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Bifidobacteria in the infant gut use human milk oligosaccharides: how does this lead to health benefits?

By Martin Frederik Laursen, Technical University of Denmark, 2022 co-recipient of Glenn Gibson Early Career Research Prize

Breast milk is the ‘gold standard’ of infant nutrition, and recently scientists have zeroed in on human milk oligosaccharides (HMOs) as key components of human milk, which through specific interaction with bifidobacteria, may improve infant health. Clarifying mechanisms by which HMOs act in concert with bifidobacteria in the infant gut may lead to better nutritional products for infants.

Back in early 2016, I was in the middle of my PhD studies working on determinants of the infant gut microbiota composition in the Licht lab at the National Food Institute, Technical University of Denmark. I had been working with fecal samples from a Danish infant cohort study, called SKOT (Danish abbreviation for “Diet and well-being of young children”), investigating how the diet introduced in the complementary feeding period (as recorded by the researchers) influences the gut microbiota development 1,2. Around the same time, Henrik Munch Roager, PostDoc in the lab, was developing a liquid chromatography mass spectrometry (LC-MS)-based method for quantifying the aromatic amino acids (AAA) and their bacterially produced metabolites in fecal samples (the 3 AAAs and 16 derivatives thereof). These bacterially produced AAA metabolites were starting to receive attention because of their role in microbiota-host cross-talk and interaction with various receptors such as the Aryl Hydrocarbon Receptor (AhR) expressed in immune cells and important for controlling immune responses at mucosal surfaces 3,4. However, virtually nothing was known about bacterial metabolism of the AAAs in the gut in an early life context. Further, the fecal samples collected from the SKOT cohort were obtained in a period of life when infants are experiencing rapid dietary changes (e.g. cessation of breastfeeding and introduction of various new foods). Thus, we wondered whether the AAA metabolites would be affected by diet and whether these metabolites might contribute to the development of the infant’s immune system. Our initial results quickly guided us on the track of breastfeeding and bifidobacteria! Here is a summary of the story, published last year in Nature Microbiology5. (See the accompanying News & Views article here.)

We initially looked at the data from a subset of 59 infants, aged 9 months, from the SKOT cohort. Here we found that both the gut microbiome and the AAA metabolome were affected by breastfeeding status (breastfed versus weaned). It is well established that certain bifidobacteria dominate the bacterial gut community in breastfed infants due to their efficient utilization of HMOs – which are abundant components of human breastmilk 6. Our data showed the same, namely enrichment of Bifidobacterium in the breastfed infants, but also indicated that the abundance of specific AAA metabolites were dependent on breastfeeding.

Trying to connect the gut microbiome and AAA metabolome, we found striking correlations between the relative abundance of Bifidobacterium and specifically abundances of three aromatic amino acid catabolites – namely indolelactic acid (ILA), phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (4-OH-PLA), collectively aromatic lactic acids. These metabolites are formed in two enzymatic reactions (a transamination followed by a hydrogenation) of the aromatic amino acids tryptophan, phenylalanine and tyrosine. However, the genes involved in this pathway were not known for bifidobacteria. Digging deeper we discovered that not all Bifidobacterium species found in the infant’s gut correlated with these metabolites. This was only true for the Bifidobacterium species enriched in the breastfed infants (e.g. B. longum, B. bifidum and B. breve), but not post-weaning/adult type bifidobacteria such as B. adolescentis and B. catenulatum group.

We decided to go back to the lab and investigate these associations by culturing representative strains of the Bifidobacterium species found in the gut of these infants. Indeed, our results confirmed that Bifidobacterium species are able to produce aromatic lactic acids, and importantly that the ability to produce them was much stronger for the HMO-utilizing (e.g. B. longum, B. bifidum and B. breve) compared to the non-HMO utilizing bifidobacteria (e.g. B. adolescentis, B. animalis and B. catenulatum). Next, in a series of experiments we identified the genetic pathway in Bifidobacterium species responsible for production of the aromatic lactic acids and performed enzyme kinetic studies of the key enzyme, an aromatic lactate dehydrogenase (Aldh), catalyzing the last step of the conversion of aromatic amino acids into aromatic lactic acids. Thus, we were able to demonstrate the genetic and enzymatic basis for production of these metabolites in Bifidobacterium species.

