Dietary Supplement Practicum 2018–Emerging Science: The Microbiome and Nutrition

Dietary Supplement Practicum 2018--Emerging Science: The Microbiome and Nutrition

I'm Barbara Sorkin from office of dietary supplements and it's my great pleasure this morning to introduce the first speaker first my office of dietary supplements colleague dr. Cindy Davis Cindy received her Bachelor of Science from Cornell University and her PhD in nutrition from the University of wisconsin-madison with a minor in human cancer biology she did postdoctoral training at NCI and then returned later to NCI and came to the office of dietary supplements from there she directs our grants and extramural activities at the office of dietary supplements well great thank you very much for that very kind introduction it's my pleasure to be here today what I'd like to do is talk a little bit about the relationship between diet at the microbiome in health so what I'd like to do is give a little bit of background on what is microbiome what is the evidence that diet can influence the microbiome and conversely how can the microbiome influence the response to dietary components and interwoven throughout this is what is the relationship between diet the microbiome and disease risk so humans actually have been considered super organisms and that's because we're a composite of many different species in addition to our human or mammalian or eukaryotic cells they're also bacterial viral are kale cells fungi phage and in fact there are estimates that there are anywhere between equal numbers to up to 10 times more microbial cells than humans the most recent estimate suggests that there are 1.3 bacterial cells for every human cells but that isn't considering the page the fungi and other microbes when we talk about the microbiota we're talking about the microbes in a specific niche so the gut microbiota are the microbes in our gastrointestinal tract and in fact that's about a hundred trillion organisms when we talk about the microbiome we're talking about their collective genome and in fact they're about 100 times as many genes from microbes as there are human genes you might also hear the term meta-genome and that's the combination about the microbial and the human genes or meta genomics and that's the study of both the microbial and human genes together so what do microbes do for us well they have many important functions they provide the ability to harvest nutrients they produce additional energy otherwise inaccessible to the host and that's mainly through the fermentation of soluble fiber to the short chain fatty-acids acetate butyrate and propria nate they produce vitamins such as folate biotin and vitamin k they metabolize carcinogens such as a heterocyclic amines formed in food during cooking they prevent colonization's by pathogens and they assist in the development of a mature immune system in the limit of time i'm only going to talk about the relationship between the microbes and diet so what is the evidence to suggest that diet can influence microbial profiles well the first evidence came from looking at the microbial composition and globally distinct populations and I'll show the landmark study on the next slide but there's also evidence to suggest that long-term food pattern consumption can influence the microbial composition and this has led to the term enterotypes and it's the idea that people that eat similar diets have similar microbial composition on regardless of where they live their age their gender and the two main enterotypes are the Bacteroides jet and Tarot type which is in people that eat a lot of protein and fat and the private ala genotype which is in people that eat a lot of dietary fiber the other source of evidence is from short-term intervention studies including low versus high fiber diets animal vs. plant food for M sources and macronutrient ratios so this is a landmark study that was first published suggesting that there's a relationship between what people consume and their microbial composition in this study they compared the microbial constant composition in children from Africa with those in Italy as you can see in Africa the main microbes are Bacteroides particularly private Ella and Xyloto doctor in contrast in Europe the product children the predominance are Firmicutes why this is actually important as the two main Bacteroides private Ella and zai Lana bacter actually have specific enzymes present in them that'll allow them to hydrolyze cellulose and xylem which allows them to actually extract as much energy as possible from the very high fiber diet that these children consumes so since they have a very low intake of energy it can maximize the amount of energy that they can get from their diet there's also evidence from short-term feeding study in this study human ten humans in a crossover design were fed either a plant-based diet or an animal-based diet now I realize this is a very complicated slide so I'll quickly walk you through the highlights the plant-based diet is shown in green the animal-based diet is shown in red and the feces were collected for four days and analyzed this is probably the most important part of the figure where they look at the similarity and differences in microbial composition among the different individuals among the different days and what I'd like to highlight is you notice that our red tend to come together and that all the green tend to come together suggesting that diet is actually more important and that when they're on these extreme diets that their microbial composition changes and that it's more similar so as I said the short term feeding alters microbial community structure and it overwhelms inter-individual differences in gene expression and in fact a modified specific metabolic pathways this idea that diet dominates host genotype in shaping the gut microbiota has also been shown in animal studies I don't usually go from the human studies to the animal studies but the human study was actually published first and in this study they compared five in braids inbred and more than 200 out bred mouse strains that were quite fed either a high-fat high-sugar diet or low-fat high-fiber diet and they looked at the microbial composition the results are shown here in this principal component analysis that