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Probiotics


Presenters: Kiran Krishnan – Microbiologist Tom Bayne D.C.

“Death sits in the bowels and bad digestion is the root of all evil.”
Hippocrates (400 B.C.)

Probiotic R

eview
? History:
– First discovered in early 1900s by Metchnikoff – Probiotic means “for life” and was coined by Lilly & Stillwell in 1960s. – Claimed to help with digestive health/discomfort and immune support

? Human Microbiome Project (HMP) through the NIH
– Greatly influenced our understanding of the function and benefits of probiotics within the last five years – Microbiome: totality of the microbes, their genetic elements (genomes) and interactions with a defined environment i.e. the human body
? Expanded our view on the role of commensal microorganisms ? Characterized microbial communities in the nasal passage, oral cavities, urogenital system and the gastrointestinal tract (GIT)

? Current Market
– 90% of leading products utilize lactobacillus sp and/or bifidobacterium sp – By 2016, the U.S. probiotic supplement market is estimated to grow to $1.5 billion in total sales (Euromonitor International 2012)

The Changing Paradigm – HMP and Probiotics
Our understanding of the form and functionality of the microbiota has changed drastically over the past 4-5 years…probiotic formulations have to change accordingly.

Sekirov I et al. Physiol Rev 2010;90:859-904
?2010 by American Physiological Society

NEW Current Understanding – The Source
? The Human Microbiome Project (HMP)
? Human flora has never been completely characterized due to limitations in past microbiology with obligate anaerobes – Metagenomics – advances in RNA/DNA sequencing allow pin-point accuracy in characterization of microbial populations. 16s ribosomal RNA often used. – Allowed for the characterization and study of the total GI microbial population as it relates to health, wellness and disease. – Launched by the NIH 5 years ago – 200 researchers, 80 research institutes

Facts: ? We are more than human – 10 trillion human cells vs. 100 trillion bacterial cells ? Over 1000 different species of commensal organisms in the gastrointestinal tract (GIT) ? Bacterial genes outnumber humans 150 to 1 ? No two individuals have the same composition--even twins ? Disease is association with disruption to ecology of GIT

Additional Facts from the HMP
? Disease is associated with a disruption to the ecology of the GIT (Crohn’s disease, IBS, asthma and even obesity) ? Pathogenic organisms are normal inhabitant of the GIT – i.e. H.pylori, staphylococcus, e.coli. Etc. ? The intestinal microbiota is not homogeneous ? GIT composition is approximately: 60% Bacteroidetes > Firmicutes> Proteobacteria> Actinobacteria ? Bacillus Spores and Lactobacillus belong to the phylum Firmicutes; Bifidobacter belongs to phylum Actinobacteria ? Intestinal types of microbiota vary on a continuum by location and tissue type

Intestinal Microbiota Composition.

Sekirov I et al. Physiol Rev 2010;90:859-904
?2010 by American Physiological Society

Additional Facts from the HMP
? Microbiota communicate with and influence human cell behavior - by signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and
mucosal immune cells.

? Not all species are able to or designed to inhabit all tissue types – i.e. significant differences between strains that inhabit and influence the epithelial, mucosal and lumen sites of the GIT. ? Colonization happens at birth and even prior to birth: ? Evidence suggests that the fetus GIT is not sterile – mother’s gut commensals have been found in amniotic fluid and cord blood – even in C-section babies. ? Vaginal microbiota dramatically changes in the first trimester – diversity decrease and dominance of lactobacillus sp. (abundance of lactobacillus johnsonii – milk digestion) – This suggests a key role of vaginal bacteria in neonate development. ? Bacterial “tutoring” is a key aspect of neonate immune development in year 1 – studies on germ free mice reveal the key function of neonate inoculum. Breast milk is a key factor (over 600 species and oligosaccharides a pre-biotics). Poor inoculum at this stage leads to chronic inflammatory conditions

Why do we need probiotics?

Change in Ecosystem

Digestive Discomfort

Malnutrition Obesity Disease Dysbiosis

Constant disruption of the gut ecology

What Are The Required Features of a Probiotic?

