In this 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.

Industry insights from NIZO 

One of the key challenges an issues facing the food industry today, is protein transition –  the growing move away from animal proteins to alternative sources. Fred van de Velde, head of NIZO’s Protein Functionality Expertise Group has more than 20 years’ professional experience in protein functionality, Fred oversees NIZO’s “source-to-society” activities in protein food technology covering the full range of protein sources. He is also professor of protein transition in food through his chair at the HAS University of Applied Sciences in the Netherlands. In this blog, which was also shown recently in Food Navigator, the protein transition was put under the microscope by asking questions to Fred.  

What protein options are there for plant-based foods? 

The protein transition is massively diversifying the range of proteins available to ingredient and food product manufacturers. Alongside the traditional animal-based sources – meat, eggs, milk – we now have many different plant-based alternatives including legumes like soybeans, peas and chickpeas, as well as maize, potatoes and oilseeds. More options are appearing all the time, from emerging sources including fava beans (also called faba or broad beans) and green leaves to future possibilities such as microalgae and proteins from single-celled organisms produced by fermentation.  

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That’s quite a bewildering array. How do I choose the best option for a new food product? 

Obviously, each protein source has its pros and cons and, when you are starting to develop a new plant-based product, understanding and evaluating them can seem an overwhelming task. But you can simplify the process by thinking about four basic considerations. 
There are the proteins that consumers already know and actively look for on the shelves. Think of milk substitutes like oats, almonds, coconut or soy. Then there are the proteins such as soy, pea and potato that product developers like to work with because they are easy to process and have the right functionality (gelling, foaming, emulsifying, etc.) to directly replace animal-based proteins.  
Next there are environmental factors such as land, water and energy usage and carbon dioxide emissions. Peas and fava beans have low emissions and water usage, as do sources that come from side streams of other products, for example rapeseed which would otherwise be waste from edible oil production. Finally, there are the nutritionists’ favourites that deliver as close as possible to a full complement of essential amino acids without causing allergy issues. For that, you could combine legume and cereal sources. 

Comparison of the water consumption for various protein sources 

So choosing a protein comes down finding the right balance of those considerations? 

Exactly. And that balance will depend on factors such as the type of product you are making and your brand image. For example, consumers are accustomed to choosing milk alternatives and drinks based on the protein types. But for semi-hard cheeses and meat substitutes, they are more likely to choose a product based on how closely it mimics the traditional, animal-based original. 
Similarly, some existing companies have built a strong brand based on soy, almond or oat milk substitutes and want to leverage that successful position as they move into new products. Meanwhile, new players in the market may want to carve out their own niche in this growing market by stressing their environmental credentials or nutritional benefits. 

And once you have decided your marketing position, your protein choice follows from there? 

Of course, you still need to consider the technical aspects of the proteins that fit your marketing decisions. Can they deliver the taste, texture and appearance you want for your product? It is worth remembering that there may be no perfect, off-the-shelf choice. 

How does that all work in practice? 

Let’s take a cream cheese substitute as an example. Consumer favourites like oats, coconut and rice are too low in protein and don’t have the technical functionality to resemble cream cheese.  
Product development favourites such as soy and potato can deliver nice textures, while peas and fava beans also bring environmental benefits. However, the sheer number of different suppliers and variants make screening the options costly and time consuming. Moreover, most legume-based proteins can bring an unwanted beany taste to the end product. Meanwhile, another environmental favourite, rapeseed has a dark colour that isn’t appropriate for a cream cheese. Finally, the nutritionists’ favourite of pulse plus cereal can lead to grittiness at high protein concentrations as well as beany and other off flavours. 

Does a plant-based cream cheese always have to be a compromise? 

Not at all. There are ways that you can improve the technical characteristics of your product. One is to combine different protein types in one product. For instance, we recently surveyed milk alternative for barista applications and found that many have a headline protein type for consumer recognition plus “hidden” secondary proteins to improve technical functionality. 
Another very promising option is fermentation, which can improve the flavour and texture of a product without adding extra “chemicals” to your ingredient list. 
Off flavours arise due to various components such as hexanal, pentanal and 2-pentylfuran. These components are common to many protein sources, but the ratios vary and define the flavour of the end product. The levels of these components in the end product can be reduced by fermentation with an appropriate culture – either in creating the protein ingredient or the final product. And we have carried out extensive taste testing to show that changing the ratios and overall levels of these components does indeed reduce the intensity of unwanted flavours. 

Comparison of various flavour components in pea proteins before fermentation (ref) and after fermentation with various cultures. 

Perceived “beany” taste of pea proteins before fermentation (ref) and after fermentation with various cultures. 

In fact, for products such as semi-hard cheeses, fermentation with different cultures could allow you to create multiple products with different flavour profiles – e.g. a gouda-like product and a cheddar-like product – from essentially the same ingredients. At the same time, fermentation can improve the firmness of your product and remove any grittiness. It can also be used to extend shelf lives without preservatives.  

Firmness of cream cheese alternatives made with pea proteins fermented with various cultures. 

There are a vast number of cultures suitable for fermentation in food production. NIZO alone has established a database of over 8000 cultures that can be screened and selected for the appropriate functionality. Many of these are lactic acid bacteria, developed for fermenting dairy products and may need to be modified for use with plant-based products. 

In short, protein choices are typically driven by marketing considerations around the type of product and your brand rather than purely technical characteristics. But if necessary, functionality, taste and texture can all be improved using carefully considered fermentation. 

