Gut microbiota topics

Here are thirty 2019 and 2020 papers related to Switch on your Nrf2 signaling pathway topics. Started gathering research on this particular theme three months ago.

There are more researchers alive today than in the sum of all history, and they’re publishing. I can’t keep up with the torrent of interesting papers.


2020 A prebiotic fructo-oligosaccharide promotes tight junction assembly in intestinal epithelial cells via an AMPK-dependent pathway

2019 Polyphenols and Intestinal Permeability: Rationale and Future Perspectives

2020 Prebiotic effect of dietary polyphenols: A systematic review

2019 Protease‐activated receptor signaling in intestinal permeability regulation

2020 Intestinal vitamin D receptor signaling ameliorates dextran sulfate sodium‐induced colitis by suppressing necroptosis of intestinal epithelial cells

2019 Intestinal epithelial cells: at the interface of the microbiota and mucosal immunity

2020 The Immature Gut Barrier and Its Importance in Establishing Immunity in Newborn Mammals

2019 Prebiotics and the Modulation on the Microbiota-GALT-Brain Axis

2019 Prebiotics, Probiotics, and Bacterial Infections

2020 Vitamin D Modulates Intestinal Microbiota in Inflammatory Bowel Diseases

2020 Microbial tryptophan metabolites regulate gut barrier function via the aryl hydrocarbon receptor

2019 Involvement of Astrocytes in the Process of Metabolic Syndrome

2020 Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice (not freely available)

2019 Akkermansia muciniphila ameliorates the age-related decline in colonic mucus thickness and attenuates immune activation in accelerated aging Ercc1−/Δ7 mice

2020 Plasticity of Paneth cells and their ability to regulate intestinal stem cells

2020 Coagulopathy associated with COVID-19 – Perspectives & Preventive strategies using a biological response modifier Glucan

2020 Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

2020 Shaping the Innate Immune Response by Dietary Glucans: Any Role in the Control of Cancer?

2020 Systemic microbial TLR2 agonists induce neurodegeneration in Alzheimer’s disease mice

2019 Prebiotic supplementation in frail older people affects specific gut microbiota taxa but not global diversity

2020 Effectiveness of probiotics, prebiotics, and prebiotic‐like components in common functional foods

2020 Postbiotics-A Step Beyond Pre- and Probiotics

2019 Pain regulation by gut microbiota: molecular mechanisms and therapeutic potential

2020 Postbiotics: Metabolites and mechanisms involved in microbiota-host interactions

2020 Postbiotics against Pathogens Commonly Involved in Pediatric Infectious Diseases

2019 Glutamatergic Signaling Along The Microbiota-Gut-Brain Axis

2019 Lipoteichoic acid from the cell wall of a heat killed Lactobacillus paracasei D3-5 ameliorates aging-related leaky gut, inflammation and improves physical and cognitive functions: from C. elegans to mice

2020 Live and heat-killed cells of Lactobacillus plantarum Zhang-LL ease symptoms of chronic ulcerative colitis induced by dextran sulfate sodium in rats

2019 Health Benefits of Heat-Killed (Tyndallized) Probiotics: An Overview

2020 New Horizons in Microbiota and Metabolic Health Research (not freely available)

Oat digestibility

A reader questioned one part of Oat species comparisons of the good stuff regarding Avena nuda hull digestibility. This 2019 study partially investigated that aspect:

“We investigated effects of proteins, lipids, and β-glucan in naked oat flour on in vitro digestibility of starch. Content of rapidly digested starch increased, and content of resistant starch decreased after removing non-starch constituents.

There are three categories of starch in accordance with the rate and degree of starch digestion, namely, rapidly digested starch, slowly digested starch, and resistant starch. Resistant starch cannot be digested. Instead, it promotes growth of beneficial colonic flora.

Digestibility of starch is influenced by size and shape of starch granules, food processing method, physical and chemical modifications, viscosity, and food matrix components. Physicochemical properties of naked oat starch and naked oat flour after removing non-starch constituents were compared to study relationships between starch digestibility and intrinsic factors:

Oats contain more proteins and lipids than other common grains. Proteins can effectively reduce starch digestibility by several mechanisms:

  • Proteins can form a protection around starch granules, restricting entry of enzymes into substrates.
  • Surface proteins can block catalytic binding of enzymes on starch granule exterior.
  • α-amylase can partially bind to proteins, reducing enzyme utilization.

By contrast, effects of lipids on starch digestibility is primarily due to forming complexes with amylose, which is better able to resist amylase.

β-glucan, particularly the extracted water-soluble fraction, can lower digestion rate of starch by increasing viscosity. β-glucan can create a complex of adjacent proteins to form a robust structure that resists amylase, resulting in a decrease in starch digestibility.” “Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch” (not freely available)

When viewing the above graphic, keep in mind that its order wasn’t sequential. So “degreased” oat flour (lipids removed, DG-NOF) wasn’t included in “deproteinized” oat flour (DP-NOF).

This in vitro study missed an opportunity to investigate human-practical aspects. Nobody eats oats without preparing them with water. But effects on digestibility from minutes and hours of soaking, boiling, microwaving, etc. weren’t analyzed.

Gut microbiota outnumber human cells 10-to-1. Treat them well with both Avena nuda resistant oat starch and indigestible hulls, and expect reciprocity.

Long-lasting benefits of a common vaccine

This 2021 review subject was effects of the 100-year-old tuberculosis vaccine:

“Bacillus Calmette-Guerin (BCG) vaccine is one of the most widely used vaccines. It protects against many non-mycobacterial infections secondary to its nonspecific immune effects.

