The Subtle Beauty of Bacillus Subtilis (Part V)

July 20, 2015 by Ann Yonetani in Health

Thanks to Bacillus subtilis fermentation, natto may be THE most concentrated known food source of Vitamin K2 (1). Vitamin K2 is increasingly being recognized as a critical micronutrient which most people are not getting enough of through our modern eating habits. Vitamin K2 is vital to both bone and cardiovascular health.

Vitamin K2 (menaquinone) is distinct from Vitamin K1 and has additional, different functions & health benefits.

Most of us appreciate already the importance of calcium in the diet; however, calcium can do as much harm than good if not appropriately transported from food (via the bloodstream) to bones. It is emerging that perhaps the most important function of Vitamin K2 is to help deliver and deposit calcium to right places in the body (skeleton). Lack of Vitamin K2 can lead to accumulation of calcium in blood vessels, (calcification) and therefore, development of heart disease and osteoporosis. A large body of credible in vitro, in vivo, and epidemiological studies support the notion that increasing dietary Vitamin K2 intake is highly beneficial in promoting both heart and bone health (2,3,4).

This video is a bit long but is an excellent, detailed discussion of Vitamin K2's health benefits...

http://articles.mercola.com/sites/articles/archive/2015/06/28/vitamin-k2-health-benefits.aspx?x_cid=youtube Natural health expert and Mercola.com founder Dr. Joseph Mercola and Dr. Dennis Goodman talk about why vitamin K2 is as important as vitamin D.

Read Part I, Part II, Part III, Part IV & Part V of our Bacillus Subtilis series.

References: (1) http://www.scientificamerican.com/article/vitamin-k-and-natto-what-s-the-connection/ (2) http://chriskresser.com/vitamin-k2-the-missing-nutrient/ (3) Tsukamoto, Y. et al.(2000) Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and gamma-carboxylated osteocalcin concentration in normal individuals. J Bone Miner Metab. 2000;18(4):216-22. http://www.ncbi.nlm.nih.gov/pubmed/10874601. (4) Beulens, JW et al. (2010) The role of menaquinones (vitamin K₂) in human health. Br J Nutr. 2013 Oct;110(8):1357-68.

The Subtle Beauty of Bacillus Subtilis (Part IV)

June 28, 2015 by Ann Yonetani in Health

Yet another gift from Bacillus subtilis found inside natto is a medicinal enzyme called "nattokinase". This protein is made by the bacteria during fermentation of natto and is not found in other soy-based foods (1). Nattokinase is commonly taken as a drug/supplement blood thinner or blood clotting inhibitor as an alternative therapy for cardiovascular disease and stroke prevention (2).

A graphic representation of the protein crystal structure of nattokinase enzyme (5)

Nattokinase (for biogeeks, despite its name) is a proteolytic enzyme (serine protease), meaning it functions in breaking down proteins (3). All proteins are made of chains of amino acids, precisely ordered in a genetically programmed sequence. Serine proteases (which themselves are also proteins) are designed to recognize and disassemble certain kinds of target proteins by cleaving them within a particular set sequence of amino acids (containing a serine residue) (4). Nattokinase has been found to target and break down target human proteins involved in blood clotting; thus acting as a natural inhibitor of blood clot formation or "blood thinner".

A view of amyloid protein fibrils (fibers) via atomic force microscopy. Image from Cambridge University PhysBio Research Group (http://www.physbio.group.cam.ac.uk)

Incredibly, nattokinase's potential usefulness doesn't stop there. Many scientists from around the world have also seen that nattokinase may also be effective at inhibiting more kinds of unwanted protein aggregations (in addition to blot clot formation) within the human body. For example, Alzheimer's disease is well known to be associated with the abnormal accumulation of amyloid protein fibers and plaques in the brain. Nattokinase has been shown to be capable of breaking down these amyloid fibrils! (5)

Nattokinase is actually being studied as a potential drug therapy for multiple human amyloid disorders involving pathogenic protein fiber formation including: Alzheimer's (beta-amyloid fibrils), diabetes (insulin fibers) and prion diseases (prion peptide polymers) (2).

Amazing to find Nattokinase pills on the shelf at Whole Foods (pictured here) as well as most health food stores--not one but multiple brands! But no natto?!