Three Required Features of a Probiotic
? Must have a position in the microbiota to be able to colonize well and afford beneficial function.
– Of 35,000 possible strains, not all strains are present in all individuals – If a strain doesn’t exist in the GIT it doesn’t have a space and won’t function in harmony – Rare that an “outside” strain will colonize and offer a probiotic benefit

? Must be supplemented in concentrations higher than what is found in the microbiota
– Work by stimulating – by critical mass, increased production of signaling biomarkers, and/or by competitive exclusion – Commonly used priobiotic strains such as lactobacillus and bifidobacterium sp found in the GIT in numbers exceeding 20 trillion – 50 billion of supplemented probiotics adds <2.5% to the concentration

– Food Standards Agency (FSA) study conclusions at Reading University

Three Required Features of a Probiotic
? Must by evolution, naturally occur in the environment (where they gain exposure to the host) and must be stable both in the environment and the GIT
– Hunter/gatherers gained soil organisms from unwashed foods – Able to survive passage through GIT and be wild-type adapted for GIT colonization – Endogenous species of GIT bacteria are mostly strict anaerobes. If they grow in production they are not wild-type and thus may no longer be suited to the life of the GIT – Microbiota are tissue specific. Epithelial cells, mucosal or lumen and those not adapted won’t have a chance – The host and the host’s flora have developed an evolutionary symbiosis. Gene functions have been selected over thousands of years, genotype has to be conserved

The Food Standards Agency (FSA) study w/ Reading University (UK)
Dr. G.R. Gibson, Dr. G. Rouzaud, Dr. J. Brostoff and Dr. N. Rayment
Purpose: 1) Evaluate the probiotic effect of commercial products in the human gut. Any affect on gut flora. 2) 35 strains from commercial products were studied. Primarily lactobacillus sp. and bifidobacterium sp. 3) Evaluate the survivability of common probiotics through the GIT.
STEP 1: Survival through gastric juices in the stomach. 20 minutes at pH 1-3
STEP 2: Survivors through the stomach were tested for survival in the upper small intestines via bile acid tolerance STEP 3: The 6 strains were then chosen for survival testing in lower intestines Of the 6 strains, 4 strains showed viability in lower intestinal conditions pH 1 = None survived with any viability. Unable to recovery pH 2-3 = 18 of the 35 showed around 50% survival Of the 18 tested, only 6 showed viability in the presence of bile acid salts and would have a chance to make it to the large intestines.

Only 4 of 35 strains would survive to enter the large intestine and the survivors would have less than 50% survival

So…What’s Nature’s Design for Supplemental Probiotics?

Bacterial Spores
? In particular Bacillus spores - most widely studied and most widely used probiotics outside of the current supplement market. ? Bacillus spores - first commercial probiotics and first prescription probiotics starting in 1958: – Enterogermina? (Sanofi-Aventis, Italy), Bacti-Subtil? (Aventis Pharma, France) ? Used extensively in agriculture and aquaculture – AlCare?, BioGrow?, BioPlus ?2B, LiquaLife?, etc. ? Most widely used and well studied strains in humans are: ? Bacillus Subtilis ? Bacillus Licheniformis ? Bacillus Coagulans ? Bacillus Clausii ? Patented Bacillus Indicus HU36?

Key Features of Bacterial Spores
? Form a robust endospore and can withstand: harsh temps, reactive oxygen, desiccation, low pH, gastric barriers, antibiotics, UV and gamma radiation, solvents, enzymes and high pressures. ? Found all over the environment: soil, vegetation, dust, rocks, aqua-environments, GIT of insects, marine life, mammals, etc. ? Remain dormant up to 50 years ? Colonize very effectively in the human GIT and have been found to colonize very effectively in the GIT of several different animals. ? Found as part of the normal human commensal flora. ? LONG history of use in industries where efficacy is closely measured (pharma, agricultural) ? Extremely safe ? Evolutionarily supported – true commensal organism.