The mechanism for these effects includes modification of innate and adaptive immunity. BCG vaccine is known to not only boost immune responses to many vaccines when they are co-administered, but also decreases severity of these infections when used alone.

Alteration in innate immunity is through histone modifications and epigenetic reprogramming of monocytes to develop an inflammatory phenotype, a process called trained immunity. Memory T cells of adaptive immunity are also responsible for resistance against secondary infections after administration of BCG vaccine, a process called heterologous immunity.

The PI3K/AKT pathway, another pathway for mediating immunity, was upregulated. This was supported by recent studies demonstrating its involvement in induction of trained immunity by both BCG and β-glucan.

BCG vaccine can modify both innate and adaptive immunity, and provide immunity not only against Mycobacterium tuberculosis but also other pathogens. Heterologous immunity and trained immunity contribute to pathophysiologic mechanisms which explain how a vaccine protects against unrelated pathogens.” “Bacillus Calmette-Guerin Vaccine and Nonspecific Immunity”

As inferred by “induction of trained immunity by both BCG and β-glucan” many of these findings also apply to yeast cell wall β-glucan treatments. See Choosing your future with β-glucan for a representative study.

Time-restricted prebiotics

My 700th curation is a 2021 rodent study that investigated time-restricted prebiotic intake combined with an unrestricted bad diet:

“Restricted prebiotic feeding during active phase induced weight-independent alleviation of liver steatosis and reduced serum cholesterol in high-fat diet (HFD) fed mice more significantly than unrestricted feeding.

The prebiotic was a mixture of resistant starch [86%], fructo-oligosaccharide [5%], inulin [7.5%], and xylooligosaccharide [1.5%]. It was administered via drinking water at 10% (w/v) for 11 weeks followed by 20% (w/v) for 4 weeks.

Data suggests that improvement in HFD-induced hepatic steatosis by prebiotics could be associated with increased production of SCFAs [short-chain fatty acids]. Findings suggest that SCFA production can also be modified by timed feeding of prebiotics. This implies that distinct alterations in gut microbiota introduced by a difference in prebiotic feeding regime might be an outcome of gut microbiota undergoing diurnal oscillation.

These results suggest that the impact of prebiotics on weight-independent alleviation of liver steatosis and cholesterol-lowering effect can be optimized by restricting prebiotic intake to active phase, and is associated with a distinct change of gut microbiota with increased SCFA production.” “Active phase prebiotic feeding alters gut microbiota, induces weight-independent alleviation of hepatic steatosis and serum cholesterol in high-fat diet-fed mice”

This study provided evidence for Rhythmicity in that:

“Active phase restricted feeding of prebiotics showed more significant effects on modulating gut microbiota, SCFA production, and metabolic response, independent of weight loss. Alterations in gut microbiota introduced by a difference in prebiotic feeding regime might be an outcome of gut microbiota undergoing diurnal oscillation.”

Subjects’ human-equivalent ages were ~15 years to start and ~30 years at the end. As findings may be applicable to humans, this study was similar to Eat oats for diabetes in that it passed on the issue of causes for detrimental effects continuing.

Eat whatever and whenever you want even though you know it will adversely affect your health? Sure, just add this prebiotic, or even better, time-restrict the prebiotic, and everything’s going to be alright.

Eat oats to prevent diabetes

This 2020 rodent study investigated Type 2 diabetics eating oats along with a bad diet:

“Type 2 diabetes (T2D) is a metabolic disease which is characterized by a state of chronic low-grade inflammation with abnormal expression and production of multiple inflammatory mediators. Insulin resistance (IR), a condition where higher-than-normal concentration of insulin is needed to maintain a normal glycemia and adequate glucose utilization in insulin target tissues, has been clinically recognized as the best indicator for diagnosis of T2D.

Increased proportion of whole grain foods in daily diet are associated with reduced prevalence of IR, which is mainly attributed to abundant non-digestible carbohydrates.”

Oat species was Avena nuda, analyzed as:

Left to right, diet compositions for basic chow diet, high-fat diet (HFD), and 49% HFD with 51% whole oat flour:

“An inflammation state characterized by high plasma TNF-α, IL-6, and IL-1β level was induced by HFD in T2D rats. Whole oats had anti-inflammatory effects by inhibiting production of proinflammatory cytokines. Our data supports a positive relationship between increased adipose proinflammatory cytokines and increased insulin resistance.

A drop in water and food intake indicated an improvement in typical clinical symptoms of T2D. Results of this study provide information about differences between individual oat products in improving T2D-related symptoms, and the role of gut microbiota.” “Effects of oat β-glucan, oat resistant starch, and the whole oat flour on insulin resistance, inflammation, and gut microbiota in high-fat-diet-induced type 2 diabetic rats”

This study’s design wasn’t influenced by It’s the fiber, not the fat evidence. A more thorough analysis of each diet’s fiber contents may have better explained this study’s results.

100% insoluble fiber (cellulose) in the It’s the fiber study didn’t help subjects’ health. Removing 2-5% soluble fiber from subjects’ diets in that study had large effects.

Although β-glucan isn’t the sole soluble fiber in Avena nuda oats, let’s use this study’s 51% whole-oat flour diet β-glucan of 2.62% as a proxy for soluble fiber:

  • Basic chow diet removed 1.73% (2.62 – 0.89) soluble fiber, and HFD removed 2.29% (2.62 – 0.33) soluble fiber.
  • Using its oat analysis, 51% whole-oat flour diet insoluble fiber due to oats was 4.31% ((13.53 – 5.08) * .51). The diet’s unanalyzed insoluble fiber of 3.31% (7.62 – 4.31) was roughly equivalent to HFD unanalyzed insoluble fiber of 3.44% (3.77 – 0.33).
  • Because composition of insoluble fiber matters to this study’s measurements – especially to gut microbiota – I won’t calculate estimates to compare basic chow diet’s unanalyzed insoluble fiber with the other diets’ unanalyzed insoluble fiber.

