Life in the Cloud:
December 10, 2018
Denver Naturopathic Clinic
I’m not sure whether to take a deep breath in or not.
The air in our house must be an invisible cloud of microorganisms today. It’s both bread baking day and tempeh day. The sourdough that has been energetically fermenting on the counter is ready to go in the oven. The soybeans for this week’s batch of tempeh are in the ‘new’ incubator. The ice chest we use to ferment the soybeans is actually old, missing a handle and latch ever since a black bear explored its contents years ago in Rocky Mountain Park, but new in that Amazon recently delivered a thermostat regulated heater plate so now its temperature is somewhat controlled. The heater was actually manufactured to keep caged lizards warm but so far appears also work great for making tempeh; the temperature hovers at 92 degrees F.
How many different bugs are we looking at here?
“Tempeh originated hundreds of years ago in Central and East Java and is now Indonesia’s most popular fermented food. It is made of cooked soybeans bound together by a white mycelium of Rhizovus mold into compact cakes and is usually served as thin sliced and fried.” [i] 
Tempeh is one of the rare, perhaps only, vegetarian foods that contain vitamin B-12. As a food science major at Cornell University, this piece of trivia came up in lectures fairly often; the initial paper about tempeh’s B-12 content came from in Stockton Hall, the building in which most of my classes were held. An aside by one of those professors in the ‘know’, that the tempeh used in the research, that contained so much B-12, was actually fermented way past the growth ‘endpoint’ at which retail products are allowed to reach; ‘the B-12 levels peak when the mold turns black but people prefer to eat tempeh while it is still white.’ Whether this is true or not, about the black mold B-12 content, I don’t know, but the thought has stuck in my memory since the late 1970s.
I nevertheless ‘harvest’ my tempeh while it’s white.
Technically Thai fish sauce and Thai shrimp sauce (at about 2.4 µg/100 g) also contain some B-12, but obviously, neither are vegetarian and who is going to eat 100 grams of either. Tempeh (which contains about 4.6 µg/100 g) is eaten in significant quantities so that 100 grams, about 4 ounces, can be eaten at a sitting.
The mold used to make tempeh is Rhizopus oligosporus. There are websites online on how to isolate wild Rhizopus and create your own tempeh starter. Given my background in food science, I opted instead to spend about ten bucks and purchased a half kilo of tempeh starter on eBay, which by my rough approximation is enough to make 96 batches.
Some online writers have become so enamored with the idea that fermented food will greatly improve human health that they actively encourage a seemingly Wild West Approach to cultures and the assumption that all of these microorganisms are our friends.
The tempeh mold is one of several dozen close relatives under the same family name. They are ubiquitous, you can find them pretty much anywhere in the world, so why pay money for a pure culture?
“Rhizopus is …. ubiquitous in soil, animal excrement, and rotting vegetation…. The genus is especially relevant to human enterprises. For example, certain species can act as plant pathogens that affect crops, some are producers of enzymes in industrial biofermentation, and others are used as fermentation agents in food production. Furthermore, certain species are causal agents of disease in animals, including humans, ….
Some Rhizopus species present a significant threat to post-harvest agricultural products by damaging the appearance and taste of crops, most notably sweet potatoes and strawberries Infection can also lead to human poisoning due to release of the phytotoxin rhizoxin, which is synthesized by endosymbiotic Burkholderia bacteria inhabiting the hyphae of some Rhizopus species…. Rhizopus is also an opportunistic agent of human and animal disease in immunocompromised individuals and causes approximately 60–80% of all disease manifestations of mucormycosis….” [ii]
Call me conservative, but the idea of starting with a commercially made tempeh starter seems like a wise choice.
The sourdough culture we are currently using was also purchased on eBay, but from a character named Zach. My impression is of a longhaired, rasta character living in a cabin in California. I could be totally wrong and perhaps he is wearing a white-lab-coat in some sterile laboratory and only pretending he can’t spell. What can I say? the bread rises and tastes pretty good. Sourdough starters are a combination of yeast and lactobacilli bacteria. The bacteria break down the starch in the flour, liberating sugars that the yeast metabolizes, releasing carbon dioxide. A typical population will include “Candida milleri, Lactobacillus brevis and Lactobacillus plantarum ”.
The fermentation produced by these yeast and bacteria produce significant changes in the chemical composition of the bread and it is thought that the chemicals formed potentially contribute to the health-promoting properties of the finished bread. A recent paper published in June 2018, identified “… 118 compounds with significantly increased levels in sourdough, including branched-chain amino acids (BCAAs) and their metabolites, small peptides with high proportion of BCAAs, microbial metabolites of phenolic acids and several other potentially bioactive compounds.” It is the release of these chemical compounds that may account for what is now called the ‘rye factor’, that intake of bread baked from whole-grain rye (Secale cereale L.) lowers postprandial insulin response”.[iii]
The chemistry and biology of these ferments are complex.
