In his latest column, NIZO Food Research Division Manager and FoodNavigator advisory panel member René Floris explores the role of in-vitro studies in substantiating health benefits of food products.

There is a growing awareness of the health benefits of food beyond its basic nutritional value. For example, specific components of food can boost the immune system, affect intestinal health, modulate the gut microbiome and even inhibit pathogens binding to cells in our digestive tract. Not surprisingly, this is attracting a lot of consumer interest. However, manufacturers can’t just make unsubstantiated health claims. They need evidence to support those claims and build consumer trust. In many cases, in vitro testing can provide important evidence to create compelling stories.

To find out more, I spoke to Anita Hartog. Anita is Senior Scientist in Nutrition and Health at NIZO and has over twenty years industrial experience studying the health and immunological impact of food.

Functional ingredient identification and substantiation via in vitro models

René Floris: What is an in vitro model?

Anita Hartog: An in vitro model is a type of scientific test performed in a laboratory. It is carefully designed and selected to mimic, for instance, digestion, intestinal functionality, the gut microbiome or combinations thereof. Models can represent different cell types (e.g. intestinal cells, immune cells), different actions (e.g. digestion, fermentation) and different populations (e.g., infants, toddlers, adults, the elderly). Crucially, each in vitro model must be thoroughly validated to ensure it accurately reproduces the conditions found within the human body.

RF: Where do in vitro models fit into health benefit substantiation?

AH: Randomized, placebo-controlled trials in humans are the gold standard. And if you want to make specific health claims, such intervention trials may be required by regulators. But they take a lot of time and money. In vitro studies are much quicker and can provide credibility for claims that a food component is biologically active and what the mode of action is. They can also guide the design of later human intervention studies to increase the chances of a significant result, which saves time and money.

You can also use in vitro studies to identify new functional components in food or to study impact of various types of processing on those components. They are also the ideal way to compare large numbers of nutritional components and to evaluate potential interactions between components that could either enhance or suppress the biological action you are looking to promote.

RF: How do you employ in vitro models to best effect?

AH: The first step is to consider what types of functional components may be in your food product and where they may act on for instance the gut or the immune system. For example, oligosaccharides can affect the gut’s microbiome composition and may also inhibit a pathogen’s ability to infect cells, modulate intestinal cell growth or affect immune cell function. Probiotics also influence the composition of your gut microbiome. Active components can address specific cell types in the intestine either directly or via metabolites. Based on that insight, plus the type of product you are making and its intended target population, you can choose the most relevant in vitro models for exploring your desired health benefits.

Usually, you will need to combine multiple in vitro models to fully understand a components action on the gut. For instance, you may need to combine immune and epithelial cell models to study the interaction between components that act on different types of cells or the interaction between the different cell types. Meanwhile combining digestion, gut fermentation and intestinal models may give a more realistic picture of how certain peptides, oligosaccharides or other food components are metabolised and absorbed.

RF: So, combining models and studies is essential to build a complete story?

AH: Absolutely. Take oligosaccharides for example. As I mentioned before, these can impact the body in various way. Consequently, many infant formula manufacturers want to include human milk oligosaccharides (HMOs) in their products to better mimic breast milk and support proper microbial, intestinal and immune development. However, over 200 HMO structures have been identified so far. Obviously, trying to add all of those to an infant formula would be prohibitively expensive. So how do you identify the best one? Or should you use a combination?

Babies can’t digest HMOs but using an epithelial cell model (perhaps combined with a gut fermentation model), we can perform an anti-adherence study to investigate how different HMOs inhibit a target pathogen from binding to cells in the intestinal wall. Figure 1 shows a study of two HMOs and we can see that “Oligo B” is better at preventing pathogen binding than both “Oligo A” and a combination of the two HMOs.

Figure 1: the results of an in vitro study of the relative suppression of pathogen binding by different HMOs.

So we might initially conclude that Oligo B is the best HMO to include in an infant formula. However, HMOs also have additional protective functions – such as potentially inhibiting a pathogen’s ability to disrupt the barrier that the gut wall represents. And by carrying out a barrier integrity assay, we see that in this case the combination is significantly more effective at protecting the gut barrier. Armed with both pieces of information, the manufacturer can make a more informed decision on which HMOs to include in their formula.


Figure 2: the results of an in vitro study on how different HMOs affect pathogen-induced barrier disruption in the human gut.

RF: And how would this apply to, say, the protein transition?

AH: The health impact of proteins is related to the levels of amino acids they deliver into the body, which can be quantified by the protein digestibility-corrected amino acid score (PDCAAS) or digestible indispensable amino acid score (DIAAS). Plant-based proteins typically score lower than animal-based proteins in both measures. Hence, there is a lot of effort in the industry to improve the digestibility of plant-based proteins either through smart processing or fermentation.

That’s where in vitro studies come in, allowing you to rapidly evaluate the quality of different proteins and the impact of various processing and fermentation steps using digestion models. There are two main types of these models. Static models are simpler, mimicking the biochemical processes in the gastrointestinal tract usually with a fixed set of initial conditions (pH, enzyme concentrations, bile salts, etc.). Dynamic models are more complex but provide a more realistic recreation of actual in vivo conditions. As always, the choice of which is the best model to use comes down to the specifics of the particular health benefit question you want to address.

Next month we will continue looking at the protein transition, this time focusing on the food safety aspects of introducing new protein ingredients.

Any questions?

Anita Hartog is happy to answer all your questions.

Meet Anita Hartog