To explore the temporal dynamics of Bifidobacteria and aromatic lactic acids and validate our findings in an early infancy context (a critical phase of immune system development), we recruited 25 infants (Copenhagen Infant Gut [CIG] cohort) from which we obtained feces from birth until six months of age. These data were instrumental for demonstrating the tight connection between specific Bifidobacterium species, HMO-utilization and production of aromatic lactic acids in the early infancy gut and further indicated that formula supplementation, pre-term delivery and antibiotics negatively influence the concentrations of these metabolites in early life.

Having established that HMO-utilizing Bifidobacterium species are key producers of aromatic lactic acids in the infant gut, we focused on the potential health implications of this. We were able to show that the capacity of early infancy feces to in vitro activate the AhR, depended on the abundance of aromatic lactic acid producing Bifidobacterium species and the concentrations of ILA (a known AhR agonist) in the fecal samples obtained from the CIG cohort. Further, using isolated human immune cells (ex vivo) we showed that ILA modulates cytokine responses in Th17 polarized cells – namely it increased IL-22 production in a dose and AhR-dependent manner. IL-22 is a cytokine important for protection of mucosal surfaces, e.g. it affects secretion of antimicrobial proteins, permeability and mucus production 7. Further, we tested ILA in LPS/INFγ induced monocytes (ex vivo), and found that ILA was able to decrease the production of the proinflammatory cytokine IL-12p70, in a manner dependent upon both AhR and the Hydroxycarboxylic Acid (HCA3) receptor, a receptor expressed in neutrophils, macrophages and monocytes and involved in mediation of anti-inflammatory processes 8,9. Overall, our data reveal potentially important ways in which bifidobacteria influence the infant’s developing immune system.

Figure 1 – HMO-utilizing Bifidobacterium species produce immuno-regulatory aromatic lactic acids in the infant gut.

Our study provided a novel link between HMO-utilizing Bifidobacterium species, production of aromatic lactic acids and immune-regulation in early life (Figure 1). This may explain previous observations that the relative abundance of bifidobacteria in the infant gut is inversely associated with development of asthma and allergic diseases 10–12 and our results, together with other recent findings13–15 are pointing towards aromatic lactic acids (especially ILA) as potentially important mediators of beneficial immune effects induced by HMO-utilizing Bifidobacterium species.

 

References

  1. Laursen, M. F. et al. Infant Gut Microbiota Development Is Driven by Transition to Family Foods Independent of Maternal Obesity. mSphere 1, e00069-15 (2016).
  2. Laursen, M. F., Bahl, M. I., Michaelsen, K. F. & Licht, T. R. First foods and gut microbes. Front. Microbiol. 8, (2017).
  3. Zelante, T. et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 39, 372–385 (2013).
  4. Sridharan, G. V. et al. Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat. Commun. 5, 1–13 (2014).
  5. Laursen, M. F. et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat. Microbiol. 6, 1367–1382 (2021).
  6. Sakanaka, M. et al. Varied pathways of infant gut-associated Bifidobacterium to assimilate human milk oligosaccharides: Prevalence of the gene set and its correlation with bifidobacteria-rich microbiota formation. Nutrients 12, 71 (2020).
  7. Keir, M. E., Yi, T., Lu, T. T. & Ghilardi, N. The role of IL-22 in intestinal health and disease. J. Exp. Med. 217, (2020).
  8. Peters, A. et al. Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3. PLoS Genet. 15, e1008145 (2019).
  9. Peters, A. et al. Hydroxycarboxylic acid receptor 3 and GPR84 – Two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells. Pharmacol. Res. 176, (2022).
  10. Fujimura, K. E. et al. Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation. Nat. Med. 22, 1187–1191 (2016).
  11. Stokholm, J. et al. Maturation of the gut microbiome and risk of asthma in childhood. Nat. Commun. 9, 141 (2018).
  12. Seppo, A. E. et al. Infant gut microbiome is enriched with Bifidobacterium longum ssp. infantis in Old Order Mennonites with traditional farming lifestyle. Allergy Eur. J. Allergy Clin. Immunol. 76, 3489–3503 (2021).
  13. Meng, D. et al. Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine. Pediatr. Res. 88, 209–217 (2020).
  14. Ehrlich, A. M. et al. Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells. BMC Microbiol. 20, 357 (2020).
  15. Henrick, B. M. et al. Bifidobacteria-mediated immune system imprinting early in life. Cell 184, 3884-3898.e11 (2021).