These researchers could have analyzed all this for soluble and insoluble fiber. The It’s the fiber study was published shortly before this study was published

I’ve replaced Avena sativa steel-cut oats for breakfast with the Avena nuda cultivar used in Sprouting hulless oats. They’re chewier when prepared the same way – 1/2 cup soaked overnight in 2 cups water, then microwaved 18 minutes in a 1000W microwave at 80% power.

Cooking this Avena nuda cultivar may be healthier because of oat bran’s contributions. Bran layers may have been removed during Avena sativa dehulling before the steel-cut process.

I prefer 3-day-old oat sprouts of the hulled Avena sativa cultivar used in Sprouting hulled oats because of their 97% germination rate and taste. The Avena nuda cultivar didn’t sprout as well or taste as good.

One step short of greatness

A 2021 rodent study investigated dietary effects of organic and conventional farming practices:

“We report results from a two-generation, dietary intervention study with male Wistar rats to identify the effects of feeds made from organic and conventional crops on growth, hormonal, and immune system parameters that are known to affect the risk of a number of chronic, non-communicable diseases in animals and humans.

Conventional, pesticide-based crop protection resulted in significantly lower fiber, polyphenol, flavonoid, and lutein, but higher lipid, aldicarb [a pesticide], and diquat [a herbicide] concentrations in animal feeds.

Conventional, mineral nitrogen, phosphorus and potassium (NPK)-based fertilization resulted in significantly lower polyphenol, but higher cadmium and protein concentrations in feeds.

Growth and other physiological parameters were only monitored for 9 weeks after weaning. It was therefore not possible to determine whether and to what extent:

  1. Differences in feed composition;
  2. Dietary intakes of compounds previously linked to obesity and chronic diseases; and/or
  3. Changes in endocrine and immune parameters in rats raised on feed crops treated with mineral fertilizers and/or pesticides,

would have resulted in higher levels of weight gain and/or diseases linked to obesity, endocrine disruption and/or changes in immune system activity/responsiveness.” “Feed Composition Differences Resulting from Organic and Conventional Farming Practices Affect Physiological Parameters in Wistar Rats—Results from a Factorial, Two-Generation Dietary Intervention Trial”

I’m always fascinated when researchers intentionally stop one step short of greatness.

It seems a main purpose of this study was to justify a 2013 study by these researchers on pretty much the same subject. The current study had a defined F0 generation, and four different F1 generations and F2 generations.

This study stopped without continuing to any F3 generations.

  • The F1 F2 OPOF line in the above graphic’s first column didn’t eat chow produced with either synthetic chemical pesticides or conventional fertilizers.
  • This line could have continued on to transgenerational great-grand offspring who would have had no direct exposure to the F0 generation’s conventionally fertilized and “protected” crop diet.
  • By continuing, these researchers could have found out what transgenerationally inherited effects on the F3 generation there may be from the F0 generation eating a conventionally-produced diet.
  • Anything found in this line’s F3 great-grand offspring may have applied to humans.

Do we ever consider our great-grandchildren?

A PhD in oats

The lead researcher of Eat oat sprouts for AVAs‘s second study made their PhD thesis freely available. It’s still informative 13 years later:

“The main objective of this research project was to obtain new knowledge on how to treat raw oat material of oat-based products in order to sustain or even increase levels of endogenous phenolic compounds, with emphasis on avenanthramides, in the final food product. Germination of oats proved to be a potential processing method for use on oats since it is relatively easy to perform, although time-consuming.”

Which of these may be better for you? 44.1 grams of 4-day-old hulless oat sprouts at the top:

Or 53.2 g of 3-day-old hulled oat sprouts at the bottom?

They both started from 20.0 g seeds, and germinated the same way up through three days. 20 grams was over 1,300 hulled oat seeds, and close to 700 hulless oat seeds.

3-day-old hulled oat sprouts taste better, have a higher germination rate, and supply more quantity of their nutrients by weight. Characteristics of hulls from the thesis:

“The hull constitutes on average approximately 25% of the total grain weight. Protein, oil, starch and water-soluble carbohydrate levels are overall relatively low.

A large number of bioactive phenolic compounds can be found, among them p-coumaric acid, ferulic acid, vanillic acid, tricin and avenanthramides. Hull constituents remain unaffected during germination.

Activity of β-glucanase increases during germination of oats, resulting in almost total degradation of β-glucan. Since β-glucan is known to have health beneficial effects in humans, degradation during germination is not desirable if oats are intended for use in food products rather than for brewing.”

I’m not going to deal with hulls. Humans can’t digest oat hulls anyway.

A case for 4-day-old hulless oat sprouts:

“Germination of oats can be a good method to sustain or increase avenanthramides and other potentially health beneficial phenolic compounds. Levels of avenanthramides can increase during germination, sometimes to a high degree.

There was no indication that the increase in avenanthramide content had reached a plateau for any cultivars at 120 h of germination, indicating that further increase could take place.

Total protein content in oats increases slightly during germination. Even though the increase is small, it is important, since essential amino acids lysine and tryptophan increase and therefore improve nutritional value.

Lipid content in oats decreases slightly during germination while content of free fatty acids increases, although there are differences between cultivars as well as between hulled and hulless cultivars.”

