108 Coronado Ct.
Suite B
Fort Collins, CO 80525

(970) 226-8649

Introduction

Rare Combinations LLC contracted Beyers Analytical Brewing Sciences (BABS) to provide an impartial and critical review of their Kombucha Alcohol Detector. BABS is an analytical laboratory based in Fort Collins, Colorado that is dedicated to developing and performing chemical and microbiological measurements for kombucha and beer 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. 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).

Methodology

BABS compared the Rare Combinations LLC Kombucha Alcohol Detector (KAD) to gas chromatography paired with flame ionization detection (GC-FID) utilizing AOAC 2016.12. Standards were purchased from Cerilliant when applicable. Quality controls were made from grain alcohol and analytically proofed using a calibrated Anton-Paar DMA4500 densitometer that provides accurate density readings to five decimal places (± 0.00001). Five kombucha samples were purchased from a local grocery store and tested by GC-FID and KAD in duplicate to allow comparison. The KAD was operated according to instructions provided by Rare Combinations LLC. The ABV read-out and the raw value read‑out were recorded for each sample measured by the KAD. All sample measurements were performed in duplicate and occurred at room temperature (22-23~C).

Results

The KAD instrument interface provides sample measurement results reported as ABV from a factory-installed calibration curve as well as the ability to make a custom calibration curve using raw data from the detector’s response. We decided to evaluate the accuracy of the instrument using the factory-installed calibration curve as well as using its capability to generate a custom calibration curve for alcohol. The table below compares the results from GC-FID, the KAD factory-installed calibration curve, and the KAD custom calibration curve.

Table 1: Comparison of alcohol values determined by GC-FID and KAD

Sample Measured ABV (%) (GC-FID) Measured ABV (%) (KAD – Factory Curve) Measured ABV (%) (KAD – Custom Curve)
Kombucha 1 0.35 ± 0.01 0.44 ± 0.05 0.32 ± 0.05
Kombucha 2 0.82 ± 0.01 0.79 ± 0.05 0.76 ± 0.05
Kombucha 3 0.60 ± 0.01 0.63 ± 0.05 0.54 ± 0.05
Kombucha 4 0.86 ± 0.01 0.78 ± 0.05 0.76 ± 0.05
Kombucha 5 0.16 ± 0.01 0.25 ± 0.05 0.14 ± 0.05
QC 0.75% 0.74 ± 0.01 0.68 ± 0.05 0.77 ± 0.05

 

Discussion

The KAD version that we used can provide comparable results to the GC-FID using either the factory-installed calibration curve or the custom calibration curve. However, we would recommend that each customer who purchases the KAD take the time to build their own calibration curve on site and determine what curve fit works best for them.

Final Recommendations

The KAD can be a powerful tool for spot-checking alcohol concentrations in day-to-day operations. Taking the time to develop an on-site, instrument-specific calibration curve for each KAD can potentially increase accuracy. Any instrument is only as good as the operator running it. Training, accurate standards, and quality control are required to achieve reliable measurements. Proximity to the threshold concentration of 0.5% is also an important consideration. Testing of final product with GC-FID using method AOAC 2016.12 is still recommended to ensure legal compliance.

Disclosure and Disclaimer

BABS received payment from Rare Combinations LLC to perform this instrument review and validation. Rare Combinations LLC wanted a completely impartial review and did not make payment contingent upon any specific or desired outcomes. BABS does not make any guarantees or promises to the efficacy of the KAD for individual users or the reproducibility or robustness of the instrument over time. The writing in this document is the result of a small‑scale study performed by BABS and is the opinion of BABS only. The data, opinions, observations, and anything of the like should not be used as legal guidance. Past performance is no guarantee of future results. The KAD cannot replace GC-FID as an absolute analytical method.

