Compared to microbial platforms, plants, with their intrinsic metabolic capabilities and the support of genetic engineering, can potentially produce a broader variety of complex sugars found in breast milk, paving the way for significant applications in human health.

The extensive literature on the properties of breast milk highlights what scientists consider its principal component, the “Human Milk Oligosaccharides” (HMOs). These are present in a wide variety and are crucial in the valuable process of establishing the intestinal microbiota in infants [source: “Human Milk Oligosaccharides and Microbiome Homeostasis” – Comprehensive Glycoscience, 2021].

According to UNICEF data on breastfeeding rates in the first six months of life, in 2023, 48% of infants globally were breastfed, with the remaining 52% fed with powdered formula. However, formula milk lacks human milk oligosaccharides or, at best, contains only one or two types out of the over two hundred found in breast milk. This limitation restricts the positive impact of HMOs on the health of the infants receiving it [source: “Human Milk Oligosaccharides: Health Benefits, Potential Applications in Infant Formulas, and Pharmacology” – National Library of Medicine, 2020].

How, then, can current biotechnological research and the subsequent commercial production of human milk oligosaccharides address this deficiency by enriching infant foods? Let’s explore this step by step.

Currently, Human Milk Oligosaccharides (HMOs) are produced to enhance and improve the healthiness of infant formula using microbial cell factories created from selected bacteria and fermented within bioreactors.
Research conducted by two Californian universities highlights that microbial fermentation-based production of HMOs can only produce a limited number of strains. They propose an alternative approach that utilises plant sugar metabolism and recombinant DNA technology, achieving up to eleven different HMO strains.
In the future, advancements in plant-derived systems could lead to the discovery of new human milk oligosaccharides from genetically modified plants. These could potentially serve as prebiotics for treating gastrointestinal and immune system disorders in adults.

The biological function of human milk oligosaccharides

Human Milk Oligosaccharides are complex sugars present in high concentrations in breast milk. Approximately 200 different structures have been identified so far, with their composition varying from woman to woman due to genetic factors.

Interestingly, HMOs do not provide direct nutritional value to the infant. Since they are indigestible, they reach the intestine intact, where they «act as prebiotics, promoting the growth of beneficial gut bacteria and generating short-chain fatty acids, essential for overall health». They are also involved in modulating immune responses and can selectively reduce «the binding of pathogenic bacteria and viruses to the intestinal epithelium», thereby preventing infections and diseases [source: “Human milk oligosaccharides: Shaping the infant gut microbiota and supporting health” – Journal of Functional Foods, September 2020].

The prebiotic function of human milk oligosaccharides was first described in 1954. Over the decades, scientists have increasingly focused on their various biological, physiological, and protective activities.

In recent years, research has also examined the role of HMOs in adulthood, recognising their potential as prebiotics to enhance gut flora, alleviate gastrointestinal inflammation, and treat irritable bowel syndrome [source: “The Effects of Human Milk Oligosaccharides on Gut Microbiota, Metabolite Profiles and Host Mucosal Response in Patients with Irritable Bowel Syndrome” – Nutrients, September 2021].

Production of human milk oligosaccharides via microbial fermentation

The traditional method for producing human milk oligosaccharides (HMOs) to make artificial milk more similar to breast milk relies on microbial systemsEscherichia coli has emerged as the preferred platform for creating microbial cell factories, alongside Lactococcus lactis and the yeast Saccharomyces cerevisiae.

In detail, the production process involves microbial fermentation within bioreactorsSimple sugars such as lactose (readily available and inexpensive) serve as substrates for the microbial cells and are converted into complex sugars [source: “Production of HMOs using microbial hosts – from cell engineering to large scale production” – Current Opinion in Biotechnology, April 2019].

«The microbial cell factories operate at relatively low temperatures, do not require expensive enzymes, utilise simple raw materials, and have the high specificity of biocatalysts, making them excellent means for industrial-scale production of human milk oligosaccharides», emphasise the authors of a study published in Molecules in February 2023 (“Microbial Production of Human Milk Oligosaccharides“). They note that Human Milk Oligosaccharides are primarily composed of five basic monosaccharides (D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid). Nearly all of these oligosaccharides contain a “lactose motif” at the end, which can be “extended” by adding, for instance, L-fucose and N-acetylneuraminic acid, thereby forming various complex sugar structures found in human milk.

