded here.

References to 1H qNMR Applied to Fruit Juices and Alcoholic Beverages

May 2, 2016 by process nmr Beer, Cider, Craft Beverage, NMR, NMR Test Methods, qNMR

qNMR

“Validation of quantitative NMR”, F Malz, H Jancke, J Pharm Biomed Anal, 38, 813–823, (2005)

“Quantitative ¹H NMR spectroscopy”, S K Bharti, R Roy, Trends in Anal Chem, 35, 5-26 (2012)

“NMR-based plant metabolomics: where do we stand, where do we go?” H-K Kim, Y-H Choi, R Verpoorte Trends Biotech, 29(6), 267 (2011).

“Universal quantitative NMR analysis of complex natural samples”, C Simmler, J G Napolitano, J B McAlpine, S-N Chen and G F Pauli, Current Opinion Biotech, 25, 51–59 (2014)

“Quantitative ¹H NMR: Development and Potential of a Method for Natural Products Analysis” G F Pauli, B U Jaki, D C Lankin, J. Nat. Prod., 68, 133-149 (2005)

Juices

“High-Field Proton NMR Studies of Apple Juices”, P S Belton, I Delgadillo, A M Gil, P Roma, F Casuscelli, I J Colquhoun, M J Dennis M Spraul, Mag Res Chem, 35, S52-S60 (1997)

“Quantitative determination of (-)-epicatechin in cider apple juices by ¹H NMR”, I Berregi , J I Santos , G del Campo , J I Miranda, Talanta 61 (2003) 139-145

“Use of the ¹H Nuclear Magnetic Resonance Spectra Signals from Polyphenols and Acids for Chemometric Characterization of Cider Apple Juices”, G del Campo, J. I Santos, N Iturriza, I Berregi, A Munduate, J Agric Food Chem, 54, 3095−3100 (2006)

“Untargeted NMR-Based Methodology in the Study of Fruit Metabolites”, A P Sobolev, L Mannina, N Proietti, S Carradori, M Daglia, A M Giusti, R Antiochia and D Capitani, Molecules, 20, 4088-4108 (2015)

“Quantitative determination of formic acid in apple juices by ¹H NMR spectrometry”, I Berregi, G del Campo, R Caracena, J I Miranda, Talanta, 72, 1049–1053 (2007)

“Tracking the degradation of fresh orange juice and discrimination of orange varieties: An example of NMR in coordination with chemometrics analyses”, C R de Oliveira, R L Carneiro, A G Ferreira, Food Chem, 164, 446–453 (2014)

“Quantitation determination of chlorogenic acid in cider apple juices by ¹H NMR spectrometry”, I Berregi, J I Santos, G del Campo, J I Miranda, J M Aizpurua, Anal Chim Acta, 486, 269–274 (2003)

“NMR-based multi parametric quality control of fruit juices: SGF profiling”, M Spraul, B Schuetz, P Rinke, S Koswig, E Humpfer, H Schaefer, Nutrients, 1, 148 (2009)

“Evolving window zone selection method followed by independent component analysis as useful chemometric tools to discriminate between grapefruit juice, orange juice and blends”, M Cuny, G Le Gall, I J Colquhoun, M Lees, D N Rutledge, Anal Chim Acta, 597, 203 (2007)

“Fast NMR juice identification based on sugars and other plant metabolites from fruits”, M Balan, A Nicolescu, C Deleanu, C Stavarache, M Ciobanu, Rev Roum Chim, 58(2-3), 175-182 (2013)

Adjuncts – Hops, Malt, Honey

“Detection of honey adulteration by sugar syrups using one-dimensional and two-dimensional high-resolution nuclear magnetic resonance”, D Bertelli, M Lolli, G Papotti, L Bortolotti, G Serra, M Plessi, J Agric Food Chem, 58, 8495 (2010)

“Identiﬁcation of components of Brazilian honey by H NMR and classiﬁcation of its botanical origin by chemometric methods”, E F Boffo, L A Tavares, A C T Tobias, M M C Ferreira, A G Ferreira, LWT – Food Sci Tech, 49, 55-63 (2012)

“Fast and global authenticity screening of honey using ¹H-NMR profiling”, M Spiteri, E Jamin, F Thomas, A Rebours, M Lees, K M Rogers, D N Rutledge, Food Chem, 189, 60-66 (2015)

“Characterization of Markers of Botanical Origin and Other Compounds Extracted from Unifloral Honeys”, E Schievano, E Morelato, C Facchin, S Mammi, J Agric Food Chem, 61(8), 1747-1755 (2013)

“NMR Characterization of Saccharides in Italian Honeys of Different Floral Sources”, R Consonni, L R Cagliani, C Cogliati, J Agric Food Chem, 60(18), 4526-4534 (2012)

