We have moved our location to Poughkeepsie New York – no more commuting 2 hours per day !!! New address is: Process NMR Associates, LLC, 84 Patrick Lane, Suite 115, Poughkeepsie, NY 12603-2936 Tel: (845) 240-1177
Chemical Fingerprints of Cider – Cidercon 2018 Technical Presentation – Recorded and Presented on the Cider Chat Podcast
John Edwards presented a talk on the chemical analysis of ciders at Cidercon 2018. The talk was recorded by Ria Windcaller who produces the Cider Chat Podcast which specializes in interviews and news from the world of commercial and amateur cider making. Here is the information to listen or watch the presentation.
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The Chemical Fingerprint of Beer from a Single Experiment with Minimum Sample Preparation – A Rapid Quantitive Analysis by 1H NMR Spectroscopy
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 âout of sensory target rangeâ 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
qNMR
âValidation of quantitative NMRâ, F Malz, H Jancke, J Pharm Biomed Anal, 38, 813â823, (2005)
âQuantitative 1H NMR spectroscopyâ, S K Bharti, R Roy, Trends in Anal Chem, 35, 5-26 (2012)
âNMR-based plant metabolomics: where do we stand, where do we go?â H-K Kim, Y-H Choi, R Verpoorte Trends Biotech, 29(6), 267 (2011).
âUniversal quantitative NMR analysis of complex natural samplesâ, C Simmler, J G Napolitano, J B McAlpine, S-N Chen and G F Pauli, Current Opinion Biotech, 25, 51â59 (2014)
âQuantitative 1H NMR: Development and Potential of a Method for Natural Products Analysisâ G F Pauli, B U Jaki, D C Lankin, J. Nat. Prod., 68, 133-149 (2005)
Juices
âHigh-Field Proton NMR Studies of Apple Juicesâ, 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)
âQuantitative determination of (-)-epicatechin in cider apple juices by 1H NMRâ, I Berregi , J I Santos , G del Campo , J I Miranda, Talanta 61 (2003) 139-145
âUse of the 1H Nuclear Magnetic Resonance Spectra Signals from Polyphenols and Acids for Chemometric Characterization of Cider Apple Juicesâ, G del Campo, J. I Santos, N Iturriza, I Berregi, A Munduate, J Agric Food Chem, 54, 3095â3100 (2006)
âUntargeted NMR-Based Methodology in the Study of Fruit Metabolitesâ, A P Sobolev, L Mannina, N Proietti, S Carradori, M Daglia, A M Giusti, R Antiochia and D Capitani, Molecules, 20, 4088-4108 (2015)
âQuantitative determination of formic acid in apple juices by 1H NMR spectrometryâ, I Berregi, G del Campo, R Caracena, J I Miranda, Talanta, 72, 1049â1053 (2007)
âTracking the degradation of fresh orange juice and discrimination of orange varieties: An example of NMR in coordination with chemometrics analysesâ, C R de Oliveira, R L Carneiro, A G Ferreira, Food Chem, 164, 446â453 (2014)
âQuantitation determination of chlorogenic acid in cider apple juices by 1H NMR spectrometryâ, I Berregi, J I Santos, G del Campo, J I Miranda, J M Aizpurua, Anal Chim Acta, 486, 269â274 (2003)
âNMR-based multi parametric quality control of fruit juices: SGF profilingâ, M Spraul, B Schuetz, P Rinke, S Koswig, E Humpfer, H Schaefer, Nutrients, 1, 148 (2009)
âEvolving window zone selection method followed by independent component analysis as useful chemometric tools to discriminate between grapefruit juice, orange juice and blendsâ, 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 â Hops, Malt, Honey
âDetection of honey adulteration by sugar syrups using one-dimensional and two-dimensional high-resolution nuclear magnetic resonanceâ, D Bertelli, M Lolli, G Papotti, L Bortolotti, G Serra, M Plessi, J Agric Food Chem, 58, 8495 (2010)
âIdentiïŹcation of components of Brazilian honey by H NMR and classiïŹcation of its botanical origin by chemometric methodsâ, 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 SĂžrensen, J Ă Duus, Magn Reson Chem, 41(9), 660-670 (2003)
Â
Cider
âCider, hard and sweet: history, traditions, and making your ownâ, Ben Watson, 2nd ed, Countryman Press, 2009
âCider making, using & enjoying sweet and hard ciderâ, Annie Proulx & Lew Nichols, 3rd Ed, Storey Publishing, 2003
âThe new cider maker’s handbook: a comprehensive guide for craft producersâ, Claude Jolicoeur, Chelsea Green Publishing, 2013