Eat oat sprouts for AVAs

Here are three oat studies, two of them specifically on oat sprouts. The first from 2019 was cited in Don’t brew oat sprouts – eat them! for oat sprouts having “up to 25-fold increase” in avenanthramides (AVAs):

“Oat seeds were germinated, extracted, and analysed, finding 28 unique AVAs. AVAs 2p, 2c, and 2f, which are commonly described as the major AVAs, represented less than 20% of total content in seedlings.

The germinator program was: soak for 20 h at 20 °C (aeration 1 min every 10 min), followed by germination for 72 h at 25 °C (RH ≥ 99%). After the first 72 h of germination, oat seedlings were incubated for another 96 h at 30 °C (RH 70–80%).

AVA content was boosted by germination, resulting in around 25 times larger quantities found in oat seedlings.

Previous studies also showed an increase in AVA content upon germination, but to a lesser extent than in our current experiment. It used different growth conditions, namely a shorter soaking time (10–14 vs. 20 h) and their seed germination phase was performed at lower temperature and different duration (120 h at 16 °C or 72 h at 20 °C vs 96 h at 30 °C).

Additionally, they quantified AVAs 2p, 2c, 2f, and 3f but also observed a large number of unknown peaks in the UV 340 nm chromatogram. Based on the wide variety of AVAs annotated in our work, many of these peaks probably corresponded to other AVAs, but they were not identified and quantified as such.” “Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa)”

No measurements were taken at three days when germination parameters changed. These researchers didn’t bother to take any samples between Hours 0 and 168. This lack of germination-stage evidence limited findings’ utility to other researchers.

Was this study designed to create a headline rather than useful germination-stage information? Why obliquely and directly fault another study in the Abstract, Results and Discussion, and Conclusion sections for being performed more than a decade earlier, without subsequent advancements in science and technology?

Contrast this study’s design with 2020 Oat sprouts analysis which took measurements under 18 different conditions (hulled / dehulled seeds of two oat varieties, for 1-to-9 days, at 12-to-20°C). Those researchers produced evidence to support many further studies, such as:

“Presence / absence of hull might determine different effects of germination conditions on α-amylase, protease, and lipase activities.”

The referenced disparaged 2007 study:

“..investigated the effect of a steeping and germination process, using a pilot plant malting system, on content of AVAs and other phenolic compounds. This was performed to gain a more collective and comparative picture of what happens to phenolic compounds in the oat kernel during germination.

Steeping and germination was performed at two different temperatures, 16 and 20°C. Three closely related North American covered oat cultivars were steeped to 45% moisture, which took 10, 12, and 14 h for Vista, Gem, and Dane, respectively.

After steeping, oat grains were drained and samples were germinated at 16°C for 120 h or at 20°C for 72 h (due to a machine breakdown, the 20°C experiment had to be stopped at 72 h of germination). Sampling during germination was carried out at set hours (for 16°C at 12, 24, 48, 72, 96 and 120 h and for 20°C at 12, 24, 48 and 72 h).

Chromatogram of AVAs and other phenols from cultivar Dane (16°C treatment). Peaks for AVAs 2c, 2p, 2f and 3f are identified. Unknown peaks are numbered in order of appearance (from 1-21). For isolation and identification of AVA 3f the commercial product SPC-flakes, purchased in a health food shop, was used.

An increase in AVA content of germinated seeds, as compared to raw grains, was observed for Dane (125%, p < 0.001) and for Vista (29%, p = 0.007). HHT [avenanthramide-synthesizing enzyme] activity increased 62% (p = 0.014) in Dane, whereas no change was detected in Vista and Gem. This increase started early in germination to reach its maximum at 96 h of germination.

Effects of temperature on AVAs 2c, 2p and 2f, and activities of HHT and PO, was less important than time of steeping, or time of germination, or cultivar. However, almost all unknown compounds were affected by temperature, indicating the importance of this factor.” “Avenanthramide content and related enzyme activities in oats as affected by steeping and germination” (not freely available)

This study’s HHT (avenanthramide-synthesizing enzyme) maximum-at-96-hours finding may be what I tasted in Sprouting hulless oats, where that variety’s sprouts improved their sweetness and enzymes between Days 3 and 4. Did an extra day improve AVA content?

So their pilot malting system broke down. They had to get an analysis standard from a health food store. And there were many unknowns due to 2007 science and technology.

Despite difficulties, germination-stage samples produced evidence and analyses other people could use more than a decade later.

A 2020 study cited the first study for basic, not headline, information:

“There are various potential mechanisms for avenanthramides (AVAs) anti-inflammatory effects, including inhibition of lipoxygenases (LOX), which catalyse oxygenation of polyunsaturated fatty acids into potent signal molecules involved in inflammatory processes.

It was found that AVAs comprising caffeic or sinapic acid exhibited significant lipoxygenase inhibition (60–90%) (P < 0.05), whereas low or no inhibition was observed with AVAs containing p-coumaric or ferulic acid.

Corresponding free cinnamic acids, AVA analogue Tranilast® and LOX inhibitor trans-resveratrol were included for comparison. Trans-resveratrol showed inhibition, whereas no difference in inhibition was seen on comparing AVAs with their free corresponding cinnamic acids, which implies that the anthranilic acid part of the avenanthramide molecule does not affect inhibition.

Whether dietary AVAs at intake levels normally achieved through consumption of oat products exert LOX inhibitory activity in vivo, and thereby inhibit production of pro-inflammatory compounds, remains to be elucidated. In this context it may also be of importance to emphasize that lipid content in groats of various oat cultivars is comparably high (49–135 g kg−1) and that LA [linoleic acid (C18:2, n-6)] and ALA [α-linolenic acid (C18:3, n-3)] comprise about 40% and 1%, respectively, of fatty acids.