“An improved NMR method for the quantification of alpha-acids in hops and hop products”, A C Hoek, A C Hermans-Lokkerbol, R Verpoorte, Phytochem Anal, 12(1), 53-57 (2001)

“Characterization of reduced iso-α-acids derived from hops (Humulus lupulus) by NMR”, L I Nord, S B Sørensen, J Ø Duus, Magn Reson Chem, 41(9), 660-670 (2003)

Cider

“Cider, hard and sweet: history, traditions, and making your own”, Ben Watson, 2nd ed, Countryman Press, 2009

“Cider making, using & enjoying sweet and hard cider”, Annie Proulx & Lew Nichols, 3rd Ed, Storey Publishing, 2003

“The new cider maker’s handbook: a comprehensive guide for craft producers”, Claude Jolicoeur, Chelsea Green Publishing, 2013

“Quantitative analysis of malic and citric acids in fruit juices using proton nuclear magnetic resonance spectroscopy”, G del Campo, I Berregi, R Caracena, J I Santos, Anal Chim Acta, 556, 462–468 (2006)

“Quantitative determination of lactic and acetic acids in cider by ¹H NMR spectrometry”, A Zuriarrain, J Zuriarrain, A I Puertas, M T Dueñas, I Berregi, Food Control, 52, 49–53 (2015)

“Glycerol metabolism in Lactobacillus collinoides: production of 3-hydroxypropionaldehyde, a precursor of acrolein”, Nicolas Sauvageot , Kamila Goufﬁ, Jean-Marie Laplace, Yanick Auffray, Int J Food Microbiol 55, 167–170, (2000)

“Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking”, G Garai-Ibabe, I Ibarburu , I Berregi , O Claisse , A Lonvaud-Funel , A Irastorza , MT Dueñas, Int J Food Microbiol, 121, 253–261, (2008)

“Quantitative determination of ethanol in cider by ¹H NMR spectrometry”, A Zuriarrain, J Zuriarrain , M Villar ,I Berregi, Food Control, 50, 758-762, (2015)

Wine

“Application of One- and Two-Dimensional NMR Spectroscopy for the Characterization of Protected Designation of Origin Lambrusco Wines of Modena”, G Papotti, D Bertelli, R Graziosi, M Silvestri, L Bertacchini, C Durante, and M Plessi, J Agric Food Chem, 61, 1741-1746 (2013)

“NMR metabolite ﬁngerprinting in grape derived products: An overview”, C Fotakis, K Kokkotou, P Zoumpoulakis, M Zervou, Food Res Int, 54, 1184–1194 (2013)

“NMR spectroscopy evaluation of direct relationship between soils and molecular composition of red wines from Aglianico grapes”, P Mazzei, N Francesca, G Moschetti , A Piccolo, Analytica Chimica Acta 673 (2010) 167–172

“NMR investigation of acrolein stability in hydroalcoholic solution as a foundation for the valid HS-SPME/GC–MS quantiﬁcation of the unsaturated aldehyde in beverages”, M Kächele, Y B Monakhova, T Kuballa, D W Lachenmeier, Anal Chim Acta, 820, 112–118 (2014)

“Wine science in the metabolomics era”, M E Alañón, M S Pérez-Coello, M L Marina, Trends Anal Chem, 74, 1–20 (2015)

“Amino acid uptake by wild and commercial yeasts in single fermentations and co-fermentations”, N Barrajón-Simancas, E Giese, M Arévalo-Villena, J Úbeda , A Briones, Food Chem, 127, 441–446 (2011)

“Chemical Profile of White Wines Produced from ‘Greco bianco’ Grape Variety in Different Italian Areas by Nuclear Magnetic Resonance (NMR) and Conventional Physicochemical Analyses”, M Caruso, F Galgano, M A C Morelli, L Viggiani, L Lencioni, B Giussani, F Favati, J Agric Food Chem, 60, 7-15 (2012)

“Metabolomic by ¹H NMR Spectroscopy Diﬀerentiates “Fiano Di Avellino” White Wines Obtained with Diﬀerent Yeast Strains”, P Mazzei, R Spaccini, N Francesca, G Moschetti, A Piccolo, J Agric Food Chem, 61, 10816-10822 (2013)

“An exploratory chemometric study of ¹H NMR spectra of table wines”, F H Larsen, F van den Berg, S B Engelsen, J. Chemometrics, 20, 198-208 (2006)

“Sensory attributes of wine inﬂuenced by variety and berry shading discriminated by NMR metabolomics”, S Rochfort , V Ezernieks , S E P Bastian , M O Downey. Food Chem, 121, 1296–1304 (2010)

“Metabolic Influence of Botrytis cinerea Infection in Champagne Base Wine”, Y-S Hong, C Cilindre, G Liger-Belair, P Jeandet, N Hertkorn, P Schmitt-Kopplin, J Agric Food Chem, 59, 7237-7245 (2011)