looks at similarities between different samples you can see that all the mice when they were fed the high-fat high-sugar diet clumped together and all those when they were fed the low-fat high-fiber clumped together and this was specifically due to an increase in Bacteroides when they were on the low fiber low-fat high-fiber diet and in increased firmicutes when they were on the high sugar high fat diet so how can we use this information what type of dietary modifications can we do to influence the microbial composition well the first is the use of probiotics and these are foods or dietary supplements that contain live bacteria usually lactobacillus or bifida bacteria prebiotics which are non digestible food ingredients which can selectively stimulate the growth of gut bacteria examples include inulin other oligosaccharides lactulose start resistant starch and in fact a number of dietary fiber Sabah's have been shown to exert a prebiotic effect a symbiotic is a combination of a probiotic with the prebiotic and the idea is that the prebiotic would actually be a food source or an energy source for the probiotic and in fact other dietary factors can also influence microbial composition microbes are actually able to metabolize a lot of the polyphenols present in tea cocoa and wine and use those as an energy source and many spices are actually antimicrobial and that's why they've been used for centuries in food preservation because they actually inhibit the growth of many different I grubs so I mentioned probiotics the World Health Organization developed defines proba allows microorganisms that when administered in adequate amounts confer a health benefit on the host the key parts and the definition of probiotics are live adequate amounts and health benefit these include everything from food to dietary supplements to prescription drugs and examples of typical probiotics include lactobacillus and Bifidobacterium but actually yeast resolve from served as a probiotic and other bacteria such as ecoli and bacillus we often think of e.coli as being pathogenic but it actually depends on the specific strain and there are large differences and there actually some that are being sold as probiotics fermented food and beverages typically do not contain live cultures due to the heat treatment filtration of food processing to improve shelf life hence they are not true probiotics examples include sourdough bread fermented meat sauerkraut wine and beer in vinegar fermented dairy products include yogurts natural cheese butter milk kefir and again these may or may not be considered probiotic and whether they are would depend on the bacterial levels when eaten and whether the bacteria have been shown to confer health benefits another important source of probiotics is dietary supplements many dietary supplement probiotics contain more than 1 billion live cells per gram and in fact many contain upwards of 10 billion live cells per gram and they're probably better in terms of their efficaciousness if they have multiple strains of bacteria this is data from the nutrition Business Journal which looks at the sales of dietary supplements in the United States and this is data published in 2017 which looks sales in 2016 that year probiotics were the third most popular dietary supplement sales were greater than 1.8 billion dollars per year and it increased over 17 percent between 2015 in 2016 in fact there are many proposed health benefits of probiotics because they could potentially can normalize the intestinal microbiota composition this can lead to colonization resistance and suppression of both endogenous and exogenous pathogens it can control irritable bowel syndrome inflammatory bowel diseases and allergy symptoms and they also have metabolic effects which can affect colon cancer risk lactose tolerance and serum cholesterol levels so what is the evidence to really suggest that probiotics do have these health effects I'm gonna utilize one meta-analysis to kind of show what are the strengths and weaknesses of some of the science out there related to probiotics and I chose this one because it was published only about two months ago in this study they do typical studies that looked at the relationship between probiotics and BMR they identified 13 subjects and when they summarize the data they found that individuals that consumed probiotics compared to those that didn't had a point to 7 decrease in BMI and this was statistically significant so of course the headline is probiotics our benefits are beneficial for your weight I want to point out though that there's some consistency in the studies as you can see some are on the website and others are statistically significant and on the right side and I also want to put it in perspective most of the studies are of short duration there's small sample size and are actually under power to actually look at the relationship even more important they utilize different probiotics probiotics vary depending on the bacteria that's being studied or at food either even as a bacteria and in fact they also vary in terms of matrix they use the doses they use we can go on and on most of the trials are not pre registered but perhaps the most important point is that this effect is not clinically significant I said that there was a change in BMI of 0.27 however if they actually limit the studies to only those individuals that were either obese or over to begin with and they found that the change was hacked so there was only a change in BMI 0.14 I don't think anyone would think that that's a really important change so where is the science related to probiotics there is preliminary evidence that some probiotics are helpful preventing diarrhea caused by infections and antibiotics and improving symptoms of irritable bowel disease however the US FDA has not approved any probiotics for preventing or treating any health problem probiotic supplements should contain at least 1 billion live cells per gram and are probably better if there are multiple strains of bacteria if people are generally healthy probiotics do have a good safety record however there have been reports linking probiotics to severe health effects such as dangerous infections in people with immune set weakened immune systems what we don't know is which probiotics are helpful and