Since probiotics ultimately provide maximum colon health, we might ask why bother with prebiotics rather than just go straight to probiotics? Better yet is to combine both probiotics and prebiotics in our diet. Wonder foods that are fermented not pickled in vinegar may help protect against infections and fungi. Fermented foods and beverages also provide fiber, which aids digestion and could help prevent serious digestive disorders that may be the root cause of many illnesses.
Here are just a few:. Has a harmonizing effect on the stomach, strengthens the acidity of the gastric juice when production lags, and reduces acidity when production is up. I would like to take this opportunity to share three interesting quotes from Dr. Royal Lee, one of the most prominent holistic practitioners of the twentieth century. Long before our current love affair with resistant starch, Dr. Pound for pound, potatoes furnish about one-third less calories than bread—so we may eat three times as much on a caloric basis. There are about calories per medium-sized raw potato, which is much less than a serving of spaghetti, pie or cake.
In addition, its superior digestibility and food value as a source of protein, vitamins and minerals make it ideal for reducing the caloric intake without sacrifice of many essential food factors. However, the nutritive value cannot be obtained on the basis of analysis alone. It is necessary to know the extent to which the various constituents are digestible.
Potatoes have very little fat or sugar and are high in potassium, phosphorus and calcium. The richer they are in protein, the more waxy they are; and the higher they are in starch, the more mealiness they have when cooked. There is an enzyme in raw milk which prevents constipation, and an enzyme in raw potatoes which does the same thing, according to clinical reports.
Certainly a piece of raw potato before retiring can do no harm, and it has produced beneficial results in cases of chronic constipation. Quite possible this is due to a reduction in his ordinary intake of potassium—the mineral which promotes normal heart rhythm. One of the enzymes found in raw potatoes is phosphatase, which promotes assimilation of calcium and iron in particular; another is tyrosinase, an essential component of the vitamin C complex and associated directly with the function of the adrenal glands.
Possibly the best and most current information I found was in my interview with Tim Steele, author of The Potato Hack. His book is primarily dedicated to the tremendous benefits of eating raw potatoes, especially to lose weight. But he also covers the many other benefits and uses of resistant starch. In my humble opinion, this is a book everyone should read and give as gift to those they love.
On page , Steele references no less than 19 phenomenal benefits of resistant starch, including its efficacy with treating of breast cancer, colorectal cancer, removal of certain pathogens from the small intestine, to name just a few! Visit him at his website, The Potato Hack Chronicle. Naturally, Steele is partial to the raw potato, as was Dr. Royal Lee. He advised me to inform my readers of the following: when trying to get RS for health benefits, eating most RS-containing foods beans, wheat, oats, nuts, etc..
A small raw potato contains 20g or more, while a green banana contains 15g or more. Steele recommends that we get at least 30—40g per day. And I always tell them to start slowly. Maybe a teaspoon at first for a week or two, then a tablespoon. There is no unsafe upper limit, but most studies use 40g as their dosage when studying RS effects.
Apparently, fungi would produce amylase as would some bacteria. Amylase busts biofilm. But would RS feed fungi? Or would amylase production be enough to keep populations in check?
So, the basic hypothesis is that RS may be forcing microbes to produce enzymes, acids and alcohols which serve to clean up shop in the small intestine, right where they live in their slimy biofilm matrix communities. Of course, you can also take amylase supplement also thought to degrade histamine , but the results may not be as well-targeted as with RS.
Yes, I wonder about fungi as well. Industrialization allows for year round starch consumption. I mean times were northern populations would have to endure low starch seasons no? So these sibo bacteria would have endured seasonal die off by virtue of winter. Could sibo result from chloride deficiency? Interesting idea William. There was definitely a seasonality to starches that we have eliminated by shipping goods and improving storage techniques.
But the idea that we introduced many more starches with the advent of farming grains 10K years ago may be the most significant overall change. This is SUCH a fascinating discussion. I am venturing into the RS territory. I am absolutely sure that my son now ten could never have handled this when he was younger — his gut was in really, really bad shape.
I wonder a lot of things. What are the best carbohydrates to re-introduce after an extended period of time spent not eating them? If stinky farts are the result of protein-fermenting bacteria, then what the heck does that mean one should do about it? I have added in RS in the form of potato starch, and additional carbs in the form of sweet potato.
