In this series of articles, NIZO Food Research Division Manager and FoodNavigator advisory panel member René Floris discusses some of the big issues in today’s food industry. This month, he continues his exploration of the protein transition by looking at the food safety for plant-based foods.

The market for meat- and dairy alternatives is growing at a massive rate. But while these plant-based products may deliver as close an eating experience as possible to their animal-based counterparts, the food safety challenge they present can be very different. To find out more, I spoke to Marjon Wells-Bennik, Principal Scientist Food Safety, who has over 20 years academic and industrial experience in food safety and microbiology.

Joined up thinking throughout the chain enables plant-based food safety

René Floris: How does the food safety picture for plant-based differ from animal-based food?

Marjon Wells-Bennik: Food safety is a much more varied challenge for plant-based foods and dairy alternatives. For example, in the dairy industry, there is basically one main ingredient – raw milk – which is fairly consistent in terms of its nutritional composition, physical characteristics and microbiological contaminants.

Plant-based proteins, on the other hand, come from many different sources. Each has its own mix of proteins, sugars etc. The variety of microbes present is also much greater – and the levels of microbes can vary greatly too. The soil where the source plant is grown, how the plant is harvested, how the plant materials are processed to obtain the protein, even how plant and resulting ingredients are stored and transported can play a role. So, you have very different microbial contamination starting points and growth conditions.

To make matters even more complex, plant-based proteins can have very different solubilities and reactions to heat, which affects how they can be processed. All this makes food safety a very challenging issue for plant-based products – one that requires a more holistic approach.

RF: It is often said that microbial contamination in plant-based foods is less well understood. Is that true?

MW-B: The dairy industry has been investigating microbial contamination for decades and, as I said, the conditions there are much more consistent. So it is not surprising that knowledge of microbial contamination in plant-based protein ingredients is less developed. But progress is being made. For example, at NIZO we have carried out our own research into microbial contamination of ingredients used to make dairy alternatives based on coconuts, oats, almond, peas and other legumes. We found that both the level and variety of microbes was higher than in raw milk. Some of the varieties found in plant-based ingredients are familiar from the dairy industry, such as Bacillus cereus and other Bacillus and Clostridia species. But some are new, and we do not know how the product composition influences their growth.

A large proportion of the total microbial count in plant-based ingredients consists of spores from bacteria such as Bacillus subtilis, B. licheniformis and B. amyloliquefaciens, but we alsofound B. cereus, and Geobacillus stearothermophilus. There can be as many as 1000 spores per gram in plant-based ingredients. These spores can survive high heat treatments and, upon germination, can go on to produce (heat-resistant) toxins that present a health risk. This contrasts with spore counts of around 1-10 per ml for raw milk, where the majority of bacteria are inactivated by pasteurisation.

Figure 1: Total aerobic and spore counts for samples of raw milk and various plant protein isolates.

RF: Are more extreme heat treatments than pasteurization required?

MW-B: That has two problems. Firstly, many plant proteins are denatured by high temperatures, affecting their taste, texture and nutritional value. And at the end of the day, the goal is to produce a food product that consumers want to eat or drink. Secondly, some of the spores identified in plant-based ingredients can survive even the most extreme heat treatments used in food processing. Novel techniques like Innovative Steam Injection (applying very high temperatures for very short times) are promising, particularly for inactivating spores in liquid end products, but they have not yet been widely applied to plant-based foods.

RF: Can we apply non-thermal techniques?

MW-B: There are techniques familiar from the dairy industry that we can apply to plant-based products, but we need to do so in a smart way and drawing on understanding of plant-based ingredients. For example, common heating processes such as pasteurization kill bacteria but not the spores. A technology like bactofugation has been successfully used to reduce bacterial spore levels in milk. This could also be attractive for plant-based products. However, it only works with liquids and the solubility of plant proteins varies greatly so you would need to consider that before developing a bactofugation-based process.

Fermentation is another possibility for improving product stability. Fermentation has been used for millennia in making cheese and yoghurt among other things, so is well understood. In most dairy fermentations, lactic acid is produced from lactose and helps preserve the food. Similar preservation can be achieved for plant-based products through fermentation or chemical acidification (for instance adding lactic acid). Spoilage by (heat-resistant) moulds is a major concern for yoghurt and cheese alternatives. Our own research has shown that rapid acidification through fermentation was more effective at preventing mould growth than chemical acidification – fermented samples of plant-based cream cheese were still mould free after six weeks at 6 °C.