which are not not all probiotics have the same effect and the effects are likely strain-specific how much of the probiotic people should take and who would most likely benefit from taking probiotics to be honest whether a probiotic is beneficial for a specific individual probably depends on the background microbiota composition however this is something that's usually not taken into effect as I said there's also evidence to suggest that many different dietary polyphenols can influence the consumption influenced microbiota consumption this was some studies that I was involved in we looked at the bacterial abundance after consumption of cocoa flavanols and we found that in pigs given the highest dose of cocoa flavanols there was a significant increase in the amount of Bifidobacterium in their feces and lactobacillus in their colon well it's easy to say your microbes changed but the question is does it have a biological effect and we actually saw that cocoa powder consumption decreased the expression and tnf-alpha and tolike receptors two four and nine gene expression showing that it was actually having an anti-inflammatory effect well i've mentioned how various dietary components can influence the number and types of microbes however this is a two sided relationship and i'd like to switch gears now and talk about the relationship of how the specific microbes that are present can influence the response to dietary components bacteria can produce new compounds from a number of food components a few of the dietary components that are metabolized by gut microbes and then metabolites are shown here in the in the limit of time i'll only focus on the top two dietary fiber choline and carnitine but I should say oftentimes these microbial metabolites are more biologically active than the parent compound so one of the dietary components that has been linked with health effects is dietary fiber dietary fiber has been associated with the decreased risk of colon cancer when the American Institute of cancer research in the world Cancer Research Fund summarized the epidemiologic evidence related to dietary fiber and cancer they found that for every 10 gram intake in dietary fiber per day there was a 10% decrease in the risk of developing colon cancer moreover this effect was on Lydia dietary fibers are fermented by colonic bacteria to form short chain fatty acids including butyrate acetate inappropriately butyrate is the most widely studied in the preferred energy source of kelana sites and in fact it has differential effects in normal versus cancer cells which I'll show on the next slide so this court cartoon shows the relationship between dietary fiber and colon cancer dietary fiber can be divided into both soluble and insoluble dietary fiber insoluble fiber actually increases the colonic transit time so there's less for carcinogens to be in contact with the aquatic cells in contrast soluble fiber is fermented to shore trained fatty acid the most important of which is butyrate enormous cells butyrate serves as an energy source so the colano sites utilize it and actually increase proliferation yet because of its effects on energy and it also increases differentiation in contrast in cancer cells they undergo something called the Warburg effect and what this means is they actually start utilizing glucose as a preferred energy source so since they're already utilizing the glucose for energy it means that the butyrate can affect other molecular targets within the cell and the most important molecular target is H DAC or histone deacetylase activity and what it actually does is inhibits H stack which leads to decrease proliferation and increase apoptosis there's a some evidence to suggest that dietary fiber can affect bacterial diversity it was found that when mice were fed a high-fat diet excuse me a high-fiber diet there's a large amount of bacterial diversity if they're maintained on this high-fiber diet for multiple generations they maintain that bacterial diversity in contrast if they're switched to a low fiber diet they reduced there's a decrease in microbial diversity and other generations there's a further and further reduction in diversity if they're switched to a high fiber diet from the low fiber diet there is some recovery but not complete recovery suggesting that you're actually having extinction of certain microbial species well why is this important well we know that diet intake of dietary fiber has been associated with decreased risk of cancer inflammatory bowel disease diabetes but we always don't know the mechanisms one of the things we do know that is in all of these diseases there is actually a decrease in microbial city so I would postulate that one of the mechanisms where dietary fiber is working to decrease the risk of many of these different diseases is through its effects on promoting microbial diversity there's also evidence to suggest that dietary fiber can protect the mucous barrier in this study nota biotic mice that is my said actually lack microbes were colonized with a cocktail of human microbes this was a cocktail of 14 different microbes that represent the five major phylum in humans and then they were either put on a fiber supplemented or a fiber deprived diet and infected with an enteric pathogen and what they found was that mice fed the high fiber diet have more fiber degrading bacteria affect mucous lining and are actually protected from the colonization with citrobacter attention in contrast animals that were fed the fiber deprived direct actually promoted expansion and activity of colonic mucosa degrading bacteria particularly at Berman SIA and they are more susceptible to the rhododendron infection and actually developed colitis while the study is so important is all the mice were colonized with the exact same bacteria and what this means is that the ability availability of potential food sources actually influences the growth of different bacteria and can affect the disease susceptibility so even if people have the exact same microbes what they eat can influence whether they might be susceptible to various diseases there's also evidence to suggest that there's a relationship between diet microbial metabolism and cardiovascular disease dietary carnitine which is present in meat and phosphatidylcholine and choline