Do polyphenols qualify as prebiotics? The latest scientific perspectives

Kristina Campbell, Consulting Communications Director, ISAPP

When the ISAPP scientific consensus definition of ‘prebiotic’ was published in 2017, the co-authors on the paper included polyphenols as potential prebiotic substances. At the time, the available data on the effect of polyphenols on the gut microbiota were insufficient to show a true prebiotic effect.

An ISAPP webinar held in April 2022, aimed to give an update on the health effects of polyphenols and their mechanisms of action, along with how well polyphenols fit the prebiotic definition. Prof. Daniele Del Rio from University of Parma, Italy, and Prof. Yves Desjardins from Université Laval, Canada, presented the latest perspectives in the field.

What are polyphenols?

Polyphenols are a group of compounds found in plants, with over 6000 types identified to date. They can be divided into two main categories, flavonoids and non-flavonoids.

Polyphenols are absorbed in two different ways in the body. A very small fraction is absorbed in the small intestine, but 95% of them reach the lower gut and interact with gut microbiota. Although polyphenols have a special capacity to influence the activities of microorganisms, some resident microorganisms, in turn, can change the chemical structure of polyphenols through enzymatic action. These interactions produce a unique array of metabolites, which may be responsible for some of polyphenols’ prebiotic effects.

What are the health effects of polyphenols?

Epidemiological studies show that polyphenols in the diet are associated with many health benefits, including prevention of cardiovascular disease, certain cancers, and metabolic disease. These effects occur through various mechanisms. However, association is not proof of causation. So how good is the evidence that polyphenols can lead to health benefits?

Numerous human studies exist, but the most robust study to date for the health benefits of polyphenols is a randomized, controlled trial of over 20,000 adults, published in 2022, which showed supplementation with cocoa extract reduced death from cardiovascular events (although it did not reduce the number of cardiovascular events).

What are the mechanisms of action for polyphenols?

Polyphenols have multiple mechanisms of action. Del Rio focuses on the metabolites produced from dietary polyphenols called flavan-3-ols, which are found in red wine, grapes, tea, berries, chocolate and other foods. Along with colleagues, he showed that the metabolites produced in response to a polyphenol-rich food occur two ‘waves’: a small wave in the first 2 hours after ingestion, and a larger wave 5-35 hours after ingestion. The second wave is produced when flavan-3-ols reach the colon and interact with gut microbiota.

Work is ongoing to link these metabolites to specific health effects. Along these lines, Del Rio described a study showing how cranberry flavan-3-ol metabolites help defend against infectious Escherichia coli in a model system of bladder epithelial cells. These polyphenols are transformed by the gut microbiota into smaller compounds that are absorbed—so the health benefit comes not from the activity of polyphenols directly, but from the molecule(s) that the gut microbiota has produced from the polyphenols.

How else do polyphenols work? Ample evidence suggests polyphenols interact in different ways with gut microbes: they have direct antimicrobial effects, they affect quorum sensing, they compete with bacteria for some minerals, and/or they modify ecology, thereby affecting biofilm formation. Desjardins explained that these interactions may occur in parallel: for example, polyphenols may exert antimicrobial effects when they reach the colon, and at the same time, microorganisms in the gut begin to degrade them.