However, plant-based ingredients have different sugars and fats as well as different proteins to milk. So you can’t just assume that you can use the same lactic acid bacteria for fermenting plant-based products as in dairy products. The presence of sugars other than lactose may allow spoilers to overgrow the fermentation culture, spoiling the product before it can be sufficiently acidified. You can avoid this by reformulating the product to adjust the sugar profile or using a different starter culture. The industry is starting to build up knowledge of which cultures work best with which plant-based ingredients, but there is still a long way to go. So today, it is often still a matter of trial and error.

RF: So how do you find the way through this complexity to a safe product?

MW-B: Food safety is always a matter of joined up thinking from ingredient to consumer. But this is even more true for plant-based products. The complexity of the microbiological situation, the different potentials for microbe growth mean you truly have to think about the whole chain holistically: where plants are grown, the extraction of proteins, transportation of ingredients, processing steps in manufacturing, the distribution and retail chains, desired shelf life, and how consumers will store and use products at home. Each of these steps could be a cause of contamination or pathogen and spoiler growth. But they can also be a potential solution.

For example, if you can’t use bactofugation in your final product because your protein isn’t soluble enough, perhaps you can prevent or remove contamination at an earlier stage in the chain. Maybe you need to consider using a protein concentrate obtained via a dry process instead of an isolate where the wet fractionation and subsequent evaporator process can encourage the growth of spore formers.

Predictive modelling is a powerful tool here. It allows you to carry out initial microbial risk assessments in silico, based on processing conditions, the intrinsic properties of the product, and the intended storage and consumption conditions. Such assessments can help you identify microbes of concern and make informed decisions on preventative measures and even product preservation strategies. For example, if you are planning a shelf-life stable product but the key microbes of concern are particular Bacillus bacteria which don’t grow at refrigeration temperatures, perhaps the best approach is to switch to a chilled product at least initially.

Figure 2: Oat drink spoiled by B. subtilis (left) and Almond drink spoiled by B. licheniformis (right).

Each new plant-based product can bring a new food safety challenge. But by taking this kind of end-to-end approach, it is possible to ensure ‘safety by design’ and deliver tasty, high-quality plant-based products.

Next month we will be discussing flavour, taste and mouthfeel of plant proteins.

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.

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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.

In the previous blog, we looked at the range of alternative protein options available and how to choose the right one for your new product. Having chosen the right protein source (or sources), the challenge turns to ensuring you can maintain the desired functionality of that protein during processing and deliver the associated benefits to your customers.
In this blog, which was also shown recently in Food Navigator, the role of processing in protein functionality was put under the microscope by asking questions to Peter de Jong, Principal Scientist Processing at NIZO. In addition to his role at NIZO, Peter is professor of dairy process technology at Van Hall Larenstein University of Applied Sciences and director of New Technology Development for Food at the Institute for Sustainable Process Technology.

Why is protein functionality a key issue for process development?

The food industry is increasingly aware of the value of protein in food products. That goes a lot further than just the amount of protein, but also the functionality it brings. For example, as Fred van de Velde explained last month, protein functionality can influence the taste and texture of a food product, affecting how attractive the product is to consumers. It can also impact production efficiency. For example, processing can cause certain proteins to coagulate, leading to fouling and regular production shutdowns for cleaning. And of course, there is the nutritional impact of proteins, not just in terms of macronutrient properties but also more subtle effects such as binding vitamins and, particularly topical right now, their impact on immune response and anti-viral activity.
The interest in these effects is growing rapidly as the protein transition opens up new / alternative protein sources, many of which offer much greater protein functionality that meat does. For example, there is a lot of interest right now in raw milk because it seems effect our immune systems and possibly reduce allergies.

What is the impact of processing on protein functionality?

The goal of processing is to deliver a food product that is tasty, nutritious and safe. Traditionally, the food industry has taken the cautious approach – using heat treatments like pasteurisation and UHT that ensure all pathogens are killed or deactivated. However, temperatures above 80 C reduce or even destroy the functionality of many proteins. A good illustration is the Maillard reaction, where heat causes sugar molecules to bind with amino acids. You might want this when searing a steak or baking a biscuit, but when you are processing milk it reduces the bioavailability of vital amino acids like lysine which in turn can reduce benefits raw milk has for the immune system (Figure 1). Consequently, there is a big drive towards milder processing that still delivers maximum food safety while leaving more of the protein functionality intact.

