Narrator:

Welcoming Professor Rafael Jiménez-Flores, Endowed Professor at the Ohio State University, USA Professor Jiménez-Flores holds M.S. and PhD degrees in food science and agricultural chemistry from Cornell University and UC Davis. He pioneered the cloning of milk proteins for structure function studies and later advanced mammary gland directed gene expression in transgenic animals. To date, he has published over 155 peer-reviewed papers, holds 10 patents, and has received numerous awards marking his significant contributions to the field. His latest endeavours focus on elucidating the role of MFGM in the gut brain axis, using cell culture techniques such as enteric neuro spheres. Please welcome Doctor Jiménez-Flores.

Dr. Rafael Jiménez-Flores:

Thank you. Thank you very much. It’s, to me, a pleasure to be here, especially that I have noticed so many of my paisano around, even though I live now in the other side of the border, but I'm thinking about coming back to Mexico — believe me. I want to, before anything, thank the organizers for inviting me to give you this talk.

But the second thing that I need to do immediately is to get the audience to realize that now you're going to be in a classroom at Ohio State, understanding why the previous lot of speakers have told us about microbiome. And I'm a dairy scientist, so I'm going to be talking to you about what is the MFGM, which is a part of nutrition that, as the talk goes on, is going to explain why my passion for dairy foods, and in specific, the Milk Fat Globule Membrane.

So, in this first part is what is the MFGM. And I know it's our food, but I didn't coin the word Milk Fat Globule Membrane. There's got to be some German person there because I think the important thing is the membrane and it should be first, but they always put the important thing —the verb, at the end. If you talk some German, you know what I'm talking about. But how do we use MFGM, what's its origin and how do we do it industrially? Here I'm going to put my hat of a technologist, and I'm going to tell you the most important thing — why am I interested on the MFGM? So, there's a lot of studies that get modulation of immune system bone mineral density.

I was just last week at the ADSA and there was a beautiful experiment that really demonstrates the importance of MFGM on bone density in mice. But I really focus on the cognitive function, on the gut barrier function, the microbiota. Why? You just heard the previous speakers talk about the microbiome. So, my question, is why is that important? How do we know this?

So first the origin of the Milk Fat Globule Membrane. And I give credit to my professors’ ancestors. They started studying the Milk Fat Globule Membrane in the 60s and 70s back when, I'm so old that I did one of my papers that you had to do the drawings yourself with pencil and ‘tinta china’, for those of you that know, you know Chinese ink. But here is one of the marvellous things. One of the first applications of electron microscope is how these little mice was in lactogenesis, how the fat, that’s the triglyceride, was getting into the milk by pulling out and actually micro-encapsulating every droplet of fat in milk is surrounded by this membrane, and the membrane comes from the epithelial cells of the mammary gland.

The other thing that I'm very proud of, I know when I was in the University of Illinois, I was witness of the first applications of freeze fracture microscopy was published. And believe it or not, one of the first ones that this technique was the fat globule and it showed, as you see, that it was a very irregular surface.

We now know what that is and I will explain in a minute. So the important thing, my dear colleagues, is the composition of the Milk Fat Globule Membrane and what it has to do with there's the gangliosides, which of course we know the [glycosphingolipids] contain sialic acid, which was one of the nutrients that we know have a big relevance on the gut-brain axis, the sphingolipids.

There is no plant that produces a sphingolipid. Why? Because plants have no brains or neurons, and the sphingolipids, found in abundance in milk, are those precursors that put the curvature in all our neurons. So, you tell me if it's important or not. The proteins, of course, that we have plenty of them. And the phospholipids, which are the structural elements of all the membranes in our system.

So here's a little bit of the contributions that I had. And this started, and my male colleagues will probably be envy of this, I spent 2 weeks working in France with 5 female scientists that were really outstanding. The group of Christelle Lopez. And our contribution is started by really looking at the structure, acid came directly out of human or cows’ milk. I must tell you that all the lactation advances, have done mostly, funded by dairy farmers. That's their business. So that's why we know so much about milk. And I make no bones about it. But right here, you can see those dark dots turn out that they are very densely packed. A single myelin, rafts or liquid ordered domains.

