Narrator:

Introducing Doctor Collado, a distinguished researcher with expertise in microbiology, food science and nutrition. Doctor Collado holds key positions in esteemed organizations, including the Executive Committee of the International Society for Research and Human Milk and Lactation and the Spanish Society of Microbiota, Probiotics and Prebiotics. She is currently a scientific researcher at the CSIC in the group of lactic acid bacteria and probiotics at the Institute of Agro Chemistry and Food Technology in Valencia, Spain.

Recognized for her contributions, Doctor Collado has received esteemed research awards, including the ESPGHAN Young Researcher Award and the Scientific Young Research and Nutrition 2021 from Fundación Jesus Serra Please welcome Doctor Collado.

Doctor Maria Carmen Collado:

Bienvenidos. Welcome for me. It's a pleasure to be here with all of you, and to talk about the research that we were doing during a long, long time and still trying to understand the magic of the breastmilk and the breastfeeding practices as the most important link between moms and infants. Well, my disclosure I would like to start my talk highlighting that the breastfeeding practices are the most relevant link between moms and babies, and it is not.

A single trait in humans is a defining characteristic in all the mammals. As you can see in the pictures and this link is beyond the nutritional aspects. Why? Because also is favoring the neurodevelopmental, the maturation, the, you know, the psychomotor development, the microbiota colonization, the immune system maturation and all these things. Just with one single act that is breastfeeding and the specific things that conveys the breast milk composition.

Well, we know that is not easy to work with milk, at least for me, because it's a really a complex matrix that contains nutrients, micro and macro nutrients. But that also contains other different characteristics. And compounds like oligosaccharides emulate compounds, poly amines, nucleotides, immune factors, microbes, proteins, peptides, etcetera, etcetera. Thanks to the development of the techniques, we are learning more and more about the composition of breast milk.

But you know, it's not an easy fluid. Why? Because it changes during the lactation according to the requirements of the of the neonate. It change from colostrum to transitional milk to mature milk decomposition, micronutrients and also bioactive compounds differs. We know that also the composition of breast milk change within a day is. The composition is different in the morning that in the afternoon that a night, for example, it contains melatonin, cortisol, serotonin and other factors that are helping the humans to start with the circadian rhythms. We know as well that the the composition change between fats and even within a firm because at the beginning or is more like liquid and at the end is the fat where the baby can feed or consume the fat. And recent studies also highlight that even the infant gender, if it's a boy or if it's a girl can modulate the composition of the breast milk, then that's the reason that is really, really complicated to study the breast milk composition and also if we compare with infant formula, the composition, as you can see here, the first as well. But thanks to the research that has been done and it's been done in, in this area, we can try to identify specific compounds that can be supplemented to infant formula, about all the bioactive compounds that you can see in this slide.

Today we are going to talk mainly about the microbes oligosaccharides and other compounds. But the most important thing is that these bioactive compounds have specific targets in the infant gut at intestinal epithelia, trying to promote intestinal homeostasis and also favoring the maturation of the immune system, etc., and also enteric nervous system, and also promoting the mucosal mucosal immune system.

Then we can consider the breast milk as a special food that contains nutrients, but also bioactive compounds that are going to be transferred to the neonates through the breastfeeding and are going to exert a beneficial effects on our neonates. Diet. Breastfeeding is diet is the first choice of infant diet, and we know, thanks to the literature that breastfeeding practices are associated with other benefits in our babies or in our population at short and long term, but also breast milk is is modulating and driving the intestinal microbiota composition.

How? Because there are some compounds. As I mentioned before, that model specific microbes in the infant gut, for example, we know that the presence of a specific Bifidobacterium, you know that Bifidobacterium are the main what is the is the gold standard as well in terms of the microbiota in the babies, I mean, the infant gut microbiota is characterized by the high abundance and presence of Bifidobacterium, are modulated mainly by breastfeeding practices.

And we know that some of these organisms may produce short chain fatty acids. And the short chain fatty acids are quite important because they are an energy source. They can regulate the intestinal homeostasis, the appetite and also society, and also can modulate the metabolic modulation and increase the vitamin and mineral absorption. But also this organism are able to produce specific vitamins.

like vitamin B and vitamin K that are relevant for our health. And important thing as well is that these Bifidobacterium are able to produce specific neurotransmitters, neurotransmitters like for example, Gabba, you know, gut brain axis. Then through these neurotransmitters. Also, the gut bacteria is modulating the brain development and the brain maturation. Then what we know about the lactation and the influence on infant microbiota.

