Webinar Wednesdays: DNA & Analyte Sequencing Study #2 Results
DATE: 2/20/19 1 pm on Zoom
WHO: Keisha Harrison & Dr. Chris Curtin
COST: Free for DNA/Analyte study participants, $20 KBI Non-Members

WEBINAR SUMMARY:  As part of the KBI-Oregon State University Genomic and Analyte Study [Summer 2018], Keisha Rose Harrison will be hosting a webinar review to summarize the study results and Kombucha fermentation science. What was the objective of the study? OSU is interesting in characterizing the diverse microecology of the commercial Kombucha SCOBY and the chemical composition of Kombucha tea. The flavor attributes and alcohol produced during the process are governed by the starter colony, a symbiotic culture of bacteria and yeast (SCOBY). The limited information about the composition of the Kombucha culture and the components of the end product make it cumbersome for producers to quantitatively troubleshoot. In the conventional brewing approach, there is no identification of inoculating cultures nor monitoring of culture consistency. Through this study we aim to identify common ecological and metabolic markers. 

WEBINAR TAKEAWAY: What will be gained from the webinar? Study participants will get an overview on how to best interpret their results. Group results will be presented in a meaningful way as to illustrate main ecological drivers and potential sources of divergence. Additionally, there will be a larger discussion on how the results may direct quantitative approaches to troubleshooting in production. Note, specific brewing questions that do not pertain to the study will not addressed.

REGISTER HERE

ABOUT THE WEBINAR HOSTS

Keisha Harrison, M.S., is a PhD candidate of Fermentation Science in the Food Science & Technology Department at Oregon State University (OSU). She received a Bachelor of Arts in Cell Biology and Biochemistry from Rice University and a Master of Science in Nutrition from the University of Houston. Keisha was drawn to Kombucha research because it is a beautifully complex system to study microbial interactions. She aims to understand the links between the microbial landscape of the Kombucha SCOBY and the sensory experience. She believes to get at the heart of Kombucha, we have to get better acquainted with it first!

 

 

Chris Curtin, Ph.D. is an Assistant Professor in the Food Science & Technology Department at Oregon State University (OSU). Prior to joining OSU in 2016 he lead the Biosciences research group at the Australian Wine Research Institute, where he was responsible for development of yeast strains and development of strategies to mitigate spoilage. The Curtin lab seeks to understand the role of yeasts and bacteria in production of fermented beverages, where often the same mix of species give us an array of possible outcomes. The Kombucha SCOBY embodies this complexity and has become a major focus of our research.
 

Beyers Analytical Brewing Sciences & KBI Sour Units Study – Part 1

 

Kombucha Brewers International & Beyers Analytical Brewing Sciences, LLC (BABS) are partnering to build a library of information correlating analytically measured sourness and sweetness to perceived sourness and sweetness of Kombucha.

The goal of the study is to create a standardized sour/sweet numerical value that can be used by producers and consumers alike. Similar to IBU (international bitterness units) we hope to create a Kombucha Sourness Units metric that will help consumers find products that fit their palate; provide additional information to producers to define styles of Kombucha and set a metric to judge Kombucha for international competitions and the like.

A two-part study, the first part is a focused effort to determine the possible ranges of titratable acidity (TA) values in kombucha, and then the second part will integrate this information with sensory data to-be-captured at KombuchaKon 2019. All data and information collected by BABS as part of this study will be shared with KBI and published as a report.  

To achieve the first part of this study, which involves hundreds of titratable acidity measurements in kombucha, we will need as many kombucha samples as we can get to make sure that the database is as comprehensive as possible. This is where YOU come in! Kombucha Brewers International members will be able to submit up to 5 samples for FREE testing (non-members & additional samples = $20/sample)

KBI Member can find the free coupon code here in the Member Forum!

BABS will analyze all submitted samples for TA and test results will be shared with individual brands..

Here are all of the details:

  • Samples can be submitted anytime between February 4, 2019 and March 29, 2019
  • TA measurements will be free for KBI members for up to 5 SKUs
    • 1 TA measurement per SKU
    • Additional TA measurements available for $20 each
  • TA measurements will be $20/sample for non-members
    • 1 TA measurement per SKU
  • Minimum volume of 150 mL (5 fl.oz) of each SKU will be needed for testing
  • Visit Beyers Analytical website to place an order and enroll in the study
  • Follow the shipping instructions after placing an order and ship samples to BABS

Beyers Analytical Brewing Sciences, LLC
108 Coronado Ct.
Suite B
Fort Collins, CO 80525

  • Reports of results will be sent out when testing is completed (generally 24-48 hours)
  • Contact BABS us with any questions!