âQuantitative analysis of malic and citric acids in fruit juices using proton nuclear magnetic resonance spectroscopyâ, G del Campo, I Berregi, R Caracena, J I Santos, Anal Chim Acta, 556, 462â468 (2006)
âQuantitative determination of lactic and acetic acids in cider by 1H NMR spectrometryâ, A Zuriarrain, J Zuriarrain, A I Puertas, M T Dueñas, I Berregi, Food Control, 52, 49â53 (2015)
âGlycerol metabolism in Lactobacillus collinoides: production of 3-hydroxypropionaldehyde, a precursor of acroleinâ, Nicolas Sauvageot , Kamila GoufïŹ, Jean-Marie Laplace, Yanick Auffray, Int J Food Microbiol 55, 167â170, (2000)
âGlycerol metabolism and bitterness producing lactic acid bacteria in cidermakingâ, G Garai-Ibabe, I Ibarburu , I Berregi , O Claisse , A Lonvaud-Funel , A Irastorza , MT Dueñas, Int J Food Microbiol, 121, 253â261, (2008)
âQuantitative determination of ethanol in cider by 1H NMR spectrometryâ, A Zuriarrain, J Zuriarrain , M Villar ,I Berregi, Food Control, 50, 758-762, (2015)
Wine
âApplication of One- and Two-Dimensional NMR Spectroscopy for the Characterization of Protected Designation of Origin Lambrusco Wines of Modenaâ, G Papotti, D Bertelli, R Graziosi, M Silvestri, L Bertacchini, C Durante, and M Plessi, J Agric Food Chem, 61, 1741-1746 (2013)
âNMR metabolite ïŹngerprinting in grape derived products: An overviewâ, C Fotakis, K Kokkotou, P Zoumpoulakis, M Zervou, Food Res Int, 54, 1184â1194 (2013)
âNMR spectroscopy evaluation of direct relationship between soils and molecular composition of red wines from Aglianico grapesâ, P Mazzei, N Francesca, G Moschetti , A Piccolo, Analytica Chimica Acta 673 (2010) 167â172
âNMR investigation of acrolein stability in hydroalcoholic solution as a foundation for the valid HS-SPME/GCâMS quantiïŹcation of the unsaturated aldehyde in beveragesâ, M KĂ€chele, Y B Monakhova, T Kuballa, D W Lachenmeier, Anal Chim Acta, 820, 112â118 (2014)
âWine science in the metabolomics eraâ, M E Alañón, M S PĂ©rez-Coello, M L Marina, Trends Anal Chem, 74, 1â20 (2015)
âAmino acid uptake by wild and commercial yeasts in single fermentations and co-fermentationsâ, N BarrajĂłn-Simancas, E Giese, M ArĂ©valo-Villena, J Ăbeda , A Briones, Food Chem, 127, 441â446 (2011)
âChemical Profile of White Wines Produced from âGreco biancoâ Grape Variety in Different Italian Areas by Nuclear Magnetic Resonance (NMR) and Conventional Physicochemical Analysesâ, M Caruso, F Galgano, M A C Morelli, L Viggiani, L Lencioni, B Giussani, F Favati, J Agric Food Chem, 60, 7-15 (2012)
âMetabolomic by 1H NMR Spectroscopy DiïŹerentiates âFiano Di Avellinoâ White Wines Obtained with DiïŹerent Yeast Strainsâ, P Mazzei, R Spaccini, N Francesca, G Moschetti, A Piccolo, J Agric Food Chem, 61, 10816-10822 (2013)
âAn exploratory chemometric study of 1H NMR spectra of table winesâ, F H Larsen, F van den Berg, S B Engelsen, J. Chemometrics, 20, 198-208 (2006)
âSensory attributes of wine inïŹuenced by variety and berry shading discriminated by NMR metabolomicsâ, S Rochfort , V Ezernieks , S E P Bastian , M O Downey. Food Chem, 121, 1296â1304 (2010)
âMetabolic Influence of Botrytis cinerea Infection in Champagne Base Wineâ, Y-S Hong, C Cilindre, G Liger-Belair, P Jeandet, N Hertkorn, P Schmitt-Kopplin, J Agric Food Chem, 59, 7237-7245 (2011)
âA Thorough Study on the Use of Quantitative 1H NMR in Rioja Red Wine Fermentation Processesâ, E Lopez-Rituerto, S Cabredo, M Lopez, A Avenoza, J H Busto, J M Peregrina, J Agric Food Chem, 57, 2112â2118 (2009)
âComparison 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 Bodyâ, K Skogerson, R Runnebaum, G Wohlgemuth, J De ROPP, H Heymann, O Fiehn, J Agric Food Chem, 57(15), 6899-6907 (2009)
âMetabolomic Characterization of Malolactic Fermentation and Fermentative Behaviors of Wine Yeasts in Grape Wineâ 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)
 â1H nuclear magnetic resonance-based metabolomic characterization of wines by grape varieties and production areasâ, 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)
âMetabolomic studies on geographical grapes and their wines using 1H NMR analysis coupled with multivariate statisticsâ, 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)
