What is the InnerBuddies personalized ingredient advice V1?

One of InnerBuddies products is the personalized ingredient advice. In V1 of this advice, the setup is the following:

• We measure the bacteria abundance of the InnerBuddies top 35 bacteria in the individual's gut microbiome (stool) sample.
• We compare the abundance of each bacteria in the InnerBuddies top 35 bacteria list with its corresponding minimum and maximum range of the InnerBuddies Healthy Cohort (initially InnerBuddies started with a healthy cohort that was inherited from the microbiology department of the University of Maastricht but nowadays InnerBuddies regularly updates its healthy cohort based on its large database of customer data).
• The top 35 list of bacteria is divided into positive, negative and other relevant bacteria. For the "positive" and "other relevant" bacteria, if the measured abundance is below the minimum value from the Healthy Cohort, this means there is a need to increase the abundance of this particular bacteria to bring it back in-line with the ranges in the Healthy Cohort (it is therefore categorized with a "be aware" flag). For the "negative" bacteria, it is exactly the other way around. Preferably you want to have the abundance of the "negative bacteria" as low as possible. So if the measured abundance is above the maximum value from the Healthy Cohort, there is a need to reduce the abundance of that negative bacteria (it is flagged as "be aware" again).
• The InnerBuddies science team has identified 7 gut health categories that we consider to be scientifically solid and proven in scientific publications. Some of the bacteria in the InnerBuddies top 35 have been linked to these 7 categories. For each of the 7 gut health categories, if any of the linked bacteria is in the "be aware" category, it means an ingredient advice is generated to help the individual to get all bacteria in that gut health category back in the "normal range".
• Each gut health category has been linked to specific "pathways" meaning certain biological processes in the gut microbiome. This has been done based on scientific research and publications. Specific food groups (consisting of individual food items) have been linked to these "pathways". Again, this has been done based on scientific research and publications. If a specific gut health category is in the "be aware" range (meaning that one or more bacteria linked to that gut health category are in the "be aware" range), that means the InnerBuddies ingredient advice will list the food items that belong to the food groups connected to the pathways associated to that specific gut health category.
• For each food item we have identified the daily advised dosage. This is based on common dietician knowledge available in our dietician team.

What is the setup of the Family & Friends test?

The InnerBuddies team wanted to perform an internal test of the performance of the ingredient advice within a small group of participants. While the whole ingredient advice is based on scientific research and knowledge, it combines the results of many different scientific publications and the InnerBuddies team wanted to internally test the performance within a small group of participants. Therefore we organized the Friends & Family test. We decided to test primarily with family and friends because that way we could monitor better whether participants would actually follow-up on the advice. The idea was that test participants from family and friends would be more willing to do their best to follow-up on the InnerBuddies personalized ingredient advice.

We decided for the following setup:

• 50 participants in total (in the end only 40 participants completed the full test, therefore only the data from 40 participants was included in the analysis)
• The actual testing period was 4 weeks during September 2022. All participants participated in the testing period at exactly the same time.
• We collected 4 stool samples for each participant:
• Stool sample 1 was collected in June 2022. This stool sample was used to generate the personalized ingredient advice for each individual participant. In 2022 the lab throughput time for the lab analysis took approximately 6 to 8 weeks (currently it is 2 to 3 weeks).
• Stool sample 2 was collected at the beginning of the 4-week test period. This was the starting value for the analysis.
• At the end of week 2, we collected stool sample 3, this was halfway during the test period.
• At the end of week 4 (the end of the testing period) we collected sample 4. This was the end value for the analysis.
• During the 4-week testing period, the InnerBuddies dietician team prepared a fixed weekly menu for each participant. This weekly menu was based on the individual's personalized ingredient advice from the InnerBuddies platform. So every individual receives his own personalized weekly menu. At the beginning of every test week, all participants received their new week menu for the next week.
• At the end of the test period, we evaluated with a questionaire to all participants to find out what they experienced, how good they had followed-up on the personalized nutrition advice and which results they had experienced themselves.
• We received common feedback from the participants that they felt better and had more energy.
• Some of the participants stopped halfway during the study because they felt it was too difficult for them to follow the weekly menu's giving their normal daily work pattern. Hence why only 40 of the 50 participants actually finalized their 4-week test program.

What are the measured results of the InnerBuddies Family & Friends test?

Again, the InnerBuddies Family & Friends test was meant to be an internal test, not a formal scientific clinical trial with medical ethical committee approval and peer review after publication in scientific publications. We first wanted to test how good everything works, before we would start testing in real clinical trials (because they cost a lot of money and time as well).

