[Music] but that I think I can call him the first speaker which is Phillip funny Marcel and and he will he will focus mostly on the microbiome and the effect which and the interaction between the microbiome and the host and I think this is what is key this morning and that word was already raised yesterday the key is I think inflammation and inflammation we'll hear a lot more about it from teal Phillip can you give it a start so first of all thank you for the kind invitation and thanks to Rick for introducing me I'm always a bit afraid when when the week starts to speak that is just certainly going to give the whole presentation because I think you like to do that so you're kind of tricky audience to me because you're a lot of nutritionists are in the room and I'm definitely not a nutritionist so yesterday I sometimes had a little bit of difficulties to really understand everything of feed tables and metabolize energy etc it's not so easy to move for me so as some kind of revenge I will talk about bacteria so but when I talk about I will talk about microbial metabolites so what do bacteria do with all the substrates and the ingredients the nutrients how do they use that and what can they produce in terms of beneficial metabolites and I will just focus on beneficial metabolites you also have a lot of harmful metabolites but you have to start somewhere to me really when I talk about intestinal health there's one thing that is of major importance and this is already said yesterday by Sergio and this is intestinal integrity okay so what you see here in this slide on the left this is actually the normal condition with epithelial cells that line the intestinal tracts that are very densely attached to each other and this is because you have these structures here this is tight junctions or desmosomes you have manatee for different intercellular junctions that should really go epithelial cells very close to each other and this intimate connection is essential because when you don't have that when you for example have cell death or loss of these intercellular junctions that is really a starting point for inflammation in the gut and what you get there at that specific moment is nutrients that leak out from the gut wall towards the intestinal lumen so this is energy loss of course and a lot of different microbial metabolites but also toxins and also bacteria that enter the gut wall and that is of course what we do not want because once bacteria or bacterial fragments attach to the basal or lateral surface of these cells they really connect with some kind of receptors that trigger an inflammatory cascade and that's how gut inflammation is really starting so some epithelial cell defects or some tight junctions that are loosened and then bacterial components that end up to the basal surface of these cells and that's how you get inflammatory response so basically what is causing this integrity losses in the animals there are many different things and yesterday it was said that LPS is triggering this intercellular junctions defects and it's not really true but they'll feel LPS is what is triggering inflammation but the initial defect is caused by something else it can be a lot of things of course in the poultry industry okay if you think about the chicken in the field there's a lot of different potential triggers that can invoke this initial intestinal damage epithelial cells can make me damaged by the studio toxins by enteric viruses but mycotoxins and as already said by Rick one of the things is sometimes when you have this over feeding you have bacterial overgrowth and the most suspicious bacterium for bacterial overgrowth disgust in your perfringens and this is toxin producer and this is really little for intestinal epithelial cells it damages intestinal cells and then you can also have a huge inflammatory cascade that is starting now you don't even have to visually see in microscopical damage because you always have some kind of subtle subclinical damage present if you look at the intestinal wall of a broiler chicken and pathologists should know we'll know that if you just take a section in the intestine of a broad chicken that you always have some kind of damage what is the consequence of this intestinal integrity losses well first of all inflammation of course right on top here on the histological pictures you see a section of the small intestinal wall offered animal with so-called dysbiosis okay now what you have here is all these well this is the normal condition these are very long slender villi which means a lot of sort of surface okay on this side here you have this very thick blunted feel I feel as fusion and all these brown dots or inflammatory cells that infiltrate the intestinal wall so this is inflammation of course initially caused by first of all some intestinal epithelial permeability increases or it definitely losses and of course these are just consequences wet litter dermatitis problems which is of course a welfare indicator also bacteria that translocate from the intestinal lumen to the bloodstream and cause some lesions at other sites in the body so for example famer femoral head necrosis cause power enterococci this is just a condition that is really arising from the gut this was just bacteria that are present in the gut lumen and due to some epithelial defects they can migrate to the bloodstream and go to all other places in the body and cause other diseases in this case lameness condition of course and what you see here is just well it's not so visible maybe it's better on the other screen this is an inflamed inflamed gut you see this reddening and the blood vessels that are congested this is a normal good so a lot of different that I can that are really a consequence of this intestine integrity losses and basically what I want to talk about today's which