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

Kimberly Lanski, Buddha’s Brew, Austin, TX, USA

Q: When did you first discover kombucha?

In 2000, a friend shared some he was making. He had gallon jars brewing all over his kitchen. He would fill wine bottles with the stuff and share it with us at a local macrobiotic restaurant! I was a long time meditator and it just resonated with me – energizing and yet relaxing and super healthy – just like meditation!

Q: Why did you start your own kombucha company?

Someone was selling kombucha at a local farmer’s market in 2005 and I felt really strongly that this would be a perfect business for me! He quit selling and I took over his spot. Everything took off quickly from there.

Q: What is your favorite flavor of kombucha? 

Fairment’s Raw Chai is great! Fairment is from Germany, they stayed with us in our Airbnb at KBI!

Q: What is your former/current other life or career?

I was living very austerely meditating for 20+ years with a spiritual group. Just making enough to get by. Before that I delivered mail for the Post Office for 12 years! Majored in Biology at a University.

Q: What is a saying that you live by?

“If we could see the miracle of a single flower clearly, our whole life would change” -Buddha

 

Jessi Hoeft, Ensign Beverage, Hastings, NE, USA

Q: When did you first discover kombucha?

I brewed my first batch of kombucha the summer I graduated high school in 1995. A friend’s aunt who was living in Colorado gave us a culture and said the drink she was making was helping her with all kinds of things including better complexion, weight loss, and less heart burn. We did not call it kombucha back then. We called it the weird mushroom drink under the sink. We had no idea how to care for it correctly. We would put a SCOBY in a sugary tea, wait for a bit of time then dip our ladle into it to pull off a few ounces to drink each day…holding our nose because it was soooo vinegary and acidic. After lots of education and many, many, many experimental batches we have learned to make a smooth, low sugar, low acid, non-alcoholic kombucha…and I don’t have to hold my nose any more when drinking it.

Q: Why did you start your own kombucha company?

While selling soap at the Nashville farmers market I had a friend who was making kombucha and selling it at the market and in small shops around town. We would often trade handmade soap for home-crafted kombucha. At one point we both had our products in Whole Foods and we reconnected. We chatted about what was happening in the kombucha market. Finally, the demand was growing for this healthy beverage option. At that time my husband and I were moving to Hastings, Nebraska with plans to start up a brewery. We saw a chance to produce both beer and kombucha. We have a tap room in Downtown Hastings and sell across the state of Nebraska. We are happy to bring this healthy beverage option to the middle of the map!!

Q: What is your favorite flavor of kombucha? 

Over the years I have tasted many different brands. On a quick trip to Colorado in 2015 I found the Rowdy Mermaid tap room. That was a game changer. It was a smooth kombucha without a lot of ginger or juice added to cover up the acids. That set the bar for us. I have recently told that story to Jamba, Founder of Rowdy Mermaid. He smiled and gave me a hug. Let me tell ya, that felt good 🙂

I should mention on that same trip, I found the Happy Leaf tap room. So delicious!! And in CANS! I knew right then and there that canning would be the sustainable way to package Ensign Beverage products.

Q: What is your former/current other life or career?

This brewery thing is full time now for myself and my husband, Nathan. We have 10 employees on staff as well. Growing the brands here in Nebraska and the region keeps us all on our toes. But, I have also had a wild career path from teaching to touring with national bands. Ask me about it sometime, I will tell you a story or two!

Q: What is a saying that you live by?

It might not be your cup of tea, but you won’t know until you try it!!

 

 

 

Mike Durighello, Whalebird Kombucha, San Luis Obispo, CA, USA

Q: When did you first discover kombucha?