Consumption of oats has been linked to a decreased risk of several chronic diseases, and AVAs contribute to protective effects. This study suggests that avenanthramides comprising caffeic acid or sinapic acid partly exert their antioxidant and anti-inflammatory effects via lipoxygenase inhibition.” “Avenanthramides as lipoxygenase inhibitors”

Tampa’s Riverwalk

Eat broccoli sprouts for your kidneys

Starting Year 7 of curating research with a 2021 review of kidney disease and sulforaphane:

“Many chronic kidney disease (CKD) patients progress to end-stage kidney disease – the ultimate in failed prevention. While increased oxidative stress is a major molecular underpinning of CKD progression, no treatment modality specifically targeting oxidative stress has been established clinically.

Pathophysiologic effects occur when there is an imbalance between oxidation and reduction – an altered redox state in which excess free radicals react with other molecules, including lipids, proteins, and nuclear DNA. Mitochondrial DNA is also susceptible to oxidative damage.

All mechanisms discussed above have been shown to be present in CKD. When levels of antioxidant agents such as SOD, CAT, GPx/glutathione, and NRF2 are reduced, harmful effects of oxidation and generation of ROS cannot be appropriately mitigated.

Data suggest continued SFN [sulforaphane] administration is needed to maintain activation of the NRF2 pathway to confer protection against oxidative damage of diabetes. Renal protective effect of SFN has been demonstrated in many other models of kidney injury.

SFN may have therapeutic potential in kidney disease by stimulating the NRF2 pathway.” “Eat Your Broccoli: Oxidative Stress, NRF2, and Sulforaphane in Chronic Kidney Disease”

Didn’t see where these researchers intended to perform a suggested “clinical study to assess the effect of SFN in CKD.” Keep reading before experimentally treating patients, please. Targets they missed included:

  • Parameters of myrosinase hydrolizing glucoraphanin;
  • “Consumption of broccoli strains with more glucoraphanin leads to higher plasma levels of SFN” and
  • “It follows that SFN could also pose similar adverse effects, particularly if taken in an isolated preparation.”

Also missing from this kidney review were connections to broccoli sprouts’ effectiveness in preventing bladder disease. Not coincidentally, isothiocyanate metabolites accumulate in the bladder.

I came across this paper from it citing Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. I curated it due to informatively citing Microwave broccoli to increase sulforaphane levels.

Week 42 of Changing to a youthful phenotype with broccoli sprouts

1. I had two wake-up calls on scale this week. The first started with A follow-on study to 3-day-old broccoli sprouts have the optimal yields that found:

“Activity of free MYR [myrosinase enzyme] was the highest at pH 5.0, and it decreased rapidly when pH was less or higher.”

myrosinase pH

Bought a pH meter and ReaLemon to adjust water pH when immersing broccoli sprouts for microwaving.

It turns out that only 3 drops of pH 3.5 ReaLemon is needed to change 100 ml of pH 7.0 filtered water to pH 5. A 100-fold pH change with a ReaLemon amount too small for my scale to measure.

The second came from Broccoli sprouts activate the AMPK pathway translating mouse experimental time frames to humans. One effect wasn’t realized after an equivalent 10 human years, and required another 12 human-equivalent years to manifest.

Patience with broccoli sprout efforts may stretch way past what I’ve imagined so far. 42 wasn’t the answer:

Thanks for all the fish!

n’t: A reader pointed out that the sulforaphane effect requiring another 12 human-equivalent years to manifest occurred in human-equivalent 42-year-olds. So 42 was the answer – in years, not weeks.

2. I finished the 3-lb. bag of Avena sativa oats used in Sprouting hulled oats after starting 20 gram batches twice a day. Amazon said that Montana farmer’s products were “Currently unavailable. We don’t know when or if this item will be back in stock.” I went to their website and emailed an inquiry.

Turns out it’s Amazon’s problem in restocking pallets that are already received! I placed an order directly with the farmer.

In the meantime, I’m trying another oat species, Avena nuda, from an Illinois farmer. I’ll reuse Degree of oat sprouting as the model, since it was also an Avena nuda oat variety. Results posted in Sprouting hulless oats.

I’ve had a 97% germination rate with these Avena sativa hulled oats. Too bad for vendors who:

  • Appear to sell substantially the same Avena sativa hulled oats I’ve sprouted, but put a ‘Not for sprouting’ disclaimer in product descriptions without explaining exactly why their product can’t be sprouted; and
  • Are clueless about what they sell, writing silliness like “Our Organic Gluten Free Oat Groats can not be sprouted due to the hull being removed.”

3. The first link of Item 1 above was also the blog post I made better this week after reader feedback. I included an important point previously excluded:

“Sulforaphane was extremely unstable during storage and it was best to enzymatically convert to sulforaphane before oral intake.

I also modified analysis of this graph:

myrosinase activity temperatures

I thought about adjusting microwave practices for 3-day-old broccoli sprouts from ≤ 60°C to 55°C (131°F) in consideration of both the ESP and the 55-to-65°C decline in myrosinase activity.

But myrosinase activity at unmeasured 60°C isn’t at the graph’s straight line drawn between measured 55°C and 65°C. A substantial decline begins after 60°C, not after 55°C as drawn.

Consider this graphic from Enhancing sulforaphane content:

Myrosinase robustly hydrolyzes glucoraphanin into sulforaphane at 60°C. There’s clearly a myrosinase deactivation cliff between 60°C and 65°C. Go up to the cliff edge if you must, but don’t go further.


It’s the fiber, not the fat

I came across this 2020 fiber-vs-fat rodent study from its citation in Gut microbiota and aging:

“Dietary intervention studies largely revolve around altering fat content. Little consideration has been given to amount of fiber and whether or not it is soluble.