 

The Kombucha Research Lab at Oregon State University, headed by Dr. Christopher Curtin, is conducting a new experiment to develop standards of identity for Kombucha products. With an increasing assortment of flavours and brands, the range of Kombucha products has been ever broadening. Kombucha Brewers International in collaboration with the Curtin Lab is determining Kombucha styles based upon organic acid and metabolite byproducts. Finished products as well as information regarding tea type and juice additives will be collected to construct a network of Kombucha composition. Nuclear magnetic resonance technology will be used to produce individual reports as well as to fulfill the study’s objective. 

What is the goal of the study?

  • ​Analyze finished products to determine amounts and types of analytes present
  • Use known properties from analytes to correlate to flavor profiles
  • Test TA/Brix to determine Kombucha Sourness Unit (KSU) scale – intended for consumers
  • Offer DNA Sequencing as an additional opportunity for those wanting more insight into which organisms are in their products at an affordable price

What is needed for participation?

In order to participate in the study, a finished product must be mailed to a laboratory personnel COLD (if possible, or room temp if too cost prohibitive). A brand may submit one or more samples for the study. For each sample submission, a questionnaire is to be filled out, containing information about tea type, flavor, juice additions, sweetening agent, and descriptors of flavor. The questionnaire must be completed for sample processing. 

When do you need to receive samples?

Sample collection begins May 25th and runs through July 30th. 

Results will be presented at Virtual KombuchaKon (VKK) – Sept 17-18, 2020 – individual results will be shared prior to VKK

How does my brand participate?

  1. Decide how many samples you’d like to have analyzed.
  2. Purchase that number of tests.
    1. KBI members = $125 per sample
    2. Non KBI members = $250 per sample
  3. Decide whether you want one or more of your sample(s) DNA sequenced, and purchase that number of tests (you will indicate which samples to DNA sequence in your questionnaire).
  4. Fill out Questionnaire (which will be emailed to you following your payment) – MUST BE COMPLETE IN ORDER TO RECEIVE REPORT – no refunds will be issued for incomplete questionnaires.
  5. Send samples on ice (if possible).
  6. Wait for results.

NEWLY ADDED DNA SEQUENCING

We need at least 35 brands to participate in the DNA Sequencing portion in order for the Curtin Lab to be able to run the tests. To help with that aim, we’ve extended the dates for sample collection. And if 50 samples or more are tested, the cost goes down (and KBI will refund excess payment).

​DNA Sequencing Cost per sample
​35 samples = $225 KBI members; $350 non-members
​50 samples = $165 KBI members; $260 non-members – SIGNIFICANT SAVINGS
​100 samples = $125 KBI members; $180 non- members – BEST PRICE

Sample Report

 

KBI Style Study

Choose your status



After you have indicated your member status and selected the number of samples you will be submitting above, choose whether to add DNA Sequencing to your sample(s) below.  DNA sequencing is an add-on to the Style Study only.  You may not select DNA sequencing, without also participating in the Style Study, but you may choose to DNA sequence just one, or a selection of, the samples you submit.  You will indicate which sample(s) you would like DNA sequenced when you complete the questionnaire you will receive after you are confirmed.

DNA Sequencing

Choose your status



Kombucha Brewers International & Oregon State University Analyte Study
A Summary of Kombucha Microbiome Research

by Keisha Rose Harrison

The Curtin lab at Oregon State University is interested in applying “-omics” approaches and cutting-edge technology to better understand fermented products. Unlike traditionally studied beverages, i.e. wine and beer, Kombucha is part of a burgeoning field of research. Brewers of all levels recognize that there is little consensus regarding the classification of Kombucha products.

The Kombucha sold on the market is widely varied in its “tea-flavour”, juice additives, residual sugar concentrations, organic acid concentrations, as well as, methods of production. There is a need to differentiate “true” Kombucha from “Kombucha-inspired” products. One of the goals of the OSU research team is to better characterize Kombucha products through chemical profiling. The KBI/OSU Analyte Study was designed with such an intention in mind. Participants were encouraged to submit a sample of “finished product” (Kombucha that is considered ready for shelves) for an analysis of non-volatile compounds. The intention of the study was to group products of a similar chemical composition to identify different styles of Kombucha currently on the market.

