By Keisha Rose Harrison, MS (PhD Candidate at Oregon State University)
FERMENTATION

Kombucha, a funky fermented tea, is a time immemorial beverage that has made appearances across the globe in various forms for eons. This beverage of unknown origin is shrouded in many mysteries. The potential health benefits, fermentation kinetics, and alcohol production are a few aspects that continue to baffle kombucha lovers and producers alike. Meanwhile, one area where fermentation researchers are making ground is in defining the microbiology of the Kombucha SCOBY.

In comparison to other fermented beverages, i.e. wine and beer, the amount of research done on the Kombucha SCOBY is sparse. It was known in even the earliest studies, from back in the 1960s that the starter colony was made up of both yeast and bacteria. However, there was little known about which organisms drove the fermentation, contributed to flavor, and fought off spoilage. Until recently, scientists relied upon culture-based methods (isolating microbes on media plates) to identify yeast and bacteria organisms. This technique, although cost-effective and accessible, is prone to error. Despite its limitations, scientists (Teoh et al, 2004; Liu et al, 1996), were able to come to consensus about the role of acetic acid bacteria (AAB) and saccharolytic (“sugar eating”) yeast.

By the early 2000s, we understood that AAB converted ethanol to acetic acid and built the SCOBY. Acetobacter and Gluconoacterobacter, genera of AAB, were claimed as staples and have since been renamed to fit under the genus Komagataeibacter. There was less agreement about which yeast cells eat cane sugar to produce simple sugar hexoses for the bacteria. There are accounts of Zygosaccharomyces, Schizosaccharomyces, Brettanomyces, and Candida. However, Teoh (2004) warns us that identifying organisms based upon isolation methods is generally unsuccessful.

The DNA sequencing boom changed the name of the game! With the advent of affordable and reliable genetic sequencing, high-resolution taxonomic identification became a thing of reality. First scientists used sequencing technology to characterize isolated organism as a more robust culture dependent approach. However, with next generation sequencing (NGS), we could finally for the first time identify both culturable and nonculturable organisms. With NGS technology, scientists confirmed Komagatebacter as the dominant Kombucha bacteria (Marsh, 2014; Chakravorty, 2016) revealed that lactic acid and thermophilic bacteria that are likely dependent upon Kombucha preparation and region. (See table below for comprehensive list of evaluated microorganisms) These studies finally give us an in-depth analysis of kombucha microflora.

What does this mean to brewers? The recent application of advanced sequencing proved that we can apply high-fidelity identification tools to kombucha. With the precedent already set, we can now focus on designing experiments that address the larger questions. For instance, “what is common to commercial SCOBY?” and “which organisms are found in kombucha with high organic acid production?” By sequencing a mass number of SCOBY from around the globe, we will have the potential to link microbes with the chemical composition of finished kombucha products. Additionally, we will get closer to curating reproducibility and troubleshooting problematic batches.

How you can get involved? KBI members have the opportunity to participate in highly discounted genetic sequencing ($125 per sample) of the Kombucha SCOBY and broth. Fermentation scientists at Oregon State University and creating a database of microbial populations from commercial kombucha. Not only will you have the opportunity to see what your SCOBY is comprised of, but you will also be able to see your powerhouse compares to the average SCOBY. This is just the first step in understanding how the microbial population relates to kombucha features.

For more information in registering, visit: https://kombuchabrewers.org/kbi-osu-scoby-genomics-analyte-study/

