- Two Good Probiotic Supplements That Can Improve Your Gut Health
- Why I Do Not Recommend AOR Probiotic 3, a Review
- Advanced Orthomolecular Research – Probiotic 3, the Good, the Bad, and the Ugly
- Clostridum Butyricum, a “Promising” Probiotic with Sickening Potential
- Enterococcus Faecium, Avoid Supplementation at All Cost
- Finally, Probiotic 3 Contains the HSO “Probiotic” Bacillus Subtilis
- Final Thoughts
- See more from this series:
- Clostridium Butyricum CGMCC0313.1 Modulates Lipid Profile, Insulin Resistance and Colon Homeostasis in Obese Mice
- Top 3+ Health Benefits of Clostridium butyricum Probiotics
- What is Clostridium butyricum?
- Health Benefits of C. butyricum
- Mouse Studies
- Insufficient Evidence For
- 2) H. pylori
- 3) Allergies
- Animal & Cell Research (Lacking Evidence)
- 4) Neuroprotection
- 5) Obesity
- 6) Liver Health
- 7) Immunity
- Cancer Research
- Mechanism of Effect
Two Good Probiotic Supplements That Can Improve Your Gut Health
Adequate microbial exposure is an essential component of an Organic Fitness Regimen. Rapid and powerful changes to human living conditions beginning with the Agricultural Revolution set the stage for a loss of “old microbial friends” and perturbations of the human microbiome.
In westernized nations today it’s unly that even those people we consider to be in perfect health harbor a truly healthy microbiome. This is because most people use broad-spectrum antibiotics sometime during their lives, eat a less than optimal diet, and live a life that is very disconnected from the natural environment.
In other words, we could probably all benefit from paying a little more attention to our microbial inhabitants, regardless of whether we feel sick or not.
I’m not a big proponent of supplements in general, but I think high-quality probiotics can be an exception, largely because getting the right types of microorganisms through other sources can be difficult.
Of course, fermented foods and other “natural” sources of beneficial bacteria are important, but probiotic supplements/drugs can often prove valuable as well. However, that doesn’t mean simply heading into the health food store to pick up a random probiotic supplement is going to do you much good.
The health effects listed on the label might seem impressive, but the fact is that most of the probiotic supplements you’ll find only provide minor benefits.
Some of the reasons why many probiotic supplements fail are:
- The majority of probiotic supplements only contain a handful of species of lactic acid bacteria, bacteria that do some good on their way through your body, but don’t necessarily take up permanent residence in your gut. In other words, the bacteria found in probiotic supplements aren’t necessarily adapted to live in the human gut. As Dr. Art Ayers says: Dairy probiotics don’t repair gut flora.
- Probiotic supplements often don’t contain the number of live bacteria listed on the jar.
- Most probiotic products on the market have never been clinically studied.
- There’s often no scientific evidence which shows that the bacteria survive through the stomach acid.
Of course, all of this doesn’t mean that every probiotic supplement you see at your favorite health food store are a waste of money, as there are certainly a wide variety of probiotic products out there.
However, if the choice is between a jar of “lactobacillus probiotics” and some homemade sauerkraut, you’re much better off with the sauerkraut. The fact is that at the moment, there aren’t really that many great probiotic supplements out there.
This is ly going to change in the near future though, as several microbiome companies are now in the process of developing biotherapeutic agents (probiotics) that are to be used in the treatment of various diseases and disorders (1).
So, should we just wait until thoroughly scientifically tested microbiome modulators are made available to the public in the future? Not necessarily. There are some probiotic supplements out there worth trying, two of which are Mutaflor and Probiotic-3.
Other popular probiotics, such as Prescript Assist and Primal Defense, can also be worth a try, but I tend to favor the two aforementioned. Another probiotic supplement, VSL#3, is one of the most scientifically studied probiotics on the market, and it is regularly used in the dietary management of IBS, IBD, and other gastrointestinal disorders.
However, in my opinion it is probably not the best option for the relatively healthy person who’s looking to boost his/her gut health long-term.
I have no affiliation with the companies that produce these products. As always, the goal is just to highlight possible solutions and strategies that you can use to improve your health. Also, since I have received some questions over the years as to which supplements I recommend, I thought it was appropriate to make this post.
Mutaflor is a probiotic comprised of a viable non-pathogenic bacteria strain named Escherichia coli Nissle 1917.