âUse of modern nuclear magnetic resonance spectroscopy in wine analysis: determination of minor compoundsâ, I J Kosir, J Kidric, Anal Chim Acta, 458, 77 (2002)
âChemometric classification of Apulian and Slovenian wines using 1H NMR and ICP-OES together with HPICE dataâ, M A Brescia, I J Kosir, V Caldarola, J Kidric, A Sacco, J Agric Food Chem, 51, 21 (2003)
âCharacterization of wines by nuclear magnetic resonance: a work study on wines from the Basilicata region in Italyâ, L Viggiani, M A C Morelli, J Agric Food Chem, 56, 8273 (2008)
âClassification of wines based on combination of 1H NMR spectroscopy and principal component analysisâ, Y-Y Du, G-Y Bai, X Zhang, M-L Liu, Chin J Chem, 25, 930 (2007)
â1H NMR-based metabonomics for the classification of Greek wines according to variety, region, and vintage. Comparison with HPLC dataâ, M Anastasiadi, A Zira, P Magiatis, S A Haroutounian, A L Skaltsounis, E Mikros, J Agric Food Chem, 57, 11067 (2009)
â1H 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)
 â1H NMR-based metabolomic approach for understanding the fermentation behaviors of wine yeast strainsâ, H-S Son, G-S Hwang, KM Kim, E-Y Kim, F van den Berg, W-M Park, Anal Chem, 81, 1137 (2009)
âGeneralized 2D-correlation NMR analysis of a wine fermentationâ, G M Kirwan, S Clark, N W Barnett, J O Niere, M J Adams, Anal Chim Acta, 629, 128 (2008)
âTime course of the evolution of malic and lactic acids in the alcoholic and malolactic fermentation of grape must by quantitative 1H NMR (qHNMR) spectroscopyâ, A Avenoz, J H Busto, N Canal, J M Peregrina, J Agric Food Chem, 54, 4715 (2006)
âNMR-based metabolomics in wine scienceâ, Y-S Hong, Magn Reson Chem, 49, S13 (2011)
âHigh- resolution NMR and diffusion-ordered spectroscopy of port wineâ, M Nilsson, I F Duarte, C Almeida, I Delgadillo, B J Goodfellow, A M Gil, J Agric Food Chem, 52, 3736 (2004)
 âQuantitative NMR spectroscopy of binary liquid mixtures (aldehyde + alcohol) Part I: Acetaldehyde + (methanol or ethanol or 1-propanol)â, S Jaubert, G Maurer, J Chem Thermo 68, 332â342 (2014)
Beer
âBeer metabolomics: molecular details of the brewing process and the differential effects of late and dry hoppinng on yeast purine metabolismâ, A R Spevacek, K H Benson, C W Bamforth, C M Slupsky, J. Inst. Brew., 122 21-28 (2016)
âComposition of beer by 1H NMR spectroscopy: effects of brewing site and date of productionâ, C Almeida, I F Duarte, A Barros, J Rodrigues, M Spraul, A M Gil, J Agric Food Chem, 54, 700 (2006)
âMultivariate analysis of NMR and FTIR data as a potential tool for the quality control of beer â, I F Duarte, A Barros, C Almeida, M Spraul, A M Gil, J Agric Food Chem, 52, 1031 (2004)
âHigh-Resolution Nuclear Magnetic Resonance Spectroscopy and Multivariate Analysis for the Characterization of Beerâ, Ă I Duarte, A Barros, P S Belton, R Righelato, M Spraul, E Humpfer, A M Gil, J Agric Food Chem, 50, 2475â2481 (2002)
âQuantiïŹcation of organic acids in beer by nuclear magnetic resonance (NMR)-based methodsâ, J E A Rodrigues , G L  Erny , A S  Barros , V I  Esteves , T BrandĂŁo , A A  Ferreira , E  Cabrita , A M  Gil, Anal Chim Acta, 674, 166â175 (2010)
âNMR methods for beer characterization and quality controlâ, J E Rodrigues, A M Gil, Magn Reson Chem, 49, S37âS45 (2011)
âQuality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysisâ, D W Lachenmeier, W Frank, E Humpfer, H Schafer, S Keller, M Mortter · M Spraul, Eur Food Res Technol, 220, 215â221 (2005)
âProbing beer aging chemistry by nuclear magnetic resonance and multivariate analysisâ, J A Rodrigues, A S Barros, B Carvalho, T BrandĂŁo, A M Gil, Anal Chim Acta, 702, 178â187 (2011)
âQuantification of Organic and Amino Acids in Beer by 1H NMR Spectroscopyâ, L I Nord, P Vaag, J Ă Duus, Anal Chem, 76 (16), 4790â4798 (2004)
âApplication of Quantitative Nuclear Magnetic Resonance Spectroscopy to Biological Acidification of Barley Mashesâ, A Dicaprio, J C Edwards, J Inst Brewing, 120(3), 207-211 (2014)
Beer Dextrins
âSeparation and NMR structural characterisation of singly branched a-dextrins which differ in the location of the branch pointâ, A Jodelet, N M Rigby, I J Colquhoun, Carb Res, 312, 139-151 (1998)