The mode of action of polyphenols Desjardins studies is the prebiotic mode of action—or as he describes it, “prebiotic with a twist”. A landmark paper from 2015 showed how cranberry polyphenols had protective effects on metabolism and obesity through the creation of mucin in the intestine, which formed a good niche for Akkermansia muciniphila, a keystone bacterial species for good metabolic health. Other polyphenols have since been shown to work the same way: by stimulating production of mucin, thereby providing ideal conditions for beneficial bacteria to grow. In this way, polyphenols appear to show small-scale effects comparable to the effects of probiotics, by inducing a host response that alters the bacterial niche.

Are the effects of polyphenols individual?

Del Rio offered some evidence that the health effects of polyphenols, via metabolites, is personalized: a study showed the existence of three distinct patterns of metabolite production in response to dietary polyphenols (ellagitannins). These may depend on the particular microbes of the gut and their ability to produce the relevant metabolites—so in essence, in each case the gut microbiota is equipped to produce a certain set of metabolites in response to polyphenols. More work is needed, however, to be able to personalize polyphenol intake.

Do polyphenols qualify as prebiotic substances?

Polyphenols clearly interact with gut microbiota to influence human health. The definition of a prebiotic is “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Given the available evidence that polyphenols are not metabolized or utilized by bacteria in all cases in the same direct way as carbohydrate prebiotics, Desjardins sees them as having a “prebiotic-like effect”. Rather, polyphenols are transformed into other biologically active molecules that ultimately provide health benefits to the host. These prebiotic-like properties of polyphenols are nicely summarized in a 2021 review paper and include decreasing inflammation, increasing bacteriocins and defensins, increasing gut barrier function (thereby reducing low-grade inflammation), modulating bile acids, and increasing gut immuno-globulins.

Overall, the speakers showed that polyphenols exert their health effects in several ways—and while the gut microbiota are important for their health effects, polyphenols, as a heterogenous group, may not strictly meet the criteria for prebiotics. Clearly, more research on polyphenols may reveal other mechanisms by which these important nutrients influence the gut microbiome and contribute to host health, and they may someday be regarded as prebiotics.

Watch the replay of the ISAPP webinar here.

Behind the publication: Understanding ISAPP’s new scientific consensus definition of postbiotics

A key characteristic of a probiotic is that it remains alive at the time of consumption. Yet scientists have known for decades that some non-living microorganisms can also have benefits for health: various studies (reviewed in Ouwehand & Salminen, 1998) have compared the health effects of viable and non-viable bacteria, and some recent investigations have tested the health benefits of pasteurized bacteria (Depommier et al., 2019).

Since non-viable microorganisms are often more stable and convenient to include in consumer products, interest in these ‘postbiotic’ ingredients has increased over the past several years. But before now, the scientific community had not yet united around a definition, nor had it precisely delineated what falls into this category.

An international group of scientists from the disciplines of probiotics and postbiotics, food technology, adult and pediatric gastroenterology, pediatrics, metabolomics, regulatory affairs, microbiology, functional genomics, cellular physiology and immunology met in 2019 to discuss the concept of postbiotics. This meeting led to a recently published consensus paper, including this definition: “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”.

Thus, a postbiotic must include some non-living microbial biomass, whether it be whole microbial cells or cell components.

Below is a Q&A with four of the paper’s seven ISAPP-linked authors, who highlight important points about the definition and explain how it will lay the groundwork for better scientific understanding of non-viable microbes and health in the years ahead.

Why was the concept of postbiotics needed?

Prof. Seppo Salminen, University of Turku, Finland:

We have known for a long time that inactivated microorganisms, not just live ones, may have health effects but the field had not coalesced around a term to use to describe such products or the key criteria applicable to them. So we felt we needed to assemble key experts in the field and provide clear definitions and criteria.