And they move, the best example I could do is if you see those pictures from the satellites of the Earth, they're like the clouds moving around the Earth. They really is fascinating to see them. Would that have a function? We don't know, but we know for certain that it’s the composition of these globules that is important. As a matter of fact, with Christelle, we found out that in human milk, the complex oligosaccharides, and you know those are the ones that the baby doesn't digest, but the microbiota does, specifically the Bifidobacterium, they do eat those complex oligosaccharides. Well, they are indeed also in cow's milk, the one that we use for making formulas, etc., but they are all attached to the Milk Fat Globule Membrane. So, I find this fascinating.

But here's the deal. Is that structure, is that thing that we're so proud of in my field that we discovered by lactogenesis, all this complexity of a tri-layer that you can see, Google MFGM and there comes tons of cartoons of the MFGM, and they’re all depicted as a tri-layer. Well, I'm here to demystify the thing. All the experiments that we've done in clinical trials or in the labs or in cell culture do not have a tri-layer.

Sophie Gallier in our lab, started finding out not 500m from our lab is where the cows were. And she went, got the milk from a single cow and came running to the lab to have as fresh milk as she had. And there it is, you see the fat globules with those dark, very dense that the fluorescent dye won’t even come in there. And she said, okay, I'm going to do a pasteurization. And what we did, we painted beta-lactoglobulin blue. So, look at that. Right after pasteurization already that very sophisticated tri-layer is changing. It’s getting its structure modified, if not destroyed. So, what is it that really makes it work? Well, you have to go and understand dairy technology. So, when we make cheese out of 10 kilos of milk, I know you measure it in litres, but let's do it for this example that you do kilos. For 10 kilos of milk, you get only one kilogram of cheese and 9 of whey.

The reason that the dairy industry today is making more money out of the whey than of the cheese, is this technology that I started in studying this in 1982 at Cornell, that's what we were doing — applying membranes. This is another type of membrane, this is an industrial membrane. For all practical purpose, think of it as a sieve of a molecular size.

And when we get the whey coming in, so the cheese went somewhere and we do the ultrafiltration, 2 things happen. That's the fractionation, the permeate, what goes through these little holes, is mostly lactose and minerals. So, in effect, if we take away the other solids, the lactose and minerals out, we concentrate the proteins. And that is our whey protein concentrate that I challenged you as I challenged my students going to any supermarket and start reading labels and you'll see that now whey proteins are just about everywhere. And if you go to the gym, you're going to find people making their whey protein to muscle up and drinking right there in there.

So the whole big idea is if you change from 10 kilodaltons to a larger pore, you see that you don't need statistics to see ‘yeah, these are larger’. So, the same whey comes in except that now through the permeate the whey proteins go through. Well, if you lose the whey proteins and you keep the big particles where all of these phospholipids, glycoproteins, glycolipids get collected, you get a nice high concentration of the MFGM.

That's a picture of the way protein concentrate and these, my dear colleagues, is what we are actually using in all the experiments and clinical trials where we add MFGM to infant formula or geriatric drinks or whatever it is that we're studying. So, I would like you, when you leave this meeting to really expunge from your minds that it's a tri-layer, and all of this complexity. It is beautiful for lactogenesis.

It explains almost, and I know that I romanticize this. It explains why we're mammals and why we have succeeded so much in this planet. But that's not what we're feeding to the babies. The other source, and it’s being developed now by several companies, is from buttermilk. Buttermilk is when you get cream, and you churn it you get butter, and you get buttermilk.

Okay? That's the ‘suero de mantequilla’ in Spanish and that is very rich in the Milk Fat Globule Membrane. But look, it really looks like this. So, what I explain is imagine that your Milk Fat Globule Membrane is your balloon with all your drawings. And when you pop that balloon, the balloon, the plastic just rolls and that's what we have in the buttermilk. And this becomes relevant for some of the examples that I will talk later on today and tomorrow. So now we're going to go to the effect of Milk Fat Globule Membrane in cellular, in neuronal culture, clinical evidence of development and the connection, I was asked to mention connections, and the connection here is to see what we have heard to why does it work?

Now, the scientific mind is always that insatiable curiosity by saying, yeah, we know that you got better, that now you don't have diarrhoea, we modify your microbiome. But why? Well, let's start explaining or trying to explain, what is happening in the neuronal system. So, I already mentioned myelination, so if you have a strictly vegan diet and let's say that you refused to give milk to your vegan baby because you think it's not right to give milk to a baby, you would be depriving that baby from myelination of the neurons.