My friend Carson introduced me to kombucha in 2006 while I was at UC Santa Cruz. He was an amazing water man and had just taken me out to surf. Back at the car he pulled out a few bottles of GT’s Kombucha. I was like ‘kombooka what?!’ He said ‘just drink it’ and so I did in the hopes it would make me a better surfer. The first sip was the worst thing I’d ever tasted, but I stomached it with a smile. The second time we went surfing he brought out a different flavor, Mystic Mango by GT’s, and I was hooked. What I later realized is that my body had never had a fermented food before which was why I was so shocked after that initial sip. Weeks later I had developed quite an expensive appetite of drinking two kombuchas a day which at the time GT’s was priced at $5 each. I would bring a bottle into the library with me and it had this amazing effect on my study habits. With coffee I felt wired and scattered, but with kombucha I had this even keel, sustained energy that put me in the zone. Not only that but it curbed my appetite which allowed me to study for hours on end without having to run and grab a snack. Little did I know, but those library memories would spark my own kombucha company 6 years later. And by the way, it didn’t make me a better surfer.

Q: Why did you start your own kombucha company?

I have a hard time being told what to do and after being fired from a few jobs for insubordination I realized that I needed a different path where I was in control of my own destiny. In 2012 my friend Alex moved in with me and said ‘Hey lets start a juicing business’ and I realized this was my opportunity. I worked evenings so while he was at work during the day I was learning how to start a juicing business. In my research I discovered that juicing required a ton of overhead (equipment and fruit) and I thought back to those library days and said to myself ‘why not kombucha?’ We learned how to make kombucha from this Russian guy on YouTube which at the time was one of the few ‘how to’s’ on kombucha. That was our baseline knowledge and after 1.5 years of customizing our fermentation and flavoring style we sold our first keg of kombucha to a local vegan cafe in the Spring of 2013.

Q: What is your favorite flavor of kombucha? 

I think Fine Feathers makes some of the best kombucha on the market. My two favorite flavors of theirs are the Jasmine Peony and Lemon Cayenne. There will always be a soft spot in my heart for GT’s Mystic Mango and Guava Goddess.

Q: What is your former/current other life or career?

Prior to kombucha I was in the restaurant biz as a server and bartender. Just before I went full time with Whalebird in 2015 I was working in business development for a local tech company. When I’m not at the brewery I spend my time trail running, mountain biking, and spear fishing.

Q: What is a saying that you live by?

Life is the ability to go from one failure to the next with no loss of enthusiasm.

 

 

 

Jordi Dalmau, Kombutxa, Mataró, Barcelona, Spain

Q: When did you first discover kombucha?

When I was 13 years old I was diagnosed with Gilbert’s Syndrome. It is not a disease but it means that your liver has some trouble detoxing some toxins. As I was getting older I had frequent headaches, always felt tired, long digestions, and so on, but I thought it was normal. Finally a nutritionist give me some dietary guidelines, and I improved quite a lot. In the end, he recommend I eat a bigger variety of fermented food, even kombucha. I had never heard about it. I did not find it in any herbalist. Finally, I got a scoby online and I did it myself. As soon I drank the first glass of kombucha I had the feeling of taking off a 20 kg backpack. Since then I have not taken any more drugs: I feel always full of energy and if I ever have any headache again I just need to drink more kombucha!

Q: Why did you start your own kombucha company?

I was so happy with the benefits on my health of the kombucha I was preparing in my kitchen that I could not stop giving it to all of my friends. At first they were surprised, but later they were enlightened with the new beverage. I had my own office of engineering, but I decided to change my life and start a kombucha brewery to share with everybody what changed my life. Three years later the company is growing and I am totally focused on the miracle of fermentation.

Q: What is your favorite flavor of kombucha? 

My favourite one is GTs Cucumber Mint Lime

Q: What is your former/current other life or career?

Now I am a fermentist, but my background is engineering and architecture. Also I am a father of two, it is a great experience!

Q: What is a saying that you live by?

Do… or do not. There is no try (Master Yoda)

 

 

Alex Ingalls, Pilot Kombucha, Brooklyn, NY, USA

Q: When did you first discover kombucha?

I was working at Whole Foods and was really hungover and supposed to be doing a demo and one of my co-workers recommended it as a hangover cure. I grabbed a bottle of GT Dave’s Cherry Chia (for calories because I was too nauseated to eat) and I was hooked. I went home that night and googled how to make it myself.

Q: Why did you start your own kombucha company?

I started my own company because all my friends and family kept drinking my home brews, I figured I may as well make some money off of them!

Q: What is your favorite flavor of kombucha? 