Could it be the fructose in the latter? What could cause this horrible symptom experienced by some in our family, otherwise known as an Intensely Itchy Butthole only we call it IAH? She seems to be doing great with sweet potato, and more carbohydrates in general, which I added in about a month ago. This is interesting to me.
We never used the drugs for her reflux, because it made no sense to turn OFF her stomach acid production! Anyway…I should stop typing now, but I really appreciated this post and the subsequent thoughtful, fascinating comments. Hi Sarabeth, Good you are interested in diet over drugs to address GI issues in your family. Never thought I had SIBO until I tried supplementing with potato starch and tapioca starch, which cause a lot of upper GI distress even in small doses.
Most starches except those low in fermentation potential trigger my own heartburn symptoms after a few days. So I plan on keeping an open mind about potato starch as we learn more. I just want to see clear evidence that this approach will help, not hurt people like you and me. Thanks for replying, Norm! I have been actively obsessively? I then switched to tapioca starch. Each time I tried, I would feel fine for the first 2 or 3 days, then I would start to get bad dyspepsia and heartburn. I would then stop the starch, and the symptoms would persist for another few days.
I have read a lot over at Dr. Food sources of starch has never seemed to bother me. I dont have GERD but bloating and have been on cabbage juice for three days now. Can say that I feel it helps much and am hoping for further results as I continue. From what I understand cabbage juice can kill off unwanted bacteria. After taking the cabbage juice the GERD did not reoccure and in the cases I did read that it came back after months or years they were able to use the cabbage juice once again and be rid of all symptoms.
Journal of Chemistry
Hi ing, Never heard of it before — interesting. If you find more info, please share. I havent been documenting my sources but here is one study.. Inhibition of growth of highly resistant bacterial and fungal pathogens by a natural product. ABSTRACT The continuous escalation of resistant bacteria against a wide range of antibiotics necessitates discovering novel unconventional sources of antibiotics. However, it has not been researched adequately for its antimicrobial activity on potential resistant pathogens.
The methanol crude extract of B. The screening method was conducted using disc diffusion assay against 22 pathogenic bacteria and fungi. It was followed by evaluation of the minimum inhibitory concentration MIC. Moreover, the antibacterial and the antifungal activities were confirmed using the minimum bactericidal concentration MBC and the minimum fungicidal concentration MFC , respectively. Remarkable, antibacterial activity was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus, Escherichia coli OH7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus.
Red cabbage is a rich source of phenolic compounds, anthocyanins being the most abundant class, which might explain its potent antimicrobial action. This extract is potentially novel for future antimicrobials, inexpensive, and readily available at a large scale for pharmaceutical companies for further investigation and processing. Also forgot to mention that I have symptoms of fungal rash that are much less after taking cabbage juice. I have taken natural antifungals and when they work they usually cause me mild headaches before seeing results..
I am not prone to headaches so I know when an antifungal is working based on this alone. Thanks for the info. Will have to check it out tonight. I have experiemented multiple times with cultured sauerkraut both organic homemade and raw organic brands and did not find any results for either skin or digestive issues.
I feel that the juice works on another plane other than providing good bacteria like fermented products. I have also tried over 50 probiotic supplements also to no avail. I assume I must have overgrowth of bacteria preventing these beneficial bacteria from implanting. The cabbage juice makes for very normal stools and digestion for me personally. I hope that my main concern -bloat- will be able to be resolved with the juice however after ten years of trying various methods I am not holding my breath. I am however optimistic because results are clearly visible. Right, I would guess the cabbage juice would be more of a weak prebiotic whereas the fermented cabbage is a probiotic.
Let us know if the bloating goes away while taking the cabbage juice. I juice one medium sized head of green cabbage everyday which yeilds me around 10 ounces of juice. I find better results if I drink on an empty stomach and wait a few hours before drinking or eating. I believe studies indicated more ounces spaced throughout the day but I have found many personal testimonies stating around ounces per day worked just as well. From what I have gathered there is not a concensus from the medical research on exactly how cabbage juice works for treating these ailments. I plan to try red cabbage next week.
One more thing I notice is that I no longer require my usual calcium supplement to relieve me of deficiency symptoms. I had a look at the paper Ing. Antibacterial substances from cabbage is an interesting idea as is the tried and true general approach of identifying antibiotics from fungi, bacteria and other marine and land plants. Some of the these sources have resulted in spectacular finds, such as the streptomycin used in the study as a positive control.