Referenced Papers

  • Coton, Monika, et al. “Unraveling Microbial Ecology of Industrial-Scale Kombucha Fermentations by Metabarcoding and Culture-Based Methods.” FEMS Microbiology Ecology, vol. 93, no. 5, 2017, pp. FEMS Microbiology Ecology, 2017, Vol. 93(5).
  • Chakravorty, Somnath, et al. “Kombucha tea fermentation: Microbial and biochemical dynamics.” International journal of food microbiology 220 (2016): 63-72.
  • El-Salam, S. S. A. (2012). 16S rRNA gene sequence detection of acetic acid bacteria isolated from tea kombucha. New York Science Journal, 5(3), 55-61.
  • Jankovic, I., Stojanovic, M., 1994. Microbial and chemical composition, growth, therapeutical and antimicrobial characteristics of tea fungus. Mikrobiologija 33, 25 – 34.
  • Liu, C-H., et al. “The isolation and identification of microbes from a fermented tea beverage, Haipao, and their interactions during Haipao fermentation.” Food Microbiology 13.6 (1996): 407-415.
  • Marsh, Alan J., et al. “Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples.” Food microbiology 38 (2014): 171-178.
  • Mayser, P., Gromme, S., Leitzmann, C., Gru¨nder, K., 1995. The yeast spectrum of the ‘tea fungus Kombucha’. Mycoses 38, 289 – 295
  • Shade, Ashley, D. H. Buckley, and S. H. Zinder. “The kombucha biofilm: a model system for microbial ecology.” Final report on research conducted during the Microbial Diversity course. Marine Biological Laboratories, Woods Hole, MA (2011).
  • Teoh, Ai Leng, Gillian Heard, and Julian Cox. “Yeast ecology of Kombucha fermentation.” International journal of food microbiology 95.2 (2004): 119-126.

 

KBI is excited to launch the next phase of the KBI OSU SCOBY Genomics Study – part two. Building on the data gathered in the first study and reported about here, we are calling for new samples of SCOBYs & starter liquid from any Kombucha producer around the world. 

Keisha-Rose Harrison, PhD student at Oregon State University is continuing the original study to learn more about the organisms present in Kombucha cultures through DNA Sequencing. In an effort to gain a deeper understanding of what the role of the various organisms may play in the fermentation process, we are also adding an analyte analysis to this new study. 

For the analyte study, we will be using nuclear magnetic resonance (NMR) technology to evaluate the chemical profile of finished kombucha products. This rare piece of equipment has the efficiency to detect any residual sugars, amino acids, organic acids, and vitamins within your brew with high fidelity. This a great opportunity to get at the heart of what defines your unique kombucha brew!

The overarching goal of this project is to take the information we have about the organisms present in the culture via the DNA Sequencing study and combine that with the knowledge of which analtyes are being produced to start to piece together how the different flavors and qualities of our brews match up with the range of chemical compounds within kombucha. Your participation will contribute to the general definition of kombucha. 

HOW TO PARTICIPATE IN THE STUDY – We are no longer accepting submissions for this study. 

We are aiming for 200+ total samples to be analyzed in order to have a sufficient pool to draw conclusions. We invite you to take advantage of this opportunity to learn what is in your culture while also contributing to the deeper body of knowledge about Kombucha as a whole. Furthermore, your submission will be kept confidential and you will receive an individualized chemical analysis report. Similar to the Kombucha Genetics Study, following the release of individual reports, a KBI blog report will be written to contextualize your results within the frame of the population.

If you are a current KBI member, the cost is only $125 per sample for each test – you may choose to participate in only the DNA Sequencing, only the Analyte Analysis or both for a discounted price of $200 per sample. Part of the cost goes directly to the university to cover the sequencing, part of it is to cover shipping of kits and the purchase of the kit supplies and the rest covers administrative costs.

Non-KBI members are also invited to participate!. The cost is $250 per sample for non-members with a discounted price of $450 for both. Or join KBI today to receive the member pricing.

DNA sequencing typically costs several hundred up to thousands of dollars per sample, so this is a significant savings for valuable information. The data will only ever be presented in an aggregate format to protect confidentiality for all participants. The analyte study will include over 20 different analytes providing a huge savings over testing them individually with private labs.

