Gaga for Goo: How probiotic is NYrture New York Natto?

October 03, 2015 by Ann Yonetani in Health

As you see here, after fermentation NYrture New York Natto is held together by an impressive matrix of "neba-neba" (in Japanese), "goo" (in American) or "biofilm" (in scientific terms). This "goo" which gives natto such a unique and unfamiliar (to the Western palate) texture is highly prized and regarded as an indicator of natto quality in Japan.

This stickiness is a substance secreted by the probiotic Bacillus subtilis during natto fermentation which help to protect them from environmental stress and connects them together, allowing communication and physical contact through a common medium. This "goo", primarily a polymer of glutamatic acid (a natural protein amino acid) (1), helps the bacteria to form a stable community as an continuous layer of cells known as a "biofilm"(2).

Ann Yonetani of NYrture New York Natto @ HMS

We wanted to find out "how probiotic is our natto?" or, in other words, "how many live bacteria" were really in there in this "goo"? So, we took our natto to labs at Harvard Medical School and got to do some real science looking at our food with some of the best tools and help available!

So many "functional foods" on the market claim to be "probiotic"; yet it is unclear how many actual live bacteria remain in these foods by the time they reach consumers. Our natto is made simply by fermenting GMO-free soybeans with a large innoculum of the healthy gut microbiome resident Bacillus subtilis, but we wanted to determine how much of these bacteria were still alive and well inside NYrture natto even after a few weeks of refrigerated aging (which improves its flavor, but what of its probiotic power)? A simple question...

which could also be answered quite simply by streaking out a sample of NYrture New York Natto "goo" on a sterile (LB) agar petri plate to see what, if anything, would grow…answer: LOTS!

Indeed, after two days at ambient temperature, robust growth of happy Bacillus subtilis originating from our natto! — Indeed, after two days at ambient temperature, robust **growth of happy Bacillus subtilis originating from our natto!**

Microscopy has always been my favorite scientific tool. We were so lucky to have the aid and goodwill of Bacillus subtilis scientists Paula Montero-Llopis, Alex Meeske and David Rudner at HMS Microbiology to expertly help us with imaging our "goo". We learned many interesting things….

First and foremost, NYrture New York Natto is absolutely teeming with happy, living, dividing and sporulation Bacillus subtilis---even a full month after fermentation/production!

Phase image of "goo" diluted/rinsed in PBS, spotted onto agar pads & viewed on inverted epifluorescence scope. Live cells are dark with bright halos, surrounded by large amounts of fainter, fragmented bacterial cell debris. Fluorescence imaging … — Phase image of "goo" diluted/rinsed in PBS, spotted onto agar pads & viewed on inverted epifluorescence scope. Live cells are dark with bright halos, surrounded by large amounts of fainter, fragmented bacterial cell debris. Fluorescence imaging of DAPI staining of DNA (not shown here) confirms live/dead cells.

Rough approximation: 15 million live probiotic bacterial cells per milliliter are present in NYrture NY Natto after four weeks in the fridge (3)! A typical "serving" might be 50-100 times this volume. The numbers are huge, and undoubtedly would be much higher still in a fresher sample.

Much more on this and other exciting findings from our lab experimentations to come….

For now, many heartfelt thanks to our friends at Harvard Medical School, especially those in David Rudner and George Church's laboratories for their curiosity, assistance and enthusiasm to help take natto inquiry to a new level. Stay tuned for more early results from Harvard and continuing studies at Columbia University!

References: (1) Bhat, A.R. et al. (2013) Bacillus subtilis natto: a non-toxic source of poly-gamma-glutamic acid that could be used as a cryoprotectant for probiotic bacteria. AMB Express. 3:36. http://link.springer.com/article/10.1186%2F2191-0855-3-36(2) Wikipedia [biofilm]: https://en.wikipedia.org/wiki/Biofilm (3) personal communication, Alex Meeske, Harvard Medical School.

Polyamorous for Polyamines

September 10, 2015 by Ann Yonetani in Health

New micronutrients are being discovered all the time, and "good nutrition" is based on so much more than merely what is listed on a food label. Natto is rich in yet another micronutrient contributing to its “superfood” status; polyamines. Polyamines are organic compounds essential for cell growth. Their exact function is unknown, but new research links high levels of polyamines with anti-aging effects! Amazingly, polyamines can be absorbed directly from the food we eat (1), like natto.

Spermidine & spermine are well studied polyamines found in natto with many potential health benefits.

High concentrations of polyamines are found in soybeans. Polyamines are also increased by fermentation….hence natto, fermented soybeans, may be the perfect whole food for providing these nutrients.

Polyamines are thought to be anti-inflammatory compounds. One theory of aging holds that our body's response to chronic inflammation is what "ages" us. Thus, decreasing the amount of inflammation in the body may delay and minimize the aging effects of time. One molecule Tumor Necrosis Factor (TNF) which is increased in certain cancers, inflammatory bowel disease and other autoimmune conditions, is inhibited by two polyamines (spermine and spermidine) by inhibiting TNF from stimulated immune system cells (2). Therefore dietary polyamine intake may decrease inflammation in the body.

We don't like to use this term, but polyamines may have "anti-aging," properties by preserving organ health and function. For example, in one experimental study in which mice were fed a polyamine rich diet lived longer and had fewer markers of aging (hardening and thickening of the kidney tubules) than did mice who were polyamine deprived. In other words, polyamine intake seemed to delay the progression of aging (3).

Mary Badon, MD MBA, who co-authored this blog post.

References: (1) J Nutr Sci Vitaminol (Tokyo). 2009 Aug;55(4):361-6. Long-term oral polyamine intake increases blood polyamine concentrations. Soda K1, Kano Y, Sakuragi M, Takao K, Lefor A, Konishi F. 2. J Exp Med. 1997 May 19;185(10):1759-68. (2) Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response. Zhang M1, Caragine T, Wang H, Cohen PS, Botchkina G, Soda K, Bianchi M, Ulrich P, Cerami A, Sherry B, Tracey KJ. 3. Exp Gerontol. 2009 Nov;44(11):727-32. doi: 10.1016/j.exger.2009.08.013. Epub 2009 Sep 6. (3) Polyamine-rich food decreases age-associated pathology and mortality in aged mice. Soda K, Dobashi Y, Kano Y, Tsujinaka S, Konishi F. (4) Biosci Biotechnol Biochem. 1997 Sep;61(9):1582-4. Polyamine content of ordinary foodstuffs and various fermented foods. Okamoto A1, Sugi E, Koizumi Y, Yanagida F, Udaka S.

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.