So I guess after reading all this text, you are curious to see the results of the Family & Friends test?

First, let's look at the progress of the participants during the 4-week testing period. For this purposes, we categorized the individuals in three different groups:

• GREEN group, these were the participants from which we knew they had followed-up on the personalized ingredient advice very well.
• YELLOW group, these were the participants from which we knew they had followed-up on the personalized ingredient advice OK-ish.
• ORANGE group, these were the participants from which we felt they only followed-up on the personalized ingredient advice in a limited way.

Again, we organized the Friends & Family test with participants that our team knew quite well so our team could judge pretty well how each of the individual participants had actually followed-up on the personalized ingredient advice. We used this knowledge to categorize them into these three different groups.

In the above picture, you can see how each of the 3 different groups (GREEN, YELLOW, ORANGE) fared across the testing period of 4 weeks (test2 was the test at the beginning of the testing period, test3 was the test half-way during the testing period and test4 was the test at the end of the testing period). On each of the three different graphs, the vertical axis displays Pielou's evenness calculation (this is a well-known scientific indicator that measures how even the total bacteria population is spread across the different bacteria <-- higher is better) and the horizontal axis displays the number of different bacterial genus that we found in the stool sample (number of OTU'S is another well-known scientific indicator that measures how many different bacterial genus have been found in the stool sample).

What can we conclude from this picture:

1. The participants on average managed to increase their number of OTU's when moving from test2 to test3 to test4. This increase happened while Pielou's evenness remained fairly constant so the increase in the number of different bacteria was fairly spread.
2. The GREEN group had a higher "evenness" than the YELLOW group and the YELLOW group again had a higher "evenness" than the ORANGE group. The well-known Shannon Index (a scientific indicator for "diversity" of the gut microbiome) basically combines "evenness" and "number of OTU's" into a single diversity calculation but "evenness" has a bigger impact on Shannon Index than the number of OTU's. This means that the participants that followed-up better on the InnerBuddies personalized ingredient advice also had a higher gut microbiome diversity. Why is this important? Because gut microbiome diversity has been linked to many different medical conditions (and has been used in 1000+ scientific publications) where higher is better.

The following picture gives a good overview of the Shannon Index evolution for each of the three different test groups across the different tests performed during the testing period.

While participants were moving from test2 to test3 to test4, you can clearly see that all three groups managed to increase their Shannon Index. You can also clearly see that the GREEN group outperforms the YELLOW group and the YELLOW group again outperforms the ORANGE group. So if participants followed-up on the InnerBuddies personalized ingredient advice better, they generally also achieved a higher Shannon Index (and therefore a higher gut microbiome diversity).

Does the InnerBuddies personalized ingredient advice allow to achieve "micro-interventions" in the gut microbiome?

This is an important question for InnerBuddies since our ambition is to move to a level of personalized nutrition advice where we can achieve very targeted "micro-interventions" in the gut microbiome to achieve specific beneficial results on a very individual level.

In the table below, we have summarized the results from the Family & Friends test which were achieved during the 4-week testing period.

As you can see in the table above, our personalized ingredient advice (if followed-up on by individuals) allows to move the gut microbiome composition in the right direction in a relatively short period of 4 weeks.

What's next?

InnerBuddies at the moment is working on an updated version of the personalized nutrition advice. With this update we target a couple of improvements:

• We will move from a bacteria abundance focus to a bacterial pathway focus. Bacterial pathways are the biological functions performed by the different bacteria. By looking at bacterial functions instead of individual bacteria we think we can move the concept of "micro interventions" to a next level. This change will allow us to achieve very targeted personalized interventions.
• Functions are more stable than bacteria across different regions and countries. Specific bacterial functions can be performed by different bacteria. While the bacterial composition can be quite different in individuals from different continents due to their very different diets and food culture, bacterial functions however remain the same since they are required for the human body to function. This will allow us to better scale-up our personalized nutrition advice to also work for individuals from different continents. Our Healthy Cohort definition will also be updated to use bacterial functions abundance instead of being focused on bacteria.
• The move to bacterial functions will allow us to generate even more personalized recommendations for individuals since we can use the full richness of the gut microbiome to base the personalized recommendations on.
• The use of bacterial functions will also allow us to better inform our customers about why they should follow-up on specific nutrition advices because it allows us to directly show the link to specific biological functions inside their body.

We currently expect this V2 update to the InnerBuddies personalized nutrition recommendations to become available in Q1 2025. We are already in the process of setting up clinical trials with some universities to then test this update in external clinical trials where the universities will also publish the results in scientific publications.