By Dr. Chris Curtin & Keisha-Rose Harrison of Oregon State University & Hannah Crum of Kombucha Brewers International & Kombucha Kamp

Thank you to everyone who participated in the first round of the KBI & Oregon State University Kombucha SCOBY DNA Sequencing study. With your help we sequenced nearly 100 samples provided by over 70 different companies from 26 states and 9 different countries around the world. The largest SCOBY DNA Sequencing Study to date! Previous studies that sought to improve our knowledge of SCOBY microbial populations have been more limited in scope. We now have more data points which can provide a clearer understanding of which microbes are common across commercial SCOBYs, and an opportunity to learn which strains may be responsible for different fermentation and flavour outcomes. 

DNA Sequencing Analysis Process

For each sample received, we blended a standardized amount of SCOBY under controlled conditions, and then extracted the DNA from all microbes present in the sample. Regions of DNA that can be interpreted as ‘barcodes’ were amplified from each sample and sequenced using Illumina Miseq technology. The sequences of these ‘barcodes’ were compared to large databases of fungi and bacteria, and assigned to Operational Taxonomic Units (OTUs). In this study OTUs were defined at a standard cutoff of 97% similarity. In other words, if two ‘barcodes’ are only 96% similar in DNA sequence they would not be grouped into the same OTU.

Is an OTU representative of a species? Sometimes yes, but often no – within many genera (particularly for bacteria) individual species cannot be reliably differentiated using current DNA ‘barcodes’. This approach is highly robust when used to provide resolution at the genus level, meaning that all OTUs for one type of organisms are grouped together. For example, if a SCOBY contained both Lactobacillus casei and Lactobacillus plantarum their DNA ‘barcodes’ would be grouped together as Lactobacillus.

 

After the samples are matched at the genus level, each ‘barcode’ detected is counted to provide the frequency with which it appears in the sample to determine the proportion at which each genus of the whole bacterial or fungal population exists. These are the numbers of the sample represented as as follows: Dekkera 0.67 means that 67% of the fungal population in your sample belongs to the Dekkera genus.

Interpreting Your Results

Each sequencing report provided participants the bacteria and fungi profile of the sampled SCOBY. Below is an example of a report of bacteria detected. The genus level is read as “g_GENUS”. The number provided indicates how much of bacteria was present  in the sample. For instance, 94.8% of this example sample is Gluconoacetobacter. Some other OTU groupings could not be reliably defined at genus level, in which case they will be named at family level but assigned as ‘Other’ or ‘g__’ at genus level. In this example, 2.7% of the bacterial population is made up of bacteria within family Acetobacteraceae that could not be assigned to a genus.

Using the values provided we can create a visual of the bacteria composition.

Please note that the taxonomic name of organisms can change. For example, Dekkera is now known only as Brettanomyces (previously the names were interchangeable), while Gluconoacteobacter xylinum and Acetobacter xylinum have been reassigned to Komagataeibacter xylinum.  We’re sorry, but this is just as confusing for seasoned microbiologists!

Q: “My report says “unknown” for some of the organisms. What does that mean?”
A: Sometimes ‘barcodes’ are not able to be definitely assigned at the genus level due to a lack of resolution or because the sample may contain a species that has not yet been identified by the microbiology community. These OTUs are then grouped at the family level and genus may be given as ‘undefined’, ‘unknown’ or ‘other’.

How do your results compare to the “average” SCOBY

Each company received an individual report listing the respective percentages of each type of bacteria and yeast found in their sample.

In the aggregate, it was found that the organisms occurred in these relative percentages:

TABLE OF ORGANISMS FROM GREATEST TO LEAST FOR YEAST & BACTERIA

 

Making Sense of the Study

DOMINANT YEAST AND BACTERIA

As presented at KombuchaKon18, results from the study confirm that there is variability in SCOBY samples collected from different brewers. However we were able to identify core microbes that are present in most SCOBYs. On average, the most common fungi were Brettanomyces/Dekkera and Starmerella, while the most common bacteria were Gluconacetobacter, Gluconobacter, and Lactobacillus.