So, can we just turn down the heat?

Unfortunately, it isn’t as simple as that. We at NIZO have analysed a lot of production processes and have found a great deal of variation in the protein functionality impact of seemingly similar process. Looking at the Maillard reaction I mentioned earlier, even processes as familiar as pasteurisation or UHT can vary in the amount of amino acid lost by a factor of two.
This shows two things. First, that there is plenty of room to optimize current processes. Second, food manufacturing processes are very complex, with multiple possible reactions between ingredients, each of which interacts differently with the process conditions. And that makes optimizing a process extremely challenging.

How do you start to optimize such a complex process?

One way is through data analytics: collecting as much data as you can from your factory and analysing it for any correlations. But this is a bit of a black box approach. It can help you identify which conditions or temperatures are linked to specific outcomes, but it doesn’t give you any insight into why or how to fix the issue. So, it isn’t really any help if you are trying to design a new process.
A better approach is through computer modelling of your factory set up. This does require deep understanding of the chemical reactions that can occur during food processing, but once you have built your model – or had it built for you – you can thoroughly explore the impact of variations in process conditions, either by manually tweaking process parameters in the model or by running simulations.
The results can be incredible. We have seen cases where manufacturers have been able to optimize process performance and improve bioavailability of nutrients by up to 30% without affecting the products physical properties or microbial quality specifications.

What other new technologies could help manufacturers retain protein functionality?

The industry is always innovating, finding new ways to make products better. One technology that I am excited by at the moment is called Innovative Steam Injection or ISI. This involves a very short blast of very high temperature – around 160 C for up to 1 second. This is enough to inactivate microorganisms in the food but, crucially, does not denature the proteins. Prototype ISI processes have been able to deliver “pasteurised” milk with shelf lives up to 60 days and just one-third the degradation of proteins such as β-lactoglobulin, immunoglobulin and lactoferrin (Figure 2). And even expert tasters couldn’t taste the difference.

And it is not just for dairy. ISI can be used with any pumpable fluid product. This could be very important for the protein transition as the microorganism contaminants in plant-based proteins are much more diverse and less well known. Currently, the plant-based food industry relies on extreme heat treatments which certainly kill off all pathogens but also destroy the desired functionality. As I said before, understanding how your protein interacts with your process allows you to find approaches that eliminate what needs to be eliminated (microorganisms, fouling, etc) but keeps what you want to keep in terms of nutrition, digestibility and flavour. The plant-based sector is starting to figure out what this means, but I think they could still learn a lot from the dairy sector.

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. 

Alternative proteins offer a perfect solution to a more sustainable food production system. Customers are ready to embrace this trend, but will do so only if taste, texture and healthfulness remain uncompromised. 

The world population keeps increasing: In 2050, there will be nearly 10 billion mouths to feed. Our current animal food production system will not suffice.  Therefore, food producers, ingredient companies and researchers are on a quest to find sustainable alternatives. Enter plant proteins. Available in abundance, they are often a side or waste-stream of existing food production processes. Moreover, they can offer additional benefits such as muscle health support, improved digestion and weight control. But while consumers are actively trying to eat more plant-based or animal-and-plant-protein blended foods (and reduce their meat consumption), they are not willing to compromise on taste and texture. Indeed, customers’ expectations are skyrocketing: They want something special, and they want it now. And it should be as transparent and sustainable as possible.

Bridging the gap

If we are to meet their expectations, we will need to bridge the gap between our understanding of animal-derived proteins and alternative ones such as plants, algae and insects. Microalgae, for instance, are known to be highly functional as foaming agents or emulsifiers. But how do we apply them without creating fishy and musty off-flavors, or green or red off-colors? We could make use of insects. After all, they can quickly produce large amounts of protein-rich biomass and have the potential to convert low-value waste into higher-value insect proteins. But as long as the Western world struggles to accept their application in a visible form, we will need to grind them, leading to brown off-coloring and protein hydrolysis. A success story of alternative proteins is presented by the extraction of RuBisCo, the world’s most abundant protein. NIZO scientists have been able to prevent off-color and solubility issues while upscaling and testing the process at a semi-industrial level.