“A Thorough Study on the Use of Quantitative ¹H NMR in Rioja Red Wine Fermentation Processes”, E Lopez-Rituerto, S Cabredo, M Lopez, A Avenoza, J H Busto, J M Peregrina, J Agric Food Chem, 57, 2112–2118 (2009)

“Comparison of Gas Chromatography-Coupled Time-of-Flight Mass Spectrometry and ¹H Nuclear Magnetic Resonance Spectroscopy Metabolite Identification in White Wines from a Sensory Study Investigating Wine Body”, K Skogerson, R Runnebaum, G Wohlgemuth, J De ROPP, H Heymann, O Fiehn, J Agric Food Chem, 57(15), 6899-6907 (2009)

“Metabolomic Characterization of Malolactic Fermentation and Fermentative Behaviors of Wine Yeasts in Grape Wine” H-S Son, G-S Hwang, W-M Park, Y-S Hong, AND C-H Lee, J Agric Food Chem, 57(11), 4801-4809 (2009)

“¹H nuclear magnetic resonance-based metabolomic characterization of wines by grape varieties and production areas”, H-S Son, K-M Kim, F Van den Berg, G-S Hwang , W-M Park, C-H Lee, J Agric Food Chem, 56, 8007 (2008)

“Metabolomic studies on geographical grapes and their wines using ¹H NMR analysis coupled with multivariate statistics”, H-S Son, G-S Hwang, KM Kim, H-J Ahn, W-M Park, F Van Den Berg, J Agric Food Chem, 57, 1481 (2009)

“Use of modern nuclear magnetic resonance spectroscopy in wine analysis: determination of minor compounds”, I J Kosir, J Kidric, Anal Chim Acta, 458, 77 (2002)

“Chemometric classification of Apulian and Slovenian wines using ¹H NMR and ICP-OES together with HPICE data”, M A Brescia, I J Kosir, V Caldarola, J Kidric, A Sacco, J Agric Food Chem, 51, 21 (2003)

“Characterization of wines by nuclear magnetic resonance: a work study on wines from the Basilicata region in Italy”, L Viggiani, M A C Morelli, J Agric Food Chem, 56, 8273 (2008)

“Classification of wines based on combination of ¹H NMR spectroscopy and principal component analysis”, Y-Y Du, G-Y Bai, X Zhang, M-L Liu, Chin J Chem, 25, 930 (2007)

“¹H NMR-based metabonomics for the classification of Greek wines according to variety, region, and vintage. Comparison with HPLC data”, M Anastasiadi, A Zira, P Magiatis, S A Haroutounian, A L Skaltsounis, E Mikros, J Agric Food Chem, 57, 11067 (2009)

“¹H NMR and chemometrics to characterize mature grape berries in four wine- growing areas in Bordeaux, France, G E Pereira, J P Gaudillere, C Van Leeuwen, G Hilbert, O Lavialle, M Maucourt, J Agric Food Chem, 53, 6382 (2005)

“¹H NMR-based metabolomic approach for understanding the fermentation behaviors of wine yeast strains”, H-S Son, G-S Hwang, KM Kim, E-Y Kim, F van den Berg, W-M Park, Anal Chem, 81, 1137 (2009)

“Generalized 2D-correlation NMR analysis of a wine fermentation”, G M Kirwan, S Clark, N W Barnett, J O Niere, M J Adams, Anal Chim Acta, 629, 128 (2008)

“Time course of the evolution of malic and lactic acids in the alcoholic and malolactic fermentation of grape must by quantitative ¹H NMR (qHNMR) spectroscopy”, A Avenoz, J H Busto, N Canal, J M Peregrina, J Agric Food Chem, 54, 4715 (2006)

“NMR-based metabolomics in wine science”, Y-S Hong, Magn Reson Chem, 49, S13 (2011)

“High- resolution NMR and diffusion-ordered spectroscopy of port wine”, M Nilsson, I F Duarte, C Almeida, I Delgadillo, B J Goodfellow, A M Gil, J Agric Food Chem, 52, 3736 (2004)

“Quantitative NMR spectroscopy of binary liquid mixtures (aldehyde + alcohol) Part I: Acetaldehyde + (methanol or ethanol or 1-propanol)”, S Jaubert, G Maurer, J Chem Thermo 68, 332–342 (2014)

Beer

“Beer metabolomics: molecular details of the brewing process and the differential effects of late and dry hoppinng on yeast purine metabolism”, A R Spevacek, K H Benson, C W Bamforth, C M Slupsky, J. Inst. Brew., 122 21-28 (2016)

“Composition of beer by ¹H NMR spectroscopy: effects of brewing site and date of production”, C Almeida, I F Duarte, A Barros, J Rodrigues, M Spraul, A M Gil, J Agric Food Chem, 54, 700 (2006)

“Multivariate analysis of NMR and FTIR data as a potential tool for the quality control of beer “, I F Duarte, A Barros, C Almeida, M Spraul, A M Gil, J Agric Food Chem, 52, 1031 (2004)