The strain was first isolated in Germany by professor Alfred Nissle in the year 1917. Today, the bacteria is grown in fermenters and is placed into enteric-coated gelatine capsules.
Mutaflor has been on the market for almost a century and has been clinically studied for use in a variety of diseases and disorders of the gastrointestinal tract. A number of European studies on E.
coli Nissle 1917 have shown positive results when used in conditions such as ulcerative colitis, Crohn’s Disease, chronic constipation, prolonged diarrhea, irritable bowel syndrome and pouchitis.
This product is fairly expensive and the shipping cost can be high; but it is ly going to be worth the money. This is one of the most potent probiotic supplements out there.
(Update: I’ve written a comprehensive article on Mutaflor that details all you need to know about this supplement.)
ProBiotic 3 contains three strains of bacteria: Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus. S. faecalis is naturally found in the gastrointestinal tract and is helpful in preventing the growth of harmful bacteria. C.
butyricum is also a natural part of the microflora, and breaks down dietary fiber that is otherwise indigestible. This breakdown produces several nutrients, including ones that are used by human cells in the digestive tract, and that reduce inflammation and intestinal permeability. B.
mesentericus supports the growth of the other two bacteria as well as the beneficial bacterial strain bifidobacterium. (3)
More info about Probiotic-3.
Some possible places to buy Probiotic-3:
Final words: As always, low exposure to harmful substances, a healthy diet rich in prebiotic fibers, and adequate exposure to microorganisms in general are what lay the foundation for a healthy microbiome. These products are more the final decorations on the cake.
Why I Do Not Recommend AOR Probiotic 3, a Review
When I started researching the Advanced Orthomolecular Research Probiotic 3 supplement for this blog post, I believed that one of the bacteria in it, Clostridium butyricum was extremely beneficial. No probiotic supplement is best for everyone.
However, when you formulate potentially pathogenic strains that are difficult to reduce if they become pathogenic in a “probiotic” supplement, something must be said.
most HSO probiotic supplements on the market (except the new version of Prescript Assist) the following probiotic supplement has helped some people recover their digestive health. Though I do not recommend it, I do not outright say it is dangerous and has no merit.
It has, however, worsened the digestive health of some people as well and has the potential of causing hospitalization from dysbiosis or worse.
Advanced Orthomolecular Research – Probiotic 3, the Good, the Bad, and the Ugly
First, I want to start by stating that I admire the company that produces Probiotic 3, Advanced Orthomolecular Research. I frequently recommend the use of some of their supplements.
Doctor Traj Nibbler started AOR because he was contacted by advocacy groups for people with AIDS to help locate hard to find supplemental formulations to improve their health. Dr. Nibbler discovered that it was difficult to source dietary supplements especially of high quality in large quantities.
He decided to start sourcing and compounding his own brand of supplements for the people in the advocacy groups. Dr. Nibbler then begun to standardize some of his formulas and eventually founded the company Advanced Orthomolecular Research to sell quality supplements around the world.
AOR brought many essential supplement advancements to the market including D-Ribose, Benfotiamine, sustained release R-lipoic acid, and oxaloacetate (benaGene). I have recommended these supplements in the past to many of my clients, and I am glad that AOR made them widely available to the public.
Clostridum Butyricum, a “Promising” Probiotic with Sickening Potential
I believed in the past that Clostridium butyricum might be an important “probiotic” for supplementation. One of the bacterial strains that are not included in most probiotic supplements, but I thought should be. The bacteria at first glance appears to be important for proper digestive health. It produces the short chain fatty acid butyrate.
Butyrate is a significant energy source for colonic cells, anti-cancerous, increases mitochondrial activity, helps maintain proper integrity of the gut mucosal barrier, reduces inflammation, and improves microbiome health by reducing the chance of developing dysbiosis. C.
butyricum is a strictly anaerobic endospore-forming Gram-positive bacteria that is a soil inhabitant in various parts of the world. C. butyricum is also found in our gut microbiome, up to 10-20% of the samples. Though C.
Butyricum has been used as a probiotic in Asian countries, it is also known to cause opportunistic infections including dysbiosis, chronic diarrhea, sepsis, foodborne illness, and rarely botulism and necrotizing enterocolitis.
“In 1986, the first infant botulism case caused by a BoNT/E-producing C. butyricum strain was reported in Italy. Since then, C. butyricum has been associated with botulism in other countries (i.e. China, India, Japan, Ireland, and the USA).“
“NEC is a devastating gastrointestinal disease-causing high morbidity and mortality, affecting predominantly preterm neonates during outbreaks.