In our team, we were studying the different factors that are influencing the infant gut microbiota development. And of course, the first one is a type of birth or the mode of delivery. We know that babies born vaginally are exposed to the maternal microbiota, mainly because they pass through the vaginal canal and also in contact with the gut microbiota of the moms and these babies.

Can the microbiota, similar to the maternal microbiota, while the babies born by C-section are exposed to the environmental microbiota? And that's the reason that the microbiota is not reflecting the maternal microbiota. But there are also other many factors, like, for example, prematurity, the use of antibiotics where the babies are going to be born, geographical location, etc. but among all of them, in our experience, the breastfeeding practices are key on the microbiota colonization in our neonates.

Even in preterm, the use of maternal own milk, and in case that it was not possible, the use of maternal milk modulates in a good way. The gut microbiota of the preterm baby. In this bigger study. Well, I love always to show this study because was led by a colleague from well, a colleague from me is Chris Christopher's Stewart.

He make a really big study in a big cohort. And they also found that the breastfeeding practices are among the most important contributors to the infant gut microbiota colonization. During the first 40 month of life. You can see in pink color health among all the different factors, that includes maternal BMI, weight gain, medication, probiotic mode of birth, preterm gender, what you can see the the big list that they have.

Breastfeeding is among the most important factor. But not just breastfeeding is influencing the gut microbiota. In our team, we were studying as well the influence of the breastfeeding practices on the oral microbiota development, because, you know, or milk also modulates the oral microbes. And we found really interesting studies. For example, we found that infants who were breastfed during long time have a different oral microbiota trajectory compared to those who were not receiving breast milk.

And also breastfeeding was associated with a lower risk of carriers and allergies later in life. Just because of this, then we can not focus our research just in the gut, because also oral microbiota and oral connections with immune system and also with the brain are still underexplored. And from here, I would like to highlight that it's important to consider these human needs as well as a target for the breastfeeding composition and breastfeeding compounds. Okay, we are going to talk a little bit more about the bioactive compounds that are present in human milk. And as I told you, I'm a food microbiologist and we are working mainly with microbes. And I had the pleasure to work with the pioneer and the father of the human milk microbiota, professor Juan Miguel Rodriguez in Spain, who have reported in 2003, more or less not a long time ago, the presence of beneficial bacteria in human milk. Okay, at that time, the people say, no, no, the milk is a sterile bacteria just because of the contamination. And he demonstrated that not the presence of Lactobacillus and Bifidobacterium in human milk. And thanks to the development of the techniques, mainly the next generation sequencing. And we identify more than 800 different species present in human milk, more that we expect to found in milk. And among all of them, most of most of the species belong to Staphylococcus and Streptococcus species. But also we can identify a presence of lactic acid bacteria Lactobacillus, Enterococcus, etc.. Bifidobacterium. But it's critical. I know that bacteria can be found as well in human milk. In terms of quantity, human milk has not a big amount of bacteria, but if we consider that babies are consuming around 800ml-one liter of milk per day, there is a constant amount of bacteria that is going to be exposed.

These baby and the bacteria would exert some effects. What type of effects can be produced by this bacteria? Well, we can divide in three levels. For example the protector protection, because the bacteria present in human milk can adhere to the intestinal epithelia, can produce some antimicrobial compounds, can stimulate the immune system, for example, at metabolic level. Because, as I told you at the beginning of this presentation, the bacteria can produce some short chain fatty acids, some vitamins, etc. that are important and also the other one, the other, the other would be like a structural because this bacteria, when adhered to the intestine, are promoting the gut homeostasis and maintaining the gut balance and good health. But also the presence of bacteria is not a single trait in humans. There are many reports in the territory that you can identify, or you can found the presence of different organisms in milk from other mammals, like goat, cow, pig milk, donkey, etc. in another site, our team was pioneer in identifying distinct fungi in human milk from healthy woman and how this happened. Really, it was an accidental and accidental finding. We were trying to plate the bacteria in a specific culture media, and we found that strange colony really creamy, big, and the smell was quite similar to the bread. Then we say, okay, maybe it's a yeast we look the morphology under the microscope and we found this lemon shape characteristic of specific yeast. Then we say, okay, this sample is contaminated. But as we are scientists, we were thinking, okay, maybe, maybe human milk. So it's a transfer of fungi and yeast at least the presence of yeast and fungi have been also reported in other mammals. Milk? Why not in humans? then we organize a nice study with culture methods and culture methods all together to confirm the presence of fungi and yeast in human milk. And then you can see our publication. We published two different works about this. The first one discovering and reporting the presence of different yeast and fungi in milk, mainly Saccharomyces, Malassezia and some Candida’s present in human milk. And then we were thinking, okay, maybe the Spanish mums were different and they are the single ones in the world that had yeast in their milk.