Why? Because you need sphingomyelin. And why is it important? Because all you know this and I just have a nice picture to show off, is that when the neurons are myelinated, the electrical impulse goes so much faster, people calculate 1000 to 10,000 times faster than on the non-myelinated neuron. That's probably why the babies take so long to really start reacting and even though I’m a new grandpa, I cannot wait to play catch with my baby grandson. But right now all he does is look at the ball and it hits him in the face, so I'm not discouraged yet.

The other thing that happens, and this was some years ago, my Irish colleagues, this is what you see in the petri dish, a neuronal culture. And the best thing you have to do to proliferate is to use the fetal calf serum. So, yes, it's got to be in the fetal state, take that serum, which I think is cruel, and you put it in your neuronal culture, and they grow moderately well. But what they did is they purified the phospholipids from milk, and they add it to the cell culture in 75, 150 and 300mg/L.

Now, I hate statistics, but to describe this result, you don't even need statistics. Just look at the difference between that and that, in blue is the nuclei, the number of cells per square micron. You can see that it works. However, the caveat is that at 300 that was toxic. So that didn't help much on the neurons. But we know that the neurons have a very important response to the phospholipids in milk.

So now here come here comes my reasoning. We go from the top down. A lot of people have studied in Peru, Indonesia, Belgium, Japan, Sweden, Sweden, China. And by the way, not all of these studies have been financed by Mead Johnson — some of them have, but not all of them. And if you add all of the babies that have gone through the treatment and measure neurodevelopment and immune system, etc., you have over a thousand babies in the experiment.

Now you can make statistics. Now you can start really looking at the truth through a scientific eye. And what was the result? And this is what is amazing. All of those studies report something similar to this. Breastfed babies on Bayley’s Scale of the brain function, cognitive — 106. Fantastic. Really good. Great. The ones that were fed infant formula without the MFGM, they didn't do good, they didn't fail but it was 101. Exactly that same formula but add in that ingredient that I just described to the formula, feeding to the baby for 6 months and then doing the test 12 months later, it was 105. So with all of those studies, there shouldn't be any question in our mind that MFGM does work, but we all, before we decide that we have the confidence, we are trained to ask why.

Well, here are some of our efforts to ask why. But before I do that, furthermore, Doctor Colombo here did a wonderful study in China with almost 500 or 800 babies. But the beautiful thing is that he followed up 5 years later. Now just think about the logistics of that is I think, at least nightmarish. And then he saw, well, what happened to those babies that we fed them MFGM 5 1/2 years later? Well, he measured the rapid brain growth, but with the aid of [MFGM], here is the impressive thing. The myelination growth rate, the rate by which the neurons of the babies fed the MFGM was much higher in those 5 years than the ones that didn't. The results, as we saw on the 12 months on cognitive language, on motor, the dark blue bars clearly indicate what happen, which is great.

But here is the real key that to me, make me a believer of this thing. Five and a half years later, it was on 5, some 6. Now we can measure, full scale IQ. We have processing speed much faster, visual and spatial. Now I ask you which ones of you are paediatricians, which of the mothers that are in your patients wouldn't really like their baby to be smarter? and a lot would want will say, I want my kid to be smarter than my husband. I think there's a lot of us. So, yes, don't get all bent out of shape, guys. That's how our wives see us. But rigor in science comes when you have a group of statisticians and do a meta-analysis.

Now, I'm not that much of a super-fan of meta-analysis. Like I said, I don't like statistics, but these people do. And they got all of those studies, and you know, when you see the result, which is the average here, close to 0, it would make, it makes no difference. But as you can see, the rhomboid is there around 4 for the grading of the babies and it favours and MFGM.

Well, if you just do the statistic and you go with the effect in the starter and standard difference, which is a more strict kind of measurement, still comes ahead. So that's good to very good. And it also balances the thing of what we consider because there's not such when language is close, motor but the executive function, and my daughter is a PhD in cognitive and infant development, so she teaches me this. When I say something that is wrong, believe me, she lets me know. All of you, if you want to know what's wrong with you, have a daughter. And to make sure, to make sure that this was true, look at the meta-analysis on breast milk versus milk with MFGM.