Further, novel microbes (that is, new species hitherto not used in foods) in foods and feeds are being introduced as live or dead preparations. The paper highlights regulatory challenges and for safety and health effect assessment for dead preparations of microbes.

Can bacterial metabolites be postbiotics?

Prof. Gabriel Vinderola, National University of Litoral, Argentina:

Postbiotics can include metabolites – for example, fermented products with metabolites and microbial cells or their components, but pure metabolites are not postbiotics.

Can you expand on what is not included in the category of postbiotics?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

The term ‘postbiotic’ today is sometimes applied to components derived from microbial growth that are purified, so no cell or cell products remain. The panel made the decision that such purified, microbe-derived substances (e.g. butyrate) should be called by their chemical names and that there was no need for a single encompassing term for them. Some people may be surprised by this. But microbe-derived substances include a whole host of purified pharmaceuticals and industrial chemicals, and these are not appropriately within the scope of ‘postbiotics’.

For something to be a postbiotic, what kinds of microorganisms can it originate from?

Prof. Gabriel Vinderola, National University of Litoral, Argentina:

A postbiotic must derive from a living microorganism on which a technological process is applied for life termination (heat, high pressure, oxygen exposure for strict anaerobes, etc). Viruses, including bacteriophages, are not considered living microorganisms, so postbiotics cannot be derived from them.

Safety and benefits must be demonstrated for its non-viable form. A postbiotic does not have to be derived from a probiotic (see here for a list of criteria required for a probiotic). So the microbe used to derive a postbiotic does not need to demonstrate a health benefit while alive. Further, a probiotic product that loses cell viability during storage does not automatically qualify as a postbiotic; studies on the health benefit of the inactivated probiotic are still required.

Vaccines or substantially purified components and products (for example, proteins, peptides, exopolysaccharides, SCFAs, filtrates without cell components and chemically synthesized compounds) would not qualify as postbiotics in their own right, although some might be present in postbiotic preparations.

What was the most challenging part of creating this definition?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

The panel didn’t want to use the term ‘inactive’ to describe a postbiotic, because clearly even though they are dead, they retain biological activity. There was a lot of discussion about the word ‘inanimate’, as it’s not so easy to translate. But the panel eventually decided it was the best option.

 Does this definition encompass all postbiotic products, no matter whether they are taken as dietary supplements or drugs?

Prof. Hania Szajewska, Medical University of Warsaw, Poland:

Indeed. However, as of today, postbiotics are found primarily in foods and dietary supplements.

Where can you currently find postbiotics in consumer products, and what are their health effects?

Prof. Hania Szajewska, Medical University of Warsaw, Poland:

One example is specific fermented infant formulas with postbiotics which have been commercially available in some countries such as Japan and in Europe, South America, and the Middle East for years. The postbiotics in fermented formulas are generally derived from fermentation of a milk matrix by Bifidobacterium, Streptococcus, and/or Lactobacillus strains.

Potential clinical effects of postbiotics include prevention of common infectious diseases such as upper respiratory tract infections and acute gastroenteritis. Moreover, fermented formulas have the potential to improve some digestive symptoms or discomfort (e.g. colic in infants). In addition, there is some rationale for immunomodulating, anti-inflammatory effects which may potentially translate into other clinical benefits, such as improving allergy symptoms. Still, while these effects are likely, more well-designed, carefully conducted trials are needed.

What do we know about postbiotic safety?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

Living microbes have the potential, especially in people with compromised health, to cause an infection. But because the microbes in postbiotics are not alive, they cannot cause infections. This risk factor, then, is removed from these preparations. Of course, the safety of postbiotics for their intended use must be demonstrated, but infectivity should not be a concern.

What are the take-home points about the postbiotics definition?

Prof. Seppo Salminen, University of Turku, Finland:

Postbiotics, which encompass inanimate microbes with or without metabolites, can be characterized, are likely to be more stable than live counterparts and are less likely to be a safety concern, since dead bacteria and yeast are not infective.

Read the postbiotic definition paper here.

See the press release about this paper here.

