Bile salts: definition, functions, enterohepatic circulation, synthesis

What are bile salts

Bile salts and bile acids are polar cholesterol derivatives, and represent the major route for the elimination of the steroid from the body.
They are molecules with similar but not identical structures, and diverse physical and biological characteristics.
They are synthesized in the liver, stored in the gallbladder, secreted into the duodenum, and finally, for the most part, reabsorbed in the ileum.
Because at physiological pH these molecules are present as anions, the terms bile acid and bile salts are used herein as synonyms.

Chemical structure of bile salts

Bile Salts
Fig. 1 – Chemical Structures of the Most Abundant Bile Acids

Bile salts have similarities and differences with cholesterol molecule.
Like the steroid, they have a nucleus composed of four fused rings: three cyclohexane rings, labeled A, B and C, and a cyclopentane ring, labeled D. This structure is the perhydrocyclopentanophenanthrene, more commonly known as steroid nucleus.
In higher vertebrates, they have 24 carbon atoms, as the side chain is three carbons shorter than the original. In lower vertebrates, bile acids have 25, 26, or 27 carbon atoms. The side chain ends with a carboxyl group, ionized at pH 7, that can be linked to the amino acid glycine or taurine (see below).
In addition to the hydroxyl group at position 3, they have hydroxyl groups at positions 7 and/or 12.
All this makes them much more polar than cholesterol.

Bile Salts
Fig. 2 – Cholic Acid Structure

Since A and B rings are fused in cis configuration, the planar structure of the steroid nucleus is curved, and it is possible to identify:

  • a concave side, which is hydrophilic because the hydroxyl groups and the carboxyl group of the side chain, with or without the linked amino acid, are oriented towards it;
  • a convex side, which is hydrophobic because the methyl groups present at position 18 and 19 are orientated towards it.

Therefore, having both polar and nonpolar groups, they are amphiphilic molecules and excellent surfactants. However, their chemical structure makes them different from many other surfactants, often composed of a polar head region and a nonpolar tail.

Primary, conjugated and secondary bile salts

Bile Salts
Fig. 3 – Conjugated Bile Acids

Primary bile acids are those synthesized directly from cholesterol in the hepatocytes. In humans, the most important are cholic acid and chenodeoxycholic acid, which make up 80% of all bile acids. Before being secreted into the biliary tree, they are almost completely conjugated, up to 98%, with the glycine or taurine, to form glycoconjugates and tauroconjugates, respectively. In particular, approximately 75% of cholic acid and chenodeoxycholic acid are conjugated with glycine, to form glycocholic acid  and glycochenodeoxycholic acid, the remaining 25% with taurine, to form taurocholic acid and taurochenodeoxycholic.
Conjugated bile acids are molecules with more hydrophilic groups than unconjugated bile acids, therefore with a increased emulsifying capacity. In fact, conjugation decreases the pKa of bile acids, from about 6, a value typical of non-conjugated molecules, to about 4 for glycocholic acid, and about 2 for taurocholic acid. This makes that conjugated bile acids are ionized in a broader range of pH to form the corresponding salts.
The hydrophilicity of the common acid and bile salts decreases in the following order: glycine-conjugated < taurine-conjugated < lithocholic acid  < deoxycholic acid  < chenodeoxycholic acid < cholic acid <ursodeoxycholic acid.
Finally, conjugation also decreases the cytotoxicity of primary bile acids.

Secondary bile acids  are formed from primary bile acids which have not been reabsorbed from the small intestine. Once they reach the colon, they can undergo several modifications by colonic microbiota to form secondary bile acids (see below). They make up the remaining 20% of the body’s bile acid pool.

Another way of categorizing bile salts is based on their conjugation with glycine and taurine and their degree of hydroxylation. On this basis, three categories are identified.

  • Trihydroxy conjugates, such as taurocholic acid and glycocholic acid.
  • Dihydroxy conjugates, such as glycodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid. They account for about 60% of bile salts present in the bile.
  • Unconjugated forms, such as cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid.

Function of bile acids

All their physiological functions are performed in the conjugated form.

  • They are the major route for the elimination of cholesterol from human body.
    Indeed, humans do not have the enzymes to break open the cyclohexane rings or  the cyclopentane ring of the steroid nucleus, nor to oxidize cholesterol to CO2 and water.
    The other mechanism to eliminate the steroid from the body is as cholesterol per se in the bile.
  • Bile salts are strong surfactants. And in particular, di- and trihydroxy conjugates are the best surfactants among bile acids, much more effective than unconjugated counterparts, since they have more polar groups.
    Once in contact with apolar lipids in the lumen of the small intestine, the convex apolar surface interacts with the apolar lipids, such as triglycerides, cholesterol esters, and ester of fat-soluble vitamins, whereas the concave polar surface interacts with the surrounding aqueous medium. This increases the dispersion of apolar lipids in the aqueous medium, as it allows the formation of tiny lipid droplets, increasing the surface area for:

lipase activity, mainly pancreatic lipase, (bile salts also play a direct role in the activation of this enzyme);

intestinal esterase activity.

Subsequently, they facilitate the absorption of lipid digestion products, as well as of fat soluble vitamins by the intestinal mucosa thanks to the formation of mixed micelles.
Bile acids perform a similar function in the gallbladder where, forming mixed micelles with phospholipids, they prevent the precipitation of cholesterol.
Note: as a consequence of the arrangement of polar and nonpolar groups, bile acids form micelles in aqueous solution, usually made up of less than 10 monomers, as long as their concentration is above the so-called critical micellar concentration or CMC.

  • At the intestinal level, they modulate the secretion of pancreatic enzymes and cholecystokinin.
  • In the small and large intestine, they have a potent antimicrobial activity, mainly deoxycholic acid, in particular against Gram-positive bacteria. This activity may be due to oxidative DNA damage, and/or to the damage of the cell membrane. Therefore, they play an important role in the prevention of bacterial overgrowth, but also in the regulation of gut microbiota composition.
  • In the last few years, it becomes apparent their regulatory role in the control of energy metabolism, and in particular for the hepatic glucose handling.

Enterohepatic circulation of bile salts

After fat intake, enteroendocrine cells of the duodenum secrete cholecystokinin into the blood stream. Hormone binding to receptors on smooth muscle cells of the gallbladder promotes their contraction; the hormone also causes the relaxation of the sphincter of Oddi. All this results in the secretion of the bile, and therefore of bile acids into the duodenum.
Under physiological conditions, human bile salt pool is constant, and equal to about 3-5 g. This is made possible by two processes:

  • their intestinal reabsorption;
  • their de novo synthesis (see below).

Up to 95% of the secreted bile salts is reabsorbed from the gut, not together with the products of lipid digestion, but through a process called enterohepatic circulation.
It is an extremely efficient recycling system, which seems to occur at least two times for each meal, and includes the liver, the biliary tree, the small intestine, the colon, and the portal circulation through which reabsorbed molecules return to the liver. Such recirculation is necessary since liver’s capacity to synthesize bile acids is limited and insufficient to satisfy intestinal needs if the bile salts were excreted in the feces in high amounts.
Most of the bile salts are reabsorbed into the distal ileum, the lower part of the small intestine, by a sodium-dependent transporter within the brush border of the enterocytes, called sodium-dependent bile acid transporter or ASBT, which carries out the cotransport of a molecule of bile acid and two sodium ions.
Within the enterocyte, it is thought that bile acids are transported across the cytosol to the basolateral membrane by the ileal bile acid-binding protein or IBABP. They cross the basolateral membrane by the organic solute transporter alpha-beta or OSTα/OSTβ, pass into the portal circulation, and, bound to albumin, reach the liver.
It should be noted that a small percentage of bile acids reach the liver through the hepatic artery.
A hepatic level, their extraction is very efficient, with a first-pass extraction fraction ranging from 50 to 90%, a percentage that depends on bile acid structure. The uptake of conjugated bile acids is mainly mediated by a Na+-dependent active transport system, that is, the sodium-dependent taurocholate cotransporting polypeptide or NTCP. However, a sodium-independent uptake can also occur, carried out by proteins of the family of organic anion transporting polypeptides or OATP, mainly OATP1B1 and OATP1B3.
The rate limiting step in the enterohepatic circulation is their canalicular secretion, largely mediated by the bile salt export pump or BSEP, in an ATP-dependent process. This pump carries monoanionic bile salts, which are the most abundant. Bile acids conjugated with glucuronic acid or sulfate, which are dianionic, are transported by different carriers, such as MRP2 and BCRP.

Note: serum levels of bile acids vary on the basis of the rate of their reabsorption, and therefore they are higher during meals, when the enterohepatic circulation is more active.

Intestinal metabolism of bile acids

Bile Salts
Fig. 4 – Intestinal Bile Acid Metabolism

Bile acids which escape ileal absorption pass into the colon where they partly undergo modifications by intestinal microbiota and are converted to secondary bile acids.
The main reactions are listed below.

  • Deconjugation
    On the side chain, hydrolysis of the C24 N-acyl amide bond can occur, with release of unconjugated bile acids and glycine or taurine. This reaction is catalyzed by bacterial hydrolases present both in the small intestine and in the colon.
  • 7α-Dehydroxylation
    Quantitatively, it is the most important reaction, carried out by colonic bacterial dehydratases that remove the hydroxyl group at position 7 to form 7-deoxy bile acids. In particular, deoxycholic acid is formed from cholic acid, and lithocholic acid, a toxic secondary bile acid, from chenodeoxycholic acid.
    It should be noted that 7α-dehydroxylation, unlike oxidation and epimerization (see below), can only occur on unconjugated bile acids, and therefore, deconjugation is an essential prerequisite.
  • Oxidation and epimerization
    They are reactions involving the hydroxyl groups at position 3, 7 and 12, catalyzed by bacterial hydroxysteroid dehydrogenases. For example, ursodeoxycholic acid derives from the epimerization of chenodeoxycholic acid.

Some of the secondary bile acids are then reabsorbed from the colon and return to the liver. In the hepatocytes, they are reconjugated, if necessary, and resecreted. Those that are not reabsorbed, are excreted in the feces.
Whereas oxidations and deconjugations are carried out by a broad spectrum of anaerobic bacteria, 7α-dehydroxylations is carried out by a limited number of colonic anaerobes.
7α-Dehydroxylations and deconjugations increase the pKa of the bile acids, and therefore their hydrophobicity, allowing a certain degree of passive absorption across the colonic wall.
The increase of hydrophobicity is also associated with an increased toxicity of these molecules. And a high concentration of secondary bile acids in the bile, blood, and feces has been associated to the pathogenesis of colon cancer.

Soluble fibers and reabsorption of bile salts

The reabsorption of bile salts can be reduced by chelating action of soluble fibers, such as those found in fresh fruits, legumes, oats and oat bran, which bind them, decreasing their uptake. In turn, this increases bile acid de novo synthesis, up-regulating the expression of the 7α-hydroxylase and sterol 12α-hydroxylase (see below), and thereby reduces hepatocyte cholesterol concentration.
The depletion of hepatic cholesterol increases the expression of the LDL receptor, and thus reduces plasma concentration of LDL cholesterol. On the other hand, it also stimulates the synthesis of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis.
Note: some anti-cholesterol drugs act by binding bile acids in the intestine, thereby preventing their reabsorption.

Synthesis of primary bile acids

Bile Salts
Fig. 5 – Primary Bile Acid Biosynthesis

Quantitatively, bile acids are the major product of cholesterol metabolism.
As previously said, enterohepatic circulation and their de novo synthesis maintain a constant bile acid pool size. In particular, de novo synthesis allows the replacement of bile salts excreted in the faces, about 5-10% of the body pool, namely ~ 0.5 g/day.
Below, the synthesis of cholic acid and chenodeoxycholic acid, and their conjugation with the amino acids taurine and glycine, is described.
There are two main pathways for bile acid synthesis: the classical pathway and the alternative pathway. In addition, some other minor pathways will also be described.

The classical or neutral pathway

In humans, up to 90% of bile salts are produced via the classical pathway (see fig. 5), also referred to as “neutral” pathway since intermediates are neutral molecules.
It is a metabolic pathway present only in the liver, that consists of reactions catalyzed by enzymes localized in the cytosol, endoplasmic reticulum, peroxisomes, and mitochondria, and whose end products are the conjugates of cholic acid and chenodeoxycholic acid.

  • The first reaction is the hydroxylation at position 7 of cholesterol, to form 7α-hydroxycholesterol. The reaction is catalyzed by cholesterol 7α-hydroxylase or CYP7A1 (E.C. 1.14.14.23). It is an enzyme localized in the endoplasmic reticulum, and catalyzes the rate-limiting step of the pathway.

Cholesterol + NADPH + H+ + O2 → 7α-Hydroxycholesterol + NADP+ + H2O

  • 7α-Hydroxycholesterol undergoes oxidation of the 3β-hydroxyl group and the shift of the double bond from the 5,6 position to the 4,5 position, to form 7α-hydroxy-4-cholesten-3-one. The reaction is catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase or HSD3B7 (E.C. 1.1.1.181), an enzyme localized in the endoplasmic reticulum.
  • 7α-Hydroxy-4-cholesten-3-one can follow two routes:

to enter the pathway that leads to the synthesis of cholic acid, through the reaction catalyzed by 7α-hydroxy-4-cholesten-3-one 12α-monooxygenase or sterol 12α-hydroxylase or CYP8B1 (E.C. 1.14.18.8), an enzyme localized in the endoplasmic reticulum;

to enter the pathway that leads the synthesis of chenodeoxycholic acid, through the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase or AKR1D1 (E.C. 1.3.1.3), a cytosolic enzyme.

It should be underlined that the activity of sterol 12α-hydroxylase determines the ratio of cholic acid to chenodeoxycholic acid, and, ultimately, the detergent capacity of bile acid pool. And in fact, the regulation of sterol 12α-hydroxylase gene transcription is one of the main regulatory step of the classical pathway.

Therefore, if 7α-hydroxy-4-cholesten-3-one proceeds via the reaction catalyzed by sterol 12α-hydroxylase, the following reactions will occur.