Its clinical presentation is characterized by abdominal distension, gastrointestinal bleeding, mucosal ulcerations and necrosis, portal venous gas, and pneumatosis intestinalis, with different degrees of severity. Despite decades of research, the pathogenesis of NEC remains elusive.
Although no aetiological microorganism has been definitively established, the most often implicated bacteria have been Clostridium species. Howard et al. first described an association between C. butyricum and NEC in 1977. They identified C. butyricum in blood and stool cultures in nine of ten preterm neonates during an outbreak of NEC.
In another study, Gorham et al. reported the presence of C. butyricum on the hands of members of the medical and nursing staff during an NEC outbreak. This was consistent with the effectiveness of preventive measures in controlling such outbreaks. Additionally, Sturm et al. demonstrated a cytotoxic effect of the supernatant of a C.
butyricum strain isolated from a preterm neonate with NEC. Recently, Smith et al.detected the presence of C. butyricum with high density in two surgical samples from preterm neonates with NEC. Both specimens were characterized by histological pneumatosis intestinalis.
In a previous study, we analysed 30 stool samples from preterm neonates with and without NEC by using 16S rRNA pyrosequencing and culture-based methods, and 163 samples by using C. butyricum qPCR ; C. butyricum was specifically associated with NEC, and culture supernatants of C. butyricum strains from preterm neonates with NEC showed significant cytotoxic activity“
“A new pathogenic C. butyricum strain (NOR33234) has recently been isolated from an elderly patient with antibiotic-associated diarrhoea; tests for C. difficile toxins gave negative results.“
“To the best of our knowledge, only one case has been reported of C. butyricum bacteraemia and sepsis, which occurred in an injecting drug user.“
There are many different individual strains of the bacteria, some containing more virulence factors than other strains. The strain used in Probiotic 3 is Clostridium butyricum TO-A. C. butyricum TO-A has been shown in one study to potentially reduce inflammation in the colon by deactivating toll- receptor 4 (TLR4) by the production and utilization of butyrate.
TLR4 is a protein when activated leads to a release of nuclear factor kappa-light-chain-enhancer of activated B cells and inflammatory cytokines that activate the innate immune system and sometimes trigger beneficial inflammation. Lipopolysaccharide (LPS) a component of the cell wall of Gram-negative bacteria activates the production of TLR4.
An overactivation of TLR4 can trigger excessive inflammation in the digestive tract leading to leaky gut (by weakening the gut junctions), inflammatory bowel disease, and even colorectal cancer if prolonged and severe enough. C.
butyricum TO-A supplementation sounds a good idea if you are dealing with leaky gut, colorectal cancer, or uncontrolled intestinal inflammation, but I do have some issues with suggesting the use of the probiotic in people suffering from these conditions.
The most common C. butyricum “probiotic” used for supplements is Clostridium butyricum MIYAIRI 588. C. butyricum MIYAIRI 588 has been tested and does not appear to contain any toxin genes and showed antibiotic sensitivity.
“Safety studies of this probiotic strain have been conducted and include assessment of antimicrobial sensitivity, documentation of the lack of Clostridium toxin genes, and evaluation of CBM 588®on reproductive and developmental toxicity in a rodent model.
With the exception of aminoglycosides, to which anaerobes are intrinsically resistant, CBM 588® showed sensitivity to all antibiotic classes important in human and animal therapeutics. In addition, analysis of the CBM 588® genome established the absence of genes for encoding for α, β, or ε toxins and botulin neurotoxins types A, B, E, or F.
There were no deleterious reproductive and developmental effects observed in mice associated with the administration of CBM 588®. “ The only study we have on its virulence ability however was founded by the pharmaceutical company that produces the probiotic strain, Miyarisan Pharmaceutical Co. LTD, Tokyo, Japan.
So before I recommend this strain more studies should be performed without the financial sponsorship of the production company before I can recommend the strain. The Clostridium strain that is in Probiotic 3, C. butyricum TO-A, has no virulence testing performed that is published to my knowledge, so it is unknown if the strain is pathogenic or contains the ability to inherit pathogency.
It might be possible for C. butyricum TO-A to obtain virulence potential from other bacteria through horizontal gene transfer.