Key Metabolic Steps

Before we jump into the research, let’s identify some of the key metabolic steps that occur during Kombucha fermentation. Kombucha is made when sweetened tea is acidulated (pH is reduced) and inoculated with a starter culture. What does these steps mean for the Kombucha brewer? As previous research at OSU has shown, a large portion of the bacterial population of the starter culture is acetic acid bacteria. The reduction in pH creates a favorable environment for the microorganisms that play a role in the transformation of sweet tea into Kombucha.

The yeast population in the starter culture initiate the breakdown of larger sugars. The rate of sucrose hydrolysis is dependent upon the composition and concentration of yeast in the starter culture. Yeast contribute to the fermentative production of ethanol and carbon dioxide. Acetic acid bacteria and lactic acid bacteria generally oxidize ethanol into organic acids, including acetic acid, lactic acid, and gluconic acids. Raw materials, such as fruit juices, can contribute to vitamin and mineral composition. Tea choice influences amino acid, catechin, and tea polyphenol composition. All of these components contribute to the variety found in Kombucha.

How Did the Study Work?

In general Kombucha metabolites include residual sugars, proteins, amino acids, minerals, organic acids, and polyphenols. To best profile Kombucha, these different groups of compounds need to be evaluated. Nuclear Magnetic Resonance (NMR) sprectroscopy allows for multiple peak measurements from a single spectra. Unlike conventional methods of GC or HPLC, multiple compounds can be measured from a single sample. Each proton is a positively charged particle whose spin is influenced by neighboring atoms. This allows for us to measure fructose and citric acid at the same time! Furthermore, NMR data has been shown to be reproducible and highly precise. Upon receipt of samples, they were normalized and loaded into an 800MHz spectrometer for H-NMR profiling. For ease in sampling handling, only volatile compounds were analyzed. The range of a few metabolic markers are displayed below.

Metabolite Concentration
Sucrose, g/L 0.0 – 42.7
Fructose, g/L 0.1 – 42.9
Caffeine, mg/L 0.0 – 66.4
Ethanol, [v/v%] 0.0 – 5.6%
Acetic acid, g/L 0.0 – 7.5
Lactic acid, g/L 0.0 – 3.2
Gluconic acid, g/L 0.0 – 24.0

Upon an initial glance, it seems unlikely that only finished Kombucha product was submitted. Ethanol levels vary from 0.0% to 5.6% weight by volume. These values suggest that high alcohol Kombucha products were included within the original analysis. Furthermore, non-significant values for organic acids, such as acetic and lactic acids, hint at the possibility that non-fermented tea was additionally submitted for analysis. Limited participant response to the study questionnaire made it challenging to confirm these explanations.

What Can Be Learned?

Without concrete information about how the sample was produced and which tea base, sugar amounts, fermentation times, etc., the meta-data is not available to make correlations between Kombucha composition and production practices. Regardless, let’s consider the original intention of the study: to approach a definition for commercial kombucha.

A principle components analysis (PCA) was the analytical tool used to approach this goal. The plot of this analysis is shown below. The axes, or “components”, represents characteristic of variability. The orientation of samples along these components explain 50.6% of group variation and the effect of varied influences on original characteristic. Furthermore, how the “dots” or samples orient along these components demonstrate the degree of “like-ness”. Samples with a similar overall compositions will appear clustered. Meanwhile, samples with disparate overall compositions will appear at opposite ends of the axes.

Let’s make sense of this. Circles were overlaid on clusters of samples or samples that appear alike. Using this method, we can identify the largest group of like samples. We will assume that these samples represent the range of the most typical Kombucha product. With the other samples removed, we can reassess the metabolite range as below. These values represent the range of a “typical” Kombucha product. With additional information about production practices and sample description, we can use this range as a guideline when defining Kombucha in its various stages.