Let's try to organize a multicenter study. Then we get samples from Finland, from China, from south Africa, and we make the same analysis. And we found the bacteria there sorry. yeast And fungi present there. Our team was also interested to understand what factors are influencing the human milk composition. And we found that most of it mainly related to the antibiotic use prematurity maternal diet. Yes. With the mom's diet we can modulate many components of human milk. I will tell you this afternoon and also other other factors like drugs and chemistry. All other teams are supporting the research to identify other organisms in milk. And they found some they found viruses, phages and even archaea present in human milk. Now we are going to move to the second big group of breast milk compounds is that human milk oligosaccharides. Human milk oligosaccharides are the family of a structurally diverse and conjugated glycans that are highly abundant and unique in human milk. There are more than 250 different oligosaccharides structures in human milk, and the most abundant one is the 2-fucosyllactose that is associated to to the presence of the 2-fucosyll transferase gene.

The human milk oligosaccharide composition also changed over the lactation in diversity and also in quantity. But the most important thing is that humans cannot use these oligosaccharides. Then who is going to use these oligosaccharides? The microbes specific microbes are going to utilize these microorganisms to grow, specifically Bifidobacterium. Also, there are other organisms like Bacteroides, akkermansia and other bacteria that also can use oligosaccharides.

And about the factors, again prematurity, month of birth, the genetic profile and again maternal diet may influence the quantity and the diversity of the human milk oligosaccharides as well as the milk is quite complex. I am not I don't have enough time to explain all the things, but think about that. If we consider that there are immune markers, platinum peptides and fatty acids in the same time, that also composition changed during the lactation, which are the relations about all these components, which are the effects to the now neonate is still more research is needed to try to understand this complex relationship and the complexity of human milk and the impact on health outcomes. And for this, I am going to give you one example is one research that we did in our team. We compare the presence of a specific one carbon metabolites, choline, betaine, methionine others. We compared a US cohort and Spanish cohort. And we analyzed these metabolites. And we found that lower methionine concentration was associated with higher weight for length see the score change I mean less betaine high risk of development obesity and overweight than we think. Okay. What would be the mechanism behind this. We run a preclinical study with an animal models, well where we provide maternal betaine supplementation to the moms. And we found a decrease in adiposity, adipose activity and also an increase in a specific bacteria. Like I mentioned here. What is doing there? Okay. Let's take a look through the microbiota data that we have in our cohort and see if also babies that consume higher amount of betaine from the moms harbored higher quantity of bifida. Oh Akkermansia sorry. And the answer was yes of course Akkermansiais not an abundant bacteria early in life, but we had the data at 12 month of life, and we found differences in those babies who consume higher amount of betaine. Higher amount of Akkermansia then if there Akkermansia Who is making this influence in the in the risk of obesity or is the Betaine. And then we run another experiment providing a Akkermansia muciniphila pasteurized to the to the pups. And we found that Akkermansia here was also influencing showing a role between Betaine specific compound in human milk and also long term metabolic effects.

What is next? Well, I already mentioned we have to do more research inside in in human milk to try to link specific components with the specific outcomes, and also trying to understand which are the factors that are able to modulate the human milk composition, how to improve the quality of maternal milk, maybe diet, dietary dietary supplementation with probiotics.

Probiotics. Well, I think we have many things to discuss later in the Q&A. And just I would like to acknowledge all the team because I am here today, but all the work that they present here was not possible without the team collaborations, funding agencies and everybody that was involved here, and mainly the mothers who kindly donate their samples to the research.

Thank you very much.