  • 7α-Hydroxy-4-cholesten-3-one is hydroxylated at position 12 by sterol 12α-hydroxylase, to form 7α,12α-dihydroxy-4-cholesten-3-one.
  • 7α,12α-Dihydroxy-4-cholesten-3-one undergoes reduction of the double bond at 4,5 position, in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase, to form 5β-cholestan-7α,12α-diol-3-one.
  • 5β-Cholestan-7α,12α-diol-3-one undergoes reduction of the hydroxyl group at position 4, in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase or AKR1C4 (EC 1.1.1.213), a cytosolic enzyme, to form 5β-cholestan-3α,7α,12α-triol.
  • 5β-Cholestan-3α,7α,12α-triol undergoes oxidation of the side chain via three reactions catalyzed by sterol 27-hydroxylase or CYP27A1 (EC 1.14.15.15). It is a mitochondrial enzyme also present in extrahepatic tissues and macrophages, which introduces a hydroxyl group at position 27. The hydroxyl group is oxidized to aldehyde, and then to carboxylic acid, to form 3α,7α,12α-trihydroxy-5β-cholestanoic acid.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoic  acid is activated to its coenzyme A ester, 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA, in the reaction catalyzed by either very long chain acyl-CoA synthetase or VLCS (EC 6.2.1.-), or bile acid CoA synthetase or BACS (EC 6.2.1.7), both localized in the endoplasmic reticulum.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoyl-CoA is transported to peroxisomes where it undergoes five successive reactions, each catalyzed by a different enzyme. In the last two reactions, the side chain is shortened to four carbon atoms, and finally cholylCoA is formed.
  • In the last step, the conjugation, via amide bond, of the carboxylic acid group of the side chain with the amino acid glycine or taurine occurs. The reaction is catalyzed by bile acid-CoA:amino acid N-acyltransferase or the BAAT (EC 2.3.1.65), which is predominantly localized in peroxisomes.
    The reaction products are thus the conjugated bile acids: glycocholic acid and taurocholic acid.

If 7α-hydroxy-4-cholesten-3-one does not proceed via the reaction catalyzed by sterol 12α-hydroxylase, it enters the pathway that leads to the synthesis of chenodeoxycholic acid conjugates, through the reactions described below.

  • 7α-Hydroxy-4-cholesten-3-one is converted to 7α-hydroxy-5β-cholestan-3-one in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase.
  • 7α-Hydroxy-5β-cholestan-3-one is converted to 5β-cholestan-3α,7α-diol in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase.

Then, the conjugated bile acids glycochenodeoxycholic acid and taurochenodeoxycholic acid are formed by modifications similar to those seen for the conjugation of cholic acid, and catalyzed mostly by the same enzymes.

Note: unconjugated bile acids formed in the intestine must reach the liver to be reconjugated.

The alternative or acidic pathway

It is prevalent in the fetus and neonate, whereas in adults it leads to the synthesis of less than 10% of the bile salts.
This pathway  (see fig. 5) differs from the classical pathway in that:

  • the intermediate products are acidic molecules, from which the alternative name “acidic pathway”;
  • the oxidation of the side chain is followed by modifications of the steroid nucleus, and not vice versa;
  • the final products are conjugates of chenodeoxycholic acid.

The first step involves the conversion of cholesterol into 27-hydroxycholesterol in the reaction catalyzed by sterol 27-hydroxylase.
27-Hydroxycholesterol can follow two routes.

Route A

  • 27-hydroxycholesterol is converted to 3β-hydroxy-5-cholestenoic acid in a reaction catalyzed by sterol 27-hydroxylase.
  • 3β-Hydroxy-5-cholestenoic acid is hydroxylated at position 7 in the reaction catalyzed by oxysterol 7α-hydroxylase or CYP7B1 (EC 1.14.13.100), an enzyme localized in the endoplasmic reticulum, to form 3β-7α-dihydroxy-5-colestenoic acid.
  • 3β-7α-Dihydroxy-5-cholestenoic acid is converted to 3-oxo-7α-hydroxy-4-cholestenoic acid, in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase.
  • 3-Oxo-7α-hydroxy-4-cholestenoic acid, as a result of side chain modifications, forms chenodeoxycholic acid, and then its conjugates.

Route B

  • 27-Hydroxycholesterol is converted to 7α,27-dihydroxycholesterol in the reaction catalyzed by oxysterol 7α-hydroxylase and cholesterol 7α-hydroxylase.
  • 7α,27-Dihydroxycholesterol is converted to 7α,26-dihydroxy-4-cholesten-3-one in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase;

7α, 26-Dihydroxy-4-cholesten-3-one can be transformed directly to conjugates of chenodeoxycholic acid, or can be converted to 3-oxo-7α-hydroxy-4-colestenoic acid,  and then undergo side chain modifications and other reactions that lead to the synthesis of the conjugates of chenodeoxycholic acid.

Minor pathways

There are also minor pathways (see fig. 5) that contribute to bile salt synthesis, although to a lesser extent than classical and alternative pathways.

For example:

  • A cholesterol 25-hydroxylase (EC 1.14.99.38) is expressed in the liver.
  • A cholesterol 24-hydroxylase or CYP46A1 (EC 1.14.14.25) is expressed in the brain, and therefore, although the organ cannot export cholesterol, it exports oxysterols.
  • A nonspecific 7α-hydroxylase has also been discovered. It is  expressed in all tissues and appears to be involved in the generation of oxysterols, which may be transported to hepatocytes to be converted to chenodeoxycholic acid.

Additionally, sterol 27-hydroxylase is expressed in various tissues, and therefore its reaction products must be transported to the liver to be converted to bile salts.

Bile salts: regulation of synthesis

Regulation of bile acid synthesis occurs via a negative feedback mechanism, particularly on the expression of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase.
When an excess of bile acids, both free and conjugated, occurs, these molecules bind to the nuclear receptor farnesoid X receptor or FRX, activating it: the most efficacious bile acid is chenodeoxycholic acid, while others, such as ursodeoxycholic acid, do not activate it.
FRX induces the expression of the transcriptional repressor small heterodimer partner or SHP, which in turn interacts with other transcription factors, such as liver receptor homolog-1 or LRH-1, and hepatocyte nuclear factor-4α or HNF-4α. These transcription factors bind to a sequence in the promoter region of 7α-hydroxylase and 12α-hydroxylase genes, region called bile acid response elements or BAREs, inhibiting their transcription.
One of the reasons why bile salt synthesis is tightly regulated is because many of their metabolites are toxic.

References

Chiang J.Y.L. Bile acids: regulation of synthesis. J Lipid Res 2009;50(10):1955-66 [PDF]

Gropper S.S., Smith J.L. Advanced nutrition and human metabolism. 6h Edition. Cengage Learning, 2012 [Google eBook]

Moghimipour E., Ameri A., and Handali S. Absorption-enhancing effects of bile salts. Molecules 2015;20(8); 14451-73 [Article]

Monte M.J., Marin J.J.G., Antelo A., Vazquez-Tato J. Bile acids: Chemistry, physiology, and pathophysiology. World J Gastroenterol 2009;15(7):804-16 [Article]

Rawn J.D. Biochimica. Mc Graw-Hill, Neil Patterson Publishers, 1990

Rosenthal M.D., Glew R.H. Medical biochemistry – Human metabolism in health and disease. John Wiley J. & Sons, Inc., Publication, 2009

Sundaram S.S., Bove K.E., Lovell M.A. and Sokol R.J. Mechanisms of Disease: inborn errors of bile acid synthesis. Nat Clin Pract Gastroenterol Hepatol 2008;5(8):456-68 [Abstract]

Human gut microbiota: definition, composition, and the effect of diet

Definition and composition of the human gut microbiota

Gut Microbiota
Fig. 1 – Lactobacillus acidophilus

The human gastrointestinal tract is one of the most fierce and competitive ecological niches. It harbors viruses, eukaryotes, bacteria, and one member of Archaebacteria, Methanobrevibacter smithii.
Bacteria vary in proportion and amount all along the gastrointestinal tract; the greatest amount is found in the colon, which contains over 400 different species belonging to 9 phyla or divisions (of the 30 recognized phyla), and hereafter you refer to them as gut microbiota.
These are the phyla and some of their most represented genera.

  • Actinobacteria (Gram-positive bacteria); Bifidobacterium, Collinsella, Eggerthella, and Propionibacterium.
  • Bacteroidetes (Gram-negative bacteria); more than 20 genera including Bacteroides, Prevotella and Corynebacterium.
  • Cyanobacteria (Gram-negative bacteria).
  • Firmicutes (Gram-positive bacteria); at least 250 genera, including Mycoplasma, Bacillus, Clostridium, Dorea, Faecalibacterium, Ruminococcus, Eubacterium, Staphylococcus, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Sporobacter, and Roseburia.
  • Fusobacteria (Gram-negative bacteria);
  • Lentisphaerae (Gram-negative bacteria).
  • Proteobacteria (Gram-negative bacteria); Escherichia, Klebsiella, Shigella, Salmonella, Citrobacter, Helicobacter, and Serratia.
  • Spirochaeates (Gram-negative bacteria).
  • Verrucomicrobia (Gram-negative bacteria).

The presence of a small subset of the bacterial world in the colon is the result of a strong selective pressure which acted, during evolution, on both the microbial colonizers, selecting organisms very well adapted to this environment, and the intestinal niche. And nevertheless, each individual harbors an unique bacterial community in his gut.
Despite the high variability existing both with regard to taxa and between individuals, it has been proposed, but not accepted by all researchers, that in most adults the bacterial gut microbiota can be classified into variants or “enterotypes”, on the basis of the ratio of the abundance of the genera Bacteroides and Prevotella. This seems to indicate that there is a limited number of well balanced symbiotic states, which could respond differently to factors such as diet, age, genetics, and drug intake (see below).

Adult’s gut harbors a large and diverse community of DNA and RNA viruses made up of about 2,000 different genotypes, none of which is dominant. Indeed, the most abundant virus accounts for only about 6% of the community, whereas in infants the most abundant virus accounts over 40% of the community. The majority of DNA viruses are bacteriophages or phages, that is, viruses that infect bacteria (they are the most abundant biological entity on earth, with an estimated population of about 1031 units), whereas the majority of RNA viruses are plant viruses.

Factors affecting gut microbiota composition and development

The intestinal bacterial community is regulated by several factors, most of which are listed below.

  • The diet of the host.
    It seems to be the most important factor.
    Traditionally considered sterile, mother’s milk harbors a rich microbiota consisting of more than 700 species, dominated by staphylococci, streptococci, bifidobacteria and lactic acid bacteria. Therefore, it is a major source for the colonization of the breastfed infant gut, and it was suggested that this mode of colonization is closely correlated with infant’s health status, because, among other functions, it could protect against infections and contribute to the maturation of the immune system. Breast milk affects intestinal microbiota also indirectly, through the presence of oligosaccharides with prebiotic activity that stimulate the growth of specific bacterial groups including staphylococci and bifidobacteria.
    A recent study has compared the intestinal microbiota of European and African children (respectively from Florence and a rural village in Burkina Faso) between the ages of 1 and 6 years old. It has highlighted the dominant role of diet over variables such as climate, geography, hygiene and health services (it was also observed the absence of significant differences in the expression of key genes regulating the immune function, which suggests a functional similarity between the two groups). Indeed infants, as long as they are breastfed, have a very similar gut microbiota, rich in Actinobacteria, mainly Bifidobacterium (see below). The subsequent introduction of solid foods in the two groups, a Western diet rich in animal fat and protein in European children, and low in animal protein but rich in complex carbohydrates in African children, leads to a differentiation in the Firmicutes/Bacteroidetes ratio between the two groups. Gram-positive bacteria, mainly Firmicutes, were more abundant than Gram-negative bacteria in European children, whereas Gram-negative bacteria, mainly Bacteroidetes, prevailed over Gram-positive bacteria in African children.
    And the long-term diets are strongly associated to the enterotype partitioning. Indeed, it has been observed that:

a diet high in animal fat and protein, i.e. a Western-type diet, leads to a gut microbiota dominated by the Bacteroides enterotype;
a diet high in complex carbohydrates, typical of agrarian societies, leads to the prevalence of the Prevotella enterotype.

Similar results emerged from the aforementioned study on children. In the Europeans, gut microbiota was dominated by taxa typical of Bacteroides enterotype, whereas in the Burkina Faso children, Prevotella enterotype dominates.
With short-term changes in the diet (10 days), such as the switch from a low-fat and high-fiber diet to a high-fat and low-fiber diet and vice versa, changes were observed in the composition of the microbiome (within 24 hours), but no stable change in the enterotype partitioning. And this underlines as a long-term diet is needed for a change in the enterotypes of the gut microbiota.
Dietary interventions can also result in changes in the gut virome, which moves to a new state, that is, changes occur in the proportions of the pre-existing viral populations, towards which subjects on the same diet converge.

  • pH, bile acids and digestive enzymes.
    The stomach, due to its low pH, is a hostile environment for bacteria, which are not present in high numbers, about 102-103 bacterial cells/gram of tissue. In addition to Helicobacter pylori, able to cause gastritis and gastric ulcers, microorganisms of the genus Lactobacillus are also present.
    Reached the duodenum, an increase in bacterial cell number occurs, 104-105 bacterial cells/gram of tissue; and similar bacterial concentrations are present in the jejunum and proximal ileum. The low number of microorganisms present in the small intestine is due to the inhospitable environment, consequent to the fact that there is the opening of the ampulla of Vater in the descending part of the duodenum, which pours pancreatic juice and bile into the duodenum, that is, pancreatic enzymes and bile acids, which damage microorganisms.
    In the terminal portion of the ileum, where the activities of pancreatic enzymes and bile acids are lower, there are about 107 bacterial cells/gram of tissue, and up to 1012-1014 bacterial cells/gram of tissue in the colon, so that bacteria represent a large proportion, about 40%, of the fecal mass.
    The distribution of bacteria along the intestine is strategic. In the duodenum and jejunum, the amount of available nutrients is much higher than that found in the terminal portion of the ileum, where just water, fiber, and electrolytes remain. Therefore, the presence of large number of bacteria in the terminal portion of the ileum, and even more in the colon, is not a problem. The problem would be to find a high bacterial concentration in the duodenum, jejunum, and proximal parts of the ileum; and there is a disease condition, called small intestinal bacterial overgrowth or SIBO, in which the number of bacteria in the small intestine increases by about 10-15 times. This puts them in a position to compete with the host for nutrients and give rise to gastrointestinal disturbances such as diarrhea.
  • The geographical position and the resulting differences in lifestyle, diet, religion etc.
    For example, a kind of geographical gradient occurs in the microbiota of European infants, with a higher number of Bifidobacterium species and some of Clostridium in Northern infants, whereas Southern infants have higher levels of Bacteroides, Lactobacillus and Eubacterium.
  • The mode of delivery (see below).
  • The genetics of the host.
  • The health status of the infant and mother.
    For example, in mothers with inflammatory bowel disease or IBD, Faecalibacterium prausnitzii, a bacterium that produces butyrate (an important source of energy for intestinal cells), and with anti-inflammatory activity is depleted, whereas there is an increase in the number of adherent Escherichia coli.
  • The treatment with antibiotics.
  • Bacterial infections and predators.
    Bacteriocins, i.e. proteins with antibacterial activity, and bacteriophages.
    Phages play an important role in controlling the abundance and composition of the gut microbiota. In particular, they could play a major role in the colonization of the newborn, infecting the dominant bacteria thus allowing to another bacterial strain to become abundant.
    This model of predator-prey dynamics, called “kill the winner”, suggests that the blooms of a specific bacterial species would lead to blooms of their corresponding bacteriophages, followed by a decline in their abundance. Therefore, the most abundant bacteriophage genotype will not be the same at different times. And although some the gene sequences present in the infant gut virome are stable over the first three months of life, dramatic changes occur in the overall composition of the viral community between the first and second week of life. During this time period also the bacterial community is extremely dynamic (see below).
  • The competition for space and nutrients.