Selecting proteins

When it comes to meeting customer demands in support health aspects such as weight control, it pays off to look into the satiety potentials of various proteins. Whey protein, for instance, hardly increases in viscosity in the stomach. It is also easily degraded; thus, readily available for consumption, making it ideal for rapid muscle recovery. Casein, on the other hand, is broken down slowly and absorbed later, making it more ideal for long-term recovery. Pea protein takes a middle position, breaking down more quickly than casein, but slower than whey.

Solving taste and texture issues

Another issue we face is the deterioration of flavor and the decrease of solubility during extraction and processing. Increasing our understanding of their impact on the quality and functionality of proteins will enable us to adjust the production process and develop ingredients that deliver the optimal sensory perception. It is worth noting that we don’t need new and expensive drying techniques to keep plant protein in its native state. By adapting critical conventional steps during extraction and spray drying, it is possible to maintain functional properties and tailor ingredients for specific applications.

Blending proteins

If we are to live up to customer expectations concerning nutritional benefits, we will need to look into protein blends. After all, many plant proteins require blending to provide a complete nutritional profile. There are various tools on the market that support the search for the right proteins to be blended (see the figure). To tailor to customers who wish to experience new food sensations, we can use protein blends to develop food products with novel texture and sensory attributes.

Processing proteins

A final challenge to overcome has to do with the impact of product properties on processing conditions. For example, an important issue of spray drying is fouling of the dryer due to stickiness of the product. This is especially true for products with a high content of carbohydrates and proteins, such as infant formula. Fouling results in shorter run times between cleaning procedures and, at worst, causes blocking of the dryer by lumps of powder. Improved process control by humidity measurements can minimize fouling, extend the time between clean-in-place procedures and avoid blockages.

Can we meet customer demand?

Can we meet customer expectations regarding flavor, texture, health and sustainability of plant-based products? The answer is yes. But there are challenges to overcome. Most importantly, we will need to expand our understanding of plant, insect and microalgal proteins. In addition, we can develop products with new texture and sensory attributes, or ensure that replacement of animal protein does not compromise taste, texture and nutritional properties. Either way, we will need to focus on the creation of synergizing protein blends and the optimization of extraction and processing methods. Although we still have ground to cover, if we continue our quest, we will be able to feed those 10 billion people in 2050 with healthy, sustainable and tasty foods.

A healthy diet is essential for preventing infection and keeping the immune system working properly. But what is a healthy diet? Clearly, if we know which nutrients and food ingredients are healthy, we can ensure we consume enough of them. For some dietary interventions and single nutrients, solid evidence from clinical research has been used to inform the public and provide dietary advice. A well-known example is the use of folic acid before and during the first weeks of pregnancy to prevent neural tube defects in new-borns.

Indeed, all nutrition and health claims used in the marketing of food products must be backed up by scientific evidence. For example, if a food manufacturer has an ingredient that looks like it might boost immunity in cultured cells, they must also prove it does the same in humans. However, food clinical trials are costly and time-consuming, especially if you’re looking for robust results. Being able to test an ingredient’s effect on health in relatively small groups and over short time periods therefore saves manufacturers both time and money.

Human challenge models as an alternative to food clinical trials

To address this need, we have designed several proof-of-concept clinical trials. Traditionally, food clinical trials test the effects of a product on the long-term health status of a large group of healthy subjects. However, NIZO’s “human challenge models” rely on a more sensitive marker of health, namely stress resilience.

So how does it work? In these models, healthy volunteers are challenged by exposing them to a moderate external “stressor”. The stressors given to volunteers include mild gut or respiratory infections to test whether a certain ingredient enhances resistance to infection, for example. Starting a few weeks preceding the infection, half of them are given the product of interest while the other half receive placebo. Researchers compare their clinical and physiological responses to see how each group responds to the challenge. Such a model has been used to demonstrate in healthy adults that an ingredient added to infant formula can increase resistance to infection with bacteria that cause diarrhoea. The results of this study were published in The Journal of Nutrition in 2016.

Our clients are always pleasantly surprised to discover that a sufficient number of people are willing to volunteer for these challenge experiments. The controlled settings offer several key advantages, including fewer subjects and shorter timelines. Crucially, these studies also meet the regulatory requirements for substantiating health benefits for the gut and immune system.

Integrating multiple areas of expertise

However, our researchers don’t only look at clinical outcomes. We also collect biological samples from participants and analyse numerous biomarkers, including microbiome profiles. The wide range of systems and models available enables them to help clients study the mechanism of action of functional ingredients.