Stage of sporulation
0
(vegetative growth)

I
(axial filament formation)

II
(polar division)

III
(engulfment)

IV to VI
(cortex and coat formation) Mother cell lysis and release of free spore

The Amazing Endospore

\

In response to nutrients, the spore will exit its dormant state and enter back into vegetative growth – 8 mins

spore

free cell

Transition back to a vegetative cell

Physiological of Bacillus Spores Probiotic functionFunctions of spores

Colonization
? Studies show that spores are well adapted to germinate grow and proliferate in the small intestines and then re-sporulate in the lower GIT ? Ability to re-sporulate is clearly an evolutionary adaptation as several functions of bacillus spores require re-sporulation in the lower GIT (GALT development) ? Bacillus spores are found in the GIT of insects, animals and humans, i.e. universal function as a commensal probiotic. ? Studies now indicate that the environment is simply a vector to transfer the bacteria from host to host. Spores are better suited for life in the GIT, but designed to be passed via the environment. (Hong et al)

Physiological of Bacillus Spores Probiotic functionFunctions of spores

Microbiota Balance
? Spores produce at least 24 potent antibiotics that control bacterial over-growth in the GIT.
? Examples: Coagulin, Subtilisin, Amicoumacin, Surfactin, Iturins A, and Bacilysin

? Spores conduct competitive exclusion (CE) of pathogenic organisms to help maintain microbiota balance.
? Accomplished by competition for space, nutrients and/or eliciting host response ? Causes host elimination of invading species

? Spores have the ability to increase the numbers of the important GIT commensals, such as lactobacillus.
? When co-cultured, Bacillus subtilis enhanced the growth and viability of L. reuteri, L. acidophilus and L. murinus (Hosoi. T et al 2000)

? Example: the capacity of B. subtilis to produce catalase and subtilisin has been reported to promote growth of Lactobacillus species.

Physiological Functions of Bacillus Spores Immunomodulation
The intestines posses the largest amount of lymphoid tissue in the human body
GALT – Gut Associated Lymphoid Tissue

Peyer’s Patch – found in the ileum of the small intestines. Maximum numbers found around age 1524. Gut bacteria is a major player in development. Plays a major role in pathogen defense and selfrecognition. PPs favor a Th1 response via INF-g, TNF-a and IL-2

Mesenteric glands – sites of amplification Immune sampling occurs in the lumen – an immune response is generated in the mucosa and then amplified in the mesenteric glands

Physiological of Bacillus Spores Probiotic functionFunctions of spores

Immunomodulation
? Critical in the development of the GALT itself by cooperation of Bacteriodes Fragilis sp. (largest population in the GIT) ? Produce potent activation and proliferation of lymphocytes in the Peyer’s patch – Promotes Th1 shift – Oral tolerance/mucosal tolerance of food proteins and nonpathogenic microbes. PP dysfunction linked to hypersensitivity, chronic Th2 activation and inflammatory conditions (celiac). ? Cause the production of important immune cytokines in cytokines in mesenteric lymph nodes (MLN) (IL-1a, IL-5, IL-6, IFN-g and TNF-a) and in the spleen (IFN-g and TNF-a) ? Interacts with Toll-like receptors(TLR). Vegetative cells of B. subtilis & other Bacillus sp. up-regulate expression of TLR2 and TLR4 ? Leads to amplified innate immune response via macrophages, monocytes, B-cells and Dendritic cells. Then push to Adaptive Immunity. Dendritic activation, important to create link between innate and adaptive immunity

Physiological Functions of Bacillus Spores

Immunomodulation
Autoimmune – Bacillus has been shown to improve adaptive immunity via PP
? ? ? activation and TLR patter recognition – important self/non-self mechanism. Over active innate linked to autoimmune conditions – need for TLR expression to make adaptive switch. Viral mimicry of self proteins invade our immune system (innoculum, vaccines antibodies, etc. Immune system controlled by GIT under development especially recognition of commensals as “self” antigens. Damage to the MHC allows pathogens loose inside the cell to attack (centromeres, DNA, RNA, and other cell products, tissue, mucous membranes etc.) Vulnerable from 0-7 years during immune development, loss of recognition of self partially responsible for low TLR activity during PP development.

?
?

Physiological of Bacillus Spores Probiotic functionFunctions of spores

Digestion: Bacillus Spores
?Produces key enzymes that help the digestion of food products and alleviate bloating, cramping and discomfort.
– includes: alpha-amylase, lactase, protease and lipase are improved with the use of Bacillus ?Resolves dysbiosis (i.e. the presence of harmful bacteria and the imbalance of good bacteria in the GIT) is the main cause of digestive disorders and small intestine bacterial overgrowth (SIBO). Can be argued as being the root cause of most disease. ?Also responsible for incomplete digestion of consumed foods. ?Studies have shown that Bacillus probiotics help prevent the growth of harmful bacteria and promotes the growth of beneficial bacteria –alleviating dysbiosis.

