Pectin and butyrate

SCFA from fibers, 1991 [1]

In 1991 a group of three investigators from representative states in the US Middle West measured short chain fatty acid production from the digestion of fivers by human intestinal bacteria.  Three healthy male donors served as the sources of the inoculum for the anaerobic fermentation of agriculture wastes as well as food grade undigestible polysaccharides.  The media included salts, yeast extract, vitamins, and the reducing agent/sulfur containing amino acid cysteine. 

Experiment 1

The fibers included

1. soy
2. sugar beet
3.  pea fiber
4. bleached oathull
5. citrus pectin

Experiment 2

1. gum Arabic
2. Arabic / guar gums mixture
3. apple pectin
4. oat fiber
5. corn bran

The scfa were monitored over the course of several days

Both butyrate and propionate were products of fermentation in all reactions.  The approximate relative amount of propionate to butyrate is written between the two columns.  In a sense, citrus pectin yields the best butyrate production without as much propionate.  Apple pectin also performed very well. 

Does the microbiome matter when fermenting orange pectin? [2]

In 2018 a Korean group continued the concept using the feces of three healthy male donors as the inoculum and citrus peel as the carbon source.  Yeast extract and L-cysteine were part of the fermentation broth.   Figure 1 examined a time dependent decrease in the total carbohydrate and an increase in hydrolysis yielding smaller units with free aldehyde groups that reacted with 3,5-dinitrosalicylic acid.  Figure 2 explored the major phyla in the three donors as well as the most abundant genera (not shown in this post).

Figure 5 documented the changes in scfa over the course of two days.  Note that butyrate is produced more than propionate in this system.  The middle figures of this publication examined differences between individual donor microbiomes and changes in the microbiomes over time in the fermentation vessels.   Make close note of the small sizes of the error bars in Figure 5.  Previous figures in this publication demonstrated considerable differences in the microbiome of the three donors that were larger than the differences between the microbiome over the course of fermentation of orange pectin. We’ll view these figures later in the most just to make a point.

Lachnospira, Roseburia, Bacteroides

This figure allows us to view individual donor variations of three genera over several hours.

There is a trend for Bacterioides to go down with the three donor samples. Can we say the same for the other taxa representatives?

The microbiome ups and downs of fermenting pectin

Figure 4 is somewhat hard to interpret because the authors never defied what they meant by “g__”, “f__”, and “o__.”  Because there were three donors, we can assume that these designations correspond to the names of the three donors. Note, this figure has been turned on its side for better viewing in this format.

Note that some of these taxa are represented by donors g and f but donor “o__” doesn’t experience time dependent changes in synch with the other donors.  The interesting thing about this study in terms of FMT is that in spite of large differences in the microbiome, the changes in scfa production when orange pectin is introduced is not that great. Going back to figures 2b and 3, we reinforce the realization that the donors are very different.

Reexamining the importance of diversity

Some of the figures comparing the differences between the donors aside from the two major phyla were not planned for this post. All that mattered was the production of scfa, right? Not only do the donor microbiomes start out different, but they stay different over the course of 18 hours.

We also are forced to ask ourselves if changes in scfa are really the ONLY goal of FMT transplantation.  We certainly have evidence that there may be other things to try first.

A cool review and turning to a new target [3]

Frank Blanco‑Pérez is the first author of a review covering the health benefits of pectin.  This post will not go into the first figures covering pectin structure.  Figure 3 is a pretty good guide for key word searching of the review and Wikipedia, of course.

TLR2 is a more general receptor for pathogen associated molecular patterns whereas TLR4 is best known as the receptor for lipopolysaccharide. It was not clear from reading the abstract of the “lemon pectin” reference if pectin binds to any of the G protein coupled receptor super family members.  A PubMed search failed to reveal any candidates.  Moving on to Figure 4 and key word/Wikipedia searching…

cscfa: not just for the TCA cycle and G protein coupled receptors

We’re developing an interest in the role of the microbiome in auto immune disorders. Here comes the start of how it works. This figure has become somewhat mutilated.

HDAC is histone deacetylase.  It became necessary to perform a PubMed search to really appreciate what the authors were trying to say.  The following is a direct quote from the abstract

“Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD⁺) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.”

Shimazu T, Hirschey MD, Newman J, He W, Shirakawa K, Le Moan N, Grueter CA, Lim H, Saunders LR, Stevens RD, Newgard CB, Farese RV Jr, de Cabo R, Ulrich S, Akassoglou K, Verdin E. (2013) Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science. 2013 Jan 11;339(6116):211-4. PMC free article

cse

The Sanchez publication is probably too complicated to thoroughly cover in this post.  Let’s create a mental image from part of the abstract

the players

• Class Switch Recombination. CSR, is a process in which naive B cells produce IgM and IgD. After acivation of antigen, they switch to producing IgG, IgA, and IgE.
• Blimp2, or the B lymphocyte induced maturation protein 1 is a protein encoded by the PRDM1 gene, is a repressing transcription factor. without Blimp1, proliferating B cells are unable to differentiate into antibody producing plasma cells, resulting in severe reduction in production of all isotypes of immunoglobulin. Other sources state that Blimp1 can serve as a transcription suppressor or inducer depending on the gene and protein context.
• The gene AICDA codes for Activation-induced cytidine deaminase. This enzyme turns cytosines to uracil, that get copied as thymine. During germinal center development of B lymphocytes, AID also generates other types of mutations, such as C:G to A:T. The Wikipedia mentioned that AICDA plays a role in CSR but did not explain how.
• HDAC is a member of the histone deacylase family. HDAC removes aceyl groups from positively charged lysine groups in histones, the protein spools around which negatively charged chromosomal DNA is wound. Inhibiting HDAC serves to keep genes from being transcribed, which ultimately leads to decreasing protein production.
• miRNA, microRNA, are 21-23 nucleotide single stranded pieces of RNA that bind to the mRNA that code for proteins in such a way as to prevent their translation into proteins and/or promote their degradation.

This is the link to the image source.

gleaning from the abstract and figures

These authors demonstrated that at low doses butyrate and propionate directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while
decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR,
somatic hypermutation and plasma cell differentiation.

1. Microbiome production of butyrate and propionate
2. butyrate and propionate inhibited HDAC activity
3. overall histone (H3K9) acetylation allowed transcription of miRNA that inhibit translation of Blimp1 and AICD mRNAs

these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impaction B cells extends to inhibition of autoantibody production and autoimmunity in mouse
lupus models.  

References

1. Titgemeyer EC, Bourquin LD, Fahey GC Jr, Garleb KA.(1991) Fermentability of various fiber sources by human fecal bacteria in vitro. Am J Clin Nutr. Jun;53(6):1418-24
2. Bang SJ, Kim G, Lim MY, Song EJ, Jung DH, Kum JS, Nam YD, Park CS, Seo DH. The influence of in vitro pectin fermentation on the human fecal microbiome. AMB Express. 2018 Jun 16;8(1):98. PMC free article
3. Blanco-Pérez F, Steigerwald H, Schülke S, Vieths S, Toda M, Scheurer S. The Dietary Fiber Pectin: Health Benefits and Potential for the Treatment of Allergies by Modulation of Gut Microbiota. Curr Allergy Asthma Rep. 2021 Sep 10;21(10):43. PMC free article
4. Sanchez HN, Moroney JB, Gan H, Shen T, Im JL, Li T, Taylor JR, Zan H, Casali P. B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids. Nat Commun. 2020 Jan 2;11(1):60. PMC free article