Horizontal gene transfer is the movement of genetic material between unicellular organisms, which can occur from bacteria to bacteria contact, transduction (bacterial DNA being transferred by a virus), or through transformation (when bacteria take components of dead bacteria or organism DNA into their genetic makeup). C. butyricum can inherit the ability to cause botulism.
“Notably, the operon encoding the BoNT/E toxin harboured by the neurotoxigenic C. butyricum strains is very similar to that carried by group II type E toxin-producing C. botulinum strains.
The presence of the BoNT/E toxin gene within either plasmids or the chromosome in different Clostridium species is consistent with horizontal transfer events mediated by plasmids or phage, and recombination events mediated by mobile genetic elements such as transposons.” Finally, it is possible for C. butyricum to acquire virulence factors from C.
difficile leading to chronic diarrhea.“Among the proteins annotated from genome sequencing of this strain, there was an enterotoxin (OA81_00270). Moreover, two annotated proteins had sequence similarity to phage holins in C. botulinum. A previous report has shown that holin- tcdE is required for exporting enterotoxins tcdA and tcdB in C. difficile. Whether the enterotoxin or holin plays a pathogenic role in C. butyricum infection remains to be examined.”
Enterococcus Faecium, Avoid Supplementation at All Cost
Enterococcus faecium is a Gram-positive bacterium that is a commonly found opportunistic bacteria in our digestive tract. E. faecium is known to cause sepsis, endocarditis, and urinary tract infections. Most strains of E.
faecium also have multi-drug antibiotic resistance and show resistance to hand washing and alcohol sanitization. Forty percent of medical intensive care units reportedly found that 80% to 90.
4%, of device-associated infections (central lines, prosthetic heart valves, ventilators, and urinary catheters) were due to vancomycin and ampicillin resistant strains of E.faecium. Enterococci bacteria account for at least 12% of nosocomial (hospital) infections in the United States.
Vancomycin and ampicillin resistant strains of E. faecium might be susceptible to the antibiotics linezolid, quinupristin-dalfopristin, and daptomycin. The strain of E. faecium used in Probiotic 3 is Enterococcus faecium TO-A.
It is unknown if Enterococcus faecium TO-A contains any virulence factors associated with E. faecium. We do know that one of the common probiotic strains of E. faecium, E.
faecium T-110 has virulence factors can produce biofilm, evade the immune system (anti-phagocytosis), produce hyaluronidase enzyme that breaks down tissue to increase virulence), and adhere to specific bodily tissues (gastrointestinal tract and the heart).
Even the supposed “non-pathogenic” strain E. faecium NRRL can produce biofilm, evade the immune system (anti-phagocytosis), and adhere to specific bodily tissues (gastrointestinal tract and the heart).
In studies antibiotic resistance has been inherited from horizontal gene transfer and maybe possibly virulence factors from other Enterococci including E. faecalis.
You might be asking yourself, why include such an “opportunistic” bacterium in a probiotic “supplement”? I thought about it for a while.
The only reason I can think of is to reduce the colonies of other “weaker” opportunistic strains that are causing digestive issues in the hope you improve.
The risk of relieving bacterial dysbiosis and potentially causing another dysbiosis that could be worse using this bacterium is too great of a risk if you ask me.
Finally, Probiotic 3 Contains the HSO “Probiotic” Bacillus Subtilis
I have written about Bacillus subtilis previously. Probiotic 3 uses the strain Bacillus subtilis TO-A and it’s virulence potential is unknown currently, therefore I do not recommend it.
Though I Advanced Orthomolecular Research and recommend many of their supplements I cannot recommend their main probiotic supplement, Probiotic 3.
The opportunistic potential of the three bacteria that are used in the probiotic, C. butyricum, E. faecium, and B. subtilis is too high.
If you have taken Probiotic 3 and it is helped or harmed your health please let me know in the comment section of the blog.
See more from this series:
Clostridium Butyricum CGMCC0313.1 Modulates Lipid Profile, Insulin Resistance and Colon Homeostasis in Obese Mice
Obesity is associated with a cluster of metabolic disorders and systemic low-grade inflammation involving multiple organs. Recent findings have suggested that intestine is a key organ altered in response to high fat diet (HFD) feeding.
Probiotics mainly lactobacillus strains have earlier been implicated in alleviating metabolic disorders. Here we aimed to examine the effects of a naturally occurring butyrate-producing probiotic clostridium butyricum CGMCC0313.1 (CB0313.1) in limiting the development of HFD-induced obesity. Mice treated with CB0313.