“High-Resolution Nuclear Magnetic Resonance Spectroscopy and Multivariate Analysis for the Characterization of Beer”, Ä I Duarte, A Barros, P S Belton, R Righelato, M Spraul, E Humpfer, A M Gil, J Agric Food Chem, 50, 2475−2481 (2002)

“Quantiﬁcation of organic acids in beer by nuclear magnetic resonance (NMR)-based methods”, J E A Rodrigues , G L Erny , A S Barros , V I Esteves , T Brandão , A A Ferreira , E Cabrita , A M Gil, Anal Chim Acta, 674, 166–175 (2010)

“NMR methods for beer characterization and quality control”, J E Rodrigues, A M Gil, Magn Reson Chem, 49, S37–S45 (2011)

“Quality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysis”, D W Lachenmeier, W Frank, E Humpfer, H Schafer, S Keller, M Mortter · M Spraul, Eur Food Res Technol, 220, 215–221 (2005)

“Probing beer aging chemistry by nuclear magnetic resonance and multivariate analysis”, J A Rodrigues, A S Barros, B Carvalho, T Brandão, A M Gil, Anal Chim Acta, 702, 178–187 (2011)

“Quantification of Organic and Amino Acids in Beer by ¹H NMR Spectroscopy”, L I Nord, P Vaag, J Ø Duus, Anal Chem, 76 (16), 4790–4798 (2004)

“Application of Quantitative Nuclear Magnetic Resonance Spectroscopy to Biological Acidification of Barley Mashes”, A Dicaprio, J C Edwards, J Inst Brewing, 120(3), 207-211 (2014)

Beer Dextrins

“Separation and NMR structural characterisation of singly branched a-dextrins which differ in the location of the branch point”, A Jodelet, N M Rigby, I J Colquhoun, Carb Res, 312, 139-151 (1998)

“¹H NMR spectroscopy for proﬁling complex carbohydrate mixtures in non-fractionated beer” B O Petersen , M Nilsson , M Bøjstrup , O Hindsgaul , S Meier, Food Chem, 150, 65–72 (2014)

“Development of brewing science in (and since) the late 19th century: Molecular proﬁles of 110–130 year old beers”, A Walther, D Ravasio, F Qin, J Wendlan , S Meier, Food Chem, 183, 227–234 (2015)

“Structural determination of some new oligosaccharides and analysis of the branching pattern of isomaltooligosaccharides from beer”, E Vinogradov, K Bock, Carb Res, 309, 57-64 (1998)

“NMR characterization of chemically synthesized branched a-dextrin model compounds”, B O Petersen, M S Motawie, B Lindberg Møller, O Hindsgaul, S Meier, Carb Res, 403, 149–156 (2105)

Sake

“NMR-Based Metabolic Profiling of Rice Wines by F2-Selective Total Correlation Spectra”, M Koda, K Furihata, F Wei, T Miyakawa, M Tanokura, J Agric Food Chem, 60(19), 4818–4825 (2012)

Vinegar

“Traditional balsamic vinegar and balsamic vinegar of Modena analyzed by nuclear magnetic resonance spectroscopy coupled with multivariate data analysis”, G Papotti, D Bertelli, R Graziosia, A Maietti, P Tedeschi, A Marchetti, M Plessi, LWT – Food Sci Tech, 60(2), 1017-1024 (2015)

“Identiﬁcation and quantiﬁcation of the main organic components of vinegars by high resolution ¹H NMR spectroscopy”, A. Caligiani , D. Acquotti , G. Palla , V. Bocchi, Anal Chim Acta, 585,110–119 (2007)

¹H NMR studies on Italian balsamic and traditional balsamic vinegars”, R Consonni, A Gatti, J Agric Food Chem, 52, 3446 (2004)

“NMR and chemometric methods: a powerful combination for characterization of Balsamic and Traditional Balsamic Vinegar of Modena”, R Consonni, L R Cagliani, F Benevelli, M Spraul, E Humpfer, M Stocchero, Anal Chim Acta, 611, 31 (2008)

Spirits

“NMR metabolite proﬁling of Greek grape marc spirits” C Fotakis , D Christodouleas , K Kokkotou , M Zervou , P Zoumpoulakis , P Moulos , M Liouni , A Calokerinos, Food Chem, 138, 1837–1846 (2013)

“NMR metabolic ﬁngerprinting and chemometrics driven authentication of Greek grape marc spirits”, C Fotakis, M Zervou, Food Chem, 196, 760-768 (2016)

“Rapid Determination of Total Thujone in Absinthe Using ¹H NMR Spectroscopy”, Y B Monakhova , T Kuballa, and D W Lachenmeier, Int J Spectroscopy, 2011, Article ID 171684, 5 pages (2011)