Physiological of Bacillus Spores Probiotic functionFunctions of spores

Key Nutrient Production: Bacillus Spores
? Production of vitamins – Bacillus probiotics have been shown to produce important amounts of menaquinones and biotin in the large intestines. Absorption of these vitamins are high as they are produced at the sight of absorption. ? Digests resistant starches and non-starch polysaccharides (NSP; major components of dietary fiber) to short-chain fatty acids (SCFA), mainly acetate, propionate, and butyrate. SCFA stimulate colonic blood flow; and fluid and electrolyte uptake. ? NEW Bacillus Indicus HU36 – produces high levels of carotenoids that are of the highest bioavailability.

Physiological of Bacillus Spores Probiotic functionFunctions of spores Additional Functions of Bacillus Spores
Detox – Bacillus has been shown to neutralize genotoxic compounds encountered in the GIT. For example: ? Vomitoxin, also known as deoxynivalenol (DON), found in wheat, corn and other grains is neutralized by bacillus in the GIT. Zearalenone (ZEA) is naturally produced by the fungus found on cattle, milk and even plants – impacts fertility and a number of other conditions. ? Cholesterol Reduction – This has been seen primarily with the consumption of Bacillus subtilis from natto. Bacillus has been shown to digest cholesterol causing fats in the GIT to reduce absorption of that form from the human. ? Weight - New research shows that some individuals have an overload of some bacteria that prevent weight loss. ? Allergies , Psoriasis, Eczema - direct correlation with GIT, especially helpful with babies born by C-Section ? Cross-Talk With Human Cells – Studies have shown that bacillus spores produce peptides that are consumed by human cells which then elicits a particular function. Example: Heatshock peptide.

Bacillus Indicus HU36? (COLORSPORE? PROJECT)
The First Carotenoid Rich Probiotic
The COLORSPORE Consortium
? Involved microbiologists, biochemists and food technologists, and its initial aim was to gain a clearer understanding of the bacterial carotenoids that have been discovered, to characterize their antioxidant activity, and bioavailability.

? Three year project from 2008-2011, 5 Million Euro funding
? Headed up and coordinated by Royal Holloway University of London – Dr. Simon Cutting ? Bacillus indicus HU36

?
? ?

Human isolate
Pigmented Strain (yellow/orange) High carotenoid producing strain

?
?

Gastric stable
Completely sequenced genome

Conclusion on Bacillus Carotenoids
? More Stable than standard carotenoids – survives the stomach

? Contains stronger antioxidants than standard carotenoids
? Bio-accessible - absorbed by intestinal cells ? Bio available (in vivo) - recovered in the main storage tissues (liver, adipose tissue).

? Bacillus Indicus HU36
? Can be used as a therapeutic agent for carotenoid supplementation. Colonization, proliferation and expression of carotenoids have been established. ? Unique approach to probiotic therapy as they also produce highly therapeutic compounds like carotenoids and ubiquinones. ? Very process stable – Major formulation studies have been completed Goethe University – Germany. Baking, acid, shelf-stable liquids, chocolate, fruit juice, etc. ? Stable at 235C for 8 min.

REVIEW

Required Features of a Probiotic

1.

Must have a position in the microbiota – its important for a probiotic to be able to colonize well to afford its beneficial function. Without a spot for it, it will not be able to compete.
Transient Bacillus Spores Qualify!

2.

Must have sufficient numbers to colonize and cause stimulation for benefit.
Because of their relatively low numbers in the GI, spores are dosed at a concentration that is 2000 times higher than the native population in the GIT.

3.

Supplemented Probiotics, by evolution, should naturally occur in the environment (where they gain exposure to the host) and be stable both in the environment and the GIT
Spores are nature’s very own probiotic. Spores use the environment as a vector for transfer from host to host and are uniquely adapted to be stable in the environment and in the GIT.

Dr. Tom Bayne
? ? ? ? Graduate National College of Chiropractic, IL 1994 Nutrition based cash practice in Glenview IL. 85% Digestive origin disorders Working with Bacillus Spore Based probiotics for 12 months ? Previously worked with typical lactobacillus and bifidobacterium based probiotics. ? Works with fermented extracts and other herbals to support the gut.