I’m obsessed with Soma’s Pear Fennel Jun.

Q: What is your former/current other life or career?

I’ve been working in the food industry since I was in college and have done it all – from short order cook to distribution to barista. My parents were very concerned about my job-hopping but I knew eventually I’d want to start a food-related business of my own, I just had no idea what it would be.

Q: What is a saying that you live by?

Do what makes you feel good!

 

Bryan Deane Bertsch, Deane’s Kombucha, Hopkins, MN, USA

Q: When did you first discover kombucha?

My wife brought kombucha home from the local Co-Op in 2006. It was love at first taste – especially how it made me feel and the fact that it was a “living” beverage. Then I found out how much she was spending on it. A friend suggested I make my own and so it began.

Q: Why did you start your own kombucha company?

When we launched in 2009 there were virtually zero local craft kombucha brewers in our market. I saw all the shelf space Whole Foods and the Co-Ops were giving it, was convinced it was going to grow exponentially, and was getting incredible feedback from friends who were drinking my home brew. Plus I figured the price point it was selling for was too much and surely I could sell for less than that. At the end of the day, Deane’s was the most expensive bottle on the shelf!

Q: What is your favorite flavor of kombucha? 

Nessalla in Madison, WI makes great kombucha They shared one of their Hop blends that I loved!

Q: What is your former/current other life or career?

Virtually every job I’ve had has prepared me to run a small kombucha business. Babysitter, paper boy, dish washer, server, bartender, door-to-door sales, media sales, food equipment sales, mortgage brokering, office manager, warehouse manager. And raising a family of 5.

Q: What is a saying that you live by?

“The further one goes, the less one knows.”

 

Becca Schepps, Mortal Kombucha, Boulder, CO, USA

Q: When did you first discover kombucha?

I first started drinking kombucha when I lived in NYC in 2008. They carried it at the corner bodega and I was looking for low sugar, flavorful alternatives. I think my first kombucha was actually a GTs with Chia Seeds. It reminded me of that Orbitz drink made by Clearly Canadian that had balls of gelatin suspended in juice. As a kid I was in awe of those bottles and loved the idea of chewing and drinking simultaneously.

Q: Why did you start your own kombucha company?

Mortal Kombucha began as a place to pour my creative energy. I wanted to make something super healthy that had a wry sense of humor and was ridiculously approachable. Mortal is the culmination of the things I believe in: Healthy doesn’t have to be serious, flavor makes life interesting, bubbles make everything better and don’t eat too much sugar, but a little bit is a-okay (i’m type 1 diabetic – so I take that last one pretty seriously).

Q: What is your favorite flavor of kombucha? 

I love that seasonal flavor that Health-Ade makes for the holidays. It’s like drinking Christmas with a bunch of ginger on top.

Q: What is your former/current other life or career?

My other life is working as a Creative Director in the advertising industry.

Q: What is a saying that you live by?

I’m currently following this motto: “Make stuff. Eat fruits. Cry a little. Be a rainbow.”

Heidi Vorrasi, RocBucha, Rochester, NY, USA

Q: When did you first discover kombucha?

I discovered kombucha while travelling to the West Coast. We were in a small town in Utah and it was on tap even in the gas station! We drank it on and off for years after that and started to brew once we were settled and wanted to experiment with more flavors.

Q: Why did you start your own kombucha company?

After home-brewing, we ran out and tried some of the local kombucha. I was unimpressed with what was available at the time (but now there are many good local ‘booch options), and thought I could do better. I wanted my community to experience good, local kombucha. It should be delicious!

Q: What is your favorite flavor of kombucha? 

I’m a big fan of buying local, and my favorite other kombucha to drink is Laxshmi by fellow local brew mistress, Catt at Happy Gut Sanctuary.

Q: What is your former/current other life or career?

While I was a former non-profit executive, my favorite time over the past 10+ years has been spent raising my three awesome kids together with my husband, John.

Q: What is a saying that you live by?

Although my kids roll their eyes when I say it (and my dad cheers): The difficult we do today, the impossible we do tomorrow. Today’s challenges are building blocks for what we can only imagine to accomplish in the future.