1 exhibited reduced lipid accumulation in liver and serum, lower circulating insulin levels and improved glucose tolerance and insulin sensitivity. Furthermore, CB0313.
1 administration reversed the HFD-induced colonic inflammation as evidenced by reduced tumor necrosis factor (TNF)-α level and increases the interleukin (IL)-10 and IL-22 levels in colon tissue. Additionally to colonic inflammation, CB0313.
1 also reduced the colon permeability by upregulating the tight junction (TJ) proteins (claudin-1 and occludin) and contributed to a decreased circulating endotoxin level. In colon content, CB0313.1 administration restored the reduced production of butyrate and other short chain fatty acids (SCFAs) caused by HFD feeding.
In adipose tissue, lower transcriptional levels of pro-inflammatory TNF-α, IL-6, IL-1β and monocyte chemotactic protein (MCP)-1 in adipose tissue were observed in CB0313.1-treated mice. Collectively, our data demonstrated that CB0313.1, targeting colon inflammation and permeability, ameliorated HFD-induced obesity, insulin resistance as well as adipose inflammation.
Citation: Shang H, Sun J, Chen YQ (2016) Clostridium Butyricum CGMCC0313.1 Modulates Lipid Profile, Insulin Resistance and Colon Homeostasis in Obese Mice. PLoS ONE 11(4): e0154373. https://doi.org/10.1371/journal.pone.0154373
Editor: Daotai Nie, Southern Illinois University School of Medicine, UNITED STATES
Received: January 22, 2016; Accepted: April 12, 2016; Published: April 28, 2016
Copyright: © 2016 Shang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data will be available on Figshare.com; DOI number is :10.6084/m9.figshare.3185320.
Funding: The present study was supported by funds from National Natural Science Foundation of China (grant NO.31471128) to YC. The present study was also supported from the National Natural Science Foundation of China (grant nos. 31400779 and 31570915; National Young 1000 Talents Plan), the Provincial Natural Science Foundation of Jiangsu (grant NO.
BK20130133), Jiangsu Province Recruitment Plan for High-level, Innovative and Entrepreneurial Talents, Jiangsu Province ‘Six Summit Talents’ Program (grant NO. 2014-SWYY-035) and Key Program of Fundamental Research Funds for the Central Universities (grant NO. JUSRP51613A) to JS.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Obesity has reached epidemic proportions and acts as a major risk factor of many metabolic diseases including type 2 diabetes (T2D).
Chronic low-grade inflammation of adipose tissue associated with increased production of inflammatory cytokines is a hallmark in the development of obesity .
Systemically enhanced cytokine productions interfere with insulin signaling pathway [2,3], resulting in systemic insulin resistance and the subsequent progression to T2D .
In addition to adipose tissues, intestine is another key site dysregulated during obesity. Leaky gut mucosal barrier causes systemic endotoxin level to increase and further enhance chronic low-grade inflammation, thereby promoting the development of obesity . A large body of evidence has shown that gut microbiota is altered during obesity and T2D.
Additionally, butyrate-producing bacterias are decreased in patients with T2D compared with healthy controls [7,8]. Manipulations of resident microbes could influence whole-body metabolism by modulating the inflammation state and gut barrier function [9,10].
A recent study shows that IL22, a cytokine that maintains gut mucosal barrier integrity within the intestine, alleviates metabolic disorders and restores mucosal immunity .
In addition, 5-aminosalicyclic acid (5-ASA), a drug with anti-inflammatory properties and that acts locally in the colon, improves gut and adipose tissue inflammation as well as systemic insulin sensitivity . These findings suggest that intestine is a novel target for therapeutic intervention in obesity and obesity-related insulin resistance.
CB0313.1 is a butyrate-producing, gram-positive bacteria and used as a probiotic for treating and preventing non-antimicrobial-induced diarrhea and irritable bowel syndrome.
Butyrate is a short chain fatty acid (SCFA) together with others (acetate, propionate) produced in large amounts from dietary fibers after fermentation in the colon. Besides being a main energy substrate for colonic epithelium , butyrate plays a key role in maintaining gut immunological homeostasis .
Butyrate helps proliferation of intestinal mucosal cells , exerts anti-inflammatory effect in rat colitis , and suppresses nuclear factor(NF)κB activation in colonocytes .
Furthermore, butyrate produced in the intestine induces differentiation of colonic regulatory T cells  and promotes peripheral regulatory T cell generation. Changes in proportions of CD4+ and Foxp3+ regulatory T cells have been shown in the colon of obese mice.