We examined age- and sex-specific effects of a refined high-fat/low soluble fiber diet (rHFD) on body weight and gut microbiota composition relative to mice fed a refined low-fat diet (rLFD) that is nutritionally and compositionally matched to rHFD.

Chow diet supplied energy as 13.4% fat, 28% protein, 57.9% carbohydrates, and 15% dietary fiber (range of total dietary fiber between 15 and 25% with 15–20% insoluble and 2–5% soluble fiber).

Two refined diets were used: rLFD supplying energy as 12% fat, 21% protein, and 67% carbohydrates; and rHFD supplying energy as 45% fat, 20% protein, and 35% carbohydrates. [Both rLFD and rHFD contained] 5% fiber in the form of insoluble cellulose.

Young adult animals consumed chow diet for 17 weeks, and 1-year aged animals consumed chow diet for 60 weeks. We included a 1-week transition period wherein all mice were fed rLFD. For the following 4 weeks, half of the animals remained on rLFD while the other half consumed rHFD.

After 4 weeks, young adult female mice showed resistance to weight gain to rHFD, consistent with previous reports. Aged females fed rHFD showed rapid body weight gain relative to rLFD-fed aged females.

Young adult and 1-year aged males showed a significant gain in body weight that was independent of refined diet formulation, suggesting that other components of the refined diet contribute to body weight gain that is independent of dietary fat.

Transition from chow diet to rLFD resulted in changes to microbiota community structure and composition in all groups, regardless of sex and age. This dietary transition was characterized by a loss within phylum Bacteroidetes and a concomitant bloom of Clostridia and Proteobacteria in a sex- and age-specific manner.

No changes to gut microbiota community structure and composition were observed between mice consuming either rLFD or rHFD, suggesting that transition to rLFD that lacks soluble fiber is the primary driver of gut microbiota alterations, with limited additional impact of dietary fat on gut microbiota.” “It’s the fiber, not the fat: significant effects of dietary challenge on the gut microbiome”

It’s alright for researchers in the Abstract and Introduction section to interpret how their rodent study may apply to humans. I appreciate when they confine their statements elsewhere to what they actually measured and found.

This study didn’t measure inflammation, behaviors, neurobiologics, metabolic parameters, immune biomarkers, or hormones. They can qualify statements with “may” all they want, but there wasn’t direct evidence for either:

“Age-specific vulnerability to diet-induced body weight gain in females may be related to aging-related changes to estrogens.”


“The lack of differences between rLFD- and rHFD-fed mice may indicate that gut microbiota structure and composition can be dissociated from body weight and systemic inflammation.”

Papers that cite this study can’t rely on its Abstract for “regulating metabolic, immune, behavioral, and neurobiological outcomes” because its experiments didn’t directly measure such outcomes.

Removing 2-5% soluble fiber from subjects’ diet had large effects. I look forward to reading human studies that are informed by this study.

Degree of oat sprouting

This 2019 study investigated oat sprout parameters:

Huskless oat ‘Gehl’ cultivated in 2016 in Canada, was used throughout the study. Grains (500 g) were sprouted at different temperatures (10, 14, 20, 25, and 30°C) and for different times (1, 2, and 3 days). Changes in vitamin C, β‐glucan, and reducing sugar were monitored, and α‐amylase activity was studied as a marker for total enzyme activity.

Mass fraction of radicle [root] and coleoptile [shoot] in grain correlated very well with β‐glucan level. A similarly good correlation was found for the much easier applicable degree of sprouting, visual assessment of coleoptile length set into relation to grain size.

Germinability after 3 days was about 99% at all temperatures. Temperatures between 20° and 25°C yielded the most dramatic changes in properties of sprouted oats.

  • At 3 days, α‐amylase activities at 20° and 25°C increased significantly to values one order of magnitude larger than those for other temperatures.
  • β‐glucan content was decreased after 3 days at all temperatures. Degradation was most pronounced at 20°C, almost halving initial β‐glucan content to 3.9%.
  • No ascorbic acid was present in native grain. Upon sprouting, a significant increase in ascorbic acid content was found – except at 30°C – with highest levels at 20°C.

Ascorbic acid content in radicles and coleoptile was four times higher than that in grain without radicles and coleoptile. Oat grains sprouted for 3 days at 20°C had an average degree of sprouting of 3; hence, radicles and coleoptile contributed about 8% of mass. These findings indicate that a fast visual determination of degree of sprouting allows to estimate, for example, ascorbic acid content without doing expensive experiments.

Around 20% of grains sprouted at 20° and 25°C had a coleoptile longer than a full grain length (degree of sprouting 5). Less long coleoptiles developed at other temperatures.

  • For the 3‐day sprouting period, the longest coleoptile was observed for sprouting at 25°C.
  • At 30°C average degree of sprouting was 1.4, and grains showed no practical radicle growth.

Coleoptile and radicle growth (input parameters for the degree of sprouting) and reducing sugars and α‐amylase activity are interdependent. Degree of sprouting could develop into a reliable characterization method for sprouted grains, usable for predicting compositional and nutritional changes of oats during sprouting.” “Sprouting of oats: A new approach to quantify compositional changes”

Relative humidity wasn’t mentioned in this study. I asked the corresponding coauthor about it, since two Sprouting oats studies stated relative humidity as a factor for sprouting oats.

I also asked them to explain their “4.5‐hr wet steeping, 19‐hr air rest, and 4‐hr steeping, all at 20°C” procedures to start germination, since I didn’t have access to the cited study. No reply yet.

This was my model study for Sprouting hulled oats.