The near constant fluctuation of organism name changes presents its own kind of challenge. In previous SCOBY & Kombucha DNA Sequencing studies, for example, there are no instances of Starmerella being detected, however, when doing cursory research, it turns out this is a newer nomenclature for many species that used to be known as different types of Candida spp. which have been identified in other studies. However, without going to the species level, it is difficult to ascertain which stains correspond to those found in previous studies versus which may be novel to Kombucha.

 

HOW DOES THIS INFLUENCE FLAVOR AND FERMENTATION?

Identifying the prevalent organisms within the SCOBY is the first step towards answering this question. We do know that different types of yeast convert sugar to alcohol at different rates. It is also known that different types of bacteria produce varying kinds of organic acids in similar fermentation models, i.e. beer, wine (see table below). There is likely some overlap in how these microbes influence kombucha.

For the next study, we aim to determine how different microbial profiles influence your finished raw product. Continued participation in these studies will go a long way to addressing these questions.

In the meantime, some of the organisms found in Kombucha are also found in wine & beer. Here are some links to charts that outline the flavor profile and characteristics of some of those organisms.

UC Davis Wine Server Database

Our yeast and bacteria are considered spoilage for beer & wine!

ARE SCOBYs MORE VARIABLE IN THEIR BACTERIA OR YEAST POPULATIONS?

On the whole, there seems to be more variability in the bacteria profiles of SCOBY samples. Bacteria are responsible for converting most of the ethanol into organic acids and for building the SCOBY. We will continue to study how different types of bacteria influence the secondary fermentation.

VARIATION BETWEEN SCOBY AND STARTER FLUID MICROBIAL POPULATIONS

For this study we only examined the SCOBY and not the starter fluid. For the second sequencing study we will look at both! As mentioned in the presentation at Kombuchakon, acetic acid bacteria build the the SCOBY. They do not have to live in the SCOBY to produce acids (e.g. vinegar is made without a SCOBY), just as yeast do not have to live in the SCOBY to degrade sugars. The SCOBY does, however, enable repeated fermentations to be started with multiple species present, something that would be difficult to achieve otherwise. The SCOBY effectively buffers microbes against what would otherwise be a fluctuating environment as the sweet tea goes through alcoholic fermentation and acetification. .

METADATA ANALYSIS

At the time of sample collection, a couple of questions were asked in order to analyze the data according to a couple of variables – namely age of the culture sampled and location. We then sent out a follow up survey for additional metadata including age of the culture sampled, type & quantity of tea; type & quantity of sugar; and batch size to see if the data would show any trends or patterns based on these variables. Not all participants answered this part of the survey and we hope to have a more thorough Metadata Analysis available in future studies. This will greatly enhance the applicability of study results to the KBI community.

REGIONAL VARIATION

Preliminary analyses suggest there may be some region-to-region variances in the bacteria and fungal composition of the SCOBY, though it should be noted that sample numbers and the amount of metadata provided were uneven. Future studies will request additional metadata in order to determine the influence of types & quantities of tea, sugar and other variables.

NEXT STEPS

KBI & OSU will be partnering on a new DNA sequencing study and will also be conducting an analyte study. We hope to receive at least as many samples as last time if not more so that our knowledge base will continue to deepen. Stay tuned for more details coming soon!

Collaborative Shelf Life Study

Contact Mike Goodrich with Cornerstone Analytical for all of the details at (888)313-1937 or
mike.goodrich@cornerstoneanalytical.com. 

KBI is excited to announce a partnership with Oregon State University to study the organisms present in Kombucha cultures from around the United States. The purpose of the study is to try sequence numerous samples from a wide variety of places to answer the question of “What is a SCOBY?”

After sequencing all of the samples, the data will be analyzed to see which organisms are common to all cultures, which are not, which are unique to specific locations. This information is the first step to gaining a deeper understanding of how the microorganisms contribute to flavor, alcohol content and more.

HOW TO PARTICIPATE IN THE STUDY

We are aiming for 200+ total samples to be analyzed in order to have a sufficient pool to draw conclusions. We invite you to take advantage of this opportunity to learn what is in your culture while also contributing to the deeper body of knowledge about Kombucha as a whole. Each participant will be provided a detailed report with all of the organisms present in their culture.