Of course you always want to know what the potency is. Another issue arises when you do get meaningful activity — what healthy bacteria are being killed and how does that affect the diversity of my entire gut microbiota. But you never know. If it works for you what can I say? Cabbage juice has a high success rate in studies for healing stomach ulcers which are also believed to have bacterial component. Fermented cabbage juice made my reflux I have the silent type, probably LPR explode in fury, also gave me serious heartburn, and a very sour stomach.
In fact, my desperate attempts to recover from it is what led me to this site. Waiting now impatiently for my copy of Fast Track Digestion Heartburn version to arrive……. Hi there Susan Coe. I am curious as to how you are responding to the FT diet. I also suspect I have LPR and i have been trialling the diet for about a month, in conjunction with apple cider vinegar before meals. Over the last few days my reflux has really kicked back in. I have been eating sauerkraut, so maybe this is this is the trigger??
Curious as to why my links to studies were deleted? I came back to the site to retrieve them for my personal reference and they are now gone. Is it possible you posted the links on the forum under the same name as this article? I suspect that is the case ing. Weird, I now see my links and previous post that I thought was missing. I have chronic klebsiella pneumoniae, as well as Sibo…. Klebsiella is a starch eating bacteria that is linked to numerous autoimmune issues like ibd and alkalosing spondylosis.
Nothing seems to help. Hi Beek, I would think that adding RS to your diet would be the last thing you would want to do. I recommend reading a recent review by Dr. Alan Ebringer on the subject. Thanks so much for the quick reply. I also wanted to get your opinion on oxalates. Doing a low oxalate diet is recommended…. What are your thoughts on this? Susan Owens is the lead researcher on this and has some really compelling insight. But it also seems that the healthier people try to eat, the sicker they get, which is usually when they start doing tons of juicing, smoothies, nuts, seeds, tubers,….
All high ox foods. Oxalate issues are linked to a deficicncy in a strain of bacteria called oxalbacter i killed the spelling, sorry , which helps degrade ox. Thanks for the article on klebsiella…. So Rs would be pretty bad, eh? What macronutrient ratio would be best for me? I suffer from bad constipation too. One thing that caught my eye was the talk on collagen in the article. I had recently tried great Lakes grassfed collagen and noticed it made my joints very sore and also caused symptoms of air hunger and Sob, and tachycardia.
The types of cross-reactive antibodies produced following Klebsiella infections will determine the anatomical location of the pathological lesions, especially in AS. Some antibodies are reacting with HLA-B27, an antigen which is expressed in most articular tissues inside the synovial joints, whilst other antibodies are reacting with types I, III, and IV of collagen, which form an important component of the spinal tissues where the pathological lesions are located. The binding of these Klebsiella cross-reactive antibodies, when present in high titres, triggers inflammatory cascades such as the complement system together with the production of various cytokines resulting in the pathological changes with consequent fibrosis, calcification, and new bone formation leading to the development of classical AS.
Moreover, the raised level of HLA-B27 antigen expressions on the targeted tissues in patients with AS  will make these molecules more accessible and hence will increase the chance of their binding to anti-Klebsiella cross-reactive antibodies. Does this mean that klebsiella also reacts with collagen supplements? I was hoping taking collagen would help my joints and pain, not inflame it. And do I also need to avoid the nonfermentable carbs listed in your Bok as to not feed the klebsiella?
Sorry for the length of my posts…. Its hard to find an expert so keen on gut and flora such as yourself! There seems to be a wealth of blog articles on the topic, but considerably less in the scientific literature which tends to make me uneasy on any topic. Please feel free to post any papers you have found on the topic. As for autoimmune responses, based on Klebsiella molecular mimicry, to collagen supplements, it would theoretically be possible if the collagen in the supplements had the same binding domains as structural collagen. Theoretically, it might be possible for collagen supplements to exacerbate that reaction as well, but I am not sure this really happens.
Beek, I would recommend cabbage juice for Klebsiella pneumoniae, I have read of fasting plus cabbage juice to rid various gut infections. Here is study with red cabbage. Hi Mary, Your first statement is more indicative of what I mean. And I am only talking about human digestion and absorption. I am interested in knowing the amount of fermentable material that persists beyond the early part of the small intestine where bacterial counts begin to increase, particularly in the case of SIBO.
