How Does SuperHMO - 2-FL, LNnT, 3-SL, 6-SL Shape Our Gut Microbiome and Benefit Our Overall Health? A Scientific Study

July 25, 2023 11 min read

How Does SuperHMO - 2-FL, LNnT, LNT, 3-SL, 6-SL Shape Our Gut Microbiome and Benefit Our Overall Health  - A Scientific Study

Not all prebiotics are created equal. Fact. There are many different types of prebiotics, and if you’re a regular reader of our blog, you’ll know that human milk oligosaccharides (HMOs) are just one example.

But a stroll down the aisle of your local health food store or even your supermarket now, will show you a whole array of prebiotic foods. An example you may already be familiar with are inulin-type fructans, such as inulin and fructo-oligosaccharides.

Just like HMOs, inulin-type fructans (ITF) are prebiotic fibres that are commonly promoted for their healthy benefits via their ability to modulate the human gut microbiome. But how do HMOs compare?

We aim to find that out in this article. Here, we’ll introduce what inulin type fructans are and how they compare to different HMO blends based on the results of Bajic et al (2023) study.

The human gut microbiome

The human gut microbiome is a complex yet unique ecosystem comprising of a plethora of archaea, bacteria, viruses and fungi. Most of the microorganisms within you are symbiotic, so you live peacefully in each other’s company. It’s a mutually beneficial relationship – you provide a place to stay, and in return, the microbes provide you with important metabolites to help sustain your health.

It's a win win.

But how exactly did this marvellous ecosystem get there in the first place?

Well, it all began when you entered this glorious world, as you made your rather dramatic entrance. If you were a natural birth, passaged through the birthing canal, you would have undergone a ‘bacterial baptism’ where you would have been ‘washed’ in your mother’s naturally occurring vaginal microbes like Lactobacillus. But don’t worry if you were born via C-section, there are other ways we acquired our early gut inhabitants, such as through breastmilk and skin-to-skin contact with our parents. And just like your fingerprint, your gut microbiota is unique to you[i].

Does your gut microbiome composition change over your lifetime?

The simple answer to this is yes. Your adult microbiome will look different to your infant one, and because it’s unique to you, it’ll likely be different to your friends, siblings and parents. The change in microbiota composition is important because scientists have shown that it is associated with aging.

For example, a study by Leite et al. (2021) found that aging was associated with a decline in bacterial diversity within the small intestine[ii]. They also found an increase in the groups of bacteria that are likely to cause disease, such as:

  • Enterococcaceae
  • Lactobacillaceae
  • Enterobacteriaceae
  • Bacteroides

But that’s not all. It’s long been known that your gut microbiome evolves with you as you get older with different bacteria species enriching the gut at different stages of life (see table).


Childhood (6-9 years)


Bifidobacterium (B. longumsubsp., B. breve, B. bifidum)

Bifidobacterium catenulatum, B. pseudocatenulatum

B. longum, B. adolescentis


These alterations in the make-up of your gut microbiome are not only important for your overall health and wellbeing, but scientists believe they may also affect the overall benefits of prebiotics.

HMOs vs inulin-type fructans

The study by Bajic et al. (2023) investigated the impact of single HMOs (2’-FL, LNnT, 3’Sialyllactose (3’SL) and 6’Sialyllactose (6’SL) and their blends on the production of microbial metabolites and composition of the microbiome compared to inulin-type fructans.

In other words, could age-dependent differences in the overall composition of the human gut microbiome have an effect on prebiotic outcomes?

What could this mean?

In the future, it could advocate the development of nutritional supplements being developed for specific ages, to achieve maximum benefit.

What are inulin-type fructans?

Inulin-type fructans are a common type of prebiotic fibre. They include short-chain fructo-oligosaccharides, oligofructose, and, of course, inulin[iii]. Although you may not be overly familiar with the term, it’s highly likely that you already regularly consume these fibres in your usual diet. That’s because they are naturally present in many fruits and vegetables.

Some research shows that ITFs have a beneficial effect on the composition and function of the human gut microbiome through the promotion of species like Bifidobacteria, Lactobacillus,and Faecalibacterium.

Their health benefits include:

  • Strengthening the gut lining
  • Lowering the risk of colon cancer
  • Controlling blood sugar levels
  • Increasing the absorption of calcium and magnesium[iv]

Like other prebiotic fibres, inulin-type fructans travel to the colon relatively untouched, where your gut microbes set to work on breaking them down through the process of fermentation.

What’s inulin?

Inulin is a member of the ITF family and is increasingly being added to foods and supplements. It naturally occurs in:

  • Artichokes
  • Asparagus
  • Bananas
  • Chicory root
  • Garlic
  • Leeks
  • Onions

So, you’ve probably been munching on inulin without even realising it for years

What are fructo-oligosaccharides (FOS)?