Here, we hypothesized that a clostridium butyricum probiotic may have beneficial effects on HFD-induced obesity and insulin resistance, by promoting SCFA production, improving colon barrier function as well as restoring colon immune homeostasis.
To this end, we investigated the effects of CB0313.1 administration on HFD-induced body weight, metabolic markers and insulin sensitivity. Potential beneficial effects of CB0313.
1 on colon homeostasis were investigated by evaluating colonic inflammation, production of SCFAs and colon permeability.
4-week male C57BL/6 mice were used in this study. All experimental protocols were approved by the Animal Ethics Committee of Jiangnan University, China, and were performed according to the ethical guidelines of the European Community guidelines (Directive 2010/63/EU).
Mice were maintained in a pathogen-free, temperature-controlled environment on a 12hr light and dark cycle at animal center of Jiangnan University. Mice were randomly divided into Normal diet (ND) group, HFD control group and HFD-CB group after acclimatization for 1 week. ND group and HFD group were administered with phosphate buffered saline (PBS). CB0313.
1 is a spore-forming probiotic and stomach acid tolerant and it (suspended in PBS, 2×108/day/mouse) was administered as soon as the HFD started.
Respiratory exchange ratio (RER, the volume ratio of oxygen consumed versus CO2 exhaled) and spontaneous locomotor activity (counts) were measured using metabolic chambers (Columbus Instruments, Columbus, OH). Activity was detected using infrared light locomotion monitoring system. Mice were individually housed and acclimated to the chambers for 24 hour before experimental measurements.
Fresh liver tissues were collected at 16 weeks of age after 12 weeks on each diet. Tissue was fixed in 10% formalin solution. Tissue slides were obtained through serial section cutting 5μm in thickness and stained with hematoxylin and eosin (H&E) as standard procedure.
Total cholesterol (TC), total triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) in serum were measured using an automatic biochemical analyzer (Mindray BS-480, Shenzhen, China). Free fatty acids (FFAs) in liver was determined by a colorimetric assay kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at the end of this experiment (GTT at 10 week, ITT at 11week).
Before the GTT test, animals were fasted overnight and 2g/ kg glucose was injected intraperitoneally. Before the ITT test, mice were fasted 4h and insulin (0.75U/kg) was injected intraperitoneally.
Blood glucose levels were determined with an Accu-chek glucosemeter (Roche Diagnostics, Almere, The Netherlands) at stated time points.
Insulin levels in serum were measured using an ELISA kit (Mouse Insulin ELISA, Mercodia, Sweden) as standard procedure.
For colonic cytokines(TNF-α, IL-10 and IL-22), the colon tissue was cut and homogenized with saline(1:19, w/v), then the homogenate was centrifuged at 4°C for 10 min at 4000g, supernatant was used for ELISA analysis (Mouse TNF-α/IL-10/IL-22 ELISA Kit, Dobio Biology Technology, Shanghai). Serum LPS levels were determined by ELISA (Mouse LPS ELISA, Xinle, Shanghai).
Total RNA was isolated from epididymal adipose tissue and colon using TRIzol (Invitrogen).
Complementary DNA was prepared by reverse transcription of 2μg total RNA using a Reverse Transcription reagent kit (RT reagent Kit with gDNA Eraser RR047A, TaKaRa, Dalian). SYBR Green PCR reagents (BIO-RAD) were used to determine the mRNA levels.
β-actin was used as a housekeeping gene. Calculations were made the comparative cycle threshold method (2-△△Ct). Primer sequences are given in Table 1.
Acetate, propionate and butyrate, present in the mice colon content were analyzed by the gas chromatography coupled mass spectrometer (GC-MS). Briefly, colon content samples (50mg) were first homogenized in 500μl of saturated NaCl solution. Thereafter, samples were acidified with 40μl 10% sulfuric acid.
1ml diethyl ether was added to the samples to extract SCFAs. Samples were then centrifuged at 14,000 g for 15 min at 4°C and supernatants were used for analysis. 1μL of supernatants were injected into Rtx-WAX capillary column (30m × 0.25mm×0.
25μm, Bellefonte, PA, USA) installed on the GC-MS-QP2010 (Shimadzu, Japan). The initial oven temperature was 100°C and increased to 140°C at a rate of 7.5°C/min. The temperature further increased to 200°C at a rate of 60°C/ min and remained for 3 min. Helium was used as the carrier gas at a flow rate of 0.