The composition of the gut microbiota throughout life

Gut Microbiota
Fig. 2 – Development of Intestinal Microflora

The development of the intestinal microbial ecosystem is a complex and crucial event in human life, highly variable from individual to individual, and influenced by the factors outlined above.
In utero, the gut is considered sterile, but is rapidly colonized by microbes at birth, as the infant is born with an immunological tolerance instructed by the mother.
However, recent studies show the presence of bacteria in the placental tissue, umbilical cord blood, fetal membranes and amniotic fluid from healthy newborns without signs of infection or inflammation. And for example, the meconium of premature infants, born to healthy mothers, contains a specific microbiota, with Firmicutes as the main phylum, and predominance of staphylococci, whereas Proteobacteria, in particular species such as Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, but also enterococci are more abundant in the faeces.
Note: the meconium is free of detectable viruses.
It seems that both vaginal and gut bacteria may gain access to the fetus, although via different route of entry: by ascending entry the vaginal ones, by dendritic cells of the immune system the gut ones. Therefore, there could exist a fetal microbiota.

Colonization occurs during delivery by a maternal inoculum, generally composed of aerobic and facultative bacteria (the newborn’s gut initially contains oxygen), then replaced by obligate anaerobes,  bacteria typically present in adulthood, to which they have created a hospitable environment.
Furthermore, there is a small number of different taxa, with a relative dominance of the phyla Actinobacteria and Proteobacteria, that remains unchanged during the first month of life, but not in the subsequent ones as there is a large increase in variability and new genetic variants. Many studies underline that the initial exposure is important in defining the “trajectories” which will lead to the adult ecosystems. Additionally, these initial communities may act as a source of protective or pathogenic microorganisms.

Mother’s vaginal and fecal microbiotas are the main sources of inoculum in vaginally delivered infants. Indeed, infants harbor microbial communities dominated by species of the genera Lactobacillus (the most abundant genus in the vaginal microbiota and early gut microbiota) Bifidobacterium, Prevotella, or Sneathia. And it seems likely that anaerobes, such as members of the phyla Firmicutes and Bacteroidetes, not growing outside of their host, rely on the close contact between mother and offspring for transmission. Finally, due to the presence of oxygen in infant gut, the transmission of strict anaerobes could occur not directly at birth but at a later stage by means of spores.
The first bacteria encountered by infants born by caesarean section are those of the skin and hospital environment, and gut microbiota is dominated by species of the genera Corynebacterium, Staphylococcus and Propionibacterium, with a lower bacterial count and diversity in first weeks of life than infants born vaginally.
Further evidence supporting the hypothesis of vertical transmission is the similarity between the microbiota of meconium and samples obtained from possible sites of contamination.
These “maternal bacteria” do not persist indefinitely, and are replaced by other populations within the first year of life.
Objects, animals, mouths and skin of relatives, and breast milk are secondary sources of inoculum; and breast milk (see below) seems to have a primary role in determining the microbial succession in the gut.
The variation and diversity among children reflect instead the individuality of these microbial exposures.
Note: the delivery mode seems also to influence the immune system during the first year of life, perhaps via the influence on the development of gut microbiota. Infants born by cesarean section have:

  • a lower bacterial count in stool samples at one month of age, mainly due to the higher number of bifidobacteria in infants born vaginally;
  • a higher number of antibody secreting cells, which could reflect an excessive antigen exposure (the intestinal barrier would be more vulnerable to the passage of antigens).

Within a days after birth, a thriving community is established. This community is less stable over time and more variable in composition than that of adults. Very soon, it will be more numerous than that of the child’s cells, evolving according to a temporal pattern highly variable from individual to individual.
Viruses, absent at birth, reach about 108 units/gram wet weight of faeces by the end of the first week of life, therefore representing a dynamic and abundant component of the developing gut microbiota. However, viral community has an extremely low diversity, like bacteria, and is dominated by phages, which probably influence the abundance and diversity of co-occurring bacteria, as seen above. The initial source of the viruses is unknown; of course, maternal and/or environmental inocula are among the possibilities. Notably, the earliest viruses could be the result of induction of prophages from the “newborn” gut bacterial flora, hypothesis supported by the observation that more than 25% of the phage sequences seem to be very similar to those of phages infecting bacteria such as Lactococcus, Lactobacillus, Enterococcus, and Streptococcus, which are abundant in breast milk.

By the end of the first month of life it is thought that the initial phase of rapid acquisition of microorganism is over.
In 1-month-old-infants, the most abundant bacteria belong to the genera Bacteroides and Escherichia, whereas Bifidobacterium, along with Ruminococcus, appear and grow to become dominant in the gastrointestinal tract of the breastfed infants between 1 and 11 months. Bifidobacteria such as Bifidobacterium longum subspecies infantis:

  • are known to be closely related to breastfeeding;
  • are among the best characterized commensal bacteria;
  • are considered probiotics, that is, microorganisms which can confer health benefits to the host.

Their abundance confers also benefits through competitive exclusion, that is, they are an obstacle to colonization by pathogens. And indeed, Escherichia and Bacteroides can become preponderant if Bifidobacterium is not adequately present in the gut.
In contrast, bacteria of the genera Escherichia (e.g. E. coli), Clostridium (e.g. C. difficile), Bacteroides (e.g. B. fragilis) and Lactobacillus are present in higher levels in formula-fed infants than in breastfed infants.
Although breast-fed infants receive only breast milk until weaning, their microbiota can show a large variability in the abundances of bacterial taxa, with differences between individuals also with regard to the temporal patterns of variation. These variations may be due to diseases, treatments with antibiotics, changes in host lifestyle, random colonization events, as well as differences in immune responses to the gut colonizing microbes. However, it is not yet clear how these factors contribute to shape infant gut microbiota.
It seems that also the virome changes rapidly after birth, as the majority of the viral sequences present in the first week of life are not found after the second week. Moreover, the repertoire expands rapidly in number and diversity during the first three months. This is in contrast with the stability observed in the adult virome, where 95% of the sequences are conserved over time.

In normal condition, towards the end of the first year of life, babies have consumed an adult-like diet for a significant time period and should have developed a microbial community with characteristics similar to those found in the adult gut, such as:

  • a more stable composition, phylogenetically more complex, and progressively more similar among different subjects;
  • a preponderance of Firmicutes and Bacteroidetes, followed by Verrucomicrobia and a very low abundance of Proteobacteria;
  • an increase in short-chain fatty acid (SCFA) levels and bacterial load in the feces;
  • an increase of genes associated with xenobiotic degradation, vitamin biosynthesis, and carbohydrate

Interestingly, the significant turnover of taxa occurring from birth to the end of the first year is accompanied by a remarkable constancy in the overall functional capabilities.
Towards the end of the first year of life also the early viral colonizers were replaced by a community specific to the child.

The gut microbiota reaches maturity at about 2.5 years of age, fully resembling the adult gut microbiota.
The selection of the most adapted bacteria is the result of various factors.

  • The transition to an adult diet.
  • An increased fitness to the intestinal environment of the taxa that typically dominate the adult gut microbiota than the early colonizers.
  • The significant changes in the intestinal environment, result of the developmental changes in the intestinal mucosa.
  • The effects of the microbiota itself.

Therefore, the first 2-3 years of life are the most critical period in which you can intervene to shape the microbiota as best as possible, and so optimize child growth and development.

From a chaotic beginning, all this leads to the establishment of the gut ecosystem typical of the young adult, which is relatively stable over time until old age (viral, archaeal and eukaryotic components included), and dominated, at least in the western population, by members of the phyla Firmicutes, about 60% of the bacterial communities, Bacteroidetes and Actinobacteria (mainly belonging to the Bifidobacterium genus), each comprising about 10% of the bacterial community, followed by Proteobacteria and Verrucomicrobia. The genera Bacteroides, Clostridium, Faecalibacterium, Ruminococcus and Eubacterium make up, together with Methanobrevibacter smithii, the large majority of the adult gut microbial community.
It should be noted that different data were obtained from analysis of populations of African rural areas, as seen above.
And the gut microbiota is sufficiently similar among subjects to allow the identification of a shared core microbiome.
Stability and resilience, however, are subject to numerous variables among which, as previously said, diet seems to be one of the most important. Therefore, in order to maintain the stability of the gut microbiota, the variables have to be kept constant, or in the case of diseases prevented (also through vaccinations). However, the stability and resilience could be harmful if the dominant community is pathogenic.

The gut microbiota undergoes substantial changes in the elderly. In a study conducted in Ireland on 161 healthy people aged 65 years and over, the gut microbiota is distinct from that of younger adults in the majority of subjects, with a composition that seems to be dominated by the phyla Bacteroidetes, the main ones, and Firmicutes, with almost inverted percentages than those found in younger adults (although large variations across subjects were observed). And there are Faecalibacterium, about 6% of the main genera, followed by species of the genera Ruminococcus, Roseburia and Bifidobacterium (the latter about 0.4%) among the most abundant genera.
Also the variability in the composition of the community is greater than in younger adults; this could be due to the increase in morbidities associated with aging and the subsequent increased intake of medications, as well as to changes in the diet.

References

Breitbart M., Haynes M., Kelley S., Angly F., Edwards R.A., Felts B., Mahaffy J.M., Mueller J., Nulton J., Rayhawk S., Rodriguez-Brito B., Salamon P., Rohwer F. Viral diversity and dynamics in an infant gut. Res Microbiol 2008:159;367-73 [Abstract]

Claesson M.J., Cusack S., O’Sullivan O., Greene-Diniz R., de Weerd H., Flannery E., Marchesi J.R., Falush D., Dinan T., Fitzgerald G., et al. Composition, variability, and temporal stability of the intestinal microbiota of the elderly. Proc Natl Acad Sci USA 2011:108(Suppl 1):4586-91 [Article]

Clemente J.C., Ursell L.K., Wegener Parfrey L., and Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148: 1258-70 [Article]

De Filippo c., Cavalieri D., Di Paola M., Ramazzotti M., Poullet J.B., Massart S., Collini S., Pieraccini G., and Lionetti P. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci 2010:107(33);14691-6 [Abstract]

Dominguez-Bello M.G., Costello E.K., Contreras M., Magris M., Hidalgo G., Fierer N., and Knight R. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci 2010:107;11971-5 [Abstract]

Fernández L., Langa S., Martín V., Maldonado A., Jiménez E., Martín R., Rodríguez J.M. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res 2013:69(1):1-10 [Abstract]

Huurre A., Kalliomäki M., Rautava S., Rinne M., Salminen S., and Isolauri E. Mode of delivery-effects on gut microbiota and humoral immunity. Neonatology 2008:93;236-40 [Abstract]

Koenig J.E., Spor A., Scalfone N., Fricker A.D., Stombaugh J., Knight R., Angenent L.T., and Ley R.E. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci 2011:108(1);4578-85 [Abstract]

Ley R.E., Peterson D.A., and Gordon J.I. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 2006:124(4);837-48 [Article]

Minot S., Sinha R., Chen J., Li H., Keilbaugh S.A., Wu G.D., Lewis J.D., and Bushman F.D. The human gut virome: inter-individual variation and dynamic response to diet. Genome Res 2011:21;1616-1625 [Abstract]

Moreno-Indias I.M., Cardona F., Tinahones F.J. and Queipo-Ortuño M.I. Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. Front Microbiol 2014:5(190);1-10 [Abstract]

Newburg D.S. & Morelli L. Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota. Pediatr Res 2015:77;115-120 [Abstract]

Palmer C., Bik E.M., DiGiulio D.B., Relman D.A., and Brown P.O. Development of the human infant intestinal microbiota. PLoS Biol 2007:5(7);e177 [Article]

Rodrıguez J.M., Murphy K., Stanton C., Ross R.P., I. Kober O.I., Juge N., Avershina E., Rudi K., Narbad A., Jenmalm M.C., Marchesi J.R. and Collado M.C. The composition of the gut microbiota throughout life, with an emphasis on early life. Microb Ecol Health Dis 2015:26;26050 [Article]

Wu G.D., Chen J., Hoffmann C., Bittinger K., Chen Y.Y., Keilbaugh S.A., Bewtra M., Knights D., Walters W.A., Knight R., et al. Linking long-term dietary patterns with gut microbial enterotypes. Science 2011:334;105-8 [Abstract]

Human microbiota: definition, composition, functions, antibiotics

What is the human microbiota?

Human Microbiota
Fig. 1 – Lactobacillus casei

It has been known for almost a century that humans harbor a microbial ecosystem, known as human microbiota, remarkably dense and diverse, made up of a  number of viruses and cells much higher than those of the human body, and that accounts for one to three percent of body weight. All the genes encoded by the human body’s microbial ecosystem, which are about 1,000 times more numerous than those of our genome, make up the human microbiome. Microorganisms colonize all the surfaces of the body that are exposed to the environment. Indeed, distinct microbial communities are found on the skin, in the vagina, in the respiratory tract, and along the whole intestinal tract, from the mouth up to rectum, the last part of the intestine.

Composition of the human microbiota

Human Microbiota
Fig. 2 – Escherichia coli

It is composed of organisms from all taxa.

  • Bacteria, at least 100 trillion (1014) cells, a number ten times greater than that of the human body. They are found in very high concentration in the intestinal tract, up to 1012-1014/gram of tissue, where they form one of the most densely populated microbial habitats on Earth. In the gut, bacteria mainly belong to the Firmicutes, Bacteroidetes and Actinobacteria phyla. Fusobacteria (oropharynx), Tenericutes, Proteobacteria, and Verrucomicrobia are other phyla present in our body.
    Note: bacterial communities in a given body region resemble themselves much more across individuals than those from different body regions of the same individual; for example, bacterial communities of the upper respiratory tract are much more similar across individuals than those of the skin or intestine of the same individual.
  • Viruses, by far the most numerous organisms, about quadrillion units. The genomes of all the viruses harbored in the human body make up the human virome. In the past, viruses and eukaryotes (see below) have been studied focusing on pathogenic microorganisms, but in recent years the attention has also shifted on many non-pathogenic members of these groups. And many of the viral gene sequences found are new, which suggests that there is still much to learn about the human virome. Finally, just like for bacteria, there is considerable interpersonal variability.
  • Archaebacteria, primarily those belonging to the order Methanobacteriales, with Methanobrevibacter smithii predominant in the human gut (up to 10% of all anaerobes).
  • Eukaryotes, and the parasites of the genera Giardia and Entamoeba have probably been among the first to be identified. But there is also a great abundance and diversity of fungal species, belonging to genera such as Candida, Penicillium, Aspergillus, Hemispora, Fusarium, Geotrichum, Hormodendrum, Cryptococcus, Saccharomyces, and Blastocystis.
Human Microbiota
Fig. 3 – Candida albicans

Based on the relationships with the human host, microorganisms may be classified as commensals or pathogens.