which are present in cheese and eggs can be metabolized by gut bacteria to form trimethylamine the trimethylamine travels to the liver works metabolize to trimethylamine oxide in both human and animal studies this formation of TMAO has been associated with increased risk of a number of different diseases including heart attack stroke cancer obesity etc you know it's so easy to look at a single nutrients and composition and we really need to be looking at the totality of the diet so this study looked at the relationship between dietary Allison and carnitine in the production of trimethylamine oxide most were put on about are their child diet the diet supplemented with carnitine carnitine and Allison or the child diet Nelson and for those of you that don't know Ellison is one of the bioactive sulfur compounds present and garlic the animals were on the diet for six weeks and then they were given a carnitine challenge and the results are shown here and I'll just take you to the top side if you look at the red diet the animals that were fed carnitine there's a large increase in the production of TMR all after the challenge in contrast if you look at the yellow group which is a group that got carnitine and Allisyn the ellison actually inhibited the increase in TMAO production after the carnitine exposure suggesting that we really need to look at the totality of the diet and I'll give you another example on this I think most people have heard about the French paradox the whole idea that people in France eat a lotta drink a lot of red wine eat a lot of meat that have a decreased risk of cardiovascular disease well there's also evidence to suggest from animal studies that resveratrol has a similar effect and actually inhibits the production of TMAO so perhaps this inhibition of tal production by resveratrol is actually contributing to the French paradox another important health effect is obesity as we all know obesity has been increasing over the years in the United States and is also associated or as has been suggested to be mediated by changes micro biome you know it's something that's happening more and more to all of us and Kelly and take us one of those things that more is not necessarily better but interestingly as we all know some animals can eat more and grow more than others but there are actually some animals that can eat more and grow less and this is germ-free animals those are the animals that lack a microbiome early studies suggest showed that when you compare germ-free animals to conventional animals the germ-free animals had 42% less body fat despite the fact that they consumed 29% more Chow than conventional animals however mice practiced something called caprara G that means that they'll actually eat each other species and if you take these germ-free animals and put them in the cage with the conventional animals within two weeks their body fat and their child consumption are similar to the conventional animals suggesting that these effects are being mediated through the microbiome there's also evidence from transplantation studies to suggest that the microbiome in obese animals is different than those in the lean animals and it can be transferred in these three different studies they took motor biotic animals that's animals that don't have a microbiome and they gave them the microbiome from obesity animals whether it be genetic induced obesity diet induced obesity or humanized mice that's mice getting feces from lean or obese humans and in all cases they found that those that got the obese had a significant increase in body fat compared to those that got the lean and there's also evidence to suggest that obesity can alter the gut microbial composition in these early studies when they compared both genetically induced obesity and diet induced obesity they found that the lean animals have significantly less from acute and significantly more Bacteroides compared to the obese animals these types of studies have been replicated by a number of different investigators but only when a high-fat diet was utilized not when a low-fat diet was utilized and I'll get to why that's important in a few slides but while we see these effects in animals the question is are they relevant to humans in this study that compared twelve unrelated obese subjects and they found that the obese subjects had significantly more firmicutes and significantly less vector-a duties than the leap than five lean individuals moreover if the subjects were put either on a carbohydrate and/or a fat restricted diet for a year and allowed to lose up to 25% of their body weight there was a significant increase in Bacteroides that correlate with changing weight so this evidence suggests that at least on an individual level that an increase in Bacteroides appears to be beneficial in terms of weight however when meta-analysis were done comparing several large studies including the NIH human microbiome project metahit in Europe and a lot of other large studies there was no correlation between the backdoor duties to film acute ratio and body weight and this is probably because of the large inter-individual variability among individuals as I said before you know there are a lot of studies suggesting that there's a relationship between obesity and the microbiome however part of the problem with a lot of these studies is that they compare animals that are fed a high-fat diet compared to those that are fed at cow diet and perhaps we're actually utilizing the wrong comparison so this was a wonderful study published in the last six months which really tried to disentangle the effects of diet versus obesity they have three different diets in this study a child at it only find low fat diet and refine high-fat diet as you can see the Chow diet and the refined low-fat diet had a similar macronutrient content whether we find high fat diet was very high in sat however they had very different on fibre content the child diet had 15% of the diet is fiber and the fiber came from many different sources and was a non purified diet in contrast the refined diet had 5% in the diet as fiber and it was all cellulose and this is important because cellulose is a non soluble fiber so the bacteria can't actually utilize it here is the data in terms of both body weight fat mass and glucose tolerance