Thank you Norm for your IBS book. It made sense to me as I had already put several of the pieces together through others work. Do you or anyone know what affect lactofermenting the rice and vegs starch, the fructose in fruits and prefermenting the fiber in vegs and fruit or nuts has on the FTD foods FP wise? Am eating meat and fat and have lost too much because of the D. I need the next puzzle piece to transition and have been thinking about the lactofermentation that I do in an airlocked vessel.
Was thinking of leaving the food matter and drinking the juice but after reading scads more conversations, I believe I would do better on limiting the potent good bacteria and begin consuming the plant matter, not so potent. Good thoughts Mormor. I have been doing lacto-fermentation myself this summer. I agree, wild fermentation is asking for trouble. I also use a modified mason jar cover with a fermentation lock. I have made enough beer and wine over the years, to appreciate the importance of keeping the process completely anaerobic. Oxygen is the enemy. Letting the fermentation proceed to completely is the key.
I am currently fermenting pickles and cauliflower with garlic and other herbs. I have not fermented starches including rice, but there are recipes for fermenting just about anything. And you will end up with much less fermentation potential.
What Is Resistant Starch?
Good luck and keep us informed about your experience and lessons. And immediately i felt like i was experiencing the best feelings and moments of my life. Then few months later I could not digest corn, potatoes, basmati rice which i had been digesting when i started eating clean. Hi Norm…its all been a very interesting read. I was hoping you can advise me with my uncommon problem, well at least I think its uncommon. I have been dealing with my candida for many years now, I have been Paleo for over 16 months, I obviously leave the fruit out completely, along with the nuts and seeds and all tubers.
I have taken every anti fungal out there, and I follow the diet religiously, then once a year I re-test with a stool sample to be told I still have Candida!! I have tried numerous brands including, 11 Strain, Prescript-Assist and Probiotic-3 and all that seems to happen is they cause my skin to react as in the same way it does when I used to eat the problematic foods IE sugars, carbs, nuts and seeds to mention a few. It looks like a major case of thrush all between my legs, so I cease taking the probiotics and within a couple of days its all cleared up, and its not sore or irritable.
This happens with homemade Kefir made with coconut milk sauerkraut and other fermented foods! This is most annoying as I know I need to populate my gut as to stand a chance of winning my battle with Candida, even the stool analysis results always indicate zero beneficial bacteria levels. Have you come across this problem before? Any advice you give I will be most grateful for…thanks. I have both fructose malabsortion and lactose intolerant.
Also have hemorrhoids. Just wondering, if it would be beneficial to take water kefir?
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The FP would be about 3 grams. Not too bad. This is such a good post, I have come back to it many times, as layers of symptoms and understanding unfold. In your book, you mention the need to have pasta, for instance…hot, not cold…or it is a source of fermentation carbs. Would the same hold true for potatoes and especially oatmeal??? Thanks so much!! Thanks Susan. Absolutely, consume these foods well heated but not overcooked.
Thanks for adding this clarification. Or just because it would diminish the quality of the food? Best, Susan. Yes, overcooking can increase resistant starch. It gets a little complicated not sure I fully understand all the details. Search Linearization of amylopectin. My recommendation is to use some caution about overheating. For instance with fried rice, you might limit the amount of time and heat when you add the rice to the pan. To increase RS in bread, let it get stale.
To increase RS in rice or potatoes, overcook in a dry manner. So, to best prepare foods with little formation of RS, think fresh, hot mashed potatoes or rice right out of the rice cooker. The more moisture in the finished product, the less chance of excess RS.
I made some potato starch using my Jack Lalanne Power Juicer. It was not fully dried when it went into a storage container.
The next day, I opened the container to get a scoop for my smoothie, and it looked like bleu cheese! Completely speckled with mold throughout. So, obviously this stuff is not anti-fungal. Potato juice, however, as found in a whole potato, does seem to have anti-fungal properties. It must, eh? To protect the potato for months or longer until the next growing season? So, when eating RUMPS, and a yeast or bacterial overgrowth is in play, I believe you would be fueling this overgrowth substantially.
Nearly everything we eat is also food for yeast and fungi. What food can you spritz with water that will not become moldy soon? I can think of a few: Fresh, intact fruits, vegetables, and mushrooms. However, given enough time and moisture, these, too, will get moldy. Is there a lesson there for people with significant overgrowths?