  • Commensals cause no harm to the host, with which they establish a symbiotic relationship that generally brings benefits to both.
  • On the contrary, pathogens are able to cause diseases, but fortunately represent a small percentage of the human microbiota. These microorganisms establish a symbiosis with the human host and benefit from it at the expense of the host. They can cause disease:

if they move from their niche, such as the intestine, into another one where they do not usually reside, such as the vagina or bladder (as in the case of Candida albicans, normally present in the intestine, but in very small quantities);
in patients with impaired immunological defenses, such as after an immunosuppressive therapy.

Functions of the human microbiota

Human Microbiota
Fig. 4 – Bifidobacterium longum

Sometimes referred to as “the forgotten organ“, human microbiota, mainly with its intestinal bacterial members, plays many important functions that can lead to nutritional, immunological, and developmental benefits, but can also cause diseases. Here are some examples.

  • It is involved in the development of the gastrointestinal system of the newborn, as shown by experiments carried out on germ-free animals in which, for example, the thickness of the intestinal mucosa is thinner than that of colonized animals, therefore more easily subject to rupture.
  • It contributes to energy harvest from nutrients, due to its ability to ferment indigestible carbohydrates, promote the absorption of monosaccharides and the storage of the derived energy. This has probably been a very strong evolutionary force that has played a major role in favor of the fact that these bacteria became our symbionts.
  • It contributes to the maintenance of the acidic pH of the skin and in the colon.
  • It is involved in the metabolism of xenobiotics and several polyphenols.
  • It improves water and mineral absorption in the colon.
  • It increases the speed of intestinal transit, slower in germ-free animals.
  • It has an important role in resistance to colonization by pathogens, primarily in the vagina and gut.
  • It is involved in the biosynthesis of isoprenoids and vitamins through the methylerythritol phosphate pathway.
  • It stimulates angiogenesis.
  • In the intestinal tract, it interacts with the immune system, providing signals for promoting the maturation of immune cells and the normal development of immune functions. And this is perhaps the most important effect of the symbiosis between the human host and microorganisms. Experiments carried out on germ-free animals have shown, for example, that:

macrophages, the cells that engulf pathogens and then present their antigens to the immune system, are found in much smaller amounts than those present in the colonized intestine, and if placed in the presence of bacteria they fail to find and therefore engulf them, unlike macrophages extracted from a colonized intestine;
there is not the chronic non-specific inflammation, present in the normal intestine as a result of the presence of bacteria (and of what we eat).

  • Changes in its composition can contribute to the development of obesity and metabolic syndrome.
  • It protects against the development of type I diabetes.
  • Many diseases, both in children and adults, such as stomach cancer, lymphoma of mucosa-associated lymphoid tissue, necrotizing enterocolitis (an important cause of morbidity and mortality in premature babies) or chronic intestinal diseases, are, and others seem to be, related to the gut microbiota.

In conclusion, it seems very likely that the human body represents a superorganism, result of years of evolution and made up of human cells, and the resulting metabolic and physiological capacities, as well as an additional organ, the microbiota.

Human Microbiome Project

Human Microbiota
Fig. 5- Human Microbiome Project

The bacterial component of the human microbiota is the subject of most studies including a large-scale project started in 2008 called “Human Microbiome Project“, whose aim is to characterize the microbiome associated with multiple body sites, such as the skin, mouth, nose, vagina and intestine, in 242 healthy adults. These studies have shown a great variability in the composition of the human microbiota; for example, twins share less than 50% of their bacterial taxa at the species level, and an even smaller percentage of viruses. The factors that shape the composition of bacterial communities begin to be understood: for example, the genetic characteristics of the host play an important, although this is not true for the viral community. And metagenomic studies have shown that, despite the great interpersonal variability in microbial community composition, there is a core of shared genes encoding signaling and metabolic pathways. It appears namely that the assembly and the structure of the microbial community does not occur according to the species but the more functional set of genes. Therefore, disease states of these communities might be better identified by atypical distribution of functional classes of genes.

Effects of antibiotics on the human microbiota

Human Microbiota
Fig. 6 – Clostridium difficile

The microbiota in healthy adult humans is generally stable over time. However, its composition can be altered by factors such as dietary changes, urbanization, travel, and especially the use of broad-spectrum antibiotics. Here are some examples of the effects of antibiotic treatments.

  • There is a long-term reduction in microbial diversity.
  • The taxa affected vary from individual to individual (even up to a third of the taxa).
  • Several taxa do not recover even after 6 months from treatment.
  • Once the bacterial communities have reshaped, a reduced resistance to colonization occurs. This allows foreign and/or pathogen bacteria, able to grow more than the commensals, to cause permanent changes in human microbiota structure, as well as acute diseases, such as the dangerous pseudomembranous colitis, and chronic diseases, as it is suspected for asthma following the use and abuse of antibiotics in childhood. Moreover, their repeated use has been suggested to increase the pool of antibiotic-resistance genes in our microbiome. In support of this hypothesis, a decrease in the number of antibiotic-resistant pathogens has been observed in some European countries following the reduction in the number of antibiotics prescribed.

Finally, you must not underestimate the fact that the intestinal microflora is involved in many chemical transformations, and its alteration could be implicated in the development of cancer and obesity. However, regarding use of antibiotics, you should be underlined that if western population has a life expectancy higher than in the past is also because you do not die of infectious diseases!

References

Burke C., Steinberg P., Rusch D., Kjelleberg S., and Thomas T. Bacterial community assembly based on functional genes rather than species. Proc Natl Acad Sci USA 2011;108:14288-93 [Abstract]

Clemente J.C., Ursell L.K., Wegener Parfrey L., and Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148:1258-70 [Article]

Gill S.R., Pop M., Deboy R.T., Eckburg P.B., Turnbaugh P.J., Samuel B.S., Gordon J.I., Relman D.A., Fraser-Liggett C.M., and Nelson K.E. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355-59 [Abstract]

Palmer C., Bik E.M., DiGiulio D.B., Relman D.A., and Brown P.O. Development of the human infant intestinal microbiota. PLoS Biol 2007;5(7):e177 [Article]

The Human Microbiome  Project

Turnbaugh P.J., Gordon J.I. The core gut microbiome, energy balance and obesity. J Physiol 2009;587:4153-58 [Abstract]

Zhang, T., Breitbart, M., Lee, W., Run, J.-Q., Wei, C., Soh, S., Hibberd, M., Liu, E., Rohwer, F., Ruan, Y. Prevalence of plant viruses in the RNA viral community of human feces. PLoS Biol 2006;4(1):e3 [Article]

Flavonoid biosynthesis pathway

The flavonoid biosynthetic pathway in plants

Flavonoid Biosynthesis
Fig. 1 – Fig. 1 – Biosynthesis of Flavonoids

The biosynthesis of flavonoids, probably the best characterized pathway of plant secondary metabolism, is part of the phenylpropanoid pathway that, in addition to flavonoids, leads to the formation of a wide range of phenolic compounds, such as hydroxycinnamic acids, stilbenes, lignans and lignins.
Flavonoid biosynthesis is linked to primary metabolism through both mitochondria- and plastid-derived molecules. Since it seems that most of the involved enzymes characterized to date operate in protein complexes located in the cell cytosol, these molecules must be exported to the cytoplasm to be used.
The end products are transported to different intracellular or extracellular locations, with flavonoids involved in pigmentation usually transported into the vacuoles.
The biosynthesis of this group of polyphenols requires one p-coumaroyl-CoA and three malonyl-CoA molecules as initial substrates.

Biosynthesis of p-coumaroyl-CoA

Flavonoid Biosynthesis
Fig. 2 – p-Cumaroyl-CoA

p-Coumaroyl-CoA is the pivotal branch-point metabolite in the phenylpropanoid pathway, being the precursor of a wide variety of phenolic compounds, both flavonoid and non-flavonoid polyphenols.
It is produced from phenylalanine via three reactions catalyzed by cytosolic enzymes collectively called group I or early-acting enzymes, in order of action:

  • phenylalanine ammonia lyase (EC 4.3.1.24);
  • cinnamate 4-hydroxylase or cinnamic acid 4-hydroxylase (EC 1.14.13.11);
  • 4-cumarato: CoA ligase or hydroxycinnamic: CoA ligase (EC 6.2.1.12).

They seems to be associated in a multienzyme complex anchored to the endoplasmic reticulum membrane. The anchoring is probably ensured by cinnamate 4-hydroxylase that inserts its N-terminal domain into the membrane of the endoplasmic reticulum itself. These complexes, referred to as “metabolons”, allow the product of a reaction to be channeled directly as substrate to the active site of the enzyme that catalyzes the consecutive reaction in the metabolic pathway.
With the exception of cinnamate 4-hydroxylase, the enzymes which act downstream of phenylalanine ammonia lyase are encoded by small gene families in all species analyzed so far.
The different isoenzymes show distinct temporal, tissue, and elicitor-induced patterns of expression. It seems, in fact, that each member of each family can be used mainly for the synthesis of a specific compound, thus acting as a control point for carbon flux among the metabolic pathways leading to lignan, lignin, and flavonoid biosynthesis.

Note: phenylalanine is a product of the shikimic acid pathway, which converts simple precursors derived from carbohydrate metabolism, phosphoenolpyruvate and erythrose-4-phosphate, into the aromatic amino acids phenylalanine, tyrosine and tryptophan. Unlike plants and microorganisms, animals do not possess the shikimic acid pathway, and are not able to synthesize the three above-mentioned amino acids, which are therefore essential nutrients.

Phenylalanine ammonia lyase (PAL)

Flavonoid Biosynthesis
Fig. 3 – Phenylalanine Ammonia Lyase

It is one of the most studied and best characterized enzymes of plant secondary metabolism. It requires no cofactors and catalyzes the reaction that links primary and secondary metabolism: the deamination of phenylalanine to trans-cinnamic acid, with the release of nitrogen as ammonia and introduction of a trans double bond between carbon atoms 7 and 8 of the side chain. Therefore, it directs the flow of carbon from the shikimic acid pathway to the different branches of the phenylpropanoid pathway. The released ammonia is probably fixed in the reaction catalyzed by glutamine synthetase.
The enzyme from monocots is also able to act as tyrosine ammonia lyase (EC 4.3.1.25), converting tyrosine to p-coumaric acid directly, (therefore without the 4-hydroxylation step), but with a lower efficiency.
In all plant species investigated,  several copies of phenylalanine ammonia lyase gene are found, copies that probably respond differentially to internal and external stimuli. Indeed, gene transcription, and then enzyme activity, are under the control of both internal developmental and external environmental stimuli. Here are some examples that require increased enzyme activity.

  • The flowering.
  • The  production of lignin to strengthen the secondary wall of xylem cells.
  • The production of flower pigments that attract pollinators.
  • Pathogen infections, that require the production of phenylpropanoid phytoalexins, or exposure to UV rays.

Cinnamate 4-hydroxylase (C4H)

Flavonoid Biosynthesis
Fig. 4 – Cinnamate 4-Hydroxylase

It belongs to the cytochrome P450 superfamily (EC 1.14.-.-), is a microsomal monooxygenase containing a heme cofactor, and dependent on both O2  and NADPH. It catalyzes the formation of p-coumaric acid  through the introduction of a hydroxyl group in 4-position of trans-cinnamic acid (this hydroxyl group is present in most flavonoids). This reaction is also part of the biosynthesis of hydroxycinnamic acids.
Increases in transcription rates and enzyme activity are observed in correlation with the synthesis of phytoalexins (in response to fungal infections), lignification as well as wounding.

4-Coumarate:CoA ligase (4CL)

Flavonoid Biosynthesis
Fig. 5 – 4-Coumarate:CoA Ligase

With Mg2+ as a cofactor, it catalyzes the ATP-dependent activation of the carboxyl group of p-coumaric acid and other hydroxycinnamic acids, metabolically rather inert molecules, through the formation of the corresponding CoA-thioester. Generally, p-coumaric acid and caffeic acid are the preferred substrates, followed by ferulic acid and 5-hydroxyferulic acid, low activity against trans-cinnamic acid and none against sinapic acid. These CoA-thioesters are able to enter various reactions such as:

  • reduction to alcohol (monolignols) or aldehydes;
  • stilbene and flavonoid biosynthesis;
  • transfer to acceptor molecules.

It should finally be pointed out that the activation of the carboxyl group can also be obtained through an UDP-glucose-dependent transfer to glucose.

Biosynthesis of malonyl-CoA

Malonyl-CoA does not derived from the phenylpropanoid pathway, but from the reaction catalyzed by acetyl-CoA carboxylase (EC 6.4.1.2, the cytosolic form, see below). The enzyme, with biotin and Mg2+ as cofactors, catalyzes the ATP-dependent carboxylation of acetyl-CoA, using bicarbonate as a source of carbon dioxide (CO2).

Flavonoid Biosynthesis
Fig. 6 – Acetyl-CoA Carboxylase

It is found both in the plastids, where it participates in the synthesis of fatty acids, and the cytoplasm, and is the latter that catalyzes the formation of malonyl-CoA that is used in the biosynthesis of flavonoids and other compounds. Increases in the transcription rate of the gene and enzyme activity are induced in response to stimuli that increase the biosynthesis of these polyphenols, such as exposure to pathogenic fungi or UV-rays.
In turn, acetyl-CoA is produced in plastids, mitochondria, peroxisomes and cytosol through different metabolic pathways. The molecules used in the biosynthesis of malonyl-CoA, and therefore of the flavonoids, are  the cytosolic ones, produced in the reaction catalyzed by ATP-citrate lyase (EC 2.3.3.8) that cleaves citrate, in the presence of CoA and ATP, to form oxaloacetate and acetyl-CoA, plus ADP and inorganic phosphate.

The first steps in flavonoid biosynthesis

Flavonoid Biosynthesis
Fig. 7 – Naringenin Chalcone

The first step in flavonoid biosynthesis is catalyzed by chalcone synthase (EC 2.3.1.74), an enzyme anchored to the endoplasmic reticulum and with no known cofactors.
From one p-coumaroyl-CoA and three malonyl-CoA, it catalyzes sequential condensation and decarboxylation reactions in the course of which a polyketide intermediate is formed. The polyketide undergoes cyclizations and aromatizations leading to the formation of the A ring. The product of the reactions is naringenin chalcone (2′,4,4′,6′-tetrahydroxychalcone), a 6′-hydroxychalcone and the first flavonoid to be synthesized by plants.
The reaction, cytosolic, is irreversible due to the release of three CO2 and 4 CoA.