Ducks in a row

Reverberations, harmonics, history

Catching up with Martin Armstrong from 2012:

“Corruption within the Roman Republic was certainly at its peak during the first century BC. There was a brewing debt crisis in Rome and the oligarchy was determined to keep power at any cost. Corruption was so widespread that interest rates doubled from 4% to 8% for the elections of 54 BC because there was so much bribery going on to gain votes.

Caesar was clearly a Popularis, a man of the people who stood against the corruption of the Republic. Like today, we have no real voting control over the fate of the nation. Those who are in charge of the political machine control the real political state.

Caesar knew who his enemies truly were. He clung to his belief that if the majority of the Senate were free of the Oligarchy of Cato and Cicero, they would surely see the light. To persuade them, Caesar wrote his seven books on his truly remarkable conquest of Gaul.

Cato and his Oligarchy were so intensely anti-Caesar that they were willing to do anything to anybody. But this was a moment in time where the corruption had simply gone too far.

By September 29th, 51 BC, Caesar ran out of civilized options. Crossing the Rubicon became the only option.

Janus was the symbol of a cyclical change, the departing of one era and the birth of another. His shrine consisted of two doorways that traditionally were left open in time of war and kept closed when Rome was at peace. Leaving the doors open in time of war symbolized the new era that was possible.

Property values were collapsing. Debts were excessive. Those who held mortgages refused to accept just the property back.

Caesar dealt with this major extraordinary situation in a truly astonishing manner, realizing that assets and money are in a union of opposing forces, yet bound together. Value of property is not a constant relationship for money itself is not like a ruler.

Money is more akin to a rubber band even when it may be gold or silver. Money is like everything else – subject to the whims of supply and demand.

The economy is a dynamic relationship between everything with no real constant. We are at a tremendous disadvantage because we have grown up thinking in a flat linear world that does not exist. We limit ourselves by thinking in money,

Caesar explained that he had to borrow to fund the war and it was unethical for him to cancel all debts since he himself would benefit. Generals come and go, but true economic reformers of the state to save the nation are rare indeed. Caesar paid for his economic reform with his life.

There can be no greater example of political corruption that required desperate reform than the calendar. Caesar replaced the typical lunar year and introduced his new calendar based on 365¼ solar days on January 1st, 45 BC.

Sulla [138 – 78 BC] was a highly original, gifted and skillful general, never losing a battle. His rival described Sulla as having the cunning of a fox and the courage of a lion – but that it was the former attribute that was by far the most dangerous. This mixture was later referred to by Machiavelli in his description of the ideal characteristics of a ruler.

Sulla was more interested in retaining institutes of government while eliminating people occupying them whereas Caesar was far more compelled to act to restore institutions and to spare people, even his more threatening enemies. These are not actions of a man interested in personal power, but a man interested in saving his country.

You live in an oligarchy no different today than what Caesar faced back then. One maxim always holds true; Absolute power, corrupts absolutely!”

It appears that only Julius Caeser ever understood

100-44 BC

A normal distribution

Does sulforaphane reach the colon?

This 2020 study subject was antimicrobial activity of sulforaphane:

“This study explored the role that digestion and cooking practices play in bioactivity and bioavailability, especially the rarely considered dose delivered to the colon.

A broccoli powder soup was prepared which contained 26.5 µmol of sulforaphane per 200 ml portion. Addition of 2% mustard seed powder at the cooling stage of the soup preparation process (~ 60 °C) increased the level of sulforaphane by nearly fourfold, 102 µmol per 200 ml.

Recovery of sulforaphane in ileal fluids post soup consumption was < 1% but the addition of mustard seeds increased colon-available sulforaphane sixfold. Analysis of glucosinolates composition in ileal fluids revealed noticeable inter-individual differences.

Consumption of sulforaphane-enriched broccoli soup may inhibit bacterial growth in the stomach and upper small intestine, but not in the terminal ileum or the colon.” “Sulforaphane-enriched extracts from glucoraphanin-rich broccoli exert antimicrobial activity against gut pathogens in vitro and innovative cooking methods increase in vivo intestinal delivery of sulforaphane”

My son has often asked me about adding mustard seeds to broccoli sprouts. Papers citing one of this study’s coauthors’ series of mustard seed studies include:

I bought a 74 gram container of mustard seeds at the local grocery store, and ground down a third as pictured. A level scoop of mustard seed powder weighs 1.5 grams.

1.5 g divided by my twice-daily 65 g of microwaved broccoli sprouts = 2%, matching this study’s methods. That’s a 24-day mustard seed supply for $2.19.

I’ll add mustard seed powder immediately after microwaving broccoli sprouts when they’re ≤ 60°C (140°F). Allowing the mixture to process for five minutes potentially facilitates myrosinase hydrolization of glucoraphanin and other glucosinolates into healthy isothiocyanate compounds.

Other aspects of this study:

1. I don’t consider overcooking broccoli an “innovative cooking method.” It’s more like researchers creating an effect in order to publish “increased the level of sulforaphane by nearly fourfold” which was presented numerically and emphasized twice in text.

2. A perspective on these types of studies from Epigenetic mechanisms of muscle memory:

“Underpowered studies may only be useful to check if the experiment works, but not as much for testing and estimating effects.

3. I didn’t agree with this study’s treatment of individual differences.

I read three other papers’ study design recommendations for researchers regarding inter-individual variability, but didn’t see markedly better ideas. Most of their verbiage concerned how to reduce heterogeneous effects rather than to understand causes and signals. Where are thoughtful counters to meaningless averages / standard deviations / p values / and so on?

4. “Addition of mustard seeds increased colon-available sulforaphane sixfold” was presented numerically and emphasized thrice in text. Too often for a n=11 study.