Say, garlic, for instance? Or some carrot juice? Or a big serving of any fresh veggies? I remember thinking, it might be best not to over cook fried rice. Do you recall the process that results in increased RS when cooking at higher heats, baking, frying etc.? Tim, Here is another question. There is lots in the literature about how fats interact with amylose to form a complex resistant to digestion. But what about amylopectin interaction with fats? Could the GI lowering affects of fats be dependent on amylose.
I seem to recall a paper addressing this point. It is all strictly a matter of drying, which allows a crystal pattern to begin forming in the starch granule structures. Overcooking fried rice, ie. The presence of protein matrix and cell wall material surrounding starch granules can prevent the accessibility of enzyme to hydrolyze the starch. The susceptibility of the starch granules are also greatly ffected by their amylose content, starch polymorphism, structure of amylopectin, and the presence of amylose-lipid complex in the starch granules.
Amylose negatively correlates with the susceptibility of starch to amylase hydrolysis because it intertwines with amylopectin, and holds the integrity of the starch granules. Amylopectin of the A-type starch has larger proportions of short branch-chains, which result in more open space weak points in the granule for amylase to penetrate and hydrolyze the starch.
Thus, the A-type polymorphic starch is more digestible than the B- and C-type polymorphic starches. Pre-swelling and debranching of the HA7 starch granules was used to enhance the amylose-lipid complex formation. So, you can see, food scientists are looking at ways to make RS more resistant, too, and using fat as a way to prevent enzyme degradation. In a real-world eating situation, what I think this all implies: retrograded amylose is able to withstand heating more than retragraded amylopectin.
And amylose retrogrades faster than amylopectin. For those looking to decrease RS, it would mean choosing foods higher in amylopectin and eating them freshly cooked and still warm, and not using leftovers. As to using fat to manipulate the RS content…not sure how it would work with eating real foods. Fat does effect the enzyme degradation of starch, fat added to starchy meals lowers the GI, so presumably increases FP. So, probably eating freshly cooked rice with your vegetables and meat stirred-in as opposed to stir-fried in hot oil should result in higher GI and less RS formation of all kinds.
For your protocol, it would probably make more sense to recommend casseroles over pan-fried cooking techniques. Thank you so much for this article Norm!!! This article brings clarity and confirmation to my condition. I have recently enrolled a gut healing program to treat my SIBO. However, there is a bottle of fiber in the supplements. I would love to hear your thoughts on this. I have been on the Low Fodmap diet for 2 years. It really helped but something was still missing so i finally conceded to spend the money on the MRT Leap food sensitivity test and that did help more but……still something was missing.
I wondered why according to the food sensitivity test and the Low Fodmap diet I could eat things like corn, maltodextrin, modified food starches etc. Well I just ran across you and your info and bingo it sounds like, and hopefully, I might have more of the answer if not all. I have ordered your book but have some questions meanwhile. I have some probiotics that have cellulose, Leucine and silica but no dairy.
Is the Cellulose anything that might cause a problem? Does soaking, sprouting lessen the affect of the resistant starch in nuts, grain? Does eating the whole grain flour form lessen the effects? I have been on this journey along time refusing any invasive techniques from the medical profession including pharmaceuticals just because I knew that they would do more harm than good but the kinds of food that I can eat has become increasingly, exorbitantly, stringently limited.
I have hopes that I eventually will be able to start adding foods back but as of now I would not know where to start. I would not worry about a bit of cellulose in the probiotics. This research paper on soaking beans suggests that soaking can reduce certain fibers as well as sugars and starch content. Be sure to remove and dispose of the water after soaking. At first I thought that gas was coming soon after I finished eating.
After some weeks I started feeling uncomfortable again, at first I thought it was just my body rearranging at the new food, then I realized it was the very same issue I had before, as the symptoms grew stronger and had so much gas I could hardly keep it in public.
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I spent last year and half doing experiment to understand what was causing my problem. Long story short after one year and a half I started again with my trials and removed wheat. It went better, but then I had huge issues with polenta. Removed corn. Had massive symptoms with hummus. Ok, must be carbs. I spent some more night on the web looking for people with similar issues and finally arrived here, where every single thing described is the analysis of my last year and a half. Even why I could have hot fried potatoes but not cold ones.