Where Can We Improve in Future Studies?

It is worth noting that this summary did not go into detail about the significance of all of the various metabolites displayed in the individual reports. This is because we do not know the relationship between these metabolites and Kombucha fermentation quite yet. Given how varied Kombucha practices and materials are, we can only begin to address the question of fermentation kinetics with a standardized system. These research projects are in the works and can only continue with your support. We encourage everyone to continue to participate and to answer the metadata questions as thoroughly and accurately as possible to provide clearer insights into how metabolites correspond to production process and ingredients.

Stay tuned for the next KBI/OSU Study – coming Fall 2019

An average of all the components found in all submitted samples analyzed through NMR spectroscopy can be found in the KBI Member Forum

 

KBI/OSU Genomic Study
A Summary of Kombucha Microbiome Research

By Keisha Rose Harrison

The KBI/OSU SCOBY Genomic Study took place from November 2017 to September 2018 as a two-part study. Part 1 asked the questions “what does the Kombucha SCOBY consist of?” and “how diverse is the Kombucha SCOBY?” in regards to the collective microbiota (the bacterial and fungal populations). Participants were asked to submit representative samples from a SCOBY (solid starter culture) with information regarding current location and time of use.

Part 2 was designed as a follow-up study to ask more specific questions about Kombucha starter cultures and brewing practices. Participants were asked in the Part 2 study to submit either broth (liquid starter culture) or SCOBY samples and to answer a questionnaire about brewing practices (including tea type and brewing volume). Data collected from both studies were combined to strengthen the scope of the study and conclusions about microbiota patterns. A total of 107 SCOBY and 19 Broth samples were collected and evaluated from 7 countries and 26 US states.

SIGN UP TODAY FOR KEISHA’S WEBINAR – JULY 31st 1pm PST 

timeline of KBI OSU SCOBY genomics study

 

Before diving into the results, let’s review the study methods and approach. Both parts relied upon the same DNA sequencing technique to determine microbiota from sample submissions. DNA was extracted using a method modified from traditional column-based extraction protocols. Measures were used to ensure that samples were thoroughly mixed to best represent the sample. Amplicon sequencing was the method of DNA sequencing. During this process, conserved regions of bacterial rDNA and fungal DNA were amplified and sequenced using fluorescent markers.

What exactly does that mean? Each bacterial and fungal cell has a “fingerprint” in its DNA that is generally conserved among members of a genus and species. It is this “fingerprint” that is copied and read. A bioinformatics pipeline is subsequently used to assign species-level identification for yeast and genus-level identification for bacteria. Relative abundance is determined as the number of times a copy of the “fingerprint” is recognized in proportion to all of the fingerprints. Bacterial and fungal relative abundances are reported separately.

results of DNA sequencing of SCOBYs

 

How to interpret your report? Participants received a report that included the participant ID (number used to ensure confidentiality), a list of bacteria genera with relative abundance, and a list of yeast species with relative abundance. An example of a “bacterial profile” report is depicted below. From this report, we can observe the composition of the bacterial population from a submitted sample. Only bacteria that were present at >0.01% were included in the report.  A majority, 43.7%, of the bacterial population in the sample was identified as Lactobacillus genus. The method of interpretation can be used for the fungal or yeast reports.

Bacteria (Genus) K-XXX
Sporolactobacillus 0.0700
Lactobacillus 0.4367
Oenococcus 0.1133
Acetobacter 0.1033
Gluconacetobacter 0.2767

 

Now that the relative abundance of bacteria and yeast are known, the next question is “so what does this mean?” What does a percentage tell us about the fermentation, taste, and quality of kombucha? On an individual level, each sample can serve as a building block of knowledge. Any quality assurance and control plan begins with a detailed overview of the system. Troubleshooting and monitoring starter culture health are all contingent upon knowing the composition of a “normal” and “healthy” culture. The impact of this research is strengthened when we zoom out and look at the whole sample population.