Clinical trials can help companies meet growing consumer demand for ‘functional’ food and drinks. But while ‘health’ is the goal, these are not the same as pharmaceutical trials. What do you need to know to ensure a smooth, efficient and effective project that substantiates your product’s health benefits? 

We expect more and more from our food. In 2019, half of global consumers increased their consumption of ‘functional’ foods and drinks. There was a 34.5% increase in the number of sports nutrition products launched with an immunity benefit claim. And there were twice as many snacks launched with digestive/gut health claims than in the previous year. The foods we pick have become an important part of our life goals: to live longer, live healthier, live fitter… 

Standout from the crowd 

Which means functional health benefits continue to offer an attractive way for companies to add value to their food products – and to make them stand out on supermarket shelves stocked to the brim with a never-ending selection of food and beverages to tempt consumers. 

It’s a challenge, but more than that it’s an opportunity: to find or develop new ingredients, and then scientifically demonstrate their benefits to regulators and consumers alike. 

Essential validation 

Clinical trials form a key component in this approach. They allow manufacturers to identify new approaches to their products, and to answer questions such as: What new ingredients are available for human consumption? What nutritional qualities do they have? How do they affect health? What additional benefits do existing products have on health concerns such as resistance to infection? 

By testing the food or beverage ingredients on volunteers, we can assess the proof of concept, gain insight on the impact of an ingredient, collect evidence of a health benefit, characterise ingredients by their effects, and provide measurable outcomes to meet regulatory requirements. 

Food is not pharma 

With all of our years running clinical trials for food and beverage ingredients, we at NIZO have built up experience and understanding of some of the issues to keep in mind when you need to substantiate the health benefits of your product. 

Firstly, clinical trials for food and beverage ingredients and compounds are in many ways similar to pharmaceutical trials. For example, the study designs are similar, the quality assurance requirements are strict to protect study subjects and data integrity, study protocols are reviewed by medical-ethical committees, etc. 

However, testing foods raises some significant and unique challenges. In food trials, we are not looking to cure or treat a health condition. Instead, we are seeking to evaluate how the ingredient helps prevent or mitigate symptoms, or enhance performance, for example. That means, on the one hand, we need to carry out the trials on relatively healthy individuals. But on the other hand, to show a benefit, we may need to ‘create’ a stress factor for the volunteers. 

Unlike pharmaceutical compounds, foods generally have multifactorial effects, acting through several different mechanisms at the same time. The volunteers’ own diets can impact the study results, so that needs to be closely monitored. The results also should be analysed by scientists and professionals with an in-depth knowledge of ingredient properties and food matrices. 

On a very practical level, it can be more difficult to arrange a ‘blind’ test with a food product, than with an anonymous pill. And the food ingredient has to be provided in a form that the volunteers can ingest and which is palatable: so taste, texture, solubility, freshness, etc. need to be considered in a way that is mostly not an issue for pharma trials. 

Prepare for success 

With all of this to keep in mind, for a smooth, efficient and effective clinical trial, you want to work with a company that combines food, nutrition and health expertise. Make sure that the team responsible for your trial understands how food ingredients are digested and metabolised – and most importantly, what this means for the human body. But don’t forget the importance of food technology, processing and safety. And finally, work with a testing company that provides not just data, but interpretation, guidance and consultancy, so that you can make decisions informed by data, but driven by expertise. 

Beer, bread, yoghurt. Fermented products are familiar to everyone. But fermentation can be used for a much wider range of products than just these old favourites. Looking to replace animal-derived products with plant-based ones? Fermentation is a natural way to improve the proteins for application of plant-based alternative products. 

When you are making plant-based products, it isn’t quite as straightforward as replacing the proteins with ones derived from plants. Plant-based proteins often have an unpleasant taste, and their solubility can vary depending on the source. Fermentation offers a solution, allowing you to change the characteristics of proteins ingredients. There is a huge range of microorganisms that can be used for such purposes. But not all microorganisms are suited for this. So, the first step is to find the right one for your needs by screening for the specific characteristics that your product needs. 

Another application of fermentation that I use as Product Manager for Protein Technology at NIZO, is to remove the unpleasant taste of proteins derived from plant-based sources such as pea proteins. Pea proteins often have a beany taste due to amongst others hexanal. Certain microorganisms can break down hexanal, and therefore reduce or even remove the beany taste. The same process can be used to reduce other off-flavours. 

Besides removing unwanted flavours, fermentation can also be used to create the flavours you do want. Imagine, for example, recreating the taste of dairy products in plant-based alternatives.  

It can also improve the texture of products through exopolysaccharide (EPS) production or hydrolytic breakdown of proteins. This approach can, for example, be used to improve the texture and  mouthfeel of plant-based cream cheese. 

Finally, fermentation can be used to increase food safety by preventing the growth of unwanted bacteria. This happens through, among other things, the acidification of the product during fermentation. This is the fermentation that has been known and used since olden times. However, fermentation can also be used to produce antimicrobial components such as bacteriocins. In this way, outgrowth of unwanted (pathogenic) microorganisms can be inhibited. 

These application examples are just some of the ways that fermentation can be used to create and improve plant-based ingredients and products through natural means. If you would like to know more, you can watch my coming webcast on December 3, 3.00-3.30PM CET, where I dive deeper into topics such as unwanted tastes and  improving textures through fermentation.