Focus on plants and their sugar metabolism

A research team from the departments of plant and microbial biology at the University of California, Berkeley, and the University of California, Davis, in a recent paper titled “Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides,” published in Nature Food on 13 June 2024, comments on the current microbial fermentation-based production of HMOs. This method currently allows for the market availability of only two to five oligosaccharides out of the approximately 200 present in human milk. This is insufficient, given the increased demand in recent years for infant formula and prebiotic foods for adults.

«There is an urgent need to develop biological platforms to produce a wider variety of Human Milk Oligosaccharides», the study group notes. The focus shifts to plants, «which, unlike many microbes used in fermentation, have evolved to create a wide range of polysaccharides from CO2 fixed through photosynthesis, including various nucleotide sugars».

In essence, plants utilise light and carbon dioxide to produce both simple and complex sugars. This process, coupled with the fact that plants can be cultivated, positions them as a potential new platform for large-scale HMO production. 

«Since plants already possess sugar metabolism, why not try to redirect it to produce human milk oligosaccharides?» the authors rhetorically ask.

From microbial systems to genetically modified plants

Researchers from two Californian universities have tested the unique metabolic capabilities of plants to produce a full range of human milk oligosaccharides (HMOs). Specifically, the experiment utilised a plant native to Australia, known as “Nicotiana benthamiana”, a close relative of the tobacco plant.

The authors remind us that all complex sugars, including human milk oligosaccharides, are composed of simple sugars (monosaccharides) linked together to form intricate, branched chains.

The uniqueness of HMOs lies in a set of branched chains designed to create “specific” connections between simple sugars.

Utilising genetic engineering techniques, particularly recombinant DNA technology, the team isolated and spliced the gene responsible for forming these specific connections among the simple sugars in human milk oligosaccharides, introducing it into “Nicotiana benthamiana”.

Once modified in its genome, the plant began producing eleven known strains of human milk oligosaccharides, alongside a variety of other complex sugars characterised by similar linkage patterns.

The second phase of the experiment focused on creating an optimised line of “Nicotiana benthamiana” plants with the aim of producing a single human milk oligosaccharide called Lacto-N-fucopentaose. This oligosaccharide is considered particularly beneficial for both infants and adults but had never been produced on a large scale (only in laboratories) using traditional microbial fermentation methods.

Plant-derived HMOs: purification process and evaluation of prebiotic activity

Before testing the actual prebiotic activity (pertaining to the growth and proper nourishment of intestinal flora) of the complex sugars extracted from genetically modified plants producing human milk oligosaccharides, the research team first purified the raw plant extracts, as «they may contain chemicals that interfere with bacterial growth tests».

The adopted method involved «extracting water from the plant, fermenting yeast to remove simple sugars, and using resin absorption with the disintegrating properties of polyvinylpolypyrrolidone molecules. The resulting sugar extract was rich in HMOs, with negligible amounts of simple sugars and phenolic compounds».

The prebiotic function of HMOs produced by transgenic plants was then compared with that of oligosaccharides found in human milk. This was done through a test measuring the growth curve of “Bifidobacterium longum Infantis, known to be one of the first colonisers of the infant gastrointestinal tract and a known consumer of human milk oligosaccharides. «We also included *Bifidobacterium animalis lactis* as a negative control element: it does not consume HMOs but grows on simple sugars, which could be present in plant extracts or human milk», explained the team.

Curve di crescita del Bifidobacterium longum Infantis (BLI 15697) e del ceppo di controllo Bifidobacterium animalis lactis (BAL 27536) in ambienti integrati con oligosaccaridi del latte umano (HMO) e oligosaccaridi derivati dalle piante transgeniche (pHMO) [fonte: “Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides” - University of California Berkeley e University of California Davis -].
Growth curves of Bifidobacterium longum Infantis (BLI 15697) and the control strain Bifidobacterium animalis lactis (BAL 27536) in environments enriched with human milk oligosaccharides (HMO) and plant-derived oligosaccharides (pHMO) [source: “Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides” – University of California Berkeley and University of California Davis –].