those nice february find high fat diet had significantly higher fat mass total body weight and blood glucose concentrations yet there were no differences in the refined low-fat diet compared to the ciao diet when we looked at the Pharma cuties tobacco ad DS ratio there were no significant differences between the two refined diets compared however these were both different than the ciao diet both in the colon and in the feces so what does this data mean it means that the high fat diet induce changes in mouth gut microbiota composition and these effects were due to diet not to obesity that glucose intolerance and obesity and mice is linked to a high-fat diet not to changes in the gut microbiota and one of the hypotheses out there when they started showing these differences in microbial composition in animals that the high fat diet was that they were better able to extract energy from the diet but this data actually shows that the Seco fermentation the energy harvest is decreased not increased by the high-fat diet as demonstrated demonstrated by the decreased production of short chain fatty-acids but the important take-home message from all of this is that the choice of diet is important to studies of microbiota composition as nutritionist we always believe that the best diet to utilize is an AI n 93 or an AI n 76 diet because it's a purified diet we know exactly what we're feeding however they utilize cellulose which can't be utilized by the gut bacteria and it's not going to be as an energy source for them so there's a need to really better understand what diet you should be feeding and to make sure that all groups are fed an appropriate controlled diet rather than just comparing a child with a purified diet so I hope I've convinced you that there's a dynamic relationship between the numbers and types of microbes and dietary components and energy consumption that both of these can influence the microbial metabolites and specifically disease risk but the question is is can we utilize this information to predict what type of dietary changes we should be doing to influence disease susceptibility there was a study that was published a few years that actually did this this was a study from Israel and they had a cohort of about 800 individuals and they looked at their glycemic response to different foods and what they found was that if you feed this different individuals the same food you're going to have large differences in their increase in blood sugar as a response to that food and they wanted to try and understand can we predict how different individuals will respond to different foods so they utilized a combination of that microbiome analysis blood tests questionnaires anthropometrics food diaries on continuous glucose monitoring after giving specific foods put this all into a computer and developed a computer algorithm and said can we validate these results in a validation cohort and can we do a dietary intervention study and what they found that was that there was high interpersonal variability in the post-meal glucose observed in the 800 person cohort they utilizing the personal and microbiome features enabled accurate glucose response prediction and in fact the prediction was accurate and superior to common practice in an independent cohort and most importantly when we took twenty twenty six individuals gave them a control diet based on how they estimated or predicted that they would respond they actually found that the personalized dietary interventions did successfully lower post male glucose well this is very interesting results the question is always can be can these study be reproduced in a different population well there is a company it's called day two you can go and google them and actually provide a sample and get an app if you want to find out your microbiome and what you should be eating but investigators at the Mayo Clinic what the app and did an independent study in 350 people trying to see can they reproduce these results in different people and or were they able to predict response and improve things and they found that they completely reproduced the results these studies are currently being submitted for publication and there's also a push to do the set well those studies are currently being ducted and in a longer term follow-up six to twelve months as well in patients with pre-diabetes and gestational diabetes the other question is is can you extrapolate these results to other disease conditions so I need you to remember that when you go to eat lunch today that you're not just feeding yourself you also be feeding the microbes and what they may want to eat might be different than what you want and then you need to take a matter genomic view of your dinner plate rather than the traditional view think of all the different dietary components that can be metabolized by con bacteria what these metabolites are and what their biological effects might be so the take-home message is that microbiome research is an emerging area of science and there are many research opportunities available the microbiome is integral to human physiology maintenance of health and development of disease that there's a two side relationship between diet and microbiome and that nutritionist statistician Xin the food industry need to stay actively informed about advances in this field but this field still is it at the preliminary stages and most important bacteria are your friends and I'll take questions now thank you very much [Applause] so any questions hi I need up and joining from Hopkins I had a question about short chain fatty-acids and the concentration levels so you talked about them as mostly beneficial but sometimes they can also be seen as harmful like propionic acid and butyric acid for the nervous system because they have broad range implications on gene expression and oxidative stress and inflammation and they've been associated with like autistic disorder and schizophrenia so could you comment on maybe if it's the difference in like concentrations or is it you know maybe different acting methods of action that they're that they pose I haven't really followed the pro prion ik literature as much but a lot everything the dose influences a poison also it's a specific I was really focusing more just on the butyrate and its effects