What does the data from both Part 1 and Part 2 tell us? By looking at the microbiota of multiple samples from various geographic locations, we began to answer questions about starter culture diversity. With the information provided by study participants were able to identify the most abundant Kombucha microorganisms, determine the impact of location and culture conditions on the microbiota, and classify starter culture “types”. Samples were collected from both solid and liquid starter cultures have the same common bacteria and yeast populations. Regardless of source, the most common yeast species (based upon an average of relative abundance) are Brettanomyces bruxellensis, Brettanomyces anomala, and Issatchenkia orientalis. The most common bacterial genera are Lactobacillus, Komagtaebacter, and Acetobacter. The proportions of bacteria genera vary depending upon starter culture source so that lactic acid bacteria is more abundant on average in broth than the SCOBY pellicle. Respectively, more acetic acid bacteria is abundant on average in the SCOBY pellicle than the broth. This makes sense we consider that the bacterial species, Komagaetibacter xylnium, is often credited with the formation of the physical SCOBY structure.

Taking a step back, let’s consider what this means. More of the bacteria type that produces ethanol, lactic acid, and carbon dioxide are on average found in broth. Whereas, more of the bacteria type that oxidizes ethanol and produces acetic acid is found in SCOBY. This diversity may be in part because of how the floating physical structure creates an air-liquid interface to support an oxygen-rich environment. Furthermore, we compared physical and liquid samples from the same producer to better understand the dynamic.

Let’s breakdown the figure below. The outside ring represents the SCOBY microbiota and the inside represents the BROTH microbiota. The color blocks represent top 5 microbial groups (so the most abundant bacteria and yeast genera). Blocks of the same color depict the same microbial group. The size of the block correlates the amount of that microbial group present. From the figure, we are comparing 4 BROTH and 4 SCOBY samples each BROTH/SCOBY pair is from the same location. The figure illustrates that in some instances the microbiota is comparable between SCOBY and BROTH and in some instances they differ. What does this mean? We cannot assume that the BROTH and SCOBY are interchangeable.

biofilm vs broth comparison of microorganisms composition

How to best understand ALL of this data? SCOBY sequencing revealed that there are many, many, MANY different species of yeast and bacteria in the Kombucha starter culture population. Looking at just the average SCOBY or BROTH composition is not representative of most individual samples and underscores the importance of starter culture diversity. A better way to compare starter cultures of rich diversity is to group similar microbiota profiles. In other words, by clustering samples that have similar amounts of Bacteria A and Yeast B, we are able to determine “types” of starter cultures. We ultimately used a hierarchical clustering analysis approach to sort through individual data and identify 5 groups within the overall population. These groups represent patterns of yeast and bacteria that any individual samples may fall under. The figure below depicts how specific types of microorganism are likely to appear together in a starter culture. Starter cultures with a large amount of the yeast genera, Brettanomyces, are less likely to be present in a culture with Starmerella/Candida. The “types” of starter culture can be described as follows:

 DOMINANT BACTERIADOMINANT YEAST
IAcetobacter, high – moderate levels of secondary KomagataeibacterVariable
IIKomagaetibacterBrettanomyces
IIILactobacillusBrettanomyces
IVKomagataeibacterStarmerella (Starmerella/Candida clade)
VKomagaetibacter AND Mixed Komagaeteibacter/Acetobacter Komagaeteibacter/Lactobacillus Brettanomyces

To determine how each starter culture type influences fermentation parameters, a preliminary study was undertaken in Dr. Chris Curtin’s lab at Oregon State University. Those findings will be available later in the year in the form of an academic publication that will be shared on the KBI site.

5 types of scobys

READ PART TWO OF THE STUDY

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.

 

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/

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.