“Solute Effects on the Interaction between Water and Ethanol in Aged Whiskey”, A Nose, M Hojo, M Suzuki, T Ueda, J Agric Food Chem, 52(17), 5359–5365 (2004)

“Hydrogen Bonding in Alcoholic Beverages (Distilled Spirits) and Water−Ethanol Mixtures”, A Nose, T Hamasaki, M Hojo, R Kato, K Uehara, T Ueda, J Agric Food Chem, 5(18), 7074–7081 (2005)

“Structurability: A Collective Measure of the Structural Differences in Vodkas”, N Hu, D Wu, K Cross, S Burikov, T Dolenko, S Patsaeva, D W Schaefer, J Agric Food Chem, 58(12), 7394–7401 (2010)

“Rapid Quantification of Ethyl Carbamate in Spirits Using NMR Spectroscopy and Chemometrics”, Y B Monakhova, T Kuballa, Dirk W Lachenmeier, ISRN Anal Chem, 2012, Article ID 989174, 5 pages (2012)

“Authenticity of the Traditional Cypriot Spirit “Zivania” on the Basis of ¹H NMR Spectroscopy Diagnostic Parameters and Statistical Analysis”, P Petrakis, I Touris, M Liouni, M Zervou, I Kyrikou, R Kokkinofta, C R Theocharis, T M Mavromoustakos, J Agric Food Chem, 53(13), 5293–5303 (2005)

“Investigation into the structural composition of hydroalcoholic solutions as basis for the development of multiple suppression pulse sequences for NMR measurement of alcoholic beverages”, Y B Monakhova, S P Mushtakova, T Kuballa, D W Lachenmeier, Magn Reson Chem, 52, 755-759 (2014)

“Quantitative ¹H NMR Analysis of Egg Yolk, Alcohol, and Total Sugar Content in Egg Liqueurs”, M Hohmann, V Koospal, C Bauer-Christoph, N Christoph, H Wachter, B Diehl, U Holzgrabe, J Agric Food Chem, 63(16), 4112–4119 (2015)

Quantitative 1H NMR Analysis of “Off-the-Shelf” Commercial Kombucha Beverages for Ethanol, Organic Acids and Residual Sugars Analysis

December 28, 2015 by process nmr Beer, Chemistry, Craft Beverage, NMR, NMR Test Methods, NMR Validation, qNMR Tagged: Ethanol, Kombucha, NMR, Organic Acid

Over the past few years our analytical NMR service has been developing a detailed chemical fingerprint analysis of alcoholic beverages by quantitative ¹H NMR (qHNMR). Beyond the typical analyses of beer, wine, port, hard cider, sake and spirits, we have been looking at other fermented beverages such as kombucha, kefir, kvass, mead, ginger beer and perry. As well as the final fermented beverages we have been actively investigating the various starting materials such as malt wort, apple juice, honey, grape juice, fruit juices, and tea. The NMR analysis can provide a rapid quantitative analysis without any sample preparation based on the molar ratio of integration value of unique molecular fingerprint peaks with the integrated signal of an internal standard. In our case we typically use maleic acid as an internal standard as it’s singlet signal peak appears in a non-overlapping are of the spectrum to the chemistry we are interested in following.

The information that can be derived from the NMR experiment covers a wide dynamic range of component molecule concentrations from 10-100,000 ppm. The analysis observes all fully dissolved chemical constituents and the spectral response is linear with regard to all chemical types. As a primary analytical method the chemist can utilize the well understood literature on the NMR chemical shifts and couplings that allow first principles analysis of each molecular fingerprint to identify and quantify the presence of targeted and non-targeted molecules in the complex mixture. The analysis provides quantitative information on the following chemical components: ethanol, higher (C3,C4,C5) alcohols, methanol, glycerol, organic acids (lactic, acetic, succinic, pyruvic, pyruvic hydrate, citric, malic, tartaric, quinic), free amino acids (alanine, isoleucine, valine, tyrosine, phenylalanine), carbohydrates (sucrose, glucose, fructose, sorbitol, xylose, galacuronic acid, maltose, 1,6- and 1,4-dextrin chemistry, maltotriose, lactose), polyphenols. It can also provide information on yeast metabolism products such as 2,3-butandiol (directly from Enterobacter or from the action of saccharomyces on diacetal which is a well-known beer flavor deviation), 1,3-propandiol (from yeast action on glycerol after carbohydrates have been entirely fermented from the beverage).