bacterial metabolites which metabolites that bacteria produce can affect intestinal epithelial integrity and they're quite some different ones this is what I show here I'm just going to talk about the green metabolites of these the green ones or the good bacteria the red ones or the bad bacteria that's quite easy and I want to talk about these ones which ones are the beneficial ones the positive metabolites and how can we use dietary strategies to steer this production well this is just a normal I don't even know whether it's normal you or the nutritionists I was just feeding table there are sugars proteins fat and what I want to do is just explain you just some examples of how sugars fats and proteins can yield beneficial metabolites that are produced by bacteria and thinking about the intestine and all the microbes that produce the microbes that are present in the intestinal tract you see that especially in the Sica there's a huge capability to ferment substrates that is the place where most bacteria are present 10 to the 11th of bacteria per gram of intestinal content which is huge you also have some fermentation in the crop and much less of course in the small intestine so if you think about bacterial fermentation you have some in all different sides of the intestinal tract but mainly here in the sickle compartment that is the place where different undigested substrates are being used by bacteria to be converted to different and metabolites now if you would look at the microbiota composition of an adult animal this is just an example of a human you would see that all different fibers and all different undigested substrates can be fermented by bacteria okay what is just listed here it's probably not so easy to see but these are different structures of different polysaccharides starch even pectins cellulose even Arabi knock silence and all the arrows here they just point to the bacterial enzymes that can degrade these different substrates so in principle in theory in the gut of any animal or human the capability to degrade even these very difficult to digest sugars is present okay so you can wonder even why we give Scylla mazes well of course because of in a chicken there's a very fast transit time and a chicken especially broiler chicken of course doesn't have really a full adult microbiota composition it's a baby bird and that's why it doesn't contain the microbiota containing all these different enzymes that until we can degrade all this well difficult to digest substrates so that's an issue really that we are working with animals that don't have an adult microbiota composition and that's why it's so sensitive to many different disease conditions to some pathogens etc now what we need really to degrade substrates or my metabolic networks or microbial networks and that means that ideally you need a very high microbial diversity this is because if you have this kind of complex substrate here for example are up in oksanen you need very a lot of different steps consequence steps to really decorate this complex polysaccharides to its smaller components and you need different bacterial species or ginger you need different enzymes that are being produced by different bacterial populations and that's why this microbial diversity is so important that this is we a measure for let's say for intestinal health the more first microbiota is the better for the animal and the better it's capable - well to be health-promoting that's a health promoting microbiome I will tell you a little bit later on what it is talking about sugar fermentation polysaccharides fermentation this is just a general team of policy of a polysaccharide degradation pathway by microbes and what you see here on top is just a polysaccharides the cell wall components for example of cereals including Amy seller losses such as Arabi knock silence pectins cellulose whatever kind of polysaccharide and really there are some bacteria this is just a bacterial phylum but there are a lot of bacteria that are capable to degrade these polysaccharides to smaller components so to oligosaccharides okay this is just by some enzymes that these bacteria produce and on their turn these smaller oligosaccharides are converted to the nth metabolites so this is an example of what is called cross feeding one bacterial group that is producing other substrates for other bacterial groups that are really further using this product to produce the nth metabolites and the end metabolites typically for sugar degradation these are short chain fatty acids these are gases and lactic acid that's always what you end up with when you speak about carbohydrate degradation by the microbiota of course there are different ones there are different short chain fatty acids and as recall already said in the introduction there's quite a difference between what is different short chain fatty acids are doing for example but you late and propionate we know that they are really have a very strong anti Salmonella activity while acetate is not having that so that already tells you that if you want an anti Seminole activity of a microbiota you need quite high concentrations of these two short chain fatty acids and as for example Salmonella is really residing in the Sica and this is also produced in the seeker there's some way to reduce someone Alok organization is anyway steel towards budget rate production I will come back to this in a minute now another way of cross feeding is for example production of lactate that is used by other bacteria to produce butter rate and this is a very important one and this is really a very important health promoting bot way that bacteria can carry out this is also cross fina cause in fact lactate this is of course produced by a lot of the selector pasilla even bifidobacteria but let the gas it on itself is toxic okay it's toxic