If you are a current KBI member, the cost is only $100 per sample. Part of the cost goes directly to the university to cover the sequencing, part of it is to cover shipping of kits and the purchase of the kit supplies and the rest covers administrative costs.

Non-KBI members are also invited to participate!. The cost is $250 per sample for non-members. Or join KBI today to receive the member pricing.

DNA sequencing typically costs several hundred up to thousands of dollars per sample, so this is a significant savings for valuable information. The data will only ever be presented in an aggregate format to protect confidentiality for all participants.

We have a tight timeline (all samples need to be received by October 15th extended to October 30th!), so please sign up today.

We look forward to your participation and hope to see you at KombuchaKon18 – Defining Our Culture where the results will be presented.

On Sunday, September 27th, KBI president Hannah Crum and and Heath-Ade CEO Daina Trout, head of LGO committee (Special Projects Team) presented to the Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM) at the annual conference of the Association of Official Analytical Chemists (AOAC) to establish a new Working Group with the aim of developing a new testing standard for the low levels of ethanol in kombucha. Experts across various industries specializing in manufacturing, food chemistry, and laboratories were present as well as representatives from government organizations like the TTB.    

erik konings nestle daina trout healthade hannah crum kombucha kamp 9.27.15
Erik Konings of Nestle presiding over the SPSFAM with presenters Daina Trout of Health-Ade & Hannah Crum of KBI

Hannah and Daina shared with those in attendance the explosive growth in the kombucha industry while enumerating the difficulties with the testing methods in use by regulators in the US today. The message was clear: currently, an accurate, standard method of testing that takes into consideration the complexities of kombucha such as the strands of living culture, yeast bodies, organic acids, low pH and other factors simply does not exist. The Q & A was lively with questions, support and enthusiasm from the audience for this fascinating scientific problem and very much echoed that current methodologies need to be revised.  

The stakeholder panel eagerly took up the process of formulating a Fitness for Purpose (FOP) statement. The FOP is ultimately the parameters that the Working Group will establish before sending out a request for methods to the international membership. 

Fitness for Purpose statement: Methods need to accurately and precisely measure the ethanol concentrations to comply with alcohol and non-alcohol declarations in Kombucha in-process and finished products.  

The Working Group will now proceed under co-chairs Hannah Crum and Sam Labonia of Cornerstone Labs. The goal is to hone in on the issues that will be necessary to take into consideration for determining accurate testing methods. KBI anticipates presenting the Standard Method Performance Requirements (SMPR®) to the SPSFAM Panel at the AOAC Midyear Meeting in March 2016 in Maryland. Since AOAC standards are in use by governments around the world, including the US, the TTB (Tax & Trade Bureau) has also been invited to participate in the process and are members of the Stakeholder Panel that will vote to determine the ultimate methodology that is selected.

Armen Mirzoian Cathy Halverson of TTB Daina Trout Hannah Crum
Armen Mirzoian, Chief Science Officer, and Cathy Halverson, Head of Alcohol Testing Lab, of the TTB attend the AOAC Stakeholder Panel and continue to participate in the Working Group spearheaded by KBI, Health-Ade and other KBI members

The reaction from not only the stakeholders, but the general AOAC attendees was overwhelmingly positive and enthusiastic about the direction of this research. Not only was the presentation very well received, the questions asked after were informed and thought-provoking.  Overall, KBI is thrilled to get the opportunity to work with the AOAC to find a method of testing ethanol in kombucha that produces accurate, consistent, repeatable results.  “The AOAC is pleased to be working with the kombucha industry, regulatory bodies, and other stakeholders to develop such a standard,” stated E. James Bradford, Ph.D., Executive Director of AOAC International.

The working group meets regularly (biweekly) to refine definitions and identify criteria for the SMPR® 

Get the latest updates on the Working Group in the Alcohol – Testing & Control Group on the KBI Member Forum.