Fructo-oligosaccharides are also a member of the ITF family and are naturally occurring sugars found in plant-based foods. You’ll sometimes hear it being referred to as oligofructose or oligofructan.

What’s the difference between HMOs and inulin-type fructans?

Inulin type fructans like inulin and fructo-oligosaccharides are well-known and researched types of prebiotic fibres or non-digestible carbohydrates. They are fermented by the gut and transformed into microbial metabolites such as short chain fatty acids (SCFAs) which have beneficial effects on your health including attenuating proinflammatory responses[v]. HMOs, however, are a “younger” prebiotic that are mainly made up of simple sugars or monosaccharides and glycosidic linkages. Plus, HMOs are usually consumed in high amounts exclusively by babies because they are naturally present in breast milk. The most abundant types being 2’-Fucosyllactose (2’-FL) and Lacto-N-neotetraose (LnNT).

Interestingly, these prebiotics are often added to infant formula milk to mimic the beneficial effects of naturally-occurring human milk oligosaccharides in human breast milk. Inulin-type fructans can be added alone or in combination whereas HMOs, predominantly 2’-FL are added as a single molecule. A study by Akkerman et al. (2021) investigated whether inulin could influence the fermentation of 2’-FL in infants and found that non-digestible carbohydrates may speed up the fermentation of HMOs[vi].

What was the purpose of the study by Bajic et al (2023)?

The study by Bajic et al (2023) investigated the effect of HMOs on the composition and function of the human gut microbiota in adults and children. The study assessed the effects of both single HMOs and HMO blends against the reference prebiotics inulin and fructo-oligosaccharide.

Let’s take a look at the insights gained from the study to understand the potential of HMOs in the different age groups.

How was the study by Bajic et al (2023) conducted?

The study by Bajic et al (2023) investigated how HMOs affect the gut microbiota of 6-year-old children and adults using inulin and fructooligosaccharides as reference prebiotics. The study sample included six children and six adults and used the following HMOs (and blends thereof):

  • 2’Fucosyllactose (2’FL)
  • Lacto-N-neotetraose (LNnT)
  • 3’Sialyllactose (3’SL)
  • 6’Sialyllactose (6-SL)

To conduct the study SIFR® (Systemic Intestinal Fermentation Research) technology was used. It’s a bioreactor-based model that’s used outside the human body (ex vivo) and relies on “anaerobic bioreactors with supplements that promote bacterial growth.”[vii]

In this study, each bioreactor contained 5 mL of nutritional medium-faecal inoculum blend supplemented with the test products, either single HMOs, HMO blends, or inulin-type fructans. Fresh faecal samples were donated by the participants of the study and the following ten study arms were tested for each of the 12 participants microbiota. They were:

  • No substrate control (NSC) with background medium and faecal microbiota but no products
  • Reference prebiotics – FOS and inulin
  • Single HMOs
    • 2’FL
    • LNnT
    • 3’SL
    • 6’SL
  • 3 HMO blends

Effect of HMOs and HMO blends on child and adult gut microbiota

The overall results of the study showed there were some differences in the faecal microbial composition between adults and children. The use of the SIFR® technology by the researchers meant that the age-dependent differences observed in microbiota composition could be preserved. This was important because these differences impacted on the affect of both types of prebiotics, HMOs, and inulin-type fructans, on the microbiome composition.

In general, HMOs and their blends were shown to increase α-diversity, that is the diversity of the microbiome in one sample. The study also revealed that both 2’-FL and LNnT boosted the growth of Bifidobacteriain both adults and children, but 3’SL and 6’SL were exclusively bifidogenic for children, and FOS and inulin had the same effect in adults. This is interesting and is due to the previously mentioned age-related differences. For example, the abundant Bifidobacteria species in children (B. pseudocatenulatum)is stimulated by the presence of 3’SL and 6’SL. In adults, on the other hand, the predominant species, B. adolescentis, is strongly stimulated by FOS and inulin.

The increased abundance of Bifidobacteria also resulted in a rise in microbial metabolites, including the short chain fatty acids (SCFAs) acetate, propionate, and butyrate. There was an increased abundance of the butyrate producer A. hallii,especially after LNnT administration.

This correlates with other studies findings such as that by Iribarren et al. (2020) who demonstrated that a 10g daily dose of 2’FL and LNnT increased the abundance of health promoting Bifidobacteriumspp. in irritable bowel syndrome patients without provoking symptoms. The study also revealed an increased abundance of A. hallii.

But the Bajic et al. (2023) study also resulted in an increase in other health promoting Bifidobacteria-mediated metabolites, including indole-3 lactic acid and 3-phenyl-lactic acid, both of which are associated with immune health and gamma-aminobutyric acid (GABA) which is associated with the gut brain axis. These metabolites were stimulated in both adults and children following the addition of 2’FL and the HMO blends.