Flavonoid Biosynthesis
Fig. 8 – Basic Flavonoid Skeleton

The B ring and the three-carbon bridge of the molecule originate from p-coumaroyl-CoA (and therefore from phenylalanine), the A ring from the three malonyl-CoA units.
Also 6’-deoxychalcone can be produced; its synthesis is thought to involve an additional reduction step catalyzed by polyketide reductase (EC. 1.1.1.-).
Chalcone synthase from some plant species, such as barley (Hordeum vulgare), accepts as substrates also caffeoil-CoA, feruloil-CoA and cinnamoyl-CoA.
It is the most abundant enzyme of the phenylpropanoid pathway, probably because it has a low catalytic activity, and, in fact, is considered to be the rate-limiting enzyme in flavonoid biosynthesis.
As for phenylalanine ammonia lyase, chalcone synthase gene expression is under the control of both internal and external factors. In some plants, one or two isoenzymes are found, while in others up to 9.
Chalcone synthase belongs to polyketide synthase group, present in bacteria, fungi and plants. These enzymes are able to catalyze the production of polyketide chains through sequential condensations of acetate units provided by malonyl-CoA units. They also includes stilbene synthase (EC 2.3.1.146), which catalyzes the formation of resveratrol, a non flavonoid polyphenol compound that has attracted much interest because of its potential health benefits.

Flavonoid Biosynthesis
Fig. 9 – (2S)-Naringenin

Generally, chalcones do not accumulate in plants because naringenin chalcone is converted to (2S)-naringenin, a flavanone, in the reaction catalyzed by chalcone isomerase (EC 5.5.1.6). The enzyme, the first of the flavonoid biosynthesis to be discovered, catalyzes a stereospecific isomerization and closes the C ring. Two types of chalcone isomerases are known, called type I and II. Type I enzymes use only 6′-hydroxychalcone substrates, like naringenin chalcone, while type II, prevalent in legumes, use both 6′-hydroxy- and 6′-deoxychalcone substrates.
It should be noted that with 6′-hydroxychalcones, isomerization can also occur nonenzymically to form a racemic mixture, both in vitro and in vivo, enough to allow a moderate synthesis of anthocyanins. On the contrary, under physiological conditions 6′-deoxychalcones are stable, and so the activity of type II chalcone isomerases is required to form flavanones.
The enzyme increases the rate of the reaction of 107 fold compared to the spontaneous reaction, but with a higher kinetics for the 6′-hydroxychalcones than 6′-deoxychalcones. Finally, it produces (2S)-flavanones, which are the biosynthetically required enantiomers.
As other enzymes in flavonoid biosynthesis, also chalcone isomerase gene expression is subject to strict control. And, as phenylalanine ammonia lyase and chalcone synthase, it is induced by elicitors.

Flavonoid Biosynthesis
Fig. 10 – Dihydrokaempferol

In the reaction catalysed by flavanone-3β-hydroxylase (EC 1.14.11.9), (2S)-flavanones undergo a stereospecific isomerization that converts them into the respective (2R,3R)-dihydroflavonols. In particular, naringenin is converted into dihydrokaempferol.
The enzyme is a cytosolic non-heme-dependent dioxygenase, dependent on Fe2+ and 2-oxoglutarate, and therefore belonging to the family of 2-oxoglutarate-dependent dioxygenase (which distinguishes them from the other hydroxylases of the flavonoid biosynthetic pathway which are cytochrome P450 enzymes).
Naringenin chalcone, (2S)-naringenin, and dihydrokaempferol are central intermediates in flavonoid biosynthesis, since they act as branch-point compounds from which the synthesis of distinct flavonoid subclasses can occur. For example, directly or indirectly:

Not all of these side metabolic pathways are present in every plant species, or are active within each tissue type of a given plant. Like enzymes previously seen, the activity of those involved in these “side-routes” is subject to strict control, resulting in a tissue-specific profile of flavonoid compounds. For example, grape seeds, flesh and skin have distinct anthocyanin, catechin, flavonol and condensed tannin profiles, whose synthesis and accumulation are strictly and temporally coordinated during the ripening process.

Bibliografia

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Heldt H-W. Plant biochemistry – 3th Edition. Elsevier Academic Press, 2005

Vogt T. Phenylpropanoid biosynthesis. Mol Plant 2010;3(1):2-20 [Abstract]

Wink M. Biochemistry of plant secondary metabolism – 2nd Edition. Annual plant reviews (v. 40), Wiley J. & Sons, Inc., Publication, 2010

Lignans: definition, chemical structure, biosynthesis, metabolism, foods

What are lignans?

Lignans are a subgroup of non-flavonoid polyphenols.
They are widely distributed in the plant kingdom, being present in more than 55 plant families, where they act as antioxidants and defense molecules against pathogenic fungi and bacteria.
In humans, epidemiological and physiological studies have shown that they can exert positive effects in the prevention of lifestyle-related diseases, such as type II diabetes and cancer. For example, an increased dietary intake of these polyphenols correlates with a reduction in the occurrence of certain types of estrogen-related tumors, such as breast cancer in postmenopausal women.
In addition, some lignans have also aroused pharmacological interest. Examples are:

  • podophyllotoxin, obtained from plants of the genus Podophyllum (Berberidaceae family); it is a mitotic toxin whose derivatives have been used as chemotherapeutic agents;
  • arctigenin and tracheologin, obtained from tropical climbing plants; they have antiviral properties and have been tested in the search for a drug to treat AIDS .

Chemical structure of lignans

Lignans
Fig. 1 – Phenylpropane unit

Their basic chemical structure consists of two phenylpropane units linked by a C-C bond between the central atoms of the respective side chains (position 8 or β), also called β-β’ bond. 3-3′, 8-O-4′, or 8-3′ bonds are observed less frequently; in these cases the dimers are called neolignans. Hence, their chemical structure is referred to as (C6-C3)2, and they are included in the phenylpropanoid group, as well as their precursors: the hydroxycinnamic acids (see below).
Based on their carbon skeleton, cyclization pattern, and the way in which oxygen is incorporated in the molecule skeleton, they can be divided into 8 subgroups: furans, furofurans, dibenzylbutanes, dibenzylbutyrolactones, dibenzocyclooctadienes, dibenzylbutyrolactols, aryltetralins and arylnaphthalenes. Furthermore, there is considerable variability regarding the oxidation level of both the propyl side chains and the aromatic rings.
They are not present in the free form in nature, but linked to other molecules, mainly as glycosylated derivatives.
Among the most common lignans, secoisolariciresinol (the most abundant one), lariciresinol, pinoresinol, matairesinol and 7-hydroxymatairesinol are found.

Note: they occur not only as dimers but also as more complex oligomers, such as dilignans and sesquilignans.

Biosynthesis of lignans

Lignans
Fig. 2 – Lignan Biosynthesis

In this section, we will examine the synthesis of some of the most common lignans.
The pathway starts from 3 of the 4 most common dietary hydroxycinnamic acids: p-coumaric acid, sinapic acid, and ferulic acid (caffeic acid is not a precursor of this subgroup of polyphenols). Therefore, they arise from the shikimic acid pathway, via phenylalanine.
The first three reactions reduce the carboxylic group of the hydroxycinnamates to alcohol group, with formation of the corresponding alcohols, called monolignols, that is, p-coumaric alcohol, sinapyl alcohol and coniferyl alcohol. These molecules also enter the pathway of lignin biosynthesis.

  • The first step, which leads to the activation of the hydroxycinnamic acids, is catalysed by hydroxycinnamate:CoA ligases, commonly called p-coumarate:CoA ligases (EC 6.2.1.12), with formation of the corresponding hydroxycinnamate-CoAs, namely, feruloil-CoA, p- coumaroyl-CoA and sinapil-CoA.
  • In the second step, a NADPH-dependent cinnamoyl-CoA: oxidoreductase, also called cinnamoyl-CoA reductase (EC1.2.1.44) catalyzes the formation of the corresponding aldehydes, and the release of coenzyme A.
  • In the last step, a NADPH-dependent cinnamyl alcohol dehydrogenase, also called monolignol dehydrogenase (EC 1.1.1.195), catalyzes the reduction of the aldehyde group to an alcohol group, with the formation of the aforementioned monolignols.
Lignans
Fig. 3 – (-)-Matairesinol

The next step, the dimerization of monolignols, involves the intervention of stereoselective mechanisms, or, more precisely, enantioselective mechanisms. In fact, most of the plant lignans exists as (+)- or (-)-enantiomers, whose relative amounts can vary from species to species, but also in different organs on the same plant, depending on the type of reactions involved.
The dimerization can occur through enzymatic reactions involving laccases (EC 1.10.3.2). These enzymes catalyze the formation of radicals that, dimerizing, form a racemic mixture. However, this does not explain how the enantiomeric mixtures found in plants are formed. The most accepted mechanism to explain the stereospecific synthesis involves the action of the laccase and of a protein able to direct the synthesis toward one or the other of the two enantiomeric forms: the dirigent protein. The reaction scheme might be: the enzyme catalyzes the synthesis of phenylpropanoid radicals that are orientated in such a way to obtain the desired stereospecific coupling by the dirigent protein.
For example, pinoresinol synthase, consisting of laccase and dirigent protein, catalyzes the stereospecific synthesis of (+)-pinoresinol from two units of coniferyl alcohol. (+)-Pinoresinol, in two consecutive stereospecific reactions catalyzed by NADPH-dependent pinoresinol/lariciresinol reductase (EC 1.23.1.2), is first reduced to (+)-lariciresinol, and then to (-)-secoisolariciresinol. (-)-Secoisolariciresinol, in the reaction catalyzed by NAD(P)-dependent secoisolariciresinol dehydrogenase (EC 1.1.1.331) is oxidized to (-)-matairesinol.

Metabolism of lignans by human gut microbiota

Their importance to human health is due largely to their metabolism by colonic microbiota, which carries out deglycosylations, para-dehydroxylations, and meta-demethylations without enantiomeric inversion. Indeed, this metabolization leads to the formation molecules with a modest estrogen-like activity (phytoestrogens), a situation similar to that observed with some isoflavones, such as those of soybean, some coumarins, and some stilbenes. These active metabolites are the so-called “mammalian lignans or enterolignans”, such as the aglycones of enterodiol and enterolactone, formed from secoisolariciresinol and matairesinol, respectively.
Studies conducted on animals fed diets rich in lignans have shown their presence as intact molecules, in low concentrations, in serum, suggesting that they may be absorbed as such from the intestine. These molecules exhibit estrogen-independent actions, both in vivo and in vitro, such as inhibition of angiogenesis, reduction of diabetes, and suppression of tumor growth.
Note: the term “phytoestrogen” refers to molecules with estrogenic or antiandrogenic activity, at least in vitro.

Once absorbed, they enter the enterohepatic circulation, and, in the liver, may undergo phase II reactions and be sulfated or glucuronidated, and finally excreted in the urine.

Food rich in lignans

Lignans
Fig. 4 – (-)-Secoisolariciresinol

The richest dietary source is flaxseed (linseed), that contains mainly secoisolariciresinol, but also lariciresinol, pinoresinol and matairesinol in good quantity (for a total amount of more than 3.7 mg/100 g dry weight). They are also found in sesame seeds.
Another important source is whole grains.
They are also present in other foods, but in concentrations from one hundred to one thousand times lower than those of flaxseed. Examples are:

  • beverages, generally more abundant in red wine, followed in descending order by black tea, soy milk and coffee;
  • fruits, such as apricots, pears, peaches, strawberries;
  • among vegetables, Brassicaceae, garlic, asparagus and carrots;
  • lentils and beans.

Their presence in grains and, to a lesser extent in red wine and fruit, makes them, at least in individuals who follow a Mediterranean-style eating pattern, the main source of phytoestrogens.

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Heldt H-W. Plant biochemistry – 3th Edition. Elsevier Academic Press, 2005

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 [Abstract]

Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231-1246 [Abstract]

Satake H, Koyama T., Bahabadi S.E., Matsumoto E., Ono E. and Murata J. Essences in metabolic engineering of lignan biosynthesis. Metabolites 2015;5: 270-290 [Abstract]

van Duynhoven J., Vaughan E.E., Jacobs D.M., Kemperman R.A., van Velzen E.J.J, Gross G., Roger L.C., Possemiers S., Smilde A.K., Doré J., Westerhuis J.A.,and Van de Wiele T. Metabolic fate of polyphenols in the human superorganism. PNAS 2011;108(suppl. 1):4531-8 [Abstract]

Wink M. Biochemistry of plant secondary metabolism – 2nd Edition. Annual plant reviews (v. 40), Wiley J. & Sons, Inc., Publication, 2010

Hydroxycinnamic acids: definition, chemical structure, synthesis, foods

What are hydroxycinnamic acids?

Hydroxycinnamic acids or hydroxycinnamates are phenolic compounds belonging to non-flavonoid polyphenols.
They are present in all parts of fruits and vegetables although the highest concentrations are found in the outer part of ripe fruits, concentrations that decrease during ripening, while the total amount increases as the size of the fruits increases.

Their dietary intake has been associated with the prevention of the development of chronic diseases such as:

  • cardiovascular disease;
  • cancer;
  • type-2 diabetes.

These effects do seem to be due not only to their high antioxidant activity (that depends upon the hydroxylation pattern of the aromatic ring, see below), but also to other mechanisms of action such as, e.g., the reduction of intestinal absorption of glucose or the modulation of secretion of some gut hormones.

Chemical structure of hydroxycinnamic acids

Hydroxycinnamic Acids
Fig. 1 – C6-C3 Skeleton

Their basic structure is a benzene ring to which a three carbon chain is attached, structure that is referred to as C6-C3. Therefore they can be included in the phenylpropanoid group.
The main dietary hydroxycinnamates are:

  • caffeic acid or 3,4-dihydroxycinnamic acid;
  • ferulic acid or 4-hydroxy-3-methoxycinnamic acid;
  • sinapic acid or 4-hydroxy-3,5-dimethoxycinnamic acid;
  • p-coumaric acid or 4-coumaric acid or 4-hydroxycinnamic acid.

In nature, they are associated with other molecules to form, e.g., glycosylated derivatives or esters of tartaric acid, quinic acid, or shikimic acid. In addition, several hundreds of anthocyanins acylated with the aforementioned hydroxycinnamates have been identified (in descending order with p-coumaric acid, more than 150, caffeic acid, about 100, ferulic acid, about 60, and sinapic acid, about 25). They are rarely present in the free form, except in processed foods that have undergone fermentation, sterilization or freezing. For example, an overlong storage of blood orange fruits causes a massive hydrolysis of hydroxycinnamic derivatives to free acids, and this in turn could lead to the formation of malodorous compounds such as vinyl phenols, indicators of too advanced senescence of the fruit.