The test results demonstrated that Bifidobacterium longum Infantis grown in an environment containing plant-derived oligosaccharides exhibited growth rates similar to those of Bifidobacterium longum Infantis fed human milk, suggesting – in these early laboratory experiments – that HMOs derived from transgenic plants possess the same biological activity as human milk HMOs.

Glimpses of Futures

The study described is only the initial research into the plant-based production of Human Milk Oligosaccharides (HMOs), achieved by inserting the gene responsible for binding simple sugars in human milk into the Nicotiana benthamiana plant. Although the preliminary results are promising, there is still much work to be done before industrial production can be realised.

Anticipating possible future scenarios, we will use the STEPS matrix to analyse the potential impacts of the evolution of prebiotic supplements (naturally present in human milk) derived from genetically modified plants on various fronts.

S – SOCIAL: the research opens up intriguing possibilities centred around human health. Not only has it shown, in an initial in vitro test, that HMOs produced from transgenic plants possess significant prebiotic properties similar to those found in human milk, making them promising candidates for infant formula supplements and adult dietary supplements, but it may also lead to the discovery of new HMOs derived from other genetically modified plants. These could serve as prebiotics for treating gastrointestinal and immune system disorders in adults. «In a future scenario, the production of HMOs from plants might even enable their direct consumption as food, by ingesting the plant itself or products derived from it», the researchers suggest. Furthermore, this type of plant product, rich in substances that nourish the gut microbiota and contribute to its well-being and immune response modulation, could in the future be added to animal feed. This would create a prebiotic food chain with positive impacts on the physical well-being of both animals and humans.

T – TECNOLOGICAL: in the future, the use of artificial intelligence techniques could facilitate the rapid identification of additional distinctive features of the bonds between the simple sugars that form human milk oligosaccharides (HMOs). This would enable the identification of the responsible genes, which could then be manipulated using recombinant DNA techniques involving various plant species. The aim is to expand the range of HMO strains derived from transgenic plants as much as possible, beyond the current eleven types (human milk contains almost two hundred). To date, microbial fermentation has been able to produce a maximum of five types, demonstrating its limitations and inability to produce specific individual HMOs, such as Lacto-N-fucopentaose.

E – ECONOMIC: plant-based production platforms for large-scale human milk oligosaccharide production stand out as cost-effective. Given the growing demand for HMO-rich foods for both infants (52% of infants worldwide are fed formula milk, which lacks the valuable prebiotics abundant in breast milk) and adults (the International Probiotics Association Europe reports that the European probiotics market is growing, with Germany, France, the UK, Italy, and Spain together representing about 61% of it), if engineered plants were to entirely replace microbial fermentation in the future, it would have a significant economic impact. Consider the bioreactors – where the chemical-physical transformation of simple sugars into complex sugars by bacteria like Escherichia coli takes place – and the substantial energy required to operate them, whereas recombinant DNA technology used for HMO production in Nicotiana benthamiana is considerably less costly.

P – POLITICAL: the research presented is signed by two US universities, which have been the leading producers of genetically modified foods for over twenty-five years. In contrast, the European legal framework has always been marked by rigidity, as evidenced by Directive (EU) 2015/412 on transgenic organisms, which are restricted or entirely banned within the Union, except for imports intended only for animal feed. However, with the European Parliament’s approval on 7 February 2024 of the Commission’s proposed law on the latest genomic techniques applied to agriculture (including recombinant DNA), the EU’s stance has become more open. If this openness is maintained, in the future it could lead to the acceptance of more complete and beneficial formula milk for infants, enriched with HMOs derived from GM plants, as well as prebiotics for adults made from the same HMOs.

S – SUSTAINABILITY: the potential future production of HMOs using GM plants to be integrated into infant formula could positively impact sustainability from both environmental and social perspectives. This is because it would utilize low-energy, low-carbon-footprint facilities compared to the bioreactors typically used in microbial fermentation processes. Additionally, it could serve as a valuable tool for enriching the diets of millions of children in the world’s most vulnerable regions (according to UNICEF’s 2024 report on child malnutrition, «approximately 181 million children under the age of 5 worldwide (one in four) are experiencing severe food poverty»), providing them with beneficial substances that support their immune system health.

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