Why once I had hot pasta and the same pasta the next day made me miserable. Why few days ago I rinced my rice after cooking and I had gas all night long. And how do I go back to before this happened? Is it because of a deficiency in alpha-amylase? Is it even possible to have or investigate such deficiency? Increases in bifidobacteria  ,  ,  ,  and Bacteroides  as well as decreases in enterobacteria and Bacteroides  have been reported. Unfortunately, most studies have been performed in either in vitro systems or animal models .
To our knowledge, there were two previous studies that have used culture independent methods to characterize the effect of RS in humans in vivo. Abell and coworkers, who used denaturing gradient gel electrophoresis DGGE to study the impact of RS2 on the human gut microbiota and revealed an enrichment of phylotypes related to Ruminococcus bromii . Walker et al detected an enrichment of bacteria related to Eubacterium rectale and Ruminococcus bromii when RS3 was consumed by overweight individuals .
Recently, the ecological study of the human gastrointestinal microbiota has gained enormous momentum through the development of high throughput multiplex sequencing of 16S rRNA tags  , . This technique has been extremely valuable, for example, in the characterization of the human microbiota in terms of lean and obese physiological states, impact of antibiotics, and the importance of delivery mode at birth  ,  , .
Pyrosequencing has significant advantages over other molecular techniques currently used to study microbial communities. First, unlike probe based techniques such as fluorescence in-situ hybridization FISH , pyrosequencing allows the determination of the entire phylogenetic spectrum of the bacterial populations in one single analysis. Second, it further allows an immediate taxonomic characterization and the flexibility to analyze the communities at different taxonomic levels. Third, pyrosequencing has a markedly increased dynamic range when compared to more traditional fingerprinting techniques such as DGGE .
The goal of the present study was to obtain a community wide perspective of the impact of RS on the composition of the human gut microbiota. We were further interested to compare RS4 and RS2 in this respect because most emphasis in the literature has been placed on the latter substrate. To achieve our goal, we conducted a placebo-controlled, double-blind crossover trial with 10 human subjects and performed a comprehensive characterization of their fecal microbiota by using a combination of approaches, including pyrosequencing of 16S rRNA tags Figure 1.
Resistant Starch in Food | SpringerLink
An initial baseline period was proceeded by 3-week periods of each dietary treatment in succession interspersed by 2-week washout periods, and a final washout period. Weekly fecal samples were collected throughout the entire study. Pyrosequencing of 16S rRNA amplicons from fecal samples resulted in an average of 3, sequences per sample after quality control , sequences in total with a mean sequence length of approximately bp. The average number of operational taxonomic units OTUs identified per subject was 1, This analysis and diversity examination by Shannon's index revealed that the consumption of RS did not alter the bacterial diversity in fecal samples Figure S1B.
Among the well characterized culturable genera were Bacteroides 7. Sequence proportions determined by pyroseqeuncing were used to establish the effects of RS on the gut microbiota composition, and the groups of colonic bacteria that were significantly affected are shown in Table 1. The control crackers included in the study providing a daily dose of more than 55 gram of native starch did not have a significant impact on the fecal microbiota, as the microbial populations during administration of these crackers showed little difference to those during baseline and washout periods.
In contrast, RS significantly affected several groups of colonic bacteria, with the two types of RS exerting functional differences in terms of their ability to modulate the gut microbiota. First we identified OTUs that were affected through the dietary treatments in individual subjects. We then constructed phylogenetic trees with representative sequences of these OTUs according to phylum, which are shown in Figures 2A for Firmicutes , 2B for Actionobacteria , and 2C Bacteroidetes.
This analysis revealed that eight OTUs showed statistically significant differences among treatment groups, seven of which could be linked to known bacterial species Table 1 , Figure 2. The trees contain representative sequences of all OTUs detected to be impacted by RS in individual subjects together with sequences of related entries in the database which included both type strains of known species and sequences from molecular studies of human fecal samples.
Sequences were aligned in Muscle 3. Open-black and closed-gray symbols were used to label sequences from individual subjects. The graphs next to the trees show the abundance of OTUs and bacterial groups that were significantly altered in the treatment groups RS2, RS4, control. These graphs show mean proportions of the three individual samples taken during the treatment periods for each subject. Background refers to samples taken in periods were no crackers were consumed.