Kombucha is a complex, living product with an enormous range of styles and flavors that delight the senses and enliven the body. Mixed with juice, spices or brewed with other beverages; it is most often consumed raw but can also be filtered, force carbonated or in some cases pasteurized; and it can be fermented with a variety of substrates yielding new and exotic combinations. This amazing diversity of expression is part of the mystique of kombucha but also presents difficulty when testing for ethanol due to its inherent biodiversity and complexity, as well as the wide spectrum of serving possibilities.

Kombucha companies currently rely on individual labs to test for ethanol. The KBI Special Projects Team (sub-committee of LGO) contacted over a dozen labs with experience working with kombucha and discovered that each lab has its own method for testing for ethanol. Three main methods are being utilized for testing ethanol in kombucha – alcolyzer (refraction), densitometry and gas chromatography. Moreover, each lab will apply their own tweaks and adjustments to the process in order to account for kombucha’s unique characteristics. The inconsistency in methodology is concerning because kombucha has so many quirks that need to be accounted for and numerous decisions in the testing process can have a significant effect on the final result. With so many changing the methods ever so slightly, it is little wonder that consistent results are simply not achievable.

Oftentimes, the sample is simply run through a beer alcolyzer system designed to effortlessly detect the much higher, and therefore easier to measure, levels of ethanol. Kombucha, being far more complex than beer, contains organic acids, fermented sugars, and live culture strands that are simply not present in most commercial beers. Moreover, the unique kombucha culture is a symbiosis and the bacteria consume the ethanol created by the yeast into organic acids that may have a similar density to ethanol thereby creating false positives.

Just a handful of labs are currently using more sophisticated techniques, like headspace gas chromatography to suss out the nuances. This method shows a lot of promise as it opts to use far lower detection limits than is recommended in the AOAC method for testing beer, consistent with the lower levels of ethanol observed in kombucha. Another difference in sample handling is to not centrifuge it to avoid adulterating the analyte.

Still others utilize AOAC 983.13 method for testing wine that relies on centrifuging the kombucha sample to remove the high levels of sediment present in kombucha and then later factors in the removed sediment into the final mass percentage.  This method shows similar results to the modified 984.14 method, and shows promise when adjusted for kombucha’s low detection levels. What is abundantly clear, however,  is that there is no consensus available for the appropriate method to use when testing kombucha.

In one lab, the same bottle of kombucha was tested using the beer alcolyzer and a modified AOAC 984.14 method for testing beer. The beer alcolyzer system claimed that the kombucha contained levels of ethanol higher than 0.5%, the level above which a beverage must be marketed as alcoholic. When analyzed using the modified AOAC method, a gas chromatography method, the kombucha was tested to be well under 0.5% ethanol. Because the limit for alcohol content in non-alcoholic kombucha is so low at 0.5%, it is imperative that the testing is done accurately and precisely. When an unmarked bottle of Bud Light was tested, both the beer alcolyzer system and the gas chromatography method were highly precise, possibly because its higher alcohol content is easier to detect with higher detection limits.

The lack of consistency is evident even at the same lab. The same samples were sent with different labels to a lab and the test results for the exact same samples had a variance of +/- 1% ABV – that’s more than double the legal limit! So, how do we all get on the same page and find an accurate, repeatable, consistent test for ethanol in kombucha? Collaboration, of course! We are symbiotic after all.

The kombucha industry is reaching out to collaborate with the most educated body of scientists to establish a new testing standard for ethanol in kombucha. By partnering with AOAC, KBI participates in an ethical, scientific process for creating testing methods. Moreover, the TTB, FDA and other government, non-profit, academic and others will work together and decide through consensus on the appropriate method for accurate, consistent and repeatable results. The lack of definitive research in this field has frustrated kombucha brewers who are taking measures to brew compliant products and regulatory agencies. Working with industry experts, KBI hopes to reach a universal truth for testing kombucha to better serve and inform consumers.

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