Case Study #1

RJ is a 62 year old male with a 41 year history of cystic acne on his chin. For 41 years he has taken broad spectrum antibiotics. If he stopped he had a breakout within 4 days. On 2 occasions I asked him to wean off the antibiotics and onto a more natural plan. Both times within 4 days he had a cystic acne outbreak on his chin.

Case study #1
? Nov. 2012 patient starts spore probiotic and 2 days later stopped antibiotics ? Patient was breakout free for months. ? Update: Patient visit on 12/3/2013. Patient had been completely free of breakouts for 12 months. He had been out of his probiotic for 3 weeks and had a business trip to Vegas after 26 days off the probiotic and 5 days of very poor Vegas lifestyle he had an outbreak on his chin. He is now back on the probiotic and acne free again

Case Study #2
AA is an 18 month old infant girl that presents with eczema on arms and legs. She is very whinny and agitated through her entire visit. Mom reports very minor eczema behind the knees, which started at 6 months. At 8 months of age she took a 10 day course of Zithromax for an ear infection. 3 weeks after starting antibiotics the eczema started to spread and worsen in intensity. She was colicky and cried more than she ever had. Stools alternated constipation/diarrhea and smelled putrid

Case Study #2
I recommended she titrate the dose of spore probiotic from 1 cap every other day to 2 caps once a day over a 2 week period. Mom reported improvement with stools and demeanor within 1 week. After 6 weeks there was a 20% reduction in area that the eczema covered on her arms and legs but the lesions were not cracked and red as before. Stools were normal in texture and smell. Patient was very pleasant through her entire follow up visit. At 12 weeks eczema was reduced to only an occasional mild outbreak behind the knees. Treatment is ongoing.

Case Study #3

RC is a 18 year old male with a 10 year history of Ulcerative Colitis. He was scheduled to have 18in of his colon removed in 2 weeks. At the time patient was eating a very highly refined diet. Lots of pastas, breads, and grains. He reported that he had tried every diet to no avail. We switched to a grain free approach. Patient reported that in the past eating grain free was typically good for 2-4 weeks and then it seemed to be an irritant.

Case Study #3

Patient started on spore probiotic. After 5 days on the product the family decided to cancel the surgery. Four weeks in to the program patient reported complete resolution of bloody stools and intestinal cramping that were so debilitating to him. One year checkup the patient is a freshman at IU and living a “normal” college life. He limits his grains and takes the spore probiotic daily.

Case Study #4
WB is a 48 year old woman with a diagnosis of IBS. WB has been a patient for 6 years. When she initially presented IBS symptoms had been going on daily for 18 months straight and she was miserable with symptoms of bloating, gas, and cramping. I recommended dietary changes and a slew of nutrients, herbals, digestive aids, and lactobacillus/bifido based probiotics. Within 6 months her gut had healed to the point where she was pain free. She stayed on a maintenance plan of plant based enzymes with her meals and chlorella.

Case Study #4 In the subsequent 5 ? years she had 7 bouts of IBS. Each “outbreak” lasted for 2-3 weeks and it slowly got better with the addition of lactobacillus/bifido probiotic, aloe juice, Lglutamine, and an increase in the chlorella and plant based enzymes. In December of ’12 she presented with a protruding belly and extreme bloating and intestinal cramping.

Case Study #4

Instead of the usual routine I put her on spore probiotics alone. She emailed me 6 hours later to say her symptoms felt almost completely gone. In the morning she called and asked if it was possible that all her symptoms were gone. She has remained IBS symptom free for 12 months the longest stretch of time in the 7 years since she was diagnosed.

Case Study #5
AS is a 51 year old female that presents with a diagnosis of IBS. Symptoms began in her late 20’s but patient reports she has always had a “sensitive stomach”. In her 20’s symptoms of diarrhea and intestinal cramping worsened and began to dramatically affect her ability to function. At 29 she quit her high stress job as a fashion editor in NYC assuming the reduction in stress would allow her to heal. This lead to a ten year quest of diet modifications, supplements, and specialists from every walk of life.

Case Study #5
At the time of her first visit her symptoms are the best they have been in years. She is following the carbohydrate specific diet and taking a grocery bag of supplements. She has daily diarrhea and mild cramping episodes 4-5x a day that last 5-10 minutes. I begin her on a low dose of spore probiotics. Her initial reactions to the spore probiotic is mixed. Her stools seemed to have improved dramatically but the cramping worsens once her dose reaches 1 capsule per day for 5 or 6 consecutive days.