Nattokinase enzyme can be extracted from natto or now made by bacteria alone, and is commonly sold in pill form as shown above at Whole Foods. It's not cheap and, like probiotic supplements, it's unlikely that this shelf-stable, isolated form of nattokinase is as active as the protein coming from live Bacillus in natto food. Why, oh why not just eat fresh, delicious, nutritious and less expensive natto?

Read Part I, Part II, Part III, Part IV & Part V of our Bacillus Subtilis series.

References: (1) Fujita, M.; Nomura, K.; Hong, K.; Ito, Y.; Asada, A.; Nishimuro, S. (1993). "Purification and Characterization of a Strong Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto, a Popular Soybean Fermented Food in Japan". Biochemical and Biophysical Research Communications 197 (3): 1340–1347. doi:10.1006/bbrc.1993.2624. PMID (2) Wikipedia [Nattokinase] https://en.wikipedia.org/wiki/Nattokinase (3) Wikipedia [serine protease]: https://en.wikipedia.org/wiki/Serine_protease (4) Yanagisawa, Y.; Chatake, T.; Chiba-Kamoshida, K.; Naito, S.; Ohsugi, T.; Sumi, H.; Yasuda, I.; Morimoto, Y. (2010). "Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase fromBacillus subtilis natto". Acta Crystallographica Section F Structural Biology and Crystallization Communications 66 (12): 1670–1673. (5) Hs,u R.L. et al. (2009).Amyloid-degrading ability of nattokinase from Bacillus subtilis natto. J Agric Food Chem. 2009 Jan 28;57(2):503-8. doi: 10.1021/jf803072r.

The Subtle Beauty of Bacillus Subtilis (Part III)

June 14, 2015 by Ann Yonetani in Health

Natto is not the first product made by Bacillus subtilis to provide benefit to human health. This bacteria is also the source of one of our oldest and widely used antibiotics, bacitracin.

An interesting story: The name of the antibiotic "bacitracin" is derived from a combination of (a) its biological source (Bacillus subtilis) and (b) the person in whom it was found (M. Tracy).

This useful drug was first discovered and isolated from a hospital culture derived from an abscessing leg wound of a young girl named Margaret Tracy back in the 1940's. In trying to diagnose and treat her infected leg, doctors realized that one component from the wound had a strong protective, antibiotic effect---this turned out to be coming from Bacillus subtilis that was present in the wound. When doctors and scientists looked further, they found this antibiotic activity was due to a natural chemical bio-product which they then named bacitracin.

Here is the original scientific article on bacitracin's discovery from 1945 (3)!

References: (1) Dougherty, T. (2012) Antibiotic Discovery and Development: Volume I. Springer Publishing. (2) Wikipedia {bacitracin]: https://en.wikipedia.org/wiki/Bacitracin (3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC518064/pdf/jbacter00667-0106.pdf (4) Jonathan Dworkin, PhD. personal communication.

Bacitracin is one of the most common antibiotics in first aid over-the-counter topical antibiotic cremes, antibiotic-containing bandages, and opthalmic antibiotic ointments (to treat pink eye).

Bacitracin is a natural antibiotic molecule produced only by Bacillus subtilis bacteria. Most of our best antibiotic drugs are, in fact, substances made by microorganisms as a defense mechanism to protect themselves from other threatening species of microbes. Bacitracin works by interfering with synthesis of the essential protective cell wall, thus crippling many other (than itself) bacterial species (1, 4).

Demonstrating antibiotic activity. A petri dish culture of bacteria (brown haze) growing on agar media topped with small white discs which are filter papers impregnated with samples of other bacterial species or purified small molecule products. Ant… — *Demonstrating antibiotic activity*. A petri dish culture of bacteria (brown haze) growing on agar media topped with small white discs which are filter papers impregnated with samples of other bacterial species or purified small molecule products. Antibiotic activity is shown by the appearance of a "halo" of no bacterial growth around the source of "killing activity". The size/diameter of the "zone of death" indicates strength of activity.

Read Part I, Part II, Part III, Part IV & Part V of our Bacillus Subtilis series.