GABA is an important neurotransmitter in the brain. Neurotransmitters are chemical messengers and GABA is known as an inhibitory neurotransmitter because it blocks specific brain signals and reduces the activity of the nervous system. For example, when it attaches to its receptor it can alleviate the feelings of anxiety and stress. Research also shows that GABA can have an anticonvulsant effect[viii]. In this study, GABA production increased after treatment with inulin, 2’FL, LNnT, and two of the HMO blends. Some studies have found an association between altered GABAergic neurotransmission and several nervous system disorders, including depression[ix], pain, sleep disturbances and changes in gut motility.

GABA production was associated with B. adolescentis,the predominant species in the adult microbiota, and likely stimulated by 2’FL and LNnT. Yet, GABA could also be increased by the presence of the sialylated HMOs, 3’SL and 6’SL, in relation to Phocaeicola vulgatus,the second most potent GABA producer. Thus, indicating that HMOs could have complementary benefits on human health.

In children, the presence of 2’FL, LNnT and HMO blends 2 and 3 resulted in an increase in melatonin. Melatonin has been shown to be an effective treatment for irritable bowel syndrome (IBS)[x] and prevents gastric mucosa ulceration. Bubenik (2002) suggested melatonin could be considered for the treatment of ulcerative colitis, colorectal cancer, gastric ulcers, and childhood colic[xi].

Sialylated HMOs make up around 13% of the total HMOs found in human breast milk. 3’-SL ad 6’SL are two simple sialylated HMOs and have prebiotic effects by modulating the gut microbiome composition through the increased abundance of Bifidobacteria.They are often added to human infant formula milk and are approved as “Generally Recognised as Safe”[xii].

In the Bajic et al (2023), 6’SL had the biggest effect on different bacterial species, increasing the health-promoting F. prausnitzii, R. torques, A. halliiand C. comes.It’s likely that’s because of the cross-feeding that Bifidobacteriais renowned for – it helps to feed other species by releasing metabolites such as short chain fatty acids.

Interestingly, the study also showed that 3’SL increased adult folic acid levels. Folate or vitamin B9 is essential for the production of healthy red blood cells and during times of rapid growth. That’s why it is recommended that women supplement their folic acid intake[xiii] if they are trying to get pregnant and for the first 3 months of pregnancy[xiv]. Folate deficiency is linked with an increased risk of many cancers[xv], including breast[xvi]. Folic acid supplementation or intake is recommended for people with IBD because it regulates the overall rectal cell turnover. Therefore, the intake of 3’SL increases the levels of folate in adults which may help to prevent several types of cancer or improve IBD outcomes.

Summary of key findings

Overall, the Bajic et al. (2023) study showed that age-associated differences in gut microbiota composition had an impact on the outcomes of the prebiotics used, resulting in different benefits, and advocating the development of age-specific nutritional supplements. HMOs and their blends were shown to be potent modulators of the human gut microbiome in adults, and particularly in children. Mixing HMOs into different blends could increase their beneficial effects because they could provide a broader range of metabolites with some selectively targeting specific Bifidobacteriumspecies. This is an aspect which could be important in people who are deficient in Bifidobacteria such as the elderly, IBD patients, or those who have recently taken a course of antibiotics.

To summarise the key findings of the study were:

  • HMOs and their blends increased α-diversity
  • 2’FL and LNnT are bifidogenic for adults and children but sialylated HMOs are exclusively bifidogenic in children
  • LNnT stimulated the production of butyrate
  • 2’FL and HMO blends stimulated the production of GABA
  • 2’FL and LNnT increased melatonin in children
  • 3’SL increased folic acid levels in adults

At Layer Origin we offer a range of HMO products designed to support and maintain the gut health of all ages. For example, our SuperHMO® Prebiotic for Kids contains a superhero blend of:

  • 2’FL
  • Lacto-N-tetraose (LNT)
  • LNnT
  • 6’SL
  • 3’SL

But if you’re looking for something a little more grown up, we offer the same blend specially formulated for adults; SuperHMO® Prebiotic Mix with 5 HMOs. Or how about our PureHMO® Tri-Prebiotic Powder, a full spectrum prebiotic containing HMOs, galactooligosachharide and fructooligosaccharide?

Explore our range in our shop.


Overall, the Bajic et al. (2023) study provides valuable insights into the differential effects of prebiotics, particularly HMOs on the composition of the human gut microbiome and how this can differ according to age. It also highlights how these effects can be enhanced by blending certain HMOs together to produce a more profound benefit. These differences highlight the importance of age-dependent supplementation and also the promise of HMOs in adults as well as children.

Written byLeanne Edermaniger, M.Sc. Leanne is a professional science writer who specializes in human health and enjoys writing about all things related to the gut microbiome. 