Case Study #5 We leave her dose at 1 cap every other day for 6 weeks. In that time the consistency of the stool was normal 85% of the time. Cramping maintained at 4-5 episodes a day but was mild and did not impact her daily routine. Overall her energy felt better. By month 4 we had slowly built the dose to 2 caps daily 5x per week. She reports normal stools 95% of the time and cramping episodes 1x every 10-14 days. Treatment is ongoing.

Case Study #6

MG is a 38 year old male that presents with Ulcerative colitis. MG is presently in a “flare up”. He is having 4-6 bloody stools daily and dropping weight fast. He has had 2 previous episodes like this since his early 20’s. He is taking antibiotics, oral and suppository steroids at the time of his visit.

Case Study #6

I recommend a course of spore probiotics. He starts at full dose of 2 caps per day. After 2 weeks he reports that he is having 1-2 bloody stools per day and considerable less cramping and pain. He has a strange report. He says that once a day he passes a pink foam type stool. Light frothy stool that floats on the water.

Case Study #6

He continues to have these foam BM’s over the next 6 weeks but in a decreasing frequency. By the end of that 6 week period his bloody stools are also gone. Patient continues to take spore probiotics daily. He has remained symptom free for 5 months and counting.

Case Study #7 IK is a 68 year old female with a lifelong history of psoriasis. The lesions are everywhere except her face and her arms and legs have no spots of clear skin at all. IK has been my patient for 13 years. We have tried everything and IK was a consummate positive patient, trying new protocols and ideas from every seminar I attended. In some cases she paid for my travel so I could learn a technique that she felt would help her heal.

Case Study #7 In November of 2012 her psoriasis was unchanged from 12 years earlier when I met her. I asked her to add the spore probiotic to her routine. Over the last 13 months we have watched her psoriasis slowly recede. At present time her arms have about 20% lesions and her legs about 30% but the scaling is pink and soft and not angry red and cracked. IK is floating on air she is so excited and jokes that she should be in a bikini by July of ’14.

Case Study #8

BK is a 39 year old female and has been a patient for 12 years. She has been diagnosed with Hashimotos thyroiditis. Blood values for the last 12 years show TSH fluctuating between 1.5-3 and thyroid peroxidase (TPO) levels from 70-125. Patient experiences fluctuations in energy levels, and IBS symptoms, but her main symptom is hair loss. She had hair transplant surgery at age 33.

Case Study #8
She was directed to take spore probiotics in response to an acute IBS outbreak in December ’12. Digestive symptoms resolved over the next 10 days and BK stayed on the spore probiotic. After 3 months she had her blood drawn. For the first time in 12 years her TPO were at 25. 6 months later her TPO levels were 16. BK’s digestive issues have not returned in the 12 month period (the longest she can remember) and her energy is overall much better. She feels that the hair loss has stopped and that hair growth is slowly starting.

Bibliography
? ? ? ? Iwasaki, A. et al. Toll-like receptor control of adaptive immune response. Nature Immunology, volume 5; October 2004. Pg 987 Torsten Stein. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology (2005) 56 (4), 845–857. Tran C Dong, et al. Bacillus Probiotics. Nutra Foods 2009, 8(2) 7-14 Gabriella Casula, Simon M. Cutting. Bacillus Probiotics: Spore Germination in the Gastrointestinal Tract. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May2002, p.2344–2352 Simon M. Cutting. Bacillus probiotics – Mechanism of action and use. Protexin Healthcare. NGO THI HOA, et al. Characterization of Bacillus Species Used for Oral Bacteriotherapy and Bacterioprophylaxis of Gastrointestinal Disorders. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 10 Dec.2000, p.5241–5247. T. Kosaka et al. Effect of Bacillus subtilis spore administration on activation of macrophages and natural killer cells in mice. Veterinary Microbiology 60 (1998) 215–225. Jen-Min Huang, et al. Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 53(2008)195–203. Huynh A. Hong, et al. Defining the natural habitat of Bacillus spore-formers. Research in Microbiology 160 (2009) 375e -379. Huynh A. Hong, et al. The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29(2005)813–835. Karin E. de Visser, et al. Paradoxical roles of the immune system during cancer development. Nature JANUARY 2006 | VOLUME 6

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