In recent years kombucha has been found to contain more than 0.5% v/v ethanol which would technically lead the product to be classified as alcoholic beverages and bring the product under scrutiny and taxation by the Alcohol and Tobacco Tax and Trade Bureau which federally regulates the alcoholic beverage industry. Kombucha is a sweetened black or green tea that has been inoculated with a symbiotic culture of bacteria and yeast (SCOBY) which ferments the sugars in the drink solution in bith the manufacturing process and in the sealed bottle shipped out to stores. The drink is sold under the premise that the SCOBY provides a probiotic culture to the consumer which means that in many Kombuch products the activity of the culture is not arrested by pasteurization or by addition of sorbate. Thus, the kombucha is bottled with active yeast and bacteria present in a high sugar containing tea drink. Fermentation is then thought to occur while the product sits on shelves and leads to >0.5% ABV when the drink is purchased or consumed. We have utilized ¹H NMR to obtain quantitative ethanol concentrations on a number of kombucha beverages bought off the shelf at grocery stores. The samples we analyzed represent the entire dataset of kombuchas that we purchased and they represent the products of 5 different manufacturers. We also aged two of the products at room temperature for 7 months and analyzed them to observe the effect of long term aging on kombucha products.

Experimental: ¹H NMR spectra were acquired on a Varian Mercury-300MVX spectrometer operating at a resonance frequency of 299.67 MHz and equipped with a Varian 5mm ATB PFG probe. The experiments are performed under quantitative conditions utilizing a 10 ms (p/3 tip angle) pulse with an 8 second acquisition time and a 7 second relaxation delay. 64 transients were acquired over a spectral window of 8 kHz at a controlled temperature of 27^oC. Water suppression was achieved by pre-saturation and this can affect the quantitation of glucose in the samples under these conditions.

Sample preparation: Samples were purchased “off the shelf” at local grocery stores and were analyzed the same day that they were purchased. Samples were prepared by 1) degassing the samples by repeated vortex agitation, 2) samples are equilibrated at 27^oC before pipetting to allow a mass to volume conversion to be utilized to calculate the %ABV utilizing an ethanol density value of 0.7816 kg/L, 3) pipetting 175ml of kombucha beverage into a 5mm NMR tube, 4) adding 100ml of a 100mg/ml solution of maleic acid (99.5% – Sigma Aldrich) in D₂O (99.8%D), and 5) addition of 375ml of D₂O (99.8%D – Cambridge Isotopes Laboratories). The final samples were thoroughly mixed using a vortex mixer.

Two of the kombucha samples were purchased in duplicate and not opened immediately but stored at room temperature for 7 months before being analyzed. These stored samples were compared with the same samples that were opened and analyzed immediately after purchase.

Calculations: Component concentrations were calculated on a mg/L basis based on a knowledge of the concentration of maleic acid internal standard present in the sample (10mg) using the following equation:

Component Concentration (C) in mg/L = 0.995 x 10 x ((I_C/N_C)/(I_MA/N_MA)) x (M_C/M_MA) x (1,000,000/175)

Where 10 mg is the mass of maleic acid used as the internal standard, I_C = integral of the component peak, N_C = number of protons represented in the component peak, I_MA = integral of maleic acid internal standard, N_MA = number of protons represented in the maleic acid integral (2), M_c = molecular weight of the component, M_MA = molecular weight of maleic acid (116.1 amu). Other aspects of the equation are – 175ml of sample must be adjusted to 1 liter (1,000,000 ml), and the whole must be multiplied by 0.995 as the maleic acid can only be guaranteed to be 99.5% pure. The ethanol content is calculated based on a weight per volume basis (mg/L) and then a calculation is performed to convert this weight/volume concentration to a volume/volume basis using a density value of 0.7816 kg/L to convert the weight of ethanol to the volume of ethanol.

Results: Figures 1-7 show the ¹H NMR spectra of the 7 kombucha samples purchased and analyzed immediately. All 7 samples were found to contain ethanol and only one of them was found to contain less than 0.5%. Figure 8 shows a stacked plot comparison of the chemistry observed in a kombucha that was aged for 7 months at room temperature compared to the sample when it was initially purchased. The alcohol content rose from 1.23 %ABV to 4.25 %ABV and it can be seen that all sugars in the original drink have been consumed by the SCOBY to produce this increased alcohol content. The acetic acid content of the aged drinks also increased but it is obvious that the conversion of ethanol to acetic acid by acetobacteria present in the SCOBY does not offset the overall production of ethanol. The component concentrations of ethanol, sugars and organic acids in each of the kombucha beverages analyzed are provided in Table I.