(970) 226-8649
info@beyersanaytical.com

Details for Part 2 will be disclosed in March. Stay tuned!

 

 

 

**The views and results in the following White Paper are the property of Beyers Analytical and do not necessarily reflect the opinions of Kombucha Brewers International.**

Introduction

Beyers Analytical Brewing Sciences, LLC (BABS) is an analytical laboratory based in Fort Collins, Colorado that is dedicated to performing chemical and microbiological measurements for kombucha, beer, spirits, wine, and coffee producers.  The analysts at BABS are certified beer chemists with the Alcohol and Tobacco Tax and Trade Bureau (TTB) and are qualified to provide accurate measurements of components within beverage products.  BABS provides education for kombucha producers regarding analytical techniques that can be used to monitor their products. We are often asked what methods can be used to monitor ethanol in kombucha.

The low level of ethanol required for non-alcoholic kombucha necessitates quick, affordable, and reliable testing that can be used to measure levels at or below 0.5% alcohol by volume (ABV).  This study presents a comparison of four ethanol measurement technologies for the kombucha industry.  Eight off-the-shelf kombucha products were analyzed in a blind test for ethanol content using gas chromatography (GC), an OptiEnz Sensors ethanol sensing system, an Anton-Paar Alcolyzer, and distillation paired with an Anton-Paar densitometer.

Results and Discussion

The table below presents the advantages and disadvantages of each ethanol measurement technology.  These items are worth considering prior to using commercial laboratory testing or purchasing an instrument as part of a quality control program.

Average ethanol measurements for each kombucha sample and performance statistics for each technology are presented in the table below.

Gas Chromatography

Measurements of ethanol concentration made using GC were taken to be the target
concentration for calculations of accuracy in this study due to the technology’s widespread use as a “gold standard.”  However, high upfront costs, high maintenance costs, complicated procedures, and long measurement times prevent most kombucha producers from using this technology at their facilities.

OptiEnz Sensors Ethanol Sensing System

The OptiEnz Sensors ethanol sensing system is more affordable than GC and the Anton-Paar. Alcolyzer, is easier to use than GC, and has one of the shortest measurement times of these technologies. This technology provided ethanol measurements in kombucha that most closely matched GC measurements.

Anton-Paar Alcolyzer

The Anton-Paar Alcolyzer is the easiest technology to use, provides short measurement times, and is more affordable than GC.  Ethanol measurements made with this technology were precise, but consistently lower than GC measurements.

Distillation and Anton-Paar Densitometer

Distillation has a low upfront cost, very long sample preparation times, and requires higher-level technical training.  Ethanol measurements made with this technology were consistently lower than GC measurements.

Conclusions

All the tested technologies are capable of measuring ethanol at concentrations found in kombucha, but the advantages and disadvantages of each method need to be considered when implementing a testing program. Proximity to the threshold concentration of 0.5% is also an important consideration.  Any instrument is only as good as the operator running it.  Training, accurate standards, and quality control are required to achieve reliable measurements.

Experimental Procedures

Gas Chromatography

Headspace gas chromatography – flame ionization detection (HS/GC-FID) measurements and sample preparation were performed using AOAC methods for determination of ethanol in kombucha (AOAC 2016.12).  Analysis was completed on a HP 5890 Series II gas chromatograph (four measurements per sample) with a Restek Stabilwax-DA capillary column using nitrogen as the carrier gas, and resolution of methanol, ethanol, isopropanol, 1-propanol, and acetone was possible using this setup.  A calibration curve was constructed using Cerilliant ethanol standards purchased from Sigma-Aldrich.

OptiEnz Sensors Ethanol Sensing System

Ethanol measurements were performed using an OptiEnz Sensors ethanol sensing system. The instrument was calibrated over 10 minutes using prepared ethanol standards.  Ethanol measurements (six measurements per sample) were made by diluting 0.1 mL of sample into 50 mL of buffer, immersing the sensor probe into the dilute solution and allowing the system to stabilize for three minutes.