Biosynthesis of hydroxycinnamic acids

Hydroxycinnamic Acids
Fig. 2 – Biosynthesis of Hydroxycinnamates

Hydroxycinnamate biosynthesis consists of a series of enzymatic reactions subsequent to that catalyzed by phenylalanine ammonia lyase (PAL). This enzyme catalyzes the deamination of phenylalanine to yield trans-cinnamic acid, so linking the aromatic amino acid to the hydroxycinnamic acids and their activated forms.
In the first step, a hydroxyl group is attached at the 4-position of the aromatic ring of trans-cinnamic acid to form p-coumaric acid (reaction catalysed by cinnamic acid 4-hydroxylase). The addition of a second hydroxyl group at the 3-position of the ring of p-coumaric acid leads to the formation of caffeic acid (reaction catalysed by p-coumarate 3-hydroxylase or phenolase), while the O-methylation of the hydroxyl group at the 3-position yields ferulic acid (reaction catalyzed by catechol-O-methyltransferase). In turn, ferulic acid is converted into sinapic acid through two reactions: a hydroxylation at the 5-position to form 5-hydroxy ferulic acid (reaction catalysed by ferulate 5-hydroxylase), and the subsequent O-methylation of the same hydroxyl group (reaction catalyzed by catechol-O-methyltransferase).
Hydroxycinnamic acids are not present in high quantities since they are rapidly converted to glucose esters or coenzyme A (CoA) esters, in reactions catalyzed by O-glucosyltransferases and hydroxycinnamate:CoA ligases, respectively. These activated intermediates are branch points, being able to participate in a wide range of reactions such as condensation with malonyl-CoA to form flavonoids, or the NADPH-dependent reduction to form lignans (precursors of lignin).

The main hydroxycinnamic acids in foods

Kiwis, blueberries, plums, cherries, apples, pears, chicory, artichokes, carrots, lettuce, eggplant, wheat and coffee are among the richest sources.

Caffeic acid

Hydroxycinnamic Acids
Fig. 3 – Caffeic Acid

It is generally, both in the free form and bound to other molecules, the most abundant hydroxycinnamic acid in vegetables and most of the fruits, where it represents between 75 and 100% of the hydroxycinnamates.
The richest sources are coffee (drink), carrots, lettuce, potatoes, even sweet ones, and berries such as blueberries, cranberries and blackberries.
Smaller quantities are present in grapes and grape-derived products, orange juice, apples, plums, peaches, and tomatoes.
Caffeic acid and quinic acid bind to form chlorogenic acid, present in many fruit and in high concentration in coffee.

Ferulic acid

Hydroxycinnamic Acids
Fig. 4 – Ferulic Acid

It is the most abundant hydroxycinnamic acid in cereals, which are also its main dietary source.
In wheat grain, its content is between 0.8 and 2 g/kg dry weight, which represents up to 90% of the total polyphenols. It is found chiefly, up to 98% of the total content, in the aleurone layer and pericarp (that is, the outer parts of the grain), and therefore its content in wheat flours depends upon the degree of refining, while the main source is obviously the bran. The molecule is present mainly in the trans form, and esterified with arabinoxylans and hemicelluloses. And in fact, in wheat bran the soluble free form represents only about 10% of its total amount. Dimers were also found, which form bridge structures between chains of hemicellulose.
In fruits and vegetables, ferulic acid is much less common than caffeic acid. The main sources are asparagus, eggplant and broccoli; lower quantities are found in blackberries, blueberries, cranberries, apples, carrots, potatoes, beets, coffee and orange juice.

Sinapic acid

Hydroxycinnamic Acids
Fig. 5 – Sinapic Acid

The highest amounts are found in citrus peel and seeds (in orange juice, the amount is much lower); appreciable quantities in Chinese cabbage and in some varieties of cranberries.

p-Coumaric acid

Hydroxycinnamic Acids
Fig. 6 – p-Coumaric Acid

High amounts are present in eggplant, the richest source, broccoli and asparagus; other sources are sweet cherries, plums, blueberries, cranberries, citrus peel and seeds, and orange juice.

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 [Abstract]

Preedy V.R. Coffee in health and disease prevention. Academic Press, 2014  [Google eBook]

Zhao Z.,  Moghadasian M.H. Bioavailability of hydroxycinnamates: a brief review of in vivo and in vitro studies. Phytochem Rev 2010;9(1):133-145 [Abstract]

Polyphenols in grapes and wine: chemical composition and biological activities

Polyphenols in Grapes
Fig. 1 – Red Grapes

The consumption of grapes and grape-derived products, particularly red wine but only at meals, has been associated with numerous health benefits, which include, in addition to the antioxidant/antiradical effect, also anti-inflammatory, cardioprotective, anticancer, antimicrobial, and neuroprotective activities.
Grapes contain many nutrients such as sugars, vitamins, minerals, fiber and phytochemicals. Among the latter, polyphenols are the most important compounds in determining the health effects of the fruit and derived products.
Indeed, grapes are among the fruits with highest content in polyphenols, whose composition is strongly influenced by several factors such as:

  • cultivar;
  • climate;
  • exposure to disease;
  • processing

Nowadays, the main species of grapes cultivated worldwide are: European grapes, Vitis vinifera, North American grapes, Vitis rotundifolia and Vitis labrusca, and French hybrids.
Note: grapes are not a fruit but an infructescence, that is, an ensemble of fruits (berries): the bunch of grapes. In turn, it consists of a peduncle, a rachis, cap stems or pedicels, and berries.

What are polyphenols in grapes and wine?

Polyphenols in red grapes and wine are significantly higher, both in quantity and variety, than in white ones. This, according to many researchers, would be the basis of the more health benefits related to the consumption of red grapes and wine than white grapes and derived products.
Polyphenols in grapes and wine are a complex mixture of flavonoid compounds, the most abundant group, and non-flavonoid compounds.
Among flavonoids, they are found:

Among non-flavonoid polyphenols:

Most of the flavonoids present in wine derive from the epidermal layer of the berry skin, while 60-70% of the total polyphenols are present in the grape seeds. It should be noted that more than 70% of grape polyphenols are not extracted and remain in the pomace.
The complex chemical interactions that occur between these compounds, and between them and the other compounds of different nature present in grapes and wine, are probably essential in determining both the quality of the grapes and wine and the broad spectrum of therapeutic effects of these foods.
In wine, the mixture of polyphenols play important functions being able to influence:

  • bitterness;
  • astringency;
  • red color, of which they are among the main responsible;
  • sensitivity to oxidation, being molecules easily oxidizable by atmospheric oxygen.

Finally, they act as preservatives and are the basis of long aging.

Polyphenols in grapes and wine: anthocyanins

They are flavonoids widely distributed in fruits and vegetables.
They are primarily located in the berry skin (in the outer layers of the hypodermal tissue), to which they confer color, having a hue that varies from red to blue. In some varieties, called “teinturier”, they also accumulate in the flesh of the berry.
There is a close relationship between berry development and the biosynthesis of anthocyanins. The synthesis starts at veraison (when the berry stops growing and changes its color), causes a color change of the berry that turns purple, and reaches the maximum levels at complete ripening.
Among wine flavonoids, they are one of the most potent antioxidants.
Each grape species and cultivars has a unique composition of anthocyanins. Moreover, in grapes of Vitis vinifera, due to a mutation in the gene coding for 5-O-glucosyltransferase, mutation that determines the synthesis of an inactive enzyme, only 3-monoglucoside derivatives are synthesized, while in other species  the glycosylation at position 5 also occurs. Interestingly, 3-monoglucoside derivatives are more intensely colored than 3,5-diglucoside derivatives.

Polyphenols in Grapes
Fig. 2 – Malvidin-3-glucoside

In red grapes and wine, the most abundant anthocyanins are the 3-monoglucosides of malvidin (the most abundant one both in grapes and wine), petunidin, delphinidin, peonidin, and cyanidin. In turn, the hydroxyl group at position 6 of the glucose can be acylated with an acetyl, caffeic or coumaric group, acylation that further enhances the stability.
Anthocyanidins, namely the non-conjugated molecules, are not present in grapes and in wine, except as traces.
Anthocyanins are scarcely present in white grapes and wine.
The composition of anthocyanins in wine is highly influenced both by the type of cultivar and by processing techniques, since they are present in wine as a result of extraction by maceration/fermentation processes. For this reason, wines deriving from similar varieties of grapes can have very different anthocyanin compositions.
Together with proanthocyanidins, they are the most important polyphenols in contributing to some organoleptic properties of red wine, as they are primarily responsible for astringency, bitterness, chemical stability against oxidation, as well as of the color of the young wine. In this regard, it should be underscored that with time their concentration decreases, while the color is due more and more to the formation of polymeric pigments produced by condensation of anthocyanins both among themselves and with other molecules.
During wine aging, proanthocyanidins and anthocyanins react to produce more complex molecules that can  partially precipitate.

Polyphenols in grapes and wine: flavanols or catechins

Polyphenols in Grapes
Fig. 3 – Catechin

They are, together with condensed tannins, the most abundant flavonoids, representing up to 50% of the total polyphenols in white grapes and between 13% and 30% in red ones.
Their levels in wine depend on the type of cultivar.
Typically, the most abundant flavanol in wine is catechin, but epicatechin and epicatechin-3-gallate are also present.

Polyphenols of grapes and wine: proanthocyanidins or condensed tannins

Polyphenols in Grapes
Fig. 4 – Procyanidin C1

Composed of catechin monomers, they are present in the berry skin, seeds and rachis of the bunch of grapes as:

  • dimers: the most common are procyanidins B1-B4, but also procyanidins B5-B8 can be present;
  • trimers: procyanidin C1 is the most abundant;
  • tetramers;
  • polymers, containing up to 8 monomers.

Their levels in wine depend on the type of grape varieties and wine-making technology, and, like anthocyanins, are much more abundant in red wines, in particular in aged wines, compared to white ones.
In addition, as previously said, together with anthocyanins, condensed tannins are important in determining some organoleptic properties of the wine.

Polyphenols of grapes and wine: flavonols

They are present in a large variety of fruit and vegetables, even if in low concentrations.
They are the third most abundant group of flavonoids in grapes, after proanthocyanidins and catechins.
They are mainly present in the outer epidermis of the berry skin, where they play a role both in providing protection against UV-A and UV-B radiations and in copigmentation together with anthocyanins.
Flavanol synthesis begins in the sprout; the highest concentration is reached a few weeks after veraison, then it decreases as the berry increases in size.
Their total amount is very variable, with the red varieties often richer than the white ones.
In grapes, they are present as 3-glucosides and their composition depends on the type of grapes and cultivar:

  • the derivatives of quercetin, kaempferol and isorhamnetin are found in white grapes;
  • the derivatives of myricetin, laricitrin and syringetin are found, together with the previous ones, only in red grapes, due to the lack of expression in white grapes of the gene coding for flavonoid-3′,5′-hydroxylase.
Polyphenols in Grapes
Fig. 5 – Quercetin-3-glucoside

In general, the 3-glucosides and 3-glucuronides of quercetin are the major flavonols in most of the grape varieties. Conversely, quercetin-3-rhamnoside and quercetin aglycone are the major flavonols in muscadine grapes.
In wine and grape juice, unlike grapes, they are also found as aglycones, as a result of the acid hydrolysis that occurs during processing and storage. They are present in wine in a variable amount, and the major molecules are the glycosides of quercetin and myricetin, which alone represent 20-50% of the total flavonols in red wine.

Polyphenols in grapes and wine: hydroxycinnamates

Polyphenols in Grapes
Fig. 6 – Ferulic Acid

Hydroxycinnamic acids are the main class of non-flavonoid polyphenols in grapes and the major polyphenols in white wine.
The most important are p-coumaric, caffeic, sinapic, and ferulic acids, present in wine as esters with tartaric acid.
They have antioxidant activity and in some white varieties of Vitis vinifera, together with flavonols, are the polyphenols mainly responsible for absorbing UV radiation in the berry.

Polyphenols in grapes and wine: stilbenes

Polyphenols in Grapes
Fig. 7 – trans-Resveratrol

They are phytoalexins which are produced in low concentrations only by a few edible species, including grapevine (on the contrary, flavonoids are present in all higher plants).
Together with the other polyphenols in grapes and wine, also stilbenes, particularly resveratrol, have been associated with health benefits resulting from the consumption of wine.
Their content increases from the veraison to the ripening of the berry, and is influenced by the type of cultivar, climate, wine-making technology, and fungal pressure.
The main stilbenes present in grapes and wine are:

  • cis- and trans-resveratrol (3,5,4′-trihydroxystilbene);
  • piceid or resveratrol-3-glucopyranoside and astringin or 3′-hydroxy-trans-piceid;
  • piceatannol;
  • dimers and oligomers of resveratrol, called viniferins, of which the most important are:

α-viniferin, a trimer;
β-viniferin, a cyclic tetramer;
γ-viniferin, a highly polymerized oligomer;
ε-viniferin, a cyclic dimer.

In grapes, other glycosylated and isomeric forms of resveratrol and piceatannol, such as resveratroloside, hopeaphenol, or resveratrol di- and tri-glucoside derivatives, have been found in trace amounts.
Glycosylation of stilbenes is important for the modulation of antifungal activity, protection from oxidative degradation, and storage of the wine.
The synthesis of dimers and oligomers of resveratrol, both in grapes and wine, represents a defense mechanism against exogenous attacks or, on the contrary, the result of the action of extracellular enzymes released from pathogens in an attempt to eliminate undesirable compounds.

Polyphenols in grapes and wine: hydroxybenzoates

Polyphenols in Grapes
Fig. 8 – Gallic Acid

The hydroxybenzoic acid derivatives are a minor component in grapes and wine.
In grapes, gentisic, gallic, p-hydroxybenzoic and protocatechuic acids are the main ones.
Unlike hydroxycinnamates, which are present in wine as esters with tartaric acid, they are found in their free form.
Together with flavonols, proanthocyanidins, catechins, and hydroxycinnamates they are among the responsible of astringency of wine.

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

Basli A, Soulet S., Chaher N., Mérillon J.M., Chibane M., Monti J.P.,1 and Richard T. Wine polyphenols: potential agents in neuroprotection. Oxid Med Cell Longev 2012 [Abstract]

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Flamini R., Mattivi F.,  De Rosso M., Arapitsas P. and Bavaresco L. Advanced knowledge of three important classes of grape phenolics: anthocyanins, stilbenes and flavonols. Int J Mol Sci 2013;14:19651-19669 [Abstract]

Georgiev V., Ananga A. and Tsolova V. Recent advances and uses of grape flavonoids as nutraceuticals. Nutrients 2014;6: 391-415 [Abstract]

Guilford J.M. and Pezzuto J.M. Wine and health: a review. Am J Enol Vitic 2011;62(4):471-486 [Abstract]

He S., Sun C. and Pan Y. Red wine polyphenols for cancer prevention. Int J Mol Sci 2008;9:842-853 [Abstract]

Xia E-Q., Deng G-F., Guo Y-J. and Li H-B. Biological activities of polyphenols from grapes. Int J Mol Sci 2010;11-622-646
[Abstract]

Waterhouse A.L. Wine phenolics. Ann N Y Acad Sci 2002;957:21-36 [Abstract]

Chemical composition of olive oil

Olive oil constituents

Olive Oil
Fig. 1 – EVOO

From a chemical point of view, we can identify in the olive oil two fractions, depending on the behavior in the presence of heating and strong alkaline solutions (concentrated solutions of KOH or NaOH):

  • the saponifiable fraction, which represents 98-99% of the total weight, is composed of substances that form soaps in the above conditions;
  • the unsaponifiable fraction, which represents the remaining 1-2% of the total weight, is composed of substances that fail to form soaps in the above conditions.