Some blog writers seem to be under the impression that to make sourdough one need only trap a few “wild yeast” and let them have at it. The world isn’t that simple. I suppose I should have purchased a starter with a known lineage.
Commercially baked products claiming to be sourdough bread are now often counterfeits; the sourdough flavoring extracts produce ‘such good’ imitations that are hard to distinguish by taste from the real thing, though the imitations may not have the health benefits. Researchers are now utilizing PCR analysis to authenticate the ‘real thing’. [iv]
Sadly, my long-prized copy of Ed Wood’s classic text on sourdough baking has fallen victim to a fit of down-sizing and is no longer among my cookbook collection. Dr. Wood, MD, PhD, is the hero of sourdough ‘culture.’ He began collecting ancient sourdough cultures in Saudi Arabia decades ago when he worked at a Riyadh hospital. He returned to the United States with a bevy of sourdough cultures and began blending the art of baking with the rigor of science. He and his wife, Jean started a company that still ships sourdough cultures. I’ve had the pleasure of using some of his cultures over the years and if you can keep them segregated, you can produce strikingly different flavored bread. There is no question that a Russian rye comes from a different culture than your basic San Francisco loaf or your Yukon sourdough.
Looking in our refrigerator and cupboard, there are quite a few other foods that are the result of fermentation including vinegar, soy sauce, miso, chocolate, coffee, mustard, cheeses, sour cream, butter, and so on. Oh, wine and beer. How could I forget about them?
Curiously a paper set for publication early next year admits that when it comes to making pickles, the biology is still a bit vague. Cucumbers turn into pickles pretty reliably under set conditions (6% sodium chloride to pH 3.2) but the public wants low sodium pickles and despite a concerted effort, these may randomly degrade into unappetizing products. A wide and diverse number of bacteria are found on cucumbers but the Lactobacilli bacteria are selectively allowed to grow by this combination of salt and acidity. Lactobacilli are the probiotic most familiar to us as they are used to produce yogurt and are the predominant flora of the vagina. Pickle bacterial cultures appear to be quite diverse. One recent Indian paper lists the presence of over a dozen different types of lactic acid bacteria in a sample of pickles including: “Enterococcus faecalis, Lactobacillus plantarum, Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactococcus lactis and Enterococcus sp.” [v]
Thus, when we make pickles we can’t rely on starter cultures, we can only add the salt and trust our luck.
Both coffee and cocoa go through similar fermentation processes before making their way to market.
“Fermentation of cocoa and coffee involves a number of groups of microorganisms including fungi, yeasts, acetic acid bacteria, and lactic acid bacteria.”[vi]
Raw coffee beans are coated with a mucilaginous gunk made up of sugars and pectin, that is digested during bacterial fermentation. At the onset, the gram-negative bacteria, klebsiella, and Erwinia, dominate but over time the lactic acid bacteria, “Leuconostoc, Lactobacillus plantarum and Lactobacillus brevis” take over. [vii]
Lactobacilli bacteria are the common bacteria in most of these foods. Is that why we assume they are so good to have in our own guts? Given how many foods we eat that are chock full of lactobacilli, is it any wonder that our guts contain them? Not as many as we once thought, mind you. In the old days, when we isolated gut bacteria with agar plates, it seemed like these Lactobacilli bacteria dominated. The new technologies suggest they are in a distinct minority, outnumbered by a multitude of other bugs.
There are a lot of microorganisms living in the GI tract, old estimates exceeded 1014. This is often said to be ∼10 times more bacterial cells than the number of human cells. However, this estimate has been revised downward and the current ratio of human: bacterial cells is thought to be closer to 1:1. [viii]
The old idea that most of these bacteria were either e. coli or Lactobacilli species has also proven to be incorrect: “… there has been a general and persistent assumption that a large number of Lactobacillus species form stable and numerically significant populations in the human intestinal tract, especially in the small intestine, where they are presumed to form epithelial associations. Considering how widespread and accepted this perception is, there is surprisingly little experimental evidence that supports it.” [ix] [x]
Lactobacilli make up only a small segment of the bacterial population in the human gut. Other animals differ appreciably from us. Rodents and chickens have large populations of lactobacilli early high up in their digestive tracts, in effect inoculating the food as it passes into the gut with lactobacilli and controlling the bacterial populations downstream. [xi] When we were first educated regarding intestinal bacteria, the only available technology for identifying these bugs was to try and grow them on Petri dishes. This has proven to be highly inaccurate:
“These molecular techniques have revealed that the diversity of the gut microbiota has been greatly underestimated. Although a complete catalog of the members of the collective human gut microbiome is not yet available, more than 10,000 different species are estimated to be present, among which a large majority of these microbes are resilient to cultivation by currently available methodologies.” [xii]
The high esteem in which we hold lactobacilli bacteria health dates back to Elie Metchnikoff (1845 to 1916), who was awarded a Nobel Prize for work on phagocytosis. In his 1907 book, the Prolongation of Life, he proposed that the gut microbiota produces small amounts of toxic substances that damage the nervous and vascular systems and that this damage ultimately leads to aging.[xiii] Metchnikoff suggested that administration of fermented milk products would “implant” these beneficial, lactic acid-producing bacteria in the intestinal tract and would “arrest intestinal putrefaction and must at the same time postpone and ameliorate old age.” Metchnikoff based this theory on two observations. First, Bulgarian peasants assumed to have a long life expectancy, ate large amounts of fermented milk products. Second, the natural fermentation of food by lactic acid-producing microbes prevented the growth of putrefactive organisms. Metchnikoff concluded, “as lactic fermentation serves so well to arrest putrefaction in general, why should it not be used for the same purpose within the digestive tube?” As a result, the “Bulgarian Bacillus” of Metchnikoff became popular in western Europe Think Danon Yogurt. Or for that matter any other yogurt. Metchnikoff’s theories still influence people today and his basic conviction that lactobacilli exert important health-promoting effects remains widespread. Of all the numerous gut bacteria, Lactobacilli may be the easiest to grow in the laboratory but the long-held assumption is that they are the dominant species is wrong.