â1H NMR spectroscopy for proïŹling complex carbohydrate mixtures in non-fractionated beerâ B O Petersen , M Nilsson , M BĂžjstrup , O Hindsgaul , S Meier, Food Chem, 150, 65â72 (2014)
âDevelopment of brewing science in (and since) the late 19th century: Molecular proïŹles of 110â130 year old beersâ, A Walther, D Ravasio, F Qin, J Wendlan , S Meier, Food Chem, 183, 227â234 (2015)
âStructural determination of some new oligosaccharides and analysis of the branching pattern of isomaltooligosaccharides from beerâ, E Vinogradov, K Bock, Carb Res, 309, 57-64 (1998)
âNMR characterization of chemically synthesized branched a-dextrin model compoundsâ, B O Petersen, M S Motawie, B Lindberg MĂžller, O Hindsgaul, S Meier, Carb Res, 403, 149â156 (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), 4818â4825 (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)
âIdentiïŹcation and quantiïŹcation of the main organic components of vinegars by high resolution 1H NMR spectroscopyâ, A. Caligiani , D. Acquotti , G. Palla , V. Bocchi, Anal Chim Acta, 585,110â119 (2007)
1H NMR studies on Italian balsamic and traditional balsamic vinegarsâ, R Consonni, A Gatti, J Agric Food Chem, 52, 3446 (2004)
âNMR and chemometric methods: a powerful combination for characterization of Balsamic and Traditional Balsamic Vinegar of Modenaâ, R Consonni, L R Cagliani, F Benevelli, M Spraul, E Humpfer, M Stocchero, Anal Chim Acta, 611, 31 (2008)
Spirits
âNMR metabolite proïŹling of Greek grape marc spiritsâ C Fotakis , D Christodouleas , K Kokkotou , M Zervou , P Zoumpoulakis , P Moulos , M Liouni , A Calokerinos, Food Chem, 138, 1837â1846 (2013)
“NMR metabolic ïŹngerprinting and chemometrics driven authentication of Greek grape marc spirits”, C Fotakis, M Zervou, Food Chem, 196, 760-768 (2016)
âRapid Determination of Total Thujone in Absinthe Using 1H NMR Spectroscopyâ, 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), 5359â5365 (2004)
“Hydrogen Bonding in Alcoholic Beverages (Distilled Spirits) and WaterâEthanol Mixtures”, A Nose, T Hamasaki, M Hojo, R Kato, K Uehara, T Ueda, J Agric Food Chem, 5(18), 7074â7081 (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), 7394â7401 (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 âZivaniaâ 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), 5293â5303 (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), 4112â4119 (2015)
Quantitative 1H NMR Analysis of âOff-the-Shelfâ Commercial Kombucha Beverages for Ethanol, Organic Acids and Residual Sugars Analysis
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 itâs 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 27oC. 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 âoff the shelfâ 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 27oC 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 â 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 ((IC/NC)/(IMA/NMA)) x (MC/MMA) x (1,000,000/175)
Where 10 mg is the mass of maleic acid used as the internal standard, IC = integral of the component peak, NC = number of protons represented in the component peak, IMA = integral of maleic acid internal standard, NMA = number of protons represented in the maleic acid integral (2), Mc = molecular weight of the component, MMA = molecular weight of maleic acid (116.1 amu). Other aspects of the equation are â 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 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 2: Kombucha #2 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 3: Kombucha #3 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 4: Kombucha #4 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 5: Kombucha #5 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 6: Kombucha #6 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 7: Kombucha #7 â 1H NMR spectrum â component peaks utilized in calculations indicated.
Figure 8: Kombucha #2 â Comparison of original analyzed âfresh kombuchaâ with same purchase date bottle aged at room temperature for 7 months â 1H NMR spectrum â 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
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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