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
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.
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 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 “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 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 – 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 – 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
|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|
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.
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
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
Validation of NMR: No Need to PANIC – Workshop held February 13, 2015, La Jolla, CA, U.S.A
In conjunction with the 3rd PANIC conference in La Jolla, California, a 1-day NMR validation workshop was held that attracted approximately 80 interested participants. The agenda of the meeting is provided at this link (http://www.nmrvalidation.org/index.php/events/event-review) and registered participants can now download the presentations presented at the meeting. At the meeting it was decided to proceed with the idea of founding an organization dedicated to the development of validate NMR methods for use throughout all industry sectors.
NMR spectroscopy provides a means to evaluate material with high compound and high material specificity. Information as to the chemical structure, stereochemistry, quantity, material composition, and material identity is encoded in the NMR spectrum. The high reproducibility of NMR spectroscopy from instrument to instrument and lab to lab makes NMR an excellent tool for material validation. Approaches to utilizing NMR as a material validation tool include using (1) targeted approaches, the identification and quantification of specific components, and (2) non-targeted approaches, the use of chemometric methods to evaluate the spectrum as a whole. Efforts to increase the number and the speed of validated NMR methods are underway. This promises to move NMR technology from R&D to a mainstream analytical tool for production leading to high quality product assessment.
Quantitative NMR spectroscopy (qNMR) provides the most universally applicable form of direct purity determination without the need for reference materials of analytes or the calculation of response factors, with the only requirement being the exhibition of suitable NMR spectral properties. Due to recent advances in the technical development of NMR instruments, such as acquisition electronics and probe design, detection limits of components in liquid mixtures have been improved into the lower ppm range (approx. 5–10 ppm amount of substance).
The development of validated procedures and qualified standards will give users the tools to routinely exploit qNMR and enable them to speed up analytical method development, with the added advantage of reducing the time and financial burden of multiple analytical testing.
Over the last few years a number of efforts have been made to include NMR in routine testing and analysis – especially in regulated fields such as those operating under GMP or GLP guidelines. Unfortunately it has been observed that approval authorities, standard method organizations, and auditors prefer to take analytical routes derived from classical chromatographic methods. Since NMR represents a direct comparison analysis method such decisions clearly fail to take advantage of the benefits that NMR can provide.
The PANIC validation group proposes to become a driving force in getting NMR methods validated, publicized, and supported by documentation and qualified standards. The organization will also provide a mechanism for repeatability/reproducibility assessment of NMR methods as well as the round-robin accreditation of NMR labs. We aim to proactively promote the technology and improve its acceptance by the analytical community across all industry sectors.
What we want:
- Identify a network of NMR people concerned with validation that can ultimately assist each other through the validation process.
- Harmonize the terminology and a standard approach for NMR validations.
- Position the guidelines produced by consensus of the NMR community so that accreditation agencies can use this process.
It is expected that there will be an annual 1-day meeting in conjunction with future PANIC conferences. A website has been been created as an organizational repository. The website can be found here: http://www.nmrvalidation.org/index.php and details of future events and, eventually, contact information will be provided.