for epithelial cells and high concentrations it's not really of any benefit sounds a little bit strange but why is lactic acid or lactic acid bacteria in general why are they good for gut health that is because this lactic acid is consumed by other bacteria to produce butter rate and this is the beneficial component not lactic acid and this is also a gross feeding pathway we should need microbial diversity you need different bacteria that catalyze these different steps and this is the beneficial end product the same you have more or less with Gauss's das production means hydrogen and carbon dioxide when you degrade sugars when the microbiota is degrading sugars and on the other end you can use this hydrogen or different bacterial populations can use this hydrogen to produce other gases and as you can see here depending on which bacterial population is using the hydrogen you can have different and components and hydrogen sulfide for example is a toxic metabolite this is a toxic components for epithelial cells and I think you've seen our which direction I'm going to if you have a lot of bacteria that for example have this pathway towards Butterick acid you have a beneficial microbiota composition if you have a pathways that go for example to hydrogen sulfide you have more or less a toxic microbiota composition so it's always competition between different microbes for different substrates and the ones that are winning the competition we lilt either beneficial or harmful metabolites also lactate for example as I said it can be used by battery producers to produce butter rates but it can also be used by so-called sulfate-reducing bacteria to produce hydrogen sulfite so also here you have this very clear competition between bacteria that can go either that way or either produce this one it's very simple example you can of course expound this with ten or even hundreds of other metabolites but this competition is very very important and as I already said but your rate is a very important key fermentation acid and the reason really is because if you add butter rate to intestinal epithelial cells you can easily document that in in vitro simulations what this simple fermentation acid is doing is strengthening while it's promoting intestinal integrity so it increases expression of these tight Junction proteins that seal the epithelial cells so it makes sure that the cells are connected to each other very densely it's a very strong anti-inflammatory compound you will see that if you look into the scientific literature it's well investigated that it is really reducing pro-inflammatory cytokine and expression is very strong anti-inflammatory compounds it increases stimulation of nuisance of antimicrobial peptides it's doing reality things that are beneficial okay so it's a very very important acid now just a little bit of details on how we can promote butter rate production one of them we did some studies with the use of enzymes and actually what we did is this is number by nadya yaqoob' watch it is just taking wheat and pre digesting wheat with an enzyme mixture okay and just take the the supernate of this pre digestion and feed it at very low concentrations even to broiler chickens so what she did is really just if you take with you enzymatically treat it in humid conditions of course in water and then take the degradation products you will see that of course the degradation product has quite a low polymerization increased so it's small fragments that you give and this is for example a rabbinic assignment oligosaccharides and of course to decrease the molecular weight and also the viscosity but when you feed this to animals at even percentages below 0.5% you see that in the gut of these animals the butter rate producing bacteria from the so called Clostridium cluster 14a and these are bacteria that consume lactic acids and don't use butter rate they are increasing okay what I mean is that if you talk about enzymes you have different possibilities to explain the efficiency of enzymes you have the gage effect so the list of components outside of the the well the plant cells you have decreases in viscosity but you also create some kind of a prebiotic effect okay because the components that are released from well this wheat degradation by enzymes they promote colonization in the seeker by these bodyweight producers that's one small story another one is using wheat bran this is just some data of a series of experiments that we recently performed and people that will go to the ESPN will see the whole story by one of the PhD students of us and what she actually did was using small particle size wheat bran this was produced by people at the University of Louisville just by milling and sieving so this is the normal wheat bran structure with about 1700 micrometer particles this is the small particle wheat bran if you give this to Brewers at 1% in the diet and then just to a full 16s sequencing to determine the bacterial populations that are present in the intestine you see that again species diversity is increasing and that means that of course the potential to have much more different enzymatic activity of functions by the microbiota is much enlarged secondly what you also see is that the the presence of the butter weight produces in the gut is increasing and these families here these are really the two different families that are the key but your weight producers this was strict anaerobic bacteria that are very very abundant but also very important in terms of data and if you would look at scientific literature on any intestinal disease in humans or in other animal species even with any disease that has some kind of gut inflammation actually the abundance or the presence of these two different families or decreased so these are let's say some kind of markers for gut health more or less