[i] The microbiome [Internet]. 2022 [cited 2023 Jul 10]. Available from: 

[ii] Leite G, Pimentel M, Barlow GM, Chang C, Hosseini A, Wang J, et al. Age and the aging process significantly alter the small bowel microbiome. Cell Reports. 2021;36(13):109765. doi:10.1016/j.celrep.2021.109765 

[iii] Hughes RL, Alvarado DA, Swanson KS, Holscher HD. The Prebiotic Potential of Inulin-Type Fructans: A Systematic Review. Adv Nutr. 2022 Mar;13(2):492-529. doi: 10.1093/advances/nmab119. Epub 2023 Feb 10. PMID: 34555168; PMCID: PMC8970830.

[iv] Roberfroid MB. Inulin-type fructans: Functional Food Ingredients. The Journal of Nutrition. 2007 Nov 1;137(11). doi:10.1093/jn/137.11.2493s 

[v] Logtenberg MJ, Akkerman R, An R, Hermes GDA, de Haan BJ, Faas MM, Zoetendal EG, Schols HA, de Vos P. Fermentation of Chicory Fructo-Oligosaccharides and Native Inulin by Infant Fecal Microbiota Attenuates Pro-Inflammatory Responses in Immature Dendritic Cells in an Infant-Age-Dependent and Fructan-Specific Way. Mol Nutr Food Res. 2020 Jul;64(13):e2000068. doi: 10.1002/mnfr.202000068. Epub 2020 Jun 2. PMID: 32420676; PMCID: PMC7378940.

[vi] Akkerman R, Logtenberg MJ, Beukema M, de Haan BJ, Faas MM, Zoetendal EG, et al. Chicory inulin enhances fermentation of 2′-fucosyllactose by infant fecal microbiota and differentially influences immature dendritic cell and T-cell cytokine responses under normal and th2-polarizing conditions. Food & Function. 2021;12(19):9018–29. doi:10.1039/d1fo00893e 

[vii] Özdinç, B. (2023) SIFR® technology: Closing the gap between Preclinical and Clinical Prebiotics Research, CosmosID. Available at: (Accessed: 19 July 2023). 

[viii] Snodgrass SR. GABA and epilepsy: Their complex relationship and the evolution of our understanding. Journal of Child Neurology. 1992;7(1):77–86. doi:10.1177/088307389200700114 

[ix] Strandwitz P, Kim KH, Terekhova D, Liu JK, Sharma A, Levering J, McDonald D, Dietrich D, Ramadhar TR, Lekbua A, Mroue N, Liston C, Stewart EJ, Dubin MJ, Zengler K, Knight R, Gilbert JA, Clardy J, Lewis K. GABA-modulating bacteria of the human gut microbiota. Nat Microbiol. 2019 Mar;4(3):396-403. doi: 10.1038/s41564-018-0307-3. Epub 2018 Dec 10. PMID: 30531975; PMCID: PMC6384127.

[x] Faghih Dinevari M, Jafarzadeh F, Jabbaripour Sarmadian A, Abbasian S, Nikniaz Z, Riazi A. The effect of melatonin on irritable bowel syndrome patients with and without sleep disorders: A randomized double-blinded placebo-controlled trial study. BMC Gastroenterology. 2023;23(1). doi:10.1186/s12876-023-02760-0 

[xi] Bubenik GA. Gastrointestinal melatonin: localization, function, and clinical relevance. Dig Dis Sci. 2002 Oct;47(10):2336-48. doi: 10.1023/a:1020107915919. PMID: 12395907.

[xii] Zhu Y, Zhang J, Zhang W, Mu W. Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3′-sialyllactose and 6′-sialyllactose. Biotechnology Advances. 2023;62:108058. doi:10.1016/j.biotechadv.2022.108058

[xiii] Folate (folic acid) – vitamin B9 [Internet]. 2023 [cited 2023 Jul 21]. Available from:

[xiv] About folic acid [Internet]. NHS; [cited 2023 Jul 21]. Available from:

[xv] Pieroth R, Paver S, Day S, Lammersfeld C. Folate and Its Impact on Cancer Risk. Curr Nutr Rep. 2018 Sep;7(3):70-84. doi: 10.1007/s13668-018-0237-y. PMID: 30099693; PMCID: PMC6132377.

[xvi] Ren X, Xu P, Zhang D, Liu K, Song D, Zheng Y, Yang S, Li N, Hao Q, Wu Y, Zhai Z, Kang H, Dai Z. Association of folate intake and plasma folate level with the risk of breast cancer: a dose-response meta-analysis of observational studies. Aging (Albany NY). 2020 Nov 4;12(21):21355-21375. doi: 10.18632/aging.103881. Epub 2020 Nov 4. PMID: 33146633; PMCID: PMC7695428.

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