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!

Kombucha is a traditionally fermented, low alcohol, non-intoxicating beverage that is most commonly consumed raw, meaning unpasteurized, to protect the probiotics and nutrients in a living form from being damaged.

Unpasteurized means not subject to pasteurization via heat or chemical means. As a result of remaining a raw product, it can experience slight shifts in ethanol as the fermentation process continues in the bottle.

To arrest that process, raw Kombucha must be kept cold at all times to maintain the integrity of the product and to prevent over fermentation in the bottle.

Cold storage ought to be maintained at 34-40º F /1.1-4.4ºC to slow the fermentation of the Kombucha throughout the supply chain.

Shelf life of Kombucha is determined by how long the product can remain in cold storage before the ethanol level goes above the prescribed legal limit (will vary based on location).

Products that claim to be both shelf-stable and raw are not currently recognized as stable without further evidence vis a vis shelf life testing by a third party lab to verify that the ethanol level remains within the legal limit for that location. No such products currently exist in the US marketplace as all raw Kombucha is subject to refrigeration.

 

Rev.090517 Approved KBI Board October 2017

webinar stills

KBI is delighted to report a wildly successful first webinar in our “Ask Us Anything” series – Fast Track to Distribution” (May 18, 2016). All of the attendees enjoyed the detailed and Kombucha-specific advice offered by our expert speakers. 

“Thank you so much for the webinar today! It was packed full of great information. The panel was so gracious with their time and experience. They gave very detailed answers to some really good questions. I really appreciated the real-life, real small business growing pain examples – especially mistakes made. I could easily see myself walking down that same path and making those same mistakes – thank you for saving me the expense and the agony!” -Elizabeth, BIP

Chris Montelius of Non-Prophet shared tips and advice from his former life experience as a beer distributor. Many members had written in with several questions and he graciously answered them all in a slide presentation.

Jared and Deanne of Kombucha on Tap covered distribution from their perspective as distributors of kegged kombucha in Southern California. They covered everything from equipment choices to costs involved in self-distribution vs. utilizing an outside distributor’s assistance, as well as keg-specific information regarding kegerators, keg washing, and more.

“Thanks for putting the Distribution webinar together, and thanks for injecting my questions into the conversation so late in the game. I found it tremendously informative, helpful, and timely as DZB begins to plan out the early phases of keg distribution. It’s also great to know that there are some tremendously knowledgeable and generous members of the Kombucha industry willing to shed some light on these topics. From our perspective, it has seemed a little isolating and unfriendly at times, but this webinar has proved contrary to that notion. Thanks again for facilitating this, and good luck with the future ones. I’ll be sure to tune in to the rest of the series.” -Joe, Dean’s Zesty Booch

Shane Dickman of High Country recounted the brand’s journey from tiny start-up in the late 2000s to its current size as a thriving national brand. From the tough early years of self-distribution, which sometimes involved drivers sleeping in the van on runs from Colorado to California, to the common mistakes many brands might make early on, with specific advice on how to avoid losing money on distribution deals, Shane’s experience provided a lot of insight into the lengths that sometimes must be traveled on the road to success.

“Loved the dist. webinar yesterday! Thanks for providing exceptional tools and resources to KBI community.” -Gayle, FedBrew

Did you miss this webinar? You’re in luck! The recorded version (as well as an audio-only version) is available in the member forums! Not a member? Join today!

 

On the heels of this successful first seminar, KBI is pleased to announce the second webinar in our “Ask Us Anything” series:

The Secrets of Successful Flavoring, June 21 from 1-3PM PST.

original

REGISTER HERE

only $20 for KBI members, $200 for non-members

Buy 3 webinars, receive the 4th one free!

Speakers

Darius Subatis of Health-Ade

Born and raised in Boston, Darius came out to California in 2006 to pursue a career in film. After spending some time working in production for NBC Universal he decided it was time for a change. Darius joined the Health-Ade team as employee hire #1 in 2013 and has used his skills from film production to run the brewery for Health-Ade. His passion for efficiency and doing things right has helped grow Health-Ade over 10x what it was just three years ago. If he’s not brewing kombucha for Health-Ade, Darius is enjoying life with his beautiful wife, Amanda, and their dog, Riley, in Santa Monica.

 

Alla Shapiro of NessAlla

Alla joined business partner Vanessa with the common vision of helping the community gain a deeper knowledge of health and well being. Both certified herbalists, we began teaching kombucha brewing classes in Madison, Wisconsin in 2007. We became well known for our kombucha knowledge and word of our expertise spread. Workshops sold out and we soon had a SCOBY farm growing! We decided to work together to create one of the nation’s first small, local kombucha breweries.

Hannah Crum the Kombucha Mamma of Kombucha Kamp

Hannah Crum of Kombucha Kamp

Hannah Crum is the Kombucha Mamma. Originator of the Kombucha Kamp workshop,she partnered with her husband Alex LaGory, aka Alex Kombucha, to create the top informational site for Kombucha in the world KombuchaKamp.com Their mission is to “change the world, one gut at a time.” Hannah is a popular speaker about Kombucha, fermentation and bacteriosapiens at corporate and health conferences, fermentation festivals and events around the world. As an outgrowth of their commercial consulting, industry reporting and marketing efforts, Alex & Hannah co-founded Kombucha Brewers International in 2014 to unite and advocate for the commercial Kombucha bottling industry around the world. Through Kombucha Kamp’s videos, blog posts and online support communities, they have mentored 100,000’s of homebrewers all over the world. Their newly released “Big Book of Kombucha” from Storey Publishing (featuring 260 “Flavor Inspirations”) has been met with critical acclaim and praise. With this impressive wealth of information, it is no surprise that they are the leading Kombucha experts in the world.

The Secrets to Successful Flavoring

Every brewer flavors in a different way- that’s one of the ways brands differentiate their products. Have you always wanted to try out some new ideas, but weren’t sure how to start? Looking for some new flavor inspirations? Had some frustrating setbacks and not sure how to proceed? This webinar is for you!

Webinar Includes:

When to Flavor

– Primary

– Secondary

– Reasons for flavoring in primary vs. secondary


Teas

How to Flavor with Tea

– Flavoring and potency of tea types

– Tea Blends

– Brand distinction with tea alone

– Preparing ingredients

fresh_fruit

How to Flavor with Fruit

– Preparing ingredients

– Whole or Sliced Fruit, Juice, Dried, Concentrate, Freeze Dried, Frozen Fruit

– Advantages and Disadvantages

How to Flavor with Herbs, Flowers and Spices

– How to prepare herbal infusions (dried vs. fresh)

essential oils and medical flowers herbs

How to Flavor with Essential Oils

How to Flavor with Different Types of Sugars

– How each type affects flavor

-White sugar, brown sugar, honey, Stevia, etc.

Unwanted flavors and solutions (ex. water quality)

– Herbal or fruit flavors that might compromise probiotic content

– Nutrition facts skewed by fruit inclusion (having each flavor tested and labeled)

– How to know when you’ve messed up. Can it be fixed?

Join us, and ask the speakers your questions in advance by emailing Morgan!

 

REGISTER HERE

only $20 for KBI members, $200 for non-members

Buy 3 webinars, receive the 4th one free!

Upcoming Webinars:

Control Your Brew- Reducing Alcohol Levels in Under-21 Kombucha

Alcohol Testing Methods- What Works, What Doesn’t, & How to Do It for Less

Under Pressure- Kegs, Cans, & Bottles