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. 

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”, according to our clinical expert Anita Hartog.

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 (see Brattinga et al, 2017).

 Contact Jose Escher if you want to enhance the flavour of your product with the naturally occurring aroma of the same product!

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.

Global demographic shifts and a world population projected to reach 9.7 billion by 2050 lead to an increasing demand for high-quality nutritional products, especially for elderly people. This will put pressure on food production and protein supply. To meet this growing demand, producers are combining vegetable and animal proteins and finding innovative ways to use available proteins more efficiently. The biggest challenge is developing high-protein applications with the right properties (structure, texture, flavour, digestibility) without increasing the food product’s carbon footprint.

Everyone needs protein

Elderly people need to maintain their muscle mass, so their diet must contain enough highly digestible protein. Other consumers need foods that help control weight and reduce the risk of obesity. For them, too, high-protein products can play an important role because they create a relatively high sense of satiety.

While proteins have obvious benefits, products with a high protein content can be undesirable from a sensory perspective: too viscous and hard to swallow in liquids, or too chewy in a solid state. Texture problems can also occur in low-fat products where fat has been replaced with extra protein. These products are perceived as drier and less juicy. To improve consumer acceptance of high-protein products, it is crucial to control their viscosity and texture. This means decreasing liquid products’ viscosity and improving the overall mouthfeel of both liquid and solid products.

Optimal digestibility

To meet the growing demand for protein, food manufacturers are turning to high-quality proteins from alternative sources, such as plants, algae and insects. The industry is also increasingly interested in pea protein, which has a good amino acid composition. However, digestibility is also of importance. Using Nizo’s SIMPHYD platform for in vitro modeling of food’s behaviour in the stomach, the digestibility of pea protein was compared with that of whey and casein proteins. Whey protein hardly increases in viscosity in the stomach, is easily degraded and hence readily available for absorption, making it ideal for rapid muscle recovery (as a ‘fast’ dietary protein). Casein, on the other hand, becomes significantly more viscous and is broken down slowly and absorbed later, making it more suitable for long-term recovery (a ‘slow’ dietary protein). Compared to whey and casein, pea protein turned out to be exactly in the middle. It creates some viscosity and breaks down more quickly than casein, but slower than whey. In other words, it is a moderately fast protein. This knowledge is essential for developing products than promote rapid recovery after a workout or products that can help maintain long-term muscle function.

In vivo measurement

Laboratory animals are sometimes used to test digestibility in an in vivo situation. As digestion takes place in the small intestine, this is also the spot where we would prefer to measure digestibility of proteins in humans.

The small intestine is essential for our health as this is where about 90% of nutrients are absorbed and key signals are generated to control our metabolism and immune system. There is increasing scientific evidence that an imbalance in our intestinal microbiome can lead to a number of diseases, including metabolic and immunological disorders such as obesity, diabetes, and inflammatory diseases. However, most studies focus on the fecal microbiome in the large intestine and pay scant attention to the possible role of the small intestinal microbiome. However, taking samples from the small intestine is a highly invasive procedure, requiring insertion of a tube through the esophagus and stomach.

At Nizo we recently made significant progress in solving this problem by developing the IntelliCap, a minimally invasive technology for taking samples from the small intestine. This measurement device, which had already been CE-marked for drug delivery and real-time measurement of temperature and pH in the gastrointestinal tract, was also approved for the aspiration of fluids. Therefore, the IntelliCap CR system can now also sample the small intestinal microbiota. Philips spin-off Medimetrics Personalized Drug Delivery BV developed the sampling device, which measures only 11mm by 26mm. This capsule has proven its worth as a means of measuring the microbiome in the small intestine, and could possibly also measure in vivo digestion of protein in the small intestine. This enables a more accurately assessment of digestibility, and hence the quality of different types of protein.


Just as the source of protein affects its digestibility, the way of processing could also have an impact on digestibility and hence the quality of proteins. Go4Dairy, a recently launched research programme, focuses on the impact of glycation on protein’s nutritional value. In the literature, Maillard products are associated with negative health effects, including hypersensitivity of the immune system, reduced digestibility, reduced bioavailability of lysine in particular, changes in gut bacteria and even carcinogenic effects. Much remains unknown, however. Go4Dairy’s aim is to reduce and control the glycation of proteins so as to optimise their nutritional value.

Proteins for the future

High-quality proteins are essential in our diet and are crucial for healthy ageing. In view of the impending protein shortage, current technological developments in plant protein structure, texture, taste and digestibility are all important steps toward creating a sustainable protein supply for a growing world population.

Fermentation & flavour formation

Recently, an interesting article in the Economist about the high diversity of yeast in cacao and coffee bean fermentations caught my eye. Being a fermentation expert at Nizo and before that at The Coca-Cola Company, knowing that the final products of both coffee and cacao are the result of fermentation seems like basic information. However, most consumers are under the impression that both products are directly derived from the plant and are only industrially processed. They are not aware that the final product is a result of natural fermentation, which is essential to final flavour formation.

Both for cacao and coffee beans, fermentation is performed by a mixture of yeasts that produce a variety of flavours, whilst the most well-known product of yeast, alcohol, is either produced in low amounts or evaporates during the process. The interesting observation reported in the Economist article related to the high diversity of yeast identified in the cacao and coffee beans isolated from different locations. All these different yeast are expected to produce different flavour molecules. This allowed for the possibility to use cacao yeasts from Costa Rica in a cacao fermentation executed in the Dominican Republic, thereby adding specific flavours normally only present in Costa Rica cacao beans to cacao beans from the Dominican Republic.

All very exciting, but the dairy industry together with Nizo has done the same things for over 65 years now. Cheese production is one of the best known examples of fermentation. Using mixtures of bacteria results in a seemingly limitless range of flavours and they are responsible for the characteristic of – for instance – gouda, cheddar and Swiss-type cheeses. The addition of fungi results in very different types again such as brie or blue cheese, all to be found in supermarkets all over Europe and the US. All these cheