Episode 60: The Drip by AQUALAB: Probiotics Explained with Anđela Martinović

About the guest 

Dr. Anđela Martinović holds a PhD in Food Systems from the University of Milan, with a specialization in probiotics and gut health. She is the co-founder and program development coordinator at epiSTEMe Hub, where she promotes science, innovation, and technology in Montenegro. Through her research and work, Anđela focuses on advancing probiotic science and exploring emerging trends like AI applications in the field, making her a leading voice in the future of gut health and functional foods.

Transcript

Zachary Cartwright :
Have you ever picked up a bottle of probiotic supplement or probiotic food and wondered, is this really doing anything for my gut? Welcome to the Drip where we keep your mind hydrated with some science music and a mantra. I'm your host, Zachary Cartwright, lead food scientist at AQUALAB by Addium. Recently at my job I've been getting lots of different questions about probiotics. So in today's episode we'll dive into the key challenges surrounding probiotics, focusing on their survival and functionality, starting from the production process all the way through their journey into the human GI system. We'll discuss how to assess probiotic survival, the critical factors to consider, and the role of multidisciplinary research in driving innovations with the probiotic ecosystem. Today my guest is Anđela Martinović and she holds a PhD in Food Systems from the University of Milan with a specialization in probiotics and gut health. Throughout her career she has led numerous scientific projects including the design and execution of clinical trials to evaluate the health benefits of probiotics, as well as the development of of innovative food products aimed at optimizing human health. Angela is also a co founder and program development coordinator at Epistem Hub where she works to advance the development and popularization of science, innovation and technology through STEM fields with a particular focus in Montenegro.

Zachary Cartwright :
She is passionate about promoting healthy lifestyles, longevity, sports and strategies that enhance overall well being. So thank you so much for being here today. What exactly are probiotics and what do you see as the key challenges in this field?

Anđela Martinović :
Yeah, first of all Zach, thank you for having me here and on the opportunity to share my knowledge about probiotics. So probiotics according to definitions are the live microorganisms which administrative in adequate amount they confer health benefit to the host. In this very simple sentence we can actually find a lot of challenges that both probiotics and also probiotic producers face during the production and two keywords I would like to highlight are live so that probiotics starting from the very start, so from their isolation, through the production process and through our git tract or gastrointestinal tract, they actually need to survive until they reach the size of their action and also they must be inadequate amount so the people actually have the benefits of another challenge. I see this field the absence of reliable molecular tools to identify probiotics, especially when we talk about in vivo clinical trials. And the third most challenge I see are the regulatory aspects because on the global scale we don't have harmonized regulation about probiotics. And this is especially Challenge for probiotic producers and also for example, especially for the small companies that want to introduce dairy their probiotic products on more markets on a global scale.

Zachary Cartwright :
And can you walk us through the production process? I know there's several different stages, but maybe start to finish briefly, what does that process look like?

Anđela Martinović :
So the production of probiotics is divided into three main stages. Usually we talk as the main stage, we think about the fermentation. But before fermentation there is upstream phases where we usually prepare fermentation media and sterilize it. Then there is a fermentation phase where probiotics or one probiotic pending on a formulation and desired probiotic products are inoculated in a fermentation media which is afterwards also centrifugated. And this is the initial stage of the downstream process where after centrifugation usually the different cryo lioprotectant compounds are added. For example, we add those are very compounds to protect the probiotic cells. During the next stages, for example, the stage of the lyophil sterilization, but also during the later stages through the passage of our gastrointestinal tract. After this stage, usually probiotics are put in the form of sachet or for example capsules where we add different excipients.

Anđela Martinović :
And sometimes also they are added in the food products. And then probiotics go on our shelves so we as a consumer can actually buy them.

Zachary Cartwright :
And I know the goal overall is to really preserve probiotic survivability and functionality. How was this done during the production process? What are. What are specific challenges in production where you're trying to focus on really preserving survivability and functionality.

Anđela Martinović :
So actually there are a lot of factors that during this production process can affect both probiotic survival and also functionality. And the main factors include the composition of fermentation or the growth media. For example, some nutrients that we add are crucial aspects that impact probiotic growth. Also survival later and functionality. Then also for example, some environmental factors such as ph, temperature, also oxygen factors are important. And particularly my experience was related to the optimization of fermentation media where I tried to optimize production process by using different fermentation media for different probiotics tray by changing some compounds such as for example yeast extracts or similarly in order to to increase probiotic growth efficiency and obtain the desired cell density in the end. And also in these compounds, we also put some protective agents to preserve probiotic or proteins or some other compounds that are essential for their probiotic traits. Also I had experience working with different cryoprotectants as that I mentioned later.

Anđela Martinović :
And these cryoprotectants can actually be very simple ones, such as, for example, some or some amino acids. But there are also more complex ones, such as, for example, skim milk, that is usually added in the production process. And these cryoprotectant, as I mentioned before, actually preserve membrane integrity of the probiotics, but also inhibits the naturation of some proteins. But also for what the cryoprotectants are important is also later on the passage, as I mentioned earlier, through our git tract.

Zachary Cartwright :
Yeah, and I was hoping you could talk about that a little bit more. You know, what are some of the challenges actually once probiotics are administered to a host organism? What challenges do you see there?

Anđela Martinović :
For example, before? I would also like to tackle on the water activity. That is also one of the most important aspects in terms of this podcast. So probiotics, once they are on the shelves, before that, probiotic producers actually must ensure the water activity, which is considered to be the best one. If the probiotics have the water activity below 0.25, because this gives also stability. If we would have the higher water activity, the stability would not be better because we will have some speed up of the metabolic activity and also some chemical or enzymatic reactions, which we don't want to be at that time, but we want during the passage, the probiotics actually reach our gut. But once they reach, so starting from the mouth, through the stomach steno, also upper and lower part of the gut, they actually have a multiple factors that affect their both viability, but also and survival and their functionality. And with this one, we want to consider the most significant ones. So our stomach acidity, that is actually that it can see pH around 1.5 to 3.5, depending also on the host or the subject that is administrating the the probiotic.

Anđela Martinović :
And this also impacts also membrane integrity, also denaturates proteins that are important for probiotic traits. And once the probiotic reach the site of the action, again, they are faced with a lot of factors, such as, for example, our indigenous gut microbiome. So the already present microorganisms there. Because probiotics must compete not just for the space, but also for the nutrients that we also ingest and that they are important for their traits, but also their growth and usually what the industry is doing. They encapsulate, for example, probiotics so that probiotics are preserved during this acidic environment in the stomach and then they are released when the PH is alkaline. In our lower part of the gut. Another strategy is also to put probiotics that will also have a better benefit for the probiotic survival or functionality. And also as a first strategy, the industry what is important is actually producers are doing is to actually put overdosage of the probiotic organisms, so you account for those losses that will happen during the GIT transit.

Zachary Cartwright :
And I understand that some of your PhD work was involving probiotics and you conducted some in vivo clinical trials. And I was hoping that you could just talk a little bit about what that graduate research looked like and some of your main takeaways from those types of projects.

Anđela Martinović :
Yeah, so during my PhD I performed in vivo clinical trial that was based on some in vitro results that I previously done by my research group at the University of Milan in Italy. Particularly in this project, we were evaluating the ability of one strain of Streptococcus thermophilus to survive the human GIT tract when administering the two different formulations differing just in cryoprotectant. So we assumed based on the previous in vitro study that actually in one of the formulation these probiotics strain with better survive. And this study was the crossover design. So, except the variability the subjects we had opportunity to see variability within the same subject. So intra subject variability. And the results of the study showed that the same survival for both formulations. So we had to reject our assumption.

Anđela Martinović :
And this also highlights how important is to perform as a final study in vivo clinical trials. Especially in this stage, you prove or reject your hypothesis and assumptions that you had in in vitro study. Also we performed some functionality experiments and assessment. And what is contrary to the survival here we observe some differences in functionality aspects. That is, I can freely say and highlight. This is usually underestimated in industrial conditions because the industry is usually focused on providing the viable cells. When you already examined some of their, let's say, functionality, some formulations, but just by changing one compound in this case was cryoprotextant. We observed a statistically significant level the functionality and functionality that we observe that is very important for this stray is the level of the URASE activity.

Anđela Martinović :
So we quantify the level of the activity in the fecal samples of the subjects. And in one of formulation we observe statistically significant decrease in the level of the URASE activity. And this is very important activity for Streptococcus thermophilus. And I will highlight that Streptococcus thermophilus is the only lactic acid bacteria that has significant level of Dura's activity and is very important for metabolism and for probiotic traits of these bacteria because is the level of the glycolytic enzymes. This is very important for the some technological features of the fermented dairy products, for example yogurt for which Streptococcus thermophilus is used. And this decreased level of urase activity is observed in previous studies that was done on a murine model where the consumption of the urase positive but beneficial bacteria, such in this case probiotics inhibits the growth of the urase positive density. But harmful bacteria such as for example Proteobacteria. And maybe listeners know that proteobacteria are characterized for the patients that has the IBD symptoms, IBD states so the altered gut microbiome.

Anđela Martinović :
And from our research we have another hypothesis that our strain in one of the formulation can actually be beneficial for these patients. Of course, this should be confirmed in another in vivo clinical trials, because in this case we had the health the subjects. And what is very important is that as I said, we need to confirm this in a subject that has this altered gut microbiome. And I believe that now the research is ongoing in this direction at the University of Milan, that my actually previous research group is now working on this.

Zachary Cartwright :
And I noticed you also had a recent study that was published in the Probiotics and Antimicrobial Proteins journal and you looked at the probiotic market in Montenegro specifically. Can you share some of your insights from from that research?

Anđela Martinović :
Yes, of course. First of all, I'm very proud of this study because I had opportunity to contribute to my home country, Montenegro and here together also. Another thing that I'm very proud is that this study I done together with my brother that is an expert in AI application healthcare sector and together with my PhD supervisor, we aim to contribute our combined expertise to analyze and better understand probiotic supplement market in Montenegro. So this study was composed on the two main parts. The first part we explored some probiotic supplement products. So we tried to map as much as possible probiotic products on the Montenegrin market. And we characterized them including strains that are there, for example, some health health related claims, amounts provided, also origin of those products and so on. And the main message from this part is that I can freely say that Montenegro is operates in a gray zone related to the regulatory aspects because we don't have any specific regulations addressed to the probiotic microorganisms.

Anđela Martinović :
And I think this is a huge gap because this diminishes the consumer trust and later on the growth and also innovations in this field. And this is very important because Montenegro is a candidate for European Union and for sure it must align with the European standards. So, having set up the national standards and also regulation guidelines would enhance product quality, improve market credibility and empower consumers to make of course, informed choices and decisions which on long term would ensure the growth of this promising market. The second part of the study involved examine sales and sale patterns of probiotic supplements. And we also developed and employed machine learning model predict the future sales. This was done on a case study example on a pharmacy chain which held 45 pharmacies that were spread across all three regions of Montenegro. What we observe is that we identify a sense of pattern in probiotic supplement sales that is influenced by Montenegrin's tourism driven economy where tourism contributes to approximately 25 of the GDP. And we concluded this because we have find a strong correlation between the sales that show pick during the summer months with the foreign tourist visits during that period.

Anđela Martinović :
So after this we developed machine learning model in which we integrated various data so data from the tourists, data from antibiotic sales and also data from COVID 19 cases. Because we have this period that we analyze five years from 2018 to to 2023, which was the period in which Covid outbreak happened. And our model showed a good forecasting accuracy for both training as well as on data sets. And it was employed to predict the probiotic supplement sales for the whole next year. So the development of this AI tool is very crucial because it can offer healthcare organizations, for example in this case pharmacy chain, significant benefits, for example supply chain optimization and to respond to the growing demand of these products. And this also can be applied for another industry such as drug or cosmetics. Also to enable better inventory management, cost reduction and in the end, sustainable market growth. At the end, this study highlights combining robust regulatory frameworks with cutting edge tools such as AI.

Anđela Martinović :
And with these tools we can support a sustainable market growing through an improve public health outcome outcomes.

Zachary Cartwright :
Yeah, I thought it was so interesting how you implemented that AI tool and how you used it here in the probiotic field. And there's so many potential applications and you touched on them just a little bit. But overall, how important is multidisciplinary research and accelerating innovation, especially within the probiotic ecosystem?

Anđela Martinović :
So a multidisciplinary research, I think it is absolutely essential for accelerating innovation in probiotic actually in all the fields. But this case we speak specifically for a probiotic field. And probiotics actually exist at the crossroad of multiple scientific and industrial domains, for example, including food science, medicine Biotechnology, artificial intelligence and even regulation sector and its actors. So when experts from all of these field and disciplines collaborate, we gain a more much comprehensive understanding of all probiotic field and can make advancements that would I think be nearly impossible if each field work in insulation. So again, let me go back on our research. So in this AI study it was multidisciplinary because we combine expertise from scientists and research from various fields, so including food and probiotic field with AI field. And also we correlated with stakeholders, for example, in this case was the pharmacy chain. And to be more clear, I will say that for studies like this the collaboration is crucial as I could never for sure develop the machine learning model by myself.

Anđela Martinović :
But neither AI expert could interpret and put into the context all the update data. Also that should be highlighted. I think that the whole study was not even possible if we did not have any data that was provided by this pharmacy cha. To which I want to say thanks on this occasion. And I think that this collaboration is a great example on how different fields come together to make significant both scientific, but also we have the practical application for the industry in this case. Beyond that we also need other stakeholders, for example NGOs which I try to do through my NGO Episteme Hubb to play a role in promoting awareness and educating consumers. So everyone from researchers and industry players to policymakers has a role in shaping the future of probiotic field. And that's why multidisciplinary collaboration isn't just important, but it's absolutely crucial.

Anđela Martinović :
Crucial.

Zachary Cartwright :
And you touched on it there for a second. But really, from your perspective, what are the emerging trends and maybe the future directions that you see for the probiotic field?

Anđela Martinović :
So the emerging trends in the probiotic field are things that we already touched on during this podcast, including multidisciplinary research, scientific breakthroughs and technological integration. Particularly with tools like AI, we will likely see major advancement in personalized probiotic solution. As technology and science continue to evolve, probiotics will increase. We target specific gut related conditions like IBS or ibd. And this precision will make probiotics more effective and tailored to individual needs. Another trend we already starting to observe and one I expect will grow, is the increasing the knowledge between gut and other organs that we will call the gut organ axis plus our overall health, for example gut brain or gut liver axis. And I believe we will have much more robust data to explore and understand the these bi directional relationships. Then I see a big focus on developing next generation probiotics strains like for example Akkermansia muciniphila, and other order offer unique metabolic activities will play a key role in advancing probiotic research.

Anđela Martinović :
And alongside these there will be efforts to improve of course probiotic delivery systems because we talk about the probiotic survival and for example another innovation in encapsulation technology. But even in 3D printing we launch ensure precise delivery and greater efficiency. Also, I think it is crucial to develop standardized methods and protocols for evaluating probiotics whatever is their survival functionality in clinical studies or the molecular tools we use just for their identification. And finally, I think we must prioritize consumer education. It's essential to provide people with realistic science based information so they can make actually informed choices. Educational compounds are are key to this and I see this podcast as the part of that effort so to help to share evidence based insights and bridge the gap between the science and the public. So I think that there is a lot of happening but these are some of the key areas I see driving the field warfare.

Zachary Cartwright :
Thank you so much for just really diving into probiotics with us today and then coming onto the show. I I want to switch gears a little bit and ask you, you know, when you're working or when you're in the lab or you're trying to do these research projects, what is music that you like to listen to? What. What music did you bring with you today?

Anđela Martinović :
Yeah, so actually depending on my mood on the day but usually I like to listen some motivating music. For example, I sometimes I like to listen just the jazz in the background or some intros of the of the songs. But actually my favorite group is the group Queen. It's considered rock but I that there are a lot of genres that they have performed in the in the past. So usually these days I listen that and because of that I suggested that this should be one of their songs that is also big motivation should be mentioned in this podcast. But depending on the mood but some motivating music. You need motivation in the research?

Zachary Cartwright :
Of course. Yes, for definitely for research you do. It's a very, I don't know, very. A tough, tough thing to do at times. Do you have a mantra? Do you have a saying or a quote? Something that you say to yourself? What do you bring or what do you use to bring some inspiration to you? What, what quote did you bring with you today?

Anđela Martinović :
So nurture the good and it will transform the world around you. This is mantra ties into the idea of probiotics that foster a balanced and thriving gut environment which metaphorically resonates with nutrient positivity and growth in it actually emphasize how small things and deep paths with consistent effort, creating broader change both internally but also externally in the world.

Zachary Cartwright :
That's perfect. I love that you tied it back to probiotics and you're right. Really small things can have such a big impact. So thank you for bringing that. Thank you for your mantra, your music, and coming on and discussing a little bit of science behind probiotics with us today. Today we I really appreciate your time and we had an episode before on Probiotics that really focused on water activity, but I'm glad we could have you today to really talk about the production process and some of the other challenges that we had skipped over before. So thank you so much for your time. Even though we're, you know, on opposite sides of the globe, I I really appreciate you being here today to go into that with us.

Anđela Martinović :
Thank you. Thank you, Zach.

Zachary Cartwright :
Today's episode is sponsored by Aqualab. In this episode we discussed probiotics. Did you know that for probiotics there's a very small range of water activity that needs to be maintained in order to preserve them between 0.15 and 0.25. However, even if you're in this range, water activity is dynamic and can change over time. The best way to predict this is using Moisture Sorption Isotherm Analysis, which is used on probiotic containing products to understand how they pick up or release moisture in changing environments. To learn about the fundamentals of isotherm analysis, a link to an application note is in the podcast description. This week, the song that I'm going to recommend that I just can't get out of my head is Push the Tempo by Subfocus and kdb. Subfocus delivers yet another high energy banger with Push the Tempo.

Zachary Cartwright :
Staying true to his signature drum and bass roots, this track is seamless in the way that it blends pulsating bass lines, crisp percussion and electrifying synths. The title really says it all. It's an adrenaline fueled ride that commands you to move. Perfect for late night sets or high octane workout playlists. Push the Temple proves that Sub Focus still knows exactly how to get the crowd hyped and hearts racing. To round out the episode, I'll be offering another mantra as a reminder, this can be a single word or a phrase that you repeat to yourself to feel calm, motivate yourself, or to express something that you believe in. This episode's mantra is I let go of what does not serve me with ease. I will repeat this three times and maybe you can even say it out loud Today.

Zachary Cartwright :
Today. Here we go. I let go of what does not serve me with ease. I let go of what does not serve me with ease. I let go of what does not serve me with ease. As you keep this mantra in mind, I also challenge you to think about what is one toxic thing or one toxic person in your life that you need to let go of. Thank you so much for listening to this episode. Episode? My name is Zachary Cartwright, and this has been another episode of the Drip, brought to you by Aqua Lab.

Zachary Cartwright :
Stay hydrated and see you next time.