The knowledge that Lactobacilli do not deserve this reputation dates back to almost 1960. In reality, Lactobacilli account for only about 0.01% of the total gut bacteria. Numbers vary greatly between individuals; lactobacilli are not detectable in around 25% of human fecal samples. [xiv] Many of the species of lactobacilli that we thought of as regular inhabitants of the human gut are literally just passing through, present in an occasional fecal sample but not setting up housekeeping. Instead, they were consumed in food and present for a small window of time.
For those of you who find this topic interesting, an excellent review was written about a decade back by Jens Walter. [xv]
[i] Role of Microorganisms in Tempeh Manufacture ―Isolation of vitamin B12 producing bacteria― By NORIYUKI OKADA Department of Applied Microbiology, National Food Research Institute. JARQ Vol. 22, No. 4, 1989
[ii] Andrii P. Gryganskyi, Jacob Golan, Somayeh Dolatabadi, Phylogenetic and Phylogenomic Definition of Rhizopus Species(Bethesda). 2018 Jun; 8(6): 2007–2018.Published online 2018 Apr 19.
Metabolic profiling of sourdough fermented wheat and rye breadVille M. Koistinen,corresponding author1 Outi Mattila,2 Kati Katina,3 Kaisa Poutanen,2 Anna-Marja Aura,2 and Kati Hanhineva
[iv] Erica Pontonio,1 Raffaella Di Cagno,2 Jennifer Mahony, et al. Sourdough authentication: quantitative PCR to detect the lactic acid bacterial microbiota in breadsSci Rep. 2017; 7: 624.
[v] J Monika, Savitri, Vijay Kumar, Anila Kumari, Kunzes Angmo, and Tek Chand BhallaFood Sci Technol. Isolation and characterization of lactic acid bacteria from traditional pickles of Himachal Pradesh, India2017 Jun; 54(7): 1945–1952.
[vi] O. Arunga et al. Lactic Acid Bacteria in Coffee and Cocoa FermentationThe Lactic Acid Bacteria Volume 1 pp 409-429 | Cite
[vii] Microbiological and Biochemical Study of Coffee Fermentation Article in Current Microbiology · May 2001
[viii] Sender R., Fuchs S. and Milo R. (2016) Revised estimates for the number of human and bacteria cells in the body. PLoS Biol. 2016 Aug; 14(8): e1002533.
[ix] Vélez MP1, De Keersmaecker SC, Vanderleyden J.Adherence factors of Lactobacillus in the human gastrointestinal tract.FEMS Microbiol Lett. 2007 Nov;276(2):140-8. [x] Appl Environ Microbiol. 2008 Aug;74(16):4985-96. doi: 10.1128/AEM.00753-08. Epub 2008 Jun 6.
[xi] Tannock, G. W. 1992. Lactic microbiota of pigs, mice and rats, p. 21-48. In B. J. B. Wood (ed.), The lactic acid bacteria in health and disease, vol. 1. Elsevier Applied Science, London, United Kingdom.
[xii] Frank, D. N., and N. R. Pace. 2008. Gastrointestinal microbiology enters the metagenomics era. Curr. Opin. Gastroenterol. 24:4-10.
[xiii] Metchnikoff, E. 1907. The prolongation of life. Optimistic studies. William Heinemann, London, United Kingdom.
[xiv] Tannock, G. W., K. Munro, H. J. Harmsen, G. W. Welling, J. Smart, and P. K. Gopal. 2000. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl. Environ. Microbiol. 66:2578-2588.
[xv] Jens WalterEcological Role of Lactobacilli in the Gastrointestinal Tract: Implications for Fundamental and Biomedical Research▿ Appl Environ Microbiol. 2008 Aug; 74(16): 4985–4996.