not really but yeah theoretically they can be more or less even when you dig into this at very lower taxonomic level you would probably find some of them what you also see is that the enterobacter CI are going down and these are greg- bacteria containing LPS and a lot of them or bacteria such as for example it should be clear coli which is an opportunistic pathogen of course so that means that just very simply one percent of small particle size wheat bran is really shifting the microbiota conversation to watch a beneficial bond I'm in the middle here this is just a measure that we use this is a quantitative measurement of the genes the bacterial genes that are present in the gut of the animals the bacterial genes that encode this particular enzyme and this enzyme which has a very difficult name is really the key enzyme in the pathway for better rate production okay so when I also think that this is quite a good marker for gut health this is an increase with about 1 lakh unit that means that the capability of this microbiota to produce budget rate has increased by adding this wheat bran and I didn't show this on these slides but if you give this wheat bran to broiler chickens and you use them in muscleman Ally infection model you will see that some an ally fraction is actually we'd used with multiple you need 1,000 fold to even 10,000 fold so again this is butter rate production and a decrease in all this enterobacter I see I including Salmonella e.coli and potential pathogenic bacteria so this is a beneficial shift in the microbiota composition now just explaining a bit why just administration of on a fairly marginal amounts of wheat bran is really increasing well this butter rate production when you take this wheat bran fractions these very small fractions and you incubate it in vitro in just a fermenter system you will see that some bacteria le attached to this wheat bran so we don't even know yet whether the wheat bran is really converted by this bacteria or this wheat bran is just used as some kind of a well carrier or a table to which all the bacteria or attaching and if you do that if you just do a batch fermentation you add wheat bran you spin down the wheat bran and you extract all the bacteria that have attached to the wheat bran you see that this is exactly more or less the same this is the composition in the normal fermentation fluid and this is the composition attached to the wheat bran so this wheat bran seems to be some kind of a carrier to which all these beneficial microbes attach and maybe they can also convert some of the wheat bran components to this beneficial substantive policy you see also betrayed production going up when you add wind brand to a simple fermenter system which settle in okhla is used as a microbial inoculant finally about sugars maybe the well the degraded component excited oligosaccharides which is the small oligosaccharide of course that is derived from arabic silent degradation if you add this to the feet at 0.5 percent in a diet dietary challenge modeling which we used as you can see high NSP containing compounds rye without enzymes and also some difficult to digest Rodian substrates if you do that and you add 0.5% stylo oligosaccharides to the diet you will see that these are just data from day 26 that you have an increase in body weight decrease in feed conversion and of course also go to all morphology changes this is meaning an increase in ileal village length also a decrease in the number of inflammatory cells infiltrating the gut wall so increasing absorptive surface and again if you do the analysis look at the virtual Milkyway acetate Kawai transferase which is the key enzyme in butter rate production you see this increase each dot is the value of one animal so exciti all exact right administration to poultry diets increases the capability of the microbes to produce but you rate so again this is likely why this is a beneficial compound just something well I couldn't forget proteins and fat so please tell me when I'm close to the end ok because most well known of course are all these well metabolism of polysaccharides and sugars and that's most widely studied and especially the role of but uric acid is most widely still studied I want to be don't know a lot of about microbial protein degradation there's really a lack of knowledge in the scientific literature about what bacteria do with amino acids and proteins I just have this figure here it's coming from review of Davila a few years ago and it's interesting because in this review paper you see exactly what how bacteria are using all the different amino acids okay as an example the normal amino acids you're either deaminated or decarboxylated to yield short chain fatty acids again and also branched chain fatty acids for example is of alaric acids and other compounds and also a lot of amines and poly amines and in fact as we are capable to know really if you have a certain amino acid that you give to a bacterium what end products are really produced the only thing we do not know is what all these end products are really doing on the host what is the effect of all these different end products on epithelial cells or epithelial cell integrity we don't really have too much of an idea sulfur-containing amino acid cysteine methionine often converted to hydrogen sulfite and other about bacterial proteins but aromatic amino acids are also quite important because they produce some in garlic and phenolic compounds and some of them have already been studied just one example is tryptophane abrasion essential amino acid and this is quite an interesting one because this is in fact more or less besides argument probably one that this has been studied with regard to the effects on epithelial integrity and if you look at tryptophan and the degradation products which are three different