89 ml/min, and the column head pressure was 62.7 kPa. The injector was set at 240°C. The injection mode was split and the ratio was 10:1. For mass spectrometer, ion source temperature was 220°C, interface temperature was 250°C, and the scan range was from m/z 2 to 100.
Real time analysis software GC-MS Postrun (GC-MS solution Version 2.72) was employed to calculate the concentrations of the acids.
An external standard method was employed to determine concentration of each SCFA.
For mouse colon samples, RIPA (containing protease inhibitors, beyotime, Shanghai) was used to lyse the tissues. The homogenates were centrifuged at 4°C for 15 min at 5000g and the supernatant was used for western blot analysis.
Equal amounts(50μg) of protein, as determined by a BCA protein assay (BCA Protein Assay Kit, beyotime, Shanghai) were separated using a polyacrylamide SDS–PAGE gel. After SDS–PAGE, proteins were transferred to a PVDF membrane following the manufacturer’s instructions.
The membrane was blocked with 5% (wt/vol) skim milk in Tris-buffered saline (TBS)/Tween 20 for 1h at room temperature followed by incubation overnight at 4°C with GAPDH, occludin (Santa Cruz) and claudin-1(Life technology) antibodies diluted in 1% skim milk in TBS/Tween20.
After overnight incubation, the membrane were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at a dilution of 1:2000 in 5% (wt/vol) skim milk in TBS/Tween 20 for 2 h at room temperature and subsequently developed with western lightening plus ECL (PerkinElmer) according to the manufacturer’s instructions.
All data are presented as mean ± SEM. The numbers of biological experiments were listed as n values and were specified in the figure legends. Difference was analyzed by unpaired Student’s t-test (GraphPad Prism 5). P
Top 3+ Health Benefits of Clostridium butyricum Probiotics
Clostridium butyricum is a potentially beneficial gut bacterium that may promote gut health and suppress H. pylori, but there may be some safety concerns. Learn more here.
What is Clostridium butyricum?
Clostridium butyricum is a butyric acid-producing, Gram-positive bacteria found in soil and the intestines of healthy animals and humans .
Butyrate (butyric acid) is a short-chain fatty acid (SCFA) that serves as energy for colonic epithelial cells, plays a key role in maintaining gut immunological homeostasis, and exerts anti-inflammatory effect .
Furthermore, butyrate is not restricted to the intestinal tract but can be disseminated systemically and is detected in the brain. Butyrate in the brain can exert neuroprotective effects on neurodegenerative disorders and improve behavioral deficits via the inhibition of histone deacetylases (HDACs) .
Clinical research about C. butyricum is still pretty limited, but you can check out the benefits of butyrate here.
C. butyricum has been used as a probiotic for non-antimicrobial induced diarrhea, antimicrobial-associated diarrhea, constipation, and irritable bowel syndrome .
Tablets containing C. butyricum were approved from the Japanese Ministry of Health and Welfare for human clinical use since 1970  and are widely used in Asia.
Health Benefits of C. butyricum
C. butyricum probiotic supplements have not been approved by the FDA for medical use. Supplements generally lack solid clinical research. Regulations set manufacturing standards for them but don’t guarantee that they’re safe or effective. Speak with your doctor before supplementing.
Concomitant administration of C. butyricum with antibiotics normalizes the intestinal microbiota, prevents the decrease of Bifidobacteria, effectively prevented antibiotic-associated diarrhea in 110 children .
In 80 ulcerative colitis (UC) patients with food allergy, specific immunotherapy (SIT) and C. butyricum significantly improved UC clinical symptoms, reduced the use of UC-control medicines, and suppressed the Th2 response .
Probiotic therapy with C. butyricum achieved favorable results with minimal side effects in 17 pouchitis in patients with UC who had undergone ileal pouch-anal anastomosis (IPAA) .
C. butyricum prevents acute colitis in mice through induction of IL-10, an anti-inflammatory cytokine .
C. butyricum effectively prevents bloody diarrhea and mucosal damage in rats with IBD, with or without prebiotics [8, 9].
C. butyricum increases Lactobacilli and Eubacterium, increases n-butyrate, propionate, and acetate concentrations, and alleviates colitis in rats .
Treatment with C. butyricum is at least as efficient as treatment with mesalamine in rats with colitis .