What needed further explanations were detailed causes for each individual’s responses or lack thereof. Stratifying subgroups into unresponsive:

  • What happened in Subjects 6’s and 10’s lives to make them unresponsive to any sulforaphane dose?
  • Were Subjects 1, 2, 5, and 7 instances of zero sulforaphane actually errors in measuring or processing? If not, what were individual causes for instances of no response?

And responsive:

  • Were Subjects 4, 8, 9, 11, and 12 averages meaningful? Excluding Subject 4’s 3.14 μmol, was the four remaining subjects’ 0.19 to 0.63 μmol average 332% increased response meaningful when the sulforaphane dose increased 392%?
  • What caused Subject 4’s 872% increased response when the sulforaphane dose increased 392%?

5. Findings of sulforaphane in 11 g broccoli powder not reaching the colon may not apply to 65 g broccoli sprouts due to mass and quantity differences. Broccoli sprouts definitely pass into the colon, like any other fibrous vegetable. Unhydrolyzed products are hydrolyzed by microflora there.

I create sulforaphane from broccoli sprouts shortly before eating them, and don’t depend on metabolism after the stomach to produce isothiocyanates. These findings may assist with that effort.

Clearing out the 2020 queue of interesting papers

I’ve partially read these 39 studies and reviews, but haven’t taken time to curate them.

Early Life

  1. Intergenerational Transmission of Cortical Sulcal Patterns from Mothers to their Children (not freely available)
  2. Differences in DNA Methylation Reprogramming Underlie the Sexual Dimorphism of Behavioral Disorder Caused by Prenatal Stress in Rats
  3. Maternal Diabetes Induces Immune Dysfunction in Autistic Offspring Through Oxidative Stress in Hematopoietic Stem Cells
  4. Maternal prenatal depression and epigenetic age deceleration: testing potentially confounding effects of prenatal stress and SSRI use
  5. Maternal trauma and fear history predict BDNF methylation and gene expression in newborns
  6. Adverse childhood experiences, posttraumatic stress, and FKBP5 methylation patterns in postpartum women and their newborn infants (not freely available)
  7. Maternal choline supplementation during the third trimester of pregnancy improves infant information processing speed: a randomized, double‐blind, controlled feeding study
  8. Preterm birth is associated with epigenetic programming of transgenerational hypertension in mice
  9. Epigenetic mechanisms activated by childhood adversity (not freely available)

Epigenetic clocks

  1. GrimAge outperforms other epigenetic clocks in the prediction of age-related clinical phenotypes and all-cause mortality (not freely available)
  2. Epigenetic age is a cell‐intrinsic property in transplanted human hematopoietic cells
  3. An epigenetic clock for human skeletal muscle
  4. Immune epigenetic age in pregnancy and 1 year after birth: Associations with weight change (not freely available)
  5. Vasomotor Symptoms and Accelerated Epigenetic Aging in the Women’s Health Initiative (WHI) (not freely available)
  6. Estimating breast tissue-specific DNA methylation age using next-generation sequencing data


  1. The Intersection of Epigenetics and Metabolism in Trained Immunity (not freely available)
  2. Leptin regulates exon-specific transcription of the Bdnf gene via epigenetic modifications mediated by an AKT/p300 HAT cascade
  3. Transcriptional Regulation of Inflammasomes
  4. Adipose-derived mesenchymal stem cells protect against CMS-induced depression-like behaviors in mice via regulating the Nrf2/HO-1 and TLR4/NF-κB signaling pathways
  5. Serotonin Modulates AhR Activation by Interfering with CYP1A1-Mediated Clearance of AhR Ligands
  6. Repeated stress exposure in mid-adolescence attenuates behavioral, noradrenergic, and epigenetic effects of trauma-like stress in early adult male rats
  7. Double-edged sword: The evolutionary consequences of the epigenetic silencing of transposable elements
  8. Blueprint of human thymopoiesis reveals molecular mechanisms of stage-specific TCR enhancer activation
  9. Statin Treatment-Induced Development of Type 2 Diabetes: From Clinical Evidence to Mechanistic Insights
  10. Rewiring of glucose metabolism defines trained immunity induced by oxidized low-density lipoprotein
  11. Chronic Mild Stress Modified Epigenetic Mechanisms Leading to Accelerated Senescence and Impaired Cognitive Performance in Mice
  12. FKBP5-associated miRNA signature as a putative biomarker for PTSD in recently traumatized individuals
  13. Metabolic and epigenetic regulation of T-cell exhaustion (not freely available)


  1. Molecular and cellular mechanisms of aging in hematopoietic stem cells and their niches
  2. Epigenetic regulation of bone remodeling by natural compounds
  3. Microglial Corpse Clearance: Lessons From Macrophages
  4. Plasma proteomic biomarker signature of age predicts health and life span
  5. Ancestral stress programs sex-specific biological aging trajectories and non-communicable disease risk

Broccoli sprouts

  1. Dietary Indole-3-Carbinol Alleviated Spleen Enlargement, Enhanced IgG Response in C3H/HeN Mice Infected with Citrobacter rodentium
  2. Effects of caffeic acid on epigenetics in the brain of rats with chronic unpredictable mild stress
  3. Effects of sulforaphane in the central nervous system
  4. Thiol antioxidant thioredoxin reductase: A prospective biochemical crossroads between anticancer and antiparasitic treatments of the modern era (not freely available)
  5. Quantification of dicarbonyl compounds in commonly consumed foods and drinks; presentation of a food composition database for dicarbonyls (not freely available)
  6. Sulforaphane Reverses the Amyloid-β Oligomers Induced Depressive-Like Behavior (not freely available)