A colorized electron micrograph image of actively growing rod-shaped Bacillus subtilis bacterial cells. The short ones are young cells which elongate as they grow up to a certain set size, at which point they initiate cell division at the cell cente… — A colorized electron micrograph image of actively growing rod-shaped *Bacillus subtilis* bacterial cells. The short ones are young cells which elongate as they grow up to a certain set size, at which point they initiate cell division at the cell center, constricting and splitting into two equal daughters. Beautifully simple, yet far from fully understood.

The Subtle Beauty of Bacillus Subtilis (Part II)

May 24, 2015 by Ann Yonetani in Health

Bacillus subtilis is an old friend.

The species of bacteria used to create natto, Bacillus subtilis, has long been known as a common soil- and grass-dwelling microbe around the world. Only quite recently, have scientists recognized that another one of its natural habitats is within the gut of humans (1) as well as ruminant (grass-eating) mammals. Bacillus subtilis is a member of our human microbiome (the rich community of benign microorganisms living in/on us).

At least a thousand different species of microorganisms are natural residents of the (healthy) human gut alone; many other species are native to other habitats of the human body (e.g. mouth, nose, skin, genitals). [cartoon image from https://imes.mi… — At least a thousand different species of microorganisms are natural residents of the (healthy) human gut alone; many other species are native to other habitats of the human body (e.g. mouth, nose, skin, genitals). [cartoon image from https://imes.mit.edu/about/news]

The human digestive system, particularly the intestinal tract, is home to a huge and diverse community of microorganisms, mostly bacteria. We live in symbiosis with these "good" microbes, collectively referred to as our "microbiome" (or microbiota) (2). It is believed that at least a thousand different species of bacteria reside in the healthy human gut (3), helping us to digest, access and even produce essential nutrients from the food we eat. An active area of scientific research is studying how our microbiome also affects many other diverse functions in the body including appetite and weight regulation, immune system function (relating also to autoimmune disorders & cancer) and even mental health. This NPR (National Public Radio) video gives a nice introduction to the subject...

NPR's Rob Stein narrates a story about the human microbiome, with animation by Benjamin Arthur and music and sound design by Jonathan Arthur.

In the past, many of these microorganisms regularly entered our bodies via naturally-grown and fermented foods which contain many benign bacteria. As the human diet becomes less diverse* (in terms of ingredients--species and varietals of plants & animals we gather, cultivate and consume) and more sterile (literally sterilized to eliminate pathogenic/ "bad" microbial contaminants and to promote commercial shelf life of processed foods), the health and diversity of our microbiome suffers.

In terms of individual cell numbers, the human body houses TEN TIMES MORE MICROBIAL cells THAN HUMAN cells. Of course microbial cells are way smaller than our own, so they are invisible and don't seem to take up much space. But in the gut, as much a… — In terms of individual cell numbers, the human body houses TEN TIMES MORE MICROBIAL cells THAN HUMAN cells. Of course microbial cells are way smaller than our own, so they are invisible and don't seem to take up much space. But in the gut, as much as 3 lbs./1.5 kg. of microbes may exist (2), and a significant portion of the mass of our poop is actually excreted dead gut bacteria.[cartoon from https://egtheory.wordpress.com/2014/12/12/microbiome/]

A general recognition of this problem has been accompanied by a proliferation of probiotic supplements and food additives, but it is unclear how much (if any) benefit these provide (4). Most "probiotic" food products contain only one or a small number of commercialized species of these organisms, at very low concentrations, and possibly dead after extended shelf time. It is highly unlikely that any of these probiotic bacteria will survive the digestive process (corrosive stomach acid and anaerobic conditions) and actually make it alive to colonize the intestine. Expensive probiotic supplement capsules do contain a much larger dose and varied array of probiotic organisms but, again, when delivered in a (highly compromised) freeze-dried powdered form, it is unclear how many of them will actually live to see your gut.

Humans (other animals and plants, too) dependent on symbiotic microbes living inside us. (http://www.bio.davidson.edu/genomics/2014/Naso/gut.html)

Doesn't it makes much more sense to address both the problem and the solution at the same time by trying to add a greater diversity of naturally-grown, unprocessed, living foods to our diet? Freshly fermented foods contain much (many orders of magnitude) higher concentrations of more diverse strains of healthy, live probiotic bacteria than do industrially-produced "probiotic" products. By changing the way that we eat, we can help restore and preserve the universes of microorganisms that live inside of each one of us.