C. butyricum beneficially modifies the intestinal microbiota in mice by increasing Bifidobacteria and Lactobacilli and reducing the populations of unwanted bacteria .
C. butyricum increased survival in E. coli infected mice .
Insufficient Evidence For
The following purported benefits are only supported by limited, low-quality clinical studies. There is insufficient evidence to support the use of C. butyricum probiotics for any of the below-listed uses. Remember to speak with a doctor before taking probiotics, and never use them in place of something your doctor recommends or prescribes.
2) H. pylori
The combined use of C. butyricum reduced the changes in the intestinal flora and decreased the incidence of gastrointestinal side effects in patients going through H. pylori eradication therapy .
C. butyricum prevented the side effects of H. pylori eradication therapy, including antibiotic-associated diarrhea, in 19 patients .
C. butyricum inhibited the growth of H. pylori and eradicated persistent H. pylori infection in mice .
C. butyricum alleviated gastric mucosal damage and ameliorates symptoms in mice with gastric ulcers, through its anti-oxidative and anti-inflammatory activities. This bacterium alleviates oxidative stress by increasing the activity of superoxide dismutase and catalases and decreasing malondialdehyde levels [16, 17].
C. butyricum improved asthma and serum specific IgE in the patients treated with specific immunotherapy (SIT), increases IL-10, and converts antigen-specific B cells to regulatory B cells .
C. butyricum markedly enhanced the efficacy of SIT on allergic rhinitis in 158 patients with allergies .
Administration of C. butyricum enforced the inhibitory effect of SIT on allergic inflammation in the mouse intestine .
Animal & Cell Research (Lacking Evidence)
No clinical evidence supports the use of C. butyricum for any of the conditions listed in this section. Below is a summary of the existing animal and cell-based research, which should guide further investigational efforts. However, the studies listed below should not be interpreted as supportive of any health benefit.
C. butyricum restores butyrate in the brain, increases BDNF levels, reduces neuronal cell death, and significantly attenuates the cognitive dysfunction and histopathological changes in mice with vascular dementia .
C. butyricum exerts neuroprotective effects against ischemia/reperfusion injury in mice through antioxidant and anti-apoptotic (cell-death-preventing) mechanisms, and by increasing butyrate contents in the brain .
C. butyricum attenuates cognitive impairment, cell damage and prevents cell death in diabetic mice with cerebral ischemia/reperfusion injury .
C. butyricum reduced fat accumulation in liver and blood, lowered insulin levels and improved glucose tolerance and insulin sensitivity in obese mice. Furthermore, C. butyricum administration ameliorated GI and fat tissue inflammation .
C. butyricum can reduce lipogenesis (fat production) through its metabolites such as butyrate .
6) Liver Health
C. butyricum increases cholesterol degrading enzymes and improves non-alcoholic fatty liver disease (NAFLD) in rats on a high-fat diet .
C. butyricum stimulates IgA, IgM and IgG production and activates local immunity in mice .
Heat-inactivated C. butyricum displays antitumor activity against sarcoma in mice  and inhibits the metastasis of melanoma by stimulating natural killer (NK) cell cytotoxic activity .
Furthermore, in mice, co-treatment with C. butyricum and Bacillus subtilis inhibits the development of colorectal cancer .
C. butyricum was also shown to kill bladder cancer cells. However, cell studies very rarely have any relevance for cancer therapies in animals or humans .
Mechanism of Effect
Researchers have investigated the potential mechanisms of C. butyricum’s benefits in cell and animal studies. They have found that this probiotic:
- Increased BCL-2 [3, 21] and p-AKT [3, 22] in the brain.
- Decreased BAX [3, 21] and caspase-3 [22, 21] in the brain.
- Increased IL-10 [7, 18, 2] and IL-22 .
- Decreased TNF-α [2, 17, 11, 2], IL1-β [17, 2], IL-6 , IL-23 , MCP-1 , and LBT4 .
- Increased 6-keto-PGF-1α , PPAR α/γ, LXR-α , and ANGPTL4 .
- Decreased MDA [17, 21].
- Increased SOD and CAT [21, 17].
- Decreased CGPR , DGAT2 , and TLR4 .
Note that not all C. butyricum strains are safe for consumption. Whereas non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates .
C. butyricum MIYAIRI 588 (or CBM 588) are safe for use as a probiotic in humans .
To avoid adverse effects, talk to your doctor before using C. butyricum probiotics.