ones it's in though it's in Daltrey acetates and in Daltrey propionate and this is this catabolism this degradation is done by bacteria that contain a certain enzyme a tryptophan is but especially more important is that in dole which always believed was some kind of a toxic aromatic acid whatever if you add this to a Patil yourself cultures you will see that dye Junction expression is actually increased this is also an end product that is really favoring intestinal integrity this is a beneficial and metabolite of bacteria so this is not only affecting by Junction expression also affecting anthro endocrine hormones expression I'm not going to focus too much on that is also affecting immune cell activity in a way that it promotes for example th17 cells to produce antimicrobial components at the epithelial surface so this is again an example of a metabolite in this case of protein degradation that is thought to be beneficial most metabolites from proteins are believed to be harmful especially in human the tuition is well known that many of these poly amines permit in Qatar for in many other metabolites are harmful but that is really because potentially carcinogenic which is of course not so important when you talk about broilers so we should really look at the acute effects just the final two completely fats this is also something that can potentially yield beneficial compounds now some people in the room that are much more about it than I do but just there's one conversion that I just wanted to show you as an example of a compound that is produced when you have fat well a conversion that a conversion of linoleic acid which is quite common of course in imbroglio diets and this can produce these conjugated forms that also have been proven to be beneficial and this is because this kind of compounds they can activate people camera and people gamma it's really an transcription factor that is anti-inflammatory so also fat degradation or the use of certain fat components oleic acid linoleic acid spermatic acid by bacteria can potentially yield different components that actually affect intestinal health so it's very complex that's really the story and if you look at the types of bacteria that can do this conversion you see lactic acid produces but also the beauty of species and this is quite important to me because that means that this bacteria for example is the most beauteous species they are bacteria that produce quite high levels of butter rates and are also one of the most abundant but irate producers so again these species but us bacteria produce also this kind of conjugated linoleic acid components maybe they produce also in dolls or other stuff maybe they produce a whole battery of beneficial compounds we do not know enough about this to say a lot and this is just but this is you know much more about this these are different different fats and you see that the percentage of linoleic acid T first quite a lot so that's what I mean with steer and you can probably steer the production of this kind of metabolites when playing with the feed composition final parts I just wanted to show you some slides on museum degradation because to me it's very important that all these bacteria that produce these beneficial compounds are very close in interaction with the epithelial cells and that means that probably the most important ones are just okay I have to skip the ones that are very closely attached to the epithelium because if they are sitting here the company was metabolites that are sensed by these cells that's quite ideal I think I'm not going to focus too much on this you have a lot of musing degrading bacteria and probably also their microbial networks are important in way that you need many different species to use the musings and musing degradation is potentially well it's thought to be toxic of course because some post we di can do that but also some beneficial microbes can use musings to colonize close to the epithelial cells and being close interaction with these cells so that's also quite important just to tell you that the brand studies we performed actually is resulting in an increase in certain bacterial species that are already known to be very close in interaction with the epithelial cells so they are located in the mucus and they use mucins they produce for example propionate which is quite beneficial and this is also one that we isolated ourselves that that is musing of mucosa associated to locate in the mutant layer and this has also been shown to be quite beneficial in a way that administration of this particular bacterium it's reducing the incidence of necrotic and to rise this is affecting performance and it's especially when you add this bacterium to the feet to the diet in freeze-dried form this is really resulting in a decrease of the enterobacter see a decrease in entropy okay the e.coli group the sulfate reducing hydrogen sulphide producing bacteria etc so there's either butter rate production or on the other side there's all these potentially toxic bacteria that you must this is the final slide look at you just to show you that in fact all these well they're quite a lot of bacterial metabolites that are beneficial quite a lot of potentially harmful but we do not know enough okay we don't yet have a full overview of all the different substrates that are being used by the bacteria we don't even have a full overview of all the different bacteria that are present in the gut and that's something we definitely need to work further on but I'm quite confident that we need let's say about competition between different bacterial species or groups for certain substrates is a key important defense in promoting cut-offs and this is probably a little bit more scientific talk but I expect a lot of practical questions that we definitely can reply to thank you [Applause]