“NIZO’s strength when executing clinical and preclinical health studies lies in the smart combination and integration of various areas of expertise”

Expertise Group Leader Nutrition & Health, Alwine Kardinaal

Sweetness enhancement using naturally occurring aromas is a promising way of reducing the sugar content of flavoured beverages while maintaining taste. Research at NIZO suggests that the ability of consumers to differentiate between taste and aroma is limited, and that aromas can be used to produce long-lasting sweetness-enhancing effects.

Reducing sugar content

An interest in healthier foods is growing on the part of both consumers and the food industry. The demand for products containing less sugar poses a challenge for the producers of flavoured drinks, which by tradition have high sugar content. While many drinks producers have solved this by replacing some or all of the sugar content with artificial or processed sweeteners, consumer organisations are starting to resist the wide application of such sweeteners. The additional dilemma for producers is that while consumers prefer not to compromise on taste, they are also increasingly on the lookout for products that contain natural ingredients, free of E numbers and artificial additives.

Aromas instead of sweeteners

This has led to a search for alternative strategies to keep these label-conscious consumers happy while avoiding high sugar content. One such strategy currently being explored at NIZO is to make use of so-called cross-modal effects: by being exposed to many different foods, we learn to associate food aromas with the taste they usually accompany. Therefore adding an aroma to mimic the smell of sugar-rich versions of the food increases the perceived sweetness by mere suggestion. In other words, the brain tells us the sweetness is there, even when the sweet ingredient is not. However, until now it was unclear whether the sweetness-enhancing effect of an aroma is strong enough to enhance taste perception in the longer term.

Separate stimulation of nose and tongue

Using aroma-induced sweetness enhancement in real foods is also a relatively new concept, and studying how taste experience can be improved with aromas requires specialised equipment. NIZO has at its disposal both an olfactometer – that can deliver precise amounts of aromas into a subject’s nose while they are consuming food for example – and a gustometer, used to deliver precise amounts of taste stimuli onto a subject’s tongue.

Ethyl hexanoate: a naturally-occurring aroma from apples

Such devices have allowed us to demonstrate that a drink is perceived as much sweeter if a sweet-smelling aroma is delivered to the nose at the same time. One of the aromas being tested at NIZO is ethyl hexanoate (HEX), a natural aroma component that is synthesised in apples during ripening. Interestingly, we see the same effect if we add HEX in liquid form to apple juice. While it is fairly easy to persuade people that the apple juice they are tasting contains a higher amount of sugar than it actually does – just by adding HEX to the apple juice – this cross-modal effect is the strongest in untrained test subjects who are exposed to HEX for the first time. This suggests that giving it to the same subjects repeatedly might reduce the effect, and that people can learn to tell the difference between taste and aroma.

The test panel

To determine whether or not HEX’s sweetness-enhancing effect is stable enough to support long-term application in food products, researchers at NIZO conducted a series of tasting sessions using a panel of 21 test subjects. They monitored the effect of adding HEX to apple juice, whereby the test subjects underwent two types of tasting sessions that alternated in a fixed schedule over a 6-month period. In all tasting sessions, subjects were given apple juice with or without added sugar that varied in HEX content. In the ‘evaluation’ sessions, subjects were asked to taste an unlabelled sample and rate its sweetness. In the ‘feedback’ sessions, a computer screen simply told them whether or not the apple juice they were tasting contained added sugar. This allowed them to learn whether the sweetness they experienced was due to added sugar or added aroma.

Learning to tell the difference

As expected, the effect was strongest during the initial session, i.e. before subjects had been given the opportunity to learn the difference between taste and aroma. During this ‘untrained’ session, subjects gave unsweetened apple juice with a higher HEX content a consistently higher sweetness rating. While this effect disappeared just after the feedback sessions, it recovered significantly during the final evaluation at 6 months for all but the highest HEX concentration. This suggests that the subjects in this test panel did not learn to distinguish between sugar and aroma-induced sweetness.