Table I: Concentration of Chemical Components of Kombucha Beverages

	Kombucha Sample
Component	#1	#1 Aged	#2	#2 Aged	#3	#4	#5	#6	#7
Lactic Acid (mg/L)	64	68	131	210	461	124	1809	24	248
Succinic Acid (mg/L)	74	97	116	277	142	134	110	64	131
Acetic Acid (mg/L)	3056	5637	2746	3333	387	2806	2051	3719	444
Malic Acid (mg/L)	175	190	175	190	185	515	0	0	99
Ethanol (mg/L)	10625	12640	9580	33245	11631	10938	8114	3866	10218
Ethanol (v/v)	1.36	1.62	1.23	4.25	1.49	1.40	1.04	0.49	1.31
Sucrose (mg/L)	0	0	4141	0	12261	11790	2021	27723	11386
Glucose (mg/L)	24017	24507	24460	0	15379	15645	22776	31450	20328
Fructose (mg/L)	31786	9433	23725	0	16634	18173	30155	30791	17437
Sorbate (mg/L)	0	0	0	0	0	0	0	0	0
Citrate (mg/L)	0	0	1592	1582	0	4416	0	0	0

Figure 1: Kombucha #1 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 2: Kombucha #2 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 3: Kombucha #3 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 4: Kombucha #4 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 5: Kombucha #5 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 6: Kombucha #6 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 7: Kombucha #7 – ¹H NMR spectrum – component peaks utilized in calculations indicated.

Figure 8: Kombucha #2 – Comparison of original analyzed “fresh kombucha” with same purchase date bottle aged at room temperature for 7 months – ¹H NMR spectrum – sugar peaks are consumed by the yeast to produce higher alcohol in the aged sample.

References:

Kombucha General Information: Kombucha 101: Benefits, Brewing, Recipes, Storage, And More – Lisa Williams – HappyHappyVegan.com -visited 4-11-19

Alcohol in Kombucha News Articles:

http://www.cnn.com/2015/12/09/health/kombucha-tea-alcohol-content/index.html – visited 12-13-15

http://www.wsj.com/articles/battle-brews-over-kombucha-teas-1447116607 – Visited 12-13-15

Kombucha Product Information:

Kombucha Brewers International – http://kombuchabrewers.org/ – visited 12-13-15

¹H qNMR:

“Universal quantitative NMR analysis of complex natural samples”, G C Simmler, J Napolitano, J B McAlpine, S-N Chen and G F Pauli, Current Opinion in Biotechnology 2014, 25:51–59

“Quantitative H NMR spectroscopy”, S K Bharti, R Roy, Trends in Analytical Chemistry, Vol. 35, 2012

“Validation of quantitative NMR”, F Malz , H Jancke, Journal of Pharmaceutical and Biomedical Analysis 38 (2005) 813–823

¹H qNMR Applied to Alcoholic and Non-Alcoholic Beverages

“Quantitative determination of ethanol in cider by ¹H NMR spectrometry”, A Zuriarrain , J Zuriarrain , M Villar ,I Berregi, Food Control 50 (2015) 758-762

“Quantitative determination of lactic and acetic acids in cider by ¹H NMR spectrometry”, A Zuriarrain, J Zuriarrain, A I Puertas, M Dueñas, I Berregi

“Identiﬁcation and quantiﬁcation of the main organic components of vinegars by high resolution ¹H NMR” spectroscopy, A Caligiani , D Acquotti , G Palla , V Bocchi, Analytica Chimica Acta 585 (2007) 110–119

“NMR-based metabolomics in wine science”, Y-S Hong, Magn. Reson. Chem. 2011,49, S13–S21

“¹H NMR-based metabolomic characterization during green tea (Camellia sinensis) fermentation”, J-E Lee , B-J Lee , J-O Chung , H-J Shin , S-J Lee , C-H Lee ⁎, Y-S Hong, Food Research International 44 (2011) 597–604

“NMR methods for beer characterization and quality control”, J E Rodrigues, A M Gil, Magn. Reson. Chem. 2011, 49, S37–S45

“Metabolomic proﬁling of Cheonggukjang during fermentation by ¹H NMR spectrometry and principal components analysis”, H-K Choi, J-H Yoon , Y-S Kim , D Y Kwon, Process Biochemistry 42 (2007) 263–266

“Quantiﬁcation of organic acids in beer by nuclear magnetic resonance (NMR)-based methods”, J E A Rodrigues , G L Erny , A S Barros , V I Esteves , T Brandão , A A Ferreira , E Cabrita , A M Gil, Analytica Chimica Acta 674 (2010) 166–175

“Monitoring a commercial fermentation with proton nuclear magnetic resonance spectroscopy with the aid of chemometrics”, S Clark , N W Barnett , M Adams , I B Cook , G A Dyson , G Johnston, Analytica Chimica Acta 563 (2006) 338–345

“Quality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysis”, D W Lachenmeier, W Frank, E Humpfer, H Schafer, S Keller, M Mortter, Manfred Spraul, Eur. Food Res. Technol. (2005) 220:215–221

“¹H NMR spectroscopy for proﬁling complex carbohydrate mixtures in non-fractionated beer”, B O. Petersen , M Nilsson , M Bøjstrup , O Hindsgaul , S Meier, Food Chemistry 150 (2014) 65–72.