Saponifiable fraction of olive oil

It is composed of saturated and unsaturated fatty acids, esterified almost entirely to glycerol to form triglycerides (or triacylglycerols). To a much lesser extent, diglycerides (or diacylglycerols), monoglycerides (monoacylglycerols), and free fatty acids are also found.
Unsaturated fatty acids make up 75 to 85% of the total fatty acids. Oleic (O) and linoleic (L) acids are the most abundant ones; palmitoleic, eptadecenoic, gadoleic and alpha-linolenic (Ln) acids are present in lower/trace amounts.

Oleic Acid
Fig. 2 – IOOC and Fatty Acids

Oleic acid is the major fatty acid in olive oils. According to the rules laid down by the International Olive Oil Council (IOOC), its concentration must range from 55% to 83% of total fatty acids.
Linoleic acid is the most abundant polyunsaturated fatty acid in olive oil; its concentration must vary between 2.5% and 21% (IOOC). Because of its high degree of unsaturation, it is subject to oxidation; this means that an oil high in linoleic acid becomes rancid easily, and thus it may be stored for a shorter time.
In a Mediterranean-type diet, olive oil is the main source of fat: therefore, oleic acid, among monounsaturated fatty acids, and linoleic acid, among polyunsaturated fatty acids, are the most abundant fatty acids.
alpha-Linolenic acid must be present in very low amount, according to the IOOC standards ≤1%. It is an omega-3 polyunsaturated fatty acid, which may have health benefits. However, because of to its high degree of unsaturation (higher than that of linoleic acid), it is very susceptible to oxidation, and therefore it promotes rancidity of the olive oil that contains it.
Saturated fatty acids make up 15 to 25% of the total fatty acids.
Palmitic (P) (7.5-20%) and stearic (S) acids (0.5-5%) are the most abundant saturated fatty acids; myristic, heptadecanoic, arachidic, behenic and lignoceric acids may be present in trace amounts.

The presence of fatty acids that should be absent or present in amounts different than those found is a marker of adulteration with other vegetable oils. On this regard, particular attention is paid to myristic, arachidic, behenic, lignoceric, gadoleic and alpha-linolenic acids, whose limits are set by IOOC.

Fatty acid composition is influenced by several factors.

  • The climate.
  • The latitude.
  • The zone of production.
    Italian, Spanish and Greek olive oils are high in oleic acid and low in palmitic and linoleic acids, while Tunisian olive oils are high in palmitic and linoleic acids but lower in oleic acid. Therefore, oils can be divided into two groups:

one rich in oleic acid and low in palmitic and linoleic acids;
the other high in palmitic and linoleic acids and low in oleic acid.

  • The cultivar.
  • The degree of olive ripeness at the time of oil extraction.
    It should be noted that oleic acid is formed first in the fruit, and data seem to indicate a competitive relationship between oleic acid and palmitic, palmitoleic, and linoleic acids.

Triglycerides of olive oil

Olive Oil
Fig. 3 – The sn Positions of Triglycerides

As previously said, fatty acids in olive oil are almost entirely present as triglycerides.
In small percentage, they are also present as diglycerides, monoglycerides, and in free form.
During triglyceride biosynthesis, thanks to the presence of specific enzymes, only about 2% of glycerol binds palmitic acid in the sn-2 position (also the percentage of stearic acid in the sn-2 position is very low); for the most part, the sn-2 position is occupied by oleic acid.
On the contrary, if we consider oils that have undergone a nonenzymatic esterification, the percentage of palmitic acid in the sn-2 position increases significantly.
Note: sn = stereospecific numbering

Among triglycerides present in significant proportions in olive oil, there are:

  • OOO: 40-59%;
  • POO: 12-20%;
  • OOL: 12.5-20%;
  • POL:  5.5-7%;
  • SOO: 3- 7%.

POP, POS, OLnL, OLnO, PLL, PLnO are present in smaller amounts.
Trilinolein (LLL) is a triglyceride that contains three molecules of linoleic acid. Its low content is an indicator of an oil of good quality.
Triglycerides containing three saturated fatty acids or three molecules of alpha-linolenic acid have not been reported.

Diglycerides and monoglycerides of olive oil

Their presence is due to an incomplete synthesis and/or a partial hydrolysis of triglycerides.
The content of diglycerides in virgin olive oil ranges from 1% to 2.8%. 1,2-Diglycerides prevail in fresh olive oil, representing over 80% of the diglycerides. During oil storage, isomerization occurs with a progressive increase of the more stable 1-3 isomers, which after about 10 months become the major isomers.
Therefore, the ratio 1,2/1,3-diglycerides may be used as an indicator of the age of the oil.
Monoglycerides are present in amounts lower than diglycerides, <0.25%, with 1-monoglycerides far more abundant than 2-monoglycerides.

Unsaponifiable fractions of olive oil

It is composed of a large number of different molecules, very important from a nutritional point of view, as they contribute significantly to the health effects of olive oil.
Furthermore, they are responsible for the stability and the taste of olive oil, and are also used to detect adulteration with other vegetable oils.
This fraction includes tocopherols, sterols, polyphenols, pigments, hydrocarbons, aromatic and aliphatic alcohol, triterpene acids, waxes, and minor constituents.
Their content is influenced by factors similar to those seen for fatty acid composition, such as:

  • the cultivar;
  • the degree of ripeness of the olive;
  • the zone of production;
  • the crop year and olive harvesting practices;
  • the storage time of olives;
  • the oil extraction process;
  • the storage conditions of the oil.

It should be noted that many of these compounds are not present in refined olive oils, as they are removed during the refining processes.

Polyphenols

They make up 18 to 37% of the unsaponifiable fraction.
They are a very heterogeneous group of molecules with nutritional and organoleptic properties  (for example, oleuropein and hydroxytyrosol give oil its bitter and pungent taste).
For a more extensive discussion, see: ” Polyphenols in olive oil: variability and composition.”

Hydrocarbons

Olive Oil
Fig. 4 – Squalene

They make up 30 to 50% of the unsaponifiable fraction.
Squalene and beta-carotene are the main molecules.
Squalene, isolated for the first time from shark liver, is the major constituent of the unsaponifiable fraction, and constitutes more than 90% of the hydrocarbons. Its concentration ranges from 200 to 7500 mg/kg of olive oil.
It is an intermediate in the biosynthesis of the four-ring structure of steroids, and it seems to be responsible of several health effects of olive oil.
In the hydrocarbon fraction of virgin olive oil, n-paraffins, diterpene and triterpene hydrocarbons, isoprenoidal polyolefins are also found.
Beta-carotene acts both as antioxidant, protecting oil during storage, and as dye (see below).

Sterols

They are important lipids of olive oil, and are:

  • linked to many health benefits for consumers;
  • important to the quality of the oil;
  • widely used for checking its genuineness.
    On this regard, it is to underline that sterols are species-specific molecules; for example, the presence of high concentrations of brassicasterol, a sterol typically found in Brassicaceae (Cruciferae) family, such as rapeseed, indicates adulteration of olive oil with canola oil.

Four classes of sterols are present in olive oil: common sterols, 4-methylsterols, triterpene alcohols, and triterpene dialcohols. Their content ranges from 1000 mg/kg, the minimum value required by the IOOC standard, to 2000 mg/kg. The lowest values are found in refined oils because of the refining processes may cause losses up to 25%.

Common sterols or 4α-desmethylsterols
Olive Oil
Fig. 5 – beta-Sitosterol

Common sterols are present mainly in the free and esterified form; however they have been also found as lipoproteins and sterylglucosides.
The main molecules are beta-sitosterol, which makes up 75 to 90% of the total sterol, Δ5-avenasterol, 5 to  20%, and campesterol, 4%. Other components found in lower amounts or traces are, for example, stigmasterol, 2%, cholesterol, brassicasterol, and ergosterol.

4-methylsterols

They are intermediates in the biosynthesis of sterols, and are present both in the free and esterified form. They are present in small amounts, much lower than those of common sterols and triterpene alcohols, varying between 50 and 360 mg/kg. The main molecules are obtusifoliol, cycloeucalenol, citrostadienol, and gramisterol.

Triterpene alcohols or 4,4-dimethylsterols

They are a complex class of sterols, present both in the free and esterified form. They are found in amounts ranging from 350 to 1500 mg/kg.
The main components are beta-amyrin, 24-methylenecycloartanol, cycloartenol, and butyrospermol; other molecules present in lower/trace amounts are, for example, cyclosadol, cyclobranol, germanicol, and dammaradienol.

Triterpene dialcohols

The main triterpene dialcohols found in olive oil are erythrodiol and uvaol.
Erythrodiol is present both in the free and esterified form; in virgin olive oil, its level varies between 19 and 69 mg/kg, and the free form is generally lower than 50 mg/kg.

Tocopherols

They make up 2 to 3% of the unsaponifiable fraction, and include vitamin E.
Of the eight E-vitamers, alpha-tocopherol represents about 90% of tocopherols in virgin olive oil. It is present in the free form and in very variable amount, but on average higher than 100 mg/kg of olive oil. Thanks to its in vivo antioxidant properties, its presence is a protective factor for health. Alpha-tocopherol concentration seems to be related to the high levels of chlorophylls and to the concomitant requirement for deactivation of singlet oxygen.
Beta-tocopherol, delta-tocopherol, and gamma-tocopherol are usually present in low amounts.

Pigments

In this group we find chlorophylls and carotenoids.
In olive oil, chlorophylls are present as phaeophytins, mainly  phaeophytin a (i.e. a chlorophyll from which magnesium has been removed and substituted with two hydrogen ions), and confer the characteristic green color to olive oil. They are photosensitizer molecules that contribute to the photooxidation of olive oil itself.
Beta-carotene and lutein are the main carotenoids in olive oil. Several xanthophylls are also present, such as antheraxanthin, beta-cryptoxanthin, luteoxanthin, mutatoxanthin, neoxanthin, and violaxanthin.
Olive oil’s color is the result of the presence of chlorophylls and carotenoids and of their green and yellow hues. Their presence is closely related.

 Triterpene acids

They are important components of the olive, and are present in trace amounts in the oil.
Oleanolic and maslinic acids are the main triterpene acids in virgin olive oil: they are present in the olive husk, from which they are extracted in small amount during processing.

Aliphatic and aromatic alcohols

Fatty alcohols and diterpene alcohols are the most important ones.
Aliphatic alcohols have a number of carbon atoms between 20 and 30, and are located mostly inside the olive stones, from where they are partially extracted by milling.

Fatty alcohols

They are linear saturated alcohols with more than 16 carbon atoms.
They are found in the free and esterified form and are present, in virgin olive oil, in amount not generally higher than 250 mg/kg.
Docosanol (C22), tetracosanol (C24), hexacosanol (C26), and octacosanol (C28) are the main fatty alcohols in olive oil, with tetracosanol and hexacosanol present in larger amounts.
Waxes, which are minor constituents of olive oil, are esters of fatty alcohols with fatty acids, mainly of palmitic acid and oleic acid. They can be used as a criterion to discriminate between different types of oils; for example, they must be present in virgin and extra virgin olive oil at levels <150 mg/kg, according to the IOOC standards.

 Diterpene alcohols

Geranylgeraniol and phytol are two acyclic diterpene alcohols, present in the free and esterified form. Among esters present in the wax fraction of extra virgin olive oil, oleate, eicosenoate , eicosanoate, docosanoate, and tetracosanoate have been found, mainly as phytyl derivatives.

Volatile compounds

More than 280 volatile compounds have been identified in olive oil, such as hydrocarbons, the most abundant fraction, alcohols, aldehydes, ketones, esters, acids, ethers and many others. However, only about 70 of them are present at levels higher than the perception threshold beyond which they may contribute to the aroma of virgin olive oil.

Minor components

Phospholipids are found among the minor components of olive oil; the main ones are phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol.
In the unfiltered oils, trace amounts of proteins may be found.

 References

Gunstone F.D. Vegetable oils in food technology: composition, properties and uses. 2th. Edition. Wiley J. & Sons, Inc., Publication, 2011

Pasqualone A., Sikorska E., Gomes T. Influence of the exposure to light on extra virgin olive oil quality during storage. Eur Food Res Technol 2005;221:92-8 [Abstract]

Servili M., Sordini B., Esposto S., Urbani S., Veneziani G., Di Maio I., Selvaggini R. and Taticchi A. Biological activities of phenolic compounds of extra virgin olive oil. Antioxidants 2014;3:1-23 [Abstract]

Gluten: definition, structure, properties, wheat, cereal list

What is gluten?

Gluten
Fig. 1 – Wheat

Gluten is not a single protein but a mixture of cereal proteins, about 80% of its dry weight (for example gliadins and glutenins in wheat grains), lipids, 5-7%, starch, 5-10%, water, 5-8%, and mineral substances, <2%.
It forms when components naturally present in the grain of cereals, the caryopsis, and in their flours, are joined together by means of mechanical stress in aqueous environment, i.e. during the formation of the dough.
The term is also related to the family of proteins that cause problems for celiac patients (see below).
Isolated for the first time in 1745 from wheat flour by the Italian chemist Jacopo Bartolomeo Beccari, it can be extracted from the dough by washing it gently under running water: starch, albumins and globulins, that are water-soluble, are washed out, and a sticky and elastic mass remains, precisely the gluten (it means glue in Latin).

Cereals containing gluten

It is present in:

  • wheat, such as:

durum wheat (Triticum durum); groats and semolina for dry pasta making are obtained from it;
common wheat or bread wheat (Triticum aestivum), so called because it is used in bread and fresh pasta making, and in bakery products;

  • rye (Secale cereale);
  • barley (Hordeum vulgare);
  • spelt, in the three species:

einkorn (Triticun monococcum);
emmer (Triticum dicoccum Schrank);
spelta (Triticum spelta);

  • khorasan wheat (Triticum turanicum); a variety of it is Kamut®;
  • triticale (× Triticosecale Wittmack), which is a hybrid of rye and common wheat;
  • bulgur, which is whole durum wheat, sprouted and then processed;
  • seitan, which is not a cereal, but a wheat derivative, also defined by some as “gluten steak”.

Given that most of the dietary intake of gluten comes from wheat flour, of which about 700 million tons per year are harvested, representing about 30% of the global cereal production, the following discussion will focus on wheat gluten, and mainly on its proteins.