View an infographic on the postbiotic definition here.

New publication co-authored by ISAPP board members gives an overview of probiotics, prebiotics, synbiotics, and postbiotics in infant formula

For meeting the nutritional needs of infants and supporting early development, human milk is the ideal food—and this is reflected in breastfeeding guidelines around the world, including the World Health Organization’s recommendation that babies receive human milk exclusively for the first six months of life and that breastfeeding be continued, along with complementary foods, up to two years of age or beyond. In certain cases, however, breastfeeding is challenging or may not even be an option. Then, parents rely on alternatives for feeding their infants.

A group of scientists, including three ISAPP board members, recently co-authored an article in the journal Nutrients entitled Infant Formula Supplemented with Biotics: Current Knowledge and Future Perspectives. In the review, they aimed to highlight the new technologies and ingredients that are allowing infant formula to better approximate the composition of human milk. They focused on four types of ingredients: probiotics, prebiotics, synbiotics, and postbiotics.

Co-author Gabriel Vinderola, Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at Dairy Products Institute (CONICET-UNL) in Santa Fe, Argentina says, “Modern technologies have allowed the production of specific microbes, subtrates selectively used by the host microbes, and even non-viable microbes and their metabolites and cell fragments—for which scientific evidence is available on their effects on infant health, when administered in adequate amounts. Thus, this current set of gut modulators can be delivered by infant formula when breastfeeding is limited or when it is not an option.”

The authors say a well-functioning gut microbiota is essential for the overall health and proper development of the infant, and components of human milk support the development of this microbiota. They list important human milk components and the novel ingredients that aim to mimic the functions of these components in infant formulas:

  • Human milk oligosaccharides (HMOs)

HMOs are specialized complex carbohydrates found in human milk, which are digested in the infant colon and serve as substrates for beneficial microbes, mainly bifidobacteria, residing there. In recent years, prebiotic mixtures of oligosaccharides (e.g. short-chain GOS and long-chain FOS) have been added to infant formula to recapitulate the effects of HMOs. But now that it’s possible to produce several types of HMOs synthetically, some infant formulas are enriched with purified HMOs: 2’-fucosyllactose (2’FL) or lacto-N-neotetraose (LNnT). Even 3′-galactosyllactose (3′-GL) can be naturally produced by a fermentation process in certain infant formulas.

  • Human milk microbiota

Human milk has a complex microbiota, which is an important source of beneficial bacteria to the infant. Studies support the notion that the human milk microbiota delivers bioactive components that support the development of the infant’s immune system. Probiotic strains are sometimes added to infant formula in order to substitute for important members of the milk microbiota.

  • Bacterial metabolites

Human milk also contains metabolic byproducts of bacteria called “metabolites” in addition to the bacteria themselves. These components have not been fully studied to date, but bacterial metabolites such as butyrate and other short-chain fatty acids may have important health effects for the overall development of the infant. A future area of nutritional research is likely to be the addition of ‘postbiotics’ — non-viable cells, their metabolites and cell components that, when administered in adequate amounts, promote health and well-being — to infant formulas. (ISAPP convened a scientific consensus panel on the definition of postbiotics, with publication of this definition expected by the end of 2020.)

 

The precise short- and long-term health benefits of adding the above ingredients to infant formula are still under study. One pediatric society (the ESPGHAN Committee on Nutrition) examined the data in 2011 and at that time did not recommend the routine use of infant formulas with added probiotic and/or prebiotic components until further trials were conducted. A systematic review concluded that evidence for the health benefits of fermented infant formula (compared with standard infant formula) are unclear, although improvements in infant gastrointestinal symptoms cannot be ruled out. Although infant formulas are undoubtedly improving, review co-author Hania Szajewska, MD, Professor of Paediatrics at The Medical University of Warsaw, Poland, says, “Matching human milk is challenging. Any alternative should not only match human milk composition, but should also match breastfeeding performance, including how it affects infant growth rate and other functions, such as the immune response.”