Repeated measures ANOVA in combination with a Tukey's post-hoc test were performed to indentify differences between treatment groups, and the background was not included in the statistic analysis. RS, and especially RS4, led to major changes in the composition of the gut microbiota in a subset of subjects. Numerically, the most substantial alterations were the change in the genus Bifidobacterium e. Despite these substantial population shifts, our findings clearly showed that effects of RS and their magnitude varied among individuals.
Figure 3 shows compositional changes induced by RS2 and RS4 when compared to administration of native starch for individual subjects. The data revealed that none of the community shifts induced through the two RS types were observed in all ten subjects. The most consistent alteration detected was the reduction in Firmicutes by RS4, which occurred in nine of the subjects Figure 3.
Other common alterations were the increase in Bacteroidetes seven subjects , Parabacteroides distasonis seven subjects , and Bifidobacterium adolescentis six subjects through RS4, and the increase of Eubacterium rectale through RS2 eight subjects. The sizes of the bubbles are proportional to the magnitude of the difference.
Black circles represent increases in proportions induced through RS treatment, and white circles show a decrease. The generation of community profiles from 17 individual samples per subject throughout the trial allowed insight into how RS influenced the population dynamics within the fecal microbiota.
Figure 4 shows the temporal patterns of the three main phyla Actinobacteria, Bacteroidetes, and Firmicutes and four selected species Ruminococcus bromii , Clostridium clostridioforme , Parabacteroides distasonis and Bifidobacterium adolescentis for five representative subjects. The data revealed that bacterial groups showed marked differences in the stability of their populations and in their temporal response to RS. For example, levels of Bifidobacterium adolescentis and Parabacteroides distasonis were remarkably stable in fecal samples in baseline and washout samples, and their populations returned to baseline level within one week after RS administration was stopped.
In contrast, proportions of some taxa, e. Although all these taxa were also significantly impacted by dietary RS, population dynamics were more idiosyncratic, and these bacterial groups might be more influenced by other dietary components or environmental factors. Graphs on the left show proportions of the three main phyla and four representative species Bifidobacterium adolescentis, Parabacteroides distasonis, Ruminococcus bromii and Clostridium clostridioforme as determined by pyrosequencing of 16S rRNA tags.
Gel images on the right show molecular fingerprints generated by DGGE. Bands that represent Bifidobacterium adolescentis and Parabacteroides distasonis are labeled. The analysis of population dynamics revealed that RS2 and RS4 induced changes within the fecal microbial community that differed in their temporal patterns. RS4 led to an abrupt increase in the abundance of Bifidobacterium adolescentis in some subjects with a slight mitigation throughout the three-week feeding period subject 1, 4, and 6.
Out of the six subjects that showed an increase in Bifidobacterium adolescentis with RS4, five did also manifest an increase with RS2. However, RS2 induced a slower gradual increase with higher proportions in week three of consumption when compared to week one. Quantification of bacterial taxa in human fecal samples by pyrosequencing of 16S rRNA tags has been validated and it showed a high correlation with other molecular methods such as qRT-PCR and phylogenetic microarray  , .
To investigate the impact of RS on human fecal microbiota with independent and well established methods, we analyzed all fecal samples obtained during this study with selective culture for representative bacterial groups, PCR-DGGE, and Bifidobacterium specific qRT-PCR. These bacterial taxa were selected as they were included in previous studies that concerned the effect pre- and probiotics on the human fecal microbiota, and enterococci and enterobacteria cannot be detected by pyrosequencing as they constitute a minor fraction of the total microbial community in the human gut  , .
No significant changes were detected for enterococci and lactose fermenting enterobacteria Table S1. The most consistent change detected was a significant increase in the staining intensity of a DNA fragment that represented Bifidobacterium adolescentis , which occurred in four subjects during RS4 consumption and in 3 subjects when RS2 was consumed. DGGE analysis confirmed the distinct dynamics of the Bifidobacterium adolescentis population in response to RS2 and RS4 that were detected by pyrosequencing, meaning that RS4 induced a swift and reversible increase in band intensity while RS2 caused a gradual raise Figure 4.
Therefore, DGGE confirmed the increase in bifidobacteria and Parabacteroides distaso nis in some of the subjects, but as expected, overall resolution of this technique was lower than pyrosequencing. The total cell numbers of bifidobacteria increased by more than three-fold on average through RS4, while RS2 doubled the numbers. In the three subjects with the highest response to RS4, qRT-PCR showed an increase of bifidobacteria to more than 10 11 cells per gram. In addition to microbiota analyses, we also collected data on bowel-related characteristics during the feeding and washout periods using a weekly symptoms diary that rated bowel movement, stool consistency, discomfort, flatulence, abdominal pain, and bloating on a scale from 1 best to 5 worse.