So this still favours breast milk, which gives me faith in Mother Nature. But that also gives me faith that we as technologies and scientists, we have to strive to be better and there's room for improvement. This also is analysis that you have to do if there were biases on the study, and I just want to point out Timby and Gurnida have a perfect score.

This is at 5 years, and the bias was due to missing outcome data. The problem was that it's very hard to come to the same babies 5 years later and have them all be as a unison. So that, bias probably is. So now, in the time that I have left, I'm going to go very quick to the effect of bacteria.

So, we heard that there's a kind of bacteria that are good for the for the microbiome. Erica Kosmerl picked Bifidobacterium infantis. When Bifidobacterium is exposed to MFGM, the surface chart changes and it changes because it makes an extra polysaccharide that is very important in how it attaches to the intestine. And if they have extra polysaccharide at the residence time of this bacteria in our gut, because no bacteria stays there forever, is longer.

Javier Fontecha demonstrated that the phospholipids in the Milk Fat Globule Membrane indeed formed part of the membrane in the intestinal epithelial cells. But this is the pièce de résistance right here. This is how, a normal cell culture, permeates any of the markers that we have. But for we insult that cell culture, with DSS, could be a radiation, antibiotics, the permeation goes much higher.

Graphically, those are the mucins. And when we insult with DSS, you see the mucins are gone. But not only that, you see the holes in the villi of the intestines. No wonder there's leaky gut. But if we have trained ourselves, if we feed those cells for a period with MFGM, the blue square shows us nothing happened with an insult. They look just the same. There's no fluid, and because I know doctors like histological cuts, here you can see we cut the plates that's injured. Look at that hole, anaemic and sad. Look, no blue mucin. But if we have fed those cells with MFGM, there's plenty of mucins.

Furthermore, and this is what the future brings, and believe me, this was done by an undergrad in my group, trained by a very good grad student. And we saw that there was, you just heard serotonin. We have lactic acid bacteria that produce serotonin when MFGM is present. So, what is it? I don't know, I did it with complexity. So, you just put the whole package MFGM, and they produce serotonin. And guess what, it’s tightly correlated with the producing of mucin. Now why is mucin important? Because it co plays the mucus in the intestine as we saw. So there's the story. There's serotonin. And I could go, I love the brain and everything, but I'm a food scientist. So I stay at the level of what is the influence of food where our intestine.

And of course, the most important part is the neuronal intestinal system. It’s the first, it’s the gate, it’s whatever's going to open in our vagus nerve or whatever is going to go upstream to our brain and reasoning starts there. And I'm about to finish, but not without first telling how we are studying this. We get undifferentiated neuronal cells we call neurospheres.

And then what normal people do in this is they get their growth factor, their interleukin or whatever, and then they measure how these cells differentiate into forming neurons or groups of neurons we call glia. So here is the preliminary that I just saved. These results have not been published, but I wanted to really thank the people that invited me with getting you some surprising results.

Two days of these neural spheres being exposed to the digest-, because we’re digesting the MFGM. You see, the baby doesn't see the MFGM intact, it digests it. So my students says, ‘no Dr Jimenez, we have to digests it’. So, I send them to Spain to learn how the digestion was done. And I'll just show you right here, these are the undifferentiated.

There's the neurons. And in 2 days we double the rate of differentiation. But your doctors tell me this wouldn't be important for neonates and premature babies. I mean something that helps their intestines, neurons differentiate and grow, I think is very important. This is just a picture of what the cell counter is looking between just, undifferentiated spherical neurons or the ones that are starting to look like neurons.

Take home message. Membrane attachment modification. We have evidence that the MFGM takes an important role. There is induction of mucins in the intestine by the MFGM and by the bacteria that have seen MFGM. Well, that's very important for what we have make the connection: microbiome – food. The induction of extra polysaccharides in lactic acid bacteria, which we have studied a lot in dairy technology, happens to be also very important because, by the way, those groups of beneficial bacteria, the doctor always talking about, are the ones that in our lab we select because they have the propensity of make exopolysaccharides. Connection?

I really think so. And the production of neurotransmitters in bacteria are common in the healthy microbiome. I just forgot to put a healthy, because serotonin really regulates a lot of our moods. So this is whom I want to thank, these are the real workers in the lab. Noticed they’re very young people, which gives me faith in the future.

And yes, Ohio State are American football champions. I don't let people forget about that. For your attention — thank you very much.

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