Note: the term gluten is also used to indicate the protein fraction that remains after removal of starch and soluble proteins from the dough obtained with corn flour: however, this “corn gluten” is “functionally” different from that obtained from wheat flour.

Cereal grain proteins

Gluten
Fig. 2 – Cereal Grain Proteins

The study of cereal grain proteins, as seen, began with the work of Beccari. 150 years later, in 1924, the English chemist Osborne T.B., which can rightly be considered the father of plant protein chemistry, developed a classification based on their solubility in various solvents.
The classification, still in use today, divides plant proteins into 4 families.

  • Albumins, soluble in water.
  • Globulins, soluble in saline solutions; for example avenalin of oat.
  • Prolamins, soluble in 70% alcohol solution, but not in water or absolute alcohol.
    They include:

gliadins of wheat;
zein of corn;
avenin of oats;
hordein of barley;
secalin of rye.

They are the toxic fraction of gluten for celiac patients.

  • Glutelins, insoluble in water and neutral salt solutions, but soluble in acidic and basic solutions.
    They include glutenins of wheat.

Albumins and globulins are cytoplasmic proteins, often enzymes, rich in essential amino acids, such as lysine, tryptophan and methionine. They are found in the aleurone layer and embryo of the caryopsis.
Prolamins and glutelins are the storage proteins of cereal grains. They are rich in glutamine and proline, but very low in lysine, tryptophan and methionine. They are found in the endosperm, and are the vast majority of the proteins in the grains of wheat, corn, barley, oat, and rye.
Although Osborne classification is still widely used, it would be more appropriate to divide cereal grain proteins into three groups: structural and metabolic proteins, storage proteins, and defense proteins.

Wheat gluten proteins

Proteins represent 10-14% of the weight of the wheat caryopsis (about 80% of its weight consists of carbohydrates).
According to the Osborne classification, albumins and globulins represent 15-20% of the proteins, while prolamins and glutelins are the remaining 80-85%, composed respectively of gliadins, 30-40%, and glutenins, 40-50%. Therefore, and unlike prolamins and glutelins in the grains of other cereals, gliadins and glutenins are present in similar amounts, about 40%.
Technologically, gliadins and glutenins are very important. Why?
These proteins are insoluble in water, and in the dough, that contains water, they bind to each other through a combination of intermolecular bonds, such as:

  • covalent bonds, i.e. disulfide bridges;
  • noncovalent bonds, such as hydrophobic interactions, van der Waals forces, hydrogen bonds, and ionic bonds.

Thanks to the formation of these intermolecular bonds, a three-dimensional lattice is formed. This structure entraps starch granules and carbon dioxide bubbles produced during leavening, and gives strength and elasticity to the dough, two properties of gluten widely exploited industrially.
In the usual diet of the European adult population, and in particular in Italian diet that is very rich in derivatives of wheat flour, gliadin and glutenin are the most abundant proteins, about 15 g per day. What does this mean? It means that gluten-free diet engages celiac patients both from a psychological and social point of view.

Note: the lipids of the gluten are strongly associated with the hydrophobic regions of gliadins and glutenins and, unlike what you can do with the flour, they are extracted with more difficulty (the lipid content of the gluten depends on the lipid content of the flour from which it was obtained).

Gliadins: extensibility and viscosity

Gluten
Fig. 3 – Wheat Grain Proteins

Gliadins are hydrophobic monomeric prolamins, of globular nature and with low molecular weight. On the basis of electrophoretic mobility in low pH conditions, they are separated into the following types:

  • alpha/beta, and gamma, rich in sulfur, containing cysteines, that are involved in the formation of intramolecular disulfide bonds, and methionines;
  • omega, low in sulfur, given the almost total absence of cysteine and methionine.

They have a low nutritional value and are toxic to celiac patients because of the presence of particular amino acid sequences in the primary structure, such as proline-serine-glutamine-glutamine and glutamine-glutamine-glutamine-proline.
Gliadins are associated with each other and with glutenins through noncovalent interactions; thanks to that, they act as “plasticizers” in dough making. Indeed, they are responsible for viscosity and extensibility of gluten, whose three-dimensional lattice can deform, allowing the increase in volume of the dough as a result of gas production during leavening. This property is important in bread-making.
Their excess leads to the formation of a very extensible dough.

Glutenins: elasticity and toughness

Glutenins are polymeric proteins, that is, formed of multiple subunits, of fibrous nature, linked together by intermolecular disulfide bonds. The reduction of these bonds allows to divide them, by SDS-PAGE, into two groups.

  • High molecular weight (HMW) subunits, low in sulfur, that account for about 12% of total gluten proteins. The noncovalent bonds between them are responsible for the elasticity and tenacity of the gluten protein network, that is, of the viscoelastic properties of gluten, and so of the dough.
  • Low molecular weight (LMW) subunits, rich in sulfur (cysteine residues).
    These proteins form intermolecular disulfide bridges to each other and with HMW subunits, leading to the formation of a glutenin macropolymer.

Glutenins allow dough to hold its shape during mechanical (kneading) and not mechanical stresses (increase in volume due to both the leavening and the heat of cooking that increases the volume occupied by gases present) which is submitted. This property is important in pasta making.
If in excess, glutenins lead to the formation of a strong and rigid dough.

Properties of wheat gluten

From the nutritional point of view, gluten proteins do not have a high biological value, being low in lysine, an essential amino acid. Therefore, a gluten-free diet does not cause any important nutritional deficiencies.
On the other hand, it is of great importance in food industry: the combination, in aqueous solution, of gliadins and glutenins to form a three-dimensional lattice, provides viscoelastic properties, that is, extensibility-viscosity and elasticity-tenacity, to the dough, and then, a good structure to bread, pasta, and in general, to all foods made with wheat flour.
It has a high degree of palatability.
It has a high fermenting power in the small intestine.
It is an exorphin: some peptides produced from intestinal digestion of gluten proteins may have an effect in central nervous system.

Gluten-free cereals

The following is a list of gluten-free cereals, minor cereals, and pseudocereals used as foods.

  • Cereals

corn or maize (Zea mays)
rice (Oryza sativa)

  • Minor cereals
    They are defined “minor” not because they have a low nutritional value, but because they are grown in small areas and in lower quantities than wheat, rice and maize.

Fonio (Digitaria exilis)
Millet (Panicum miliaceum)
Panic (Panicum italicum)
Sorghum (Sorghum vulgare)
Teff (Eragrostis tef)
Teosinte; it is a group of four species of the genus Zea. They are plants that grow in Mexico (Sierra Madre), Guatemala and Venezuela.

  • Pseudocereals.
    They are so called because they combine in their botany and nutritional properties characteristics of cereals and legumes, therefore of another plant family.

Amaranth; the most common species are:

Amaranthus caudatus;
Amaranthus cruentus;
Amarantus hypochondriacus.

Buckwheat (Fagopyrum esculentum)
Quinoa (Chenopodium quinoa), a pseudocereal with excellent nutritional properties, containing fibers, iron, zinc and magnesium. It belongs to Chenopodiaceae family, such as beets.

  • Cassava, also known as tapioca, manioc, or yuca (Manihot useful). It is grown mainly in the south of the Sahara and South America. It is an edible root tuber from which tapioca starch is extracted.

It should be noted that naturally gluten-free foods may not be truly gluten-free after processing. Indeed, the use of derivatives of gliadins in processed foods, or contamination in the production chain may occur, and this is obviously important because even traces of gluten are harmful for celiac patients.

Oats and gluten

Oats (Avena sativa) is among the cereals that celiac patients can eat. Recent studies have shown that it is tolerated by celiac patients, adult and child, even in subjects with dermatitis herpetiformis. Obviously, oats must be certified as gluten-free (from contamination).

References

Beccari J.B. De Frumento. De bononiensi scientiarum et artium instituto atque Academia Commentarii, II. 1745:Part I.,122-127

Bender D.A. “Benders’ dictionary of nutrition and food technology”. 8th Edition. Woodhead Publishing. Oxford, 2006

Berdanier C.D., Dwyer J., Feldman E.B. Handbook of nutrition and food. 2th Edition. CRC Press. Taylor & Francis Group, 2007

Phillips G.O., Williams P.A. Handbook of food proteins. 1th Edition. Woodhead Publishing, 2011

Shewry P.R. and Halford N.G. Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot 2002:53(370);947-958 [Abstract]

Yildiz F. Advances in food biochemistry. CRC Press, 2009

Polyphenols in olive oil: variability and chemical composition

Polyphenols in olive oil: influences of environment and extraction process

Polyphenols in Olive Oil
Fig. 1 – Olives

Olive oil, which is obtained from the pressing of the olives, the fruits of olive tree (Olea europaea), is the main source of fat in the Mediterranean diet, and a good source of polyphenols.
Polyphenols, natural antioxidants, are present in olive pulp and, following pressing, they pass into the oil.
Note: olives are also known as drupes or stone fruits.
The concentration of polyphenols in olive oil is the result of a complex interaction between various factors, both environmental and linked to the extraction process of the oil itself, such as:

  • the place of cultivation;
  • the cultivars (variety);
  • the level of ripeness of the olives at the time of harvesting.
    Their level usually decreases with over-ripening of the olives, although there are exceptions to this rule. For example, in  warmer climates, olives produce oils richer in polyphenols, in spite of their faster maturation.
  • the climate;
  • the extraction process. In this regard, it is to underscore that the content of polyphenol in refined olive oil is not significant.

Any variation of the concentration of different polyphenols influence the taste, nutritional properties and stability of olive oil. For example, hydroxytyrosol and oleuropein (see below) give extra virgin olive oil a pungent and bitter taste.

Key polyphenols in olive oil

Among polyphenols in olive oil, there are  molecules with simple structure, such as phenolic acids and alcohols, and molecules with complex structure, such as flavonoids, secoiridoids, and lignans.

Flavonoids

Flavonoids include glycosides of flavonols (rutin, also known as quercetin-3-rutinoside), flavones (luteolin-7-glucoside), and anthocyanins (glycosides of delphinidin).

Phenolic acids and phenolic alcohols

Among phenolic acids, the first polyphenols with simple structure observed in olive oil, they are found:

  • hydroxybenzoic acids, such as, gallic, protocatechuic, and 4-hydroxybenzoic acids (all with C6-C1 structure).
  • hydroxycinnamic acids, such as  caffeic, vanillin, syringic, p-coumaric, and o-coumaric acids (all with C6-C3 structure).

Among phenolic alcohols, the most abundant are hydroxytyrosol (also known as  3,4-dihydroxyphenyl-ethanol), and tyrosol [also known as 2-(4-hydroxyphenyl)-ethanol].

Hydroxytyrosol

Polyphenols in olive oil
Fig. 2 – Hydroxytyrosol

Hydroxytyrosol can be present as:

  • simple phenol;
  • phenol esterified with elenolic acid, forming oleuropein and its aglycone;
  • part of the molecule verbascoside.

It can also be present in different glycosidic forms, depending on the –OH group to which the glucoside, i.e. elenolic acid plus glucose, is bound.
It is one of the main polyphenols in olive oil, extra virgin olive oil, and olive vegetable water.
In nature, its concentration, such as that of tyrosol, increases during fruit ripening, in parallel with the hydrolysis of compounds with higher molecular weight, while the total content of phenolic molecules and alpha-tocopherol decreases. Therefore, it can be considered as an indicator of the degree of ripeness of the olives.
In fresh extra virgin olive oil, hydroxytyrosol is mostly present in esterified form, while in time, due to hydrolysis reactions, the non-esterified form becomes the predominant one.
Finally, the concentration of hydroxytyrosol is correlated with the stability of olive oil.

Secoiridoids

They are the polyphenols in olive oil with the more complex structure, and are produced from the secondary metabolism of terpenes.
They are glycosylated compounds and are characterized by the presence of elenolic acid in their structure (both in its aglyconic or glucosidic form). Elenolic acid is the molecule common to glycosidic secoiridoids of Oleaceae.
Unlike tocopherols, flavonoids, phenolic acids, and phenolic alcohols, that are found in many fruits and vegetables belonging to different botanical families, secoiridoids are present only in plants of the Oleaceae family.
Oleuropein, demethyloleuropein, ligstroside, and nuzenide are the main secoiridoids.
In particular, oleuropein and demethyloleuropein (as verbascoside) are abundant in the pulp, but they are also found in other parts of the fruit. Nuzenide is only present in the seeds.

Oleuropein

Polyphenols in Olive Oil
Fig. 3 – Oleuropein

Oleuropein, the ester of hydroxytyrosol and elenolic acid, is the most important secoiridoid, and the main olive oil polyphenol.
It is present in very high quantities in olive leaves, as also in all the constituent parts of the olive, including peel, pulp and kernel.
Oleuropein accumulates in olives during the growth phase, up to 14% of the net weight; when the fruit turns greener, its quantity reduces. Finally, when the olives turns dark brown, color due to the presence of anthocyanins, the reduction in its concentration becomes more evident.
It was also shown that its content is greater in green cultivars than in black ones.
During the reduction of oleuropein levels (and of the levels of other secoiridoids), an increase of compounds such as flavonoids, verbascosides, and simple phenols can be observed.
The reduction of its content is also accompanied by an increase in its secondary glycosylated products, that reach the highest values in black olives.

Lignans

Polyphenols in olive oil
Fig. 4 – Lignans

Lignans, in particular (+)-1- acetoxypinoresinol and (+)-pinoresinol, are another group of polyphenols in olive oil.
(+)-pinoresinol is a common molecule in the lignin fraction of many plants, such as sesame (Sesamun indicum) and the seeds of the species Forsythia, belonging to the family Oleaceae. It has been also found in the olive kernel.
(+)-1- acetoxypinoresinol and (+)-1-hydroxypinoresinol, and their glycosides, have been found in the bark of the olive tree.
Lignans are not present in the pericarp of the olives, nor in leaves and sprigs that may accidentally be pressed with the olives.
Therefore, how  they can pass into the olive oil becoming one of the main phenolic fractions is not yet known.
(+)-1- acetoxypinoresinol and (+)-pinoresinol are absent in seed oils, are virtually absent from refined virgin olive oil, while they may reach a concentration of 100 mg/kg in extra-virgin olive oil.
As seen for simple phenols and secoiridoids, there is considerable variation in their concentration among olive oils of various origin, variability probably related to differences between olive varieties,  production areas, climate, and oil production techniques.

References

Cicerale S., Lucas L. and Keast R. Biological activities of phenolic compounds present in virgin olive oil. Int. J. Mol. Sci. 2010;11: 458-479 [Abstract]

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 [Abstract]

Owen R.W., Mier W., Giacosa A., Hull W.E., Spiegelhalder B. and Bartsch H. Identification of lignans as major components in the phenolic fraction. Clin Chem 2000;46:976-988 [Abstract]

Tripoli E., Giammanco M., Tabacchi G., Di Majo D., Giammanco S. and La Guardia M. The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health. Nutr Res Rev 2005:18;98-112 2005:18;98-112 [Abstract]