No significant changes occurred in fecal pH for any of the treatments and no significant detrimental effects were observed on bowel movement, stool consistency, or discomfort, indicating that RS at doses of 33 g per day are well tolerated in human subjects. To gain a deeper understanding of the impact of two chemically different forms of RS on the composition and temporal dynamics of the fecal microbiota, we employed a study in 10 human subjects who were weekly monitored throughout a period of 17 weeks. The data revealed that RS types induced substrate specific shifts in the fecal microbial community that were tightly associated with consumption and which varied between subjects.
Our in vivo findings on RS2 were in accordance to previous studies on starch fermentation in in vitro models of the large intestine, which showed an enrichment of Bifidobacterium adolescentis , Eubacterium rectale , and Ruminococcus bromii  , . The same bacterial groups, with the exception of bifidobacteria, were also enriched in fecal samples of obese human subjects during consumption of RS3 . In contrast, our findings clearly showed that the response of the fecal microbiota to RS4 differed to that of RS2 and RS3.
For example, Parabacteroides distasonis was enriched through RS4, while Eubacterium rectale and Ruminococcus bromii showed a significant decrease. Strikingly, RS4 also led to phylum level alterations, decreasing the proportion of Firmicutes while increasing Bacteroidetes and Actinobacteria.
Such phylum level changes have not been observed in fecal samples of human subjects consuming RS2 in our study and RS3 as shown by Walker and coworkers . There was very little overlap in the bacterial groups that responded to both RS2 and RS4. This was surprising as the RS types used in this study were both starch polysaccharides that consist of glucose monomers with the same covalent bonds, although the RS4 was cross-linked by phosphorylation.
One of the bacterial groups that responded to both RS types was the genus Bifidobacterium , which increased in six subjects with RS4 and in five of the same subjects with RS2. However, the temporal dynamics of these modulations differed. RS2 led to a much slower raise in bifidobacteria, reaching comparable numbers to RS4 only in week three. This clearly showed that time is an important variable when studying dietary modulations of the human gut microbiota.
It appears that the ability to increase levels of bifidobacteria is comparable between RS2 and RS4 in the long run, but extended feeding studies will be necessary to determine the exact taxon-time patterns of responses to different forms of RS. Questions remain about the mechanisms by which different RS types selectively promote groups of colonic bacteria in humans in vivo.
Starch fermentation per se should not be selective as many bacterial genera Clostridium , Bacteroides , Bifidobacterium, Butyrivibrio , Prevotella , Roseburia , Eubacterium , Ruminococcus , etc. However, our in vivo findings showed that substrate preferences and competitive abilities exist in the gut environment. In this respect, it is important to point out that the RS types used in this study varied markedly in terms of their chemical structure.
RS2 is a granular form of high amylose corn starch, while RS4 is a chemically modified starch that is cross-linked through phosphate moieties. Therefore, it is possible that different groups of colonic bacteria produce enzymes with distinct activities towards the two RS types, promoting different dynamics in the gut ecosystem. However, it is also of note that the ability of bacterial groups to make use of RS in vivo might relate not only to the utilization but also to the binding of the substrate. It is striking that Ruminococcus bromii , Bifidobacterium adolescentis , and Eubacterium rectale , which showed the most substantial increases in the human gut in response to RS2, have also been shown to form highly selective associations with this substrate .
Therefore, the adherence of bacteria to starch granules might constitute an important first step in the utilization of this substrate, and groups of colonic bacteria might differ in their ability to adhere to granules of RS2 and RS4. The mechanisms by which different types of RS become fermented in the human colon remain an important area of future research. A significant finding of this study was the individualized responses of the gut microbiota to RS2 and RS4, which has also been shown previously for RS3 .
Out of the nineteen OTUs that were detected to respond during this study in individual subjects, eleven did not reach significance when all subjects were included in the analysis e. Bifidobacterium longum , Ruminococcus obeum , Roseburia intestinalis , Roseburia inulinivorans , and several Bacteroides spp. In addition, none of the taxa that were significantly affected by RS showed a response in all ten subjects.