Read Part I, Part II, Part III, Part IV & Part V of our Bacillus Subtilis series.

References: (1) Hong, H. A. et al. (2009) Bacillus subtilis isolated from the human gastronintestinal tract. Research in Microbiology. 160 (2):134-143. (2) Wikipedia [microbiome/microbiota] (3) Eckburg PB, Bik EM, Bernstein CN et al. (2005). "Diversity of the human intestinal microbial flora". Science 308 (5728): 1635–8. (4) Don't Be Fooled: 5 Probiotic Myths. http://www.livescience.com/46661-probiotics-myths.html.

* Many other reasons contributing to the current disruption of the human microbiome, to be addressed in future posts

The Subtle Beauty of Bacillus Subtilis (Part I)

April 28, 2015 by Ann Yonetani in Health

In my efforts to improve and scale up my natto making process, I found I had many questions about the biology of my magical ingredient and probiotic partners in this venture, Bacillus subtilis (B. subtilis).

As a geek at heart, I find that obstacles are often overcome by understanding more about the science underlying what I am doing. So I went to consult Dr. Jonathan Dworkin, a professor and microbiologist at Columbia University Medical Center in Washington Heights. Our chat provided many interesting and useful insights into how these bacteria live, and I want to share some of them with you.

Dr. Jonathan Dworkin sampling some New York Natto

Jonathan's lab and many others around the world have spent years dissecting the nature of signals and events that allow B. subtilis (and other spore-forming bacteria) to emerge from a state of dormancy to reinitiate growth and reproduction (1,2). This is as close as biology (currently) comes to resurrection from death.

The life of B. subtilis alternates between cycles of active (vegetative) growth and sporulation/dormancy followed by re-entry into active growth. Spores shown here in red (5). — The life of *B. subtilis* alternates between cycles of active (vegetative) growth and sporulation/dormancy followed by re-entry into active growth. Spores shown here in red (5).

B. subtilis is among a specialized group of microorganisms which can undergo a process called sporulation, which means that they can enter into a kind of non-living hibernation state by transforming into an inert, highly indestructible structure called a spore. Sporulation occurs as a protective response to nutrient limitation and probably other yet unidentified environmental stresses (2).

Spores are incredibly resistant to extreme temperatures, desiccation, radiation & chemical insults and can "survive" for indefinite periods of time. When environmental conditions become favorable again, spores can miraculously emerge from this dormant state and "come back to life" (2,3).

I've been told that my natto set-up at OFI bears much resemblance to a lab bench; the past has a funny way of following one.

New York Natto is made by inoculating steamed soybeans with B. subtilis in spore form. Biologically, the critical step in creating the natto is in germinating or "waking up" the spores to begin living, growing and nourishing themselves on nutrients provided by the soybeans. Exposing spores to a pulse of high heat is believed by many scientists in the field to promote spore germination (2), and this is exactly what happens when spores are rapidly mixed with steaming hot soybeans during natto preparation.

Louis Pasteur (1822-95), great French microbiologist whose discoveries led to the germ theory of disease, vaccination, and pasteurization (4).

Most non-sporulating microbes are killed by extreme temperatures. This is the basis of sterilization techniques like pasteurization which use the heat of boiling/steaming water to eliminate microbial contamination.

Nature fortuitously allows B. subtilis spores not only to survive, but thrive in response to boiling water temperature, so natto beans are effectively sterilized and simultaneously seeded only with viable B. subtilis. A brilliant marriage of biology and food safety.

Read Part I, Part II, Part III, Part IV & Part V of our Bacillus Subtilis series.

References: Dworkin lab website- http://www.microbiology.columbia.edu/faculty/dworkin.html (2) personal communication from Jonathan Dworkin, PhD (3) Wikipedia [sporulation in Bacillus subtilis] http://en.wikipedia.org/wiki/Sporulation_in_Bacillus_subtilis (4) Wikipedia [Louis Pasteur] (5) http://www.devbio.biology.gatech.edu/?page_id=15.