Sweetness enhancement effects stand the test of time

The result is encouraging since the experimental setup was – intentionally – a worst-case scenario: during the feedback sessions subjects were explicitly told whether or not the samples contained added sugar, whereas in real-life situations people are not always fully informed of a product’s content. After all, it is up to consumers themselves to read the label. It is therefore likely that if explicit sugar content provided on a computer screen during tasting does not negatively impact aroma-induced sweetness enhancement in the long term, the effect will also stand the test of time in real life. While a conclusive answer will require a longitudinal consumer study, these results clearly indicate that aromas have potential for enhancing sweetness in flavoured beverages. NIZO is looking forward to helping the food industry make use of such developments to help reduce the levels of sugar in their products while maintaining taste.

The experiments and findings described above were published in a paper presented at the 15th Weurman Flavour Research Symposium in Austria in September 2017 (Brattinga et al, 2017).

How to tackle undesirable microbes in foods, in particular bacterial spores?

High rates of spoilage before the end of shelf life of foods? Consumer complaints? Or worse, cases of foodborne illness due to consumption of your products? This must be avoided and safeguarded in production. As all foods placed on the market must be safe and of high quality, clearly, control of microbes that may cause food spoilage or that lead to foodborne illness has high-level priority in the food industry.

To control contaminants in fresh ready-to-eat foods, the food industry uses different conditions to prevent outgrowth of undesirable microbes. For example, low storage temperatures and short distribution chains. In processed foods, undesirable microbes are often eliminated by applying heat or alternative treatments that kill them. However, bacterial spores, which are the hardiest forms of life on earth, may survive such treatments. Their ubiquitous presence in the environment means that their presence in food ingredients and in food processing facilities is inevitable. As a result, sporeformers may lead to a reduced shelf life of products due to spoilage and in the case of foodborne pathogenic sporeformers, consumption of products in which growth has occurred may even lead to illness or death. Clearly, contaminants originating from ingredients or the processing environment must be controlled during and following production to achieve the required shelf life and assure food safety. But how to do that?

1. Analyse the number of pathogenic spores which are present in ingredients and finished product

To prevent potential problems with spores in finished food products that undergo heat treatments, the ingredients generally have specifications for spores. Many different methods exist to detect spores of different types of sporeformers, as not all spores behave the same. Different growth media may be used and incubation may take place at specific temperatures (e.g. psychrotrophic, mesophilic or thermophilic species) or in the presence or absence of oxygen. In addition, a distinction can be made in the heat resistance of spores by applying different heat treatments (e.g. 10 min 80°C, 30 min 100°C, 30 min 106°C). When setting specifications for spore concentrations in ingredients, it is critical to apply meaningful methods with relevant detection limits. Ideally, the methods allow for a link between the sporeformers present in ingredients and the potential defect rates in finished products after production. Knowledge about spore detection, problem species, impact of processing on different spore types and growth potential in finished products is crucial for such assessments.

2. Identify the source of contamination

Sporeformers may enter the production chain via ingredients. Another contamination route is via processing equipment containing biofilms that shed spores into the product. This may happen in holding tanks, on transport belts, around seals and even in heating equipment, such as the regenerative section of pasteurizers or in evaporators (by thermophilic species). When spores cause problems in finished product, a ‘track-and-trace’ approach of the problem microbes can identify the source of the contamination. By assessing the genetic make-up of the problem organism and of isolates in ingredients and along the production lines, a source can be found.

3. Define acceptable levels of pathogenic spores in finished products

Even low levels of spores in finished product may lead to spoilage or foodborne illness if they survive and if outgrowth can occur: one per packaging unit may be enough! When spores are present in ingredients, reduction to acceptable levels can be achieved by inactivation (e.g. by heat treatments) or by removal (in liquid products e.g. by bactofugation or ultrafiltration). The efficacy of such treatments can be assessed experimentally. To do so, it is critical to use spores with representative properties. The efficacy can also be calculated using modelling approaches when relevant data are available. If adjustment of processing conditions does not suffice, reduction at source may be necessary.

4. Select conditions that prevent outgrowth

Spores can ‘wake up’ via the process of germination in nutritious environments. This may happen during food production or in finished products. When conditions in the product (e.g. water activity, pH) and during storage (e.g. temperature) are favourable for outgrowth, the sporeformers can multiply. Traditional preservatives that are effective in preventing germination and outgrowth of spores (for instance sorbate and benzoate) are used less nowadays. New formulations and novel processing methods are being developed, for instance based on clean label preservatives and combinatory treatments. The efficacy of such formulations must be evaluated in products using relevant strains of sporeformers and spores thereof. This requires knowledge of species to inoculate and often high-throughput testing of conditions, to ensure efficacy of treatments.