Anton-Paar Alcolyzer

Density and Alcolyzer measurements were performed using an Anton-Paar DMA 4500 M-EC with Enhanced Calibration for Ethanol paired with an Alcolyzer Beer ME module and Sample Handling Unit (Xsample 22).  The instrument was calibrated with degassed, deionized water and achieved a density measurement of 0.99820 ± 0.00001 g/mL at 20ºC. Sample analysis (six measurements per sample) was performed by pumping 40 mL of sample through the system, bringing the sample temperature to 20.00 ± 0.01°C, and then collecting density and Alcolyzer measurements.

Distillation and Anton-Paar Densitometer

Kombucha samples (100 mL per sample) were distilled according to the TTB-recommended distillation-specific gravity method (AOAC 935.21).  Density measurements (one measurement per sample) were made using the Anton-Paar densitometer.

FERMENTATION MICROSCOPY: TIPS AND TRICKS FOR THE KOMBUCHA BREWER
DATE: 7/25/18 1 pm on Zoom
WHO: Keisha Harrison & Dr. Chris Curtin
COST: Free for DNA/Analyte study participants, $20 Kbi Members, $40 KBI Non-Members

WEBINAR SUMMARY

Explore the microscopic world at work when sweetened tea becomes kombucha. During this webinar session, members of KBI will receive information on the biochemistry and microbiology of standard kombucha fermentation. Webinar topics include yeast and bacteria strains, fermentation kinetics, the kombucha microbiome, chemical composition, and microbiology techniques. Learn more about how to apply laboratory techniques such as plating of cultures, isolation of microorganisms, and PCR. Enrollment is FREE for KBI members currently participating in the Oregon State University Kombucha Genetics and Analyte study.

WEBINAR TAKEAWAYS

– The general biochemistry of mixed culture fermentations

– The role of bacteria and yeast in Kombucha production

– Laboratory techniques that can be applied to Kombucha fermentation

ABOUT THE WEBINAR INSTRUCTORS

Keisha Harrison, M.S., is a PhD candidate of Fermentation Science in the Food Science & Technology Department at Oregon State University (OSU). She received a Bachelor of Arts in Cell Biology and Biochemistry from Rice University and a Master of Science in Nutrition from the University of Houston. Keisha was drawn to Kombucha research because it is a beautifully complex system to study microbial interactions. She aims to understand the links between the microbial landscape of the Kombucha SCOBY and the sensory experience. She believes to get at the heart of Kombucha, we have to get better acquainted with it first!

 

 

 

Chris Curtin, Ph.D. is an Assistant Professor in the Food Science & Technology Department at Oregon State University (OSU). Prior to joining OSU in 2016 he lead the Biosciences research group at the Australian Wine Research Institute, where he was responsible for development of yeast strains and development of strategies to mitigate spoilage. The Curtin lab seeks to understand the role of yeasts and bacteria in production of fermented beverages, where often the same mix of species give us an array of possible outcomes. The Kombucha SCOBY embodies this complexity and has become a major focus of our research.

 

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

Kombucha diversification is on the rise. As more brewers rush to the scene, new and old producers alike have to answer the question: what defines our Kombucha? The uniqueness of a kombucha product can be divided into two descriptors: the microbial community and the chemical composition. The flavor, the aroma, and the mouthfeel are all sensory attributes that can be related back to the chemical composition. Additionally, the fermentation of sweetened tea can produce compounds (i.e. organic acids and polyphenols) associated with reports of health benefits. Kombucha reportedly may consist of organic acids, ethanol, vitamins, polyphenols, catechins, amino acids, and antibiotics. The measure of these bioactive compounds is paramount in determining the consumer’s experience.

The symbiotic microbes within the Kombucha culture work in tandem to catabolize the sweetness from the starter material. Yeast possess an enzyme invertase which breaks down sucrose (cane sugar) into simpler hexose sugars. Bacteria and a few types of yeast can further oxidize glucose and fructose into organic acids, including acetic acid. The extent to which these molecules are broken down by the microbes determines the resulting chemical composition. Factors that influence these reactions include: microbial population, sugar concentration, fermentation temperature, and fermentation duration. A table of varying fermentation conditions and common compound concentrations can be found below.