on beneficial effects in the gastrointestinal tract thank you welcome yes okay in western countries and I I was born in India and I'm proud American I made observation that we don't talk about constipation in this nation at all which is in my opinion could be playing very important role in pathogenesis of many diseases including obesity and of course many others absolutely and it's probably influenced by the microbial composition and there's actually a large group at well there's a large group in a number of different societies but are saying are there standardized measures that we should be including when we're doing all different types of human studies to better understand changes in diet and how they're affecting microbial composition and part of it does include stool frequency and composition and things to be thinking about with respect to probiotics and dietary limit form how do the microbes survive the acidic environment of the stomach is it something to do with the formulation of the capsules yes so the question was with probiotics how do they survive the acidic environment in the stomach and companies put a lot of time into looking at the best way to package them to make sure they do in fact one of the issues with a lot of probiotic dietary supplements is that they have to have the stated composition at the end of the half-life so most bacterial dietary or probiotic dietary supplements actually contains significantly more alive bacteria than are on the packages just to make sure at the end of the shelf-life that they actually have that number of bacteria so I know it's noted that in your presentation you're very carefully distinguished in the mouse data certainly between microbes detected in the feces versus microbes detected actually in the colon so what are the latest data on the extent to which taking a probiotic whether in food or in capsule form can actually change the ecology and and and the the organisms living inside your gut as opposed to what's coming out the other end okay so the question was can probiotics and humans influence microbial composition and the answer is in most individuals no there have been a number of systematic reviews and meta-analysis that have actually looked at are there changes in microbial composition with probiotics and in general no that's not to say that for some individuals there aren't and it probably depends on your baseline microbiota whether how long you're taking them and the amount you're taking what you're eating in combination but that doesn't mean just because they're not colonizing that they may not be having beneficial effects because they still can be it was during biological effects while they're actually there any other questions yes um first one little comment at consumer we had we retest the probiotics on the market years ago we found that it many had only you know half or less than they claimed they've gotten much better I think they realized that they weren't shipping and storing these things properly so most recently we have we're not seeing so much of a problem my question is similar to some of the others before so some of the most common uses of probiotics now are for taking them with antibiotics to prevent the antibiotic associated diarrhea what's the is do you think that's a real effect I mean there's lots of studies mostly supported by industry that show that what do you what do you make of that and I think that's one of the instances where they've shown the most efficacy the question is still which probiotic for which individual but there definitely is when systematic reviews and when systematic reviews are done they definitely in general do show a protective effect that's but for a lot of the other health effects is not as strong I do want to comment on your first question about the analysis of probiotics I should mention that the u.s. Pharmacopeia has actually had a group of investigators right now developing protocols for analysis of probiotic dietary supplements that can be available there in development out to be able to actually analyze and show that they contain what they do contain is that breaking it down by strain yes and is that using any DNA technology or just plating and things like that it's DNA technology question back hi thank you for the talk so there are many different techniques in identifying quantifying the microbial composition in the community and my question is well how comparable are the results obtained from different techniques like say 16s RNA and DG or chip or shotgun sequencing well changing a method changed the conclusion from ute by using the same sample well so the answer is that there are many different methods and part of the problem is is some of the methods will just get at which microbes are present so if you look at 16s it will tell you which microbes are actually there if you go to metagenomics such as shotgun sequencing you can get better a better idea of biological function so they do answer different questions and you will get different results part of the problem right now and I say this is an evolving area of science is they're still trying to determine what are the best ways to do these studies and one of the biggest contributor to variability among studies is actually how the DNA is isolated so I think the important thing is within a study everything needs to be processed the same way samples need to be compared to each other whether that then gives you the ability to compare between studies and whether you get the same results it improves things but there still is variability when you go for method a method you're welcome in your opinion is it more important for us to have our patients really be vigilant of what they're eating versus the in taking probiotics or are they complementary I think it's more important to be vigilant and what you're reading I think the studies with dietary fiber show that a high fiber intake is extremely beneficial I think having a lots of fruits and vegetables whole grains it's the recommendations that you would make for almost every specific health condition in individuals and I still believe in terms of there are effects on the microbe it's still the best recommendations that you could be making to thank you very much [Applause]

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