“Regulatory Control of Energy Drinks Using ¹H NMR Spectroscopy”, Y B Monakhova, T Kuballa, H Reusch, K Wegert G Winkler, D W Lachenmeier, Lebensmittelchemie 66, 129–168 (2012)

“Qualitative and Quantitative Control of Honeys Using NMR Spectroscopy and Chemometrics”, M Ohmenhaeuser, Y B Monakhova, T Kuballa, D W Lachenmeier, ISRN Analytical Chemistry Volume 2013, Article ID 825318, “Qualitative and Quantitative Control of Carbonated Cola Beverages Using ¹H NMR Spectroscopy” P Maes, Y B Monakhova, and D W Lachenmeier, T Kuballa, H Reusch, J. Agric. Food Chem. 2012, 60, 2778−2784

A PDF Version of this Article can be found here – Kombucha NMR.pdf

Process NMR Associates quantitatively analyzes the component chemistry of craft beverages for consumers or manufacturers – for more information contact John Edwards at +1 (845) 240-1177

1H qNMR of Alcoholic Cider – Analysis of Small Molecule and Residual Sugar Chemistry

June 8, 2015 by process nmr Beer, Benchtop NMR, Chemistry, Cider, NMR, qNMR

1H quantitative NMR (qNMR) has been utilized to assess the the small molecule and carbohydrate chemistry of a number of home-brewed and commercial alcoholic ciders. A quantitative chemistry distribution of the products of the various fermentations that occur in cider making. Malolactic fermentation as well as fermentation by saccharomyces and wild yeasts occur in the cider making process which traditionally occurred without the intentional addition of yeast by the manufacturer. The distribution of small molecules produced by the yeast and bacterial metabolomes at work in the process can yield information of the sensory perception of ciders produced in different ways. An investigation of the residual sugar chemistry of commercial ciders gives some indication of the process of sweetening commercial cider products with sugar additions after fermentation is complete. These typical commercial ciders are very different in chemistry distribution compared to very dry cider styles such as those found in the Basque region of Spain where fermentation is taken to the extreme resulting in complete conversion of sugars to alcohol but also glycerols to 1,3 propandiol. Finally it was decided to determine how much quantitative chemistry information could be obtained from benchtop NMR systems operating in the 60 MHz range. These benchtop NMR systems have a price and cost-of-ownership that would allow small laboratories of manufacturers to think about their use in QA and QC roles.

1H qNMR analysis of molecular components in hard cider – targeted and non-targeted quantitative chemical analysis

PNA to Present 4 Topics at the 2015 ACS North East Regional Meeting

May 17, 2015 by process nmr Beer, Benchtop NMR, Chemometrics, Cider, Energy, Herbal Supplement, NMR, PAT, Petroleum, qNMR

John Edwards of Process NMR Associates will be presenting 4 papers at the 2015 ACS Northeast Regional Meeting that will be held in Ithaca, NY, June 10-13, 2015.

ABSTRACT ID: 2283171
ABSTRACT TITLE: 1H qNMR of Alcoholic Cider – Analysis of Small Molecule and Residual Sugar Chemistry (final paper number: 43)
SESSION: Food Chemistry
SESSION TIME: 5:00 PM – 9:00 PM
PRESENTATION FORMAT: Poster
DAY & TIME OF PRESENTATION: Wednesday, June, 10, 2015, 5:00 PM – 9:00 PM
ROOM & LOCATION: Emerson Suites – Campus Center

ABSTRACT ID: 2283063
ABSTRACT TITLE: Nutritional Supplement and Diesel Fuel Application Development for Benchtop NMR Systems Operating at 42, 60, and 80 MHz – Equivalency with Supercon NMR (final paper number: 336)
SESSION: Analytical Chemistry
SESSION TIME: 9:00 AM – 11:30 AM
PRESENTATION FORMAT: Oral
DAY & TIME OF PRESENTATION: Friday, June, 12, 2015 from 9:45 AM – 10:05 AM
ROOM & LOCATION: 222 – Williams Hall

ABSTRACT ID: 2283105
ABSTRACT TITLE: Survey of Low Field NMR Spectrometer Platforms for Successful Screening of Sexual Enhancement and Weight Loss Supplements for Adulteration with Drugs and Drug Analogs (final paper number: 386)
SESSION: Medicinal Chemistry
SESSION TIME: 1:00 PM – 3:20 PM
PRESENTATION FORMAT: Oral
DAY & TIME OF PRESENTATION: Friday, June, 12, 2015 from 2:20 PM – 2:40 PM
ROOM & LOCATION: 302 – Williams Hall

ABSTRACT ID: 2283153
ABSTRACT TITLE: From Mash to Bottle: Chemistry of the Beer Brewing Process and NMR-based Quality Control (final paper number: 284)
SESSION: Food Chemistry
SESSION TIME: 1:30 PM – 3:10 PM
PRESENTATION FORMAT: Oral
DAY & TIME OF PRESENTATION: Thursday, June, 11, 2015 from 1:35 PM – 1:55 PM
ROOM & LOCATION: 202 – Williams Hall