Ethanol and pH have become standard measurements for tracking the proper course of Kombucha fermentations. Although pH is effective in clarifying the endpoint, it fails to discern the various organic acid components. Some of the more commonly reported organic acids include, acetic acid, gluconic acid, glucuronic, lactic acid, citric acid, and formic acid. Acetic acid, typically the dominant acid, is produced when acetic acid bacteria oxidize ethanol produced by sacchrolytic yeast. It is detectable at 175mg/L and carries a distinguishable pungent sourness noticeable in flavor and aroma. Fruit notes can characteristic such as apple, black current, and pineapple. Meanwhile, lactic acid is produced when fructophilic lactic acid bacteria (LAB) anaerobically ferment carbohydrates. It has very little to no aroma and contributes a “tang” sour character, distinguishable at 400 mg/L. The less abundant citric acid gives of a “tart” sour taste at a threshold of 60 mg/L. The balance of these flavor-active organic acids determines how your Kombucha tastes and smells!

Increasing focus has been directed towards glucoronic acid (GlcUA) because of its association with health benefits. GlcUA acid is formed during glucose oxidation. According to one study by Nguyen (2015), the bacteria Gluconoacetobacter intermedius is capable of producing detectable levels of GlcUA acid which varies in response to yeast abundance. What’s so special about this organic acid? Numerous studies have found GlcUA to confer detoxifying benefits by binding to xenobiotics (toxins) in the liver and making them easier to eliminate. The amount of GlcUA produced during Kombucha fermentation depends substantially on fermentation temperature and the microbial composition. In addition to GlcUA, other health-related compounds, i.e. vitamin C, folic acid, polyphenols, and catechins, have been found in Kombucha, making it a likely treasure trove of beneficial compounds!

How can you profile your brew’s unique chemical composition? KBI is partnering with Oregon State University to conduct a Kombucha Analyte Study. By participating, you will submit a sample of finished product to be run on Nuclear Magnetic Resonance equipment to detect residual sugars, organic acids, caffeine, and additional metabolites with high fidelity. To learn more about participation, visit: https://kombuchabrewers.org/kbi-osu-scoby-genomics-analyte-study/.

References:

Chen, C., & Liu, B. Y. (2000). Changes in major components of tea fungus metabolites during prolonged fermentation. Journal of Applied Microbiology89(5), 834-839.

De Filippis, F., Troise, A. D., Vitaglione, P., & Ercolini, D. (2018). Different temperatures select distinctive acetic acid bacteria species and promotes organic acids production during Kombucha tea fermentation. Food Microbiology.

Jayabalan, R., Marimuthu, S., & Swaminathan, K. (2007). Changes in content of organic acids and tea polyphenols during kombucha tea fermentation. Food Chemistry102(1), 392-398.

Jayabalan, R., Malbaša, R. V., Lončar, E. S., Vitas, J. S., & Sathishkumar, M. (2014). A review on kombucha tea—microbiology, composition, fermentation, beneficial effects, toxicity, and tea fungus. Comprehensive Reviews in Food Science and Food Safety13(4), 538-550.

Lončar, E. S., Petrovič, S. E., Malbača, R. V., & Verac, R. M. (2000). Biosynthesis of glucuronic acid by means of tea fungus. Food/Nahrung44(2), 138-139.

Nguyen, N. K., Nguyen, P. B., Nguyen, H. T., & Le, P. H. (2015). Screening the optimal ratio of symbiosis between isolated yeast and acetic acid bacteria strain from traditional kombucha for high-level production of glucuronic acid. LWT-Food Science and Technology64(2), 1149-1155.

Sievers, M., Lanini, C., Weber, A., Schuler-Schmid, U., & Teuber, M. (1995). Microbiology and fermentation balance in a kombucha beverage obtained from a tea fungus fermentation. Systematic and Applied Microbiology18(4), 590-594.

Sparrow, J. (2015) Wild Brews, Brewer’s Publications

Velićanski, A., Cvetković, D,& Markov, S. (2013). Characteristics of Kombucha fermentation on medicinal herbs from Lamiaceae family. Romanian Biotechnological Letters18(1), 8034-8042.

Talawat, S., Ahantharik, P., Laohawiwattanakul, S., Premsuk, A., & Ratanapo, S. (2006). Efficacy of fermented teas in antibacterial

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/

You may also purchase a DNA &/or Analyte Study Kit below:


Choose your status




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! Drop an email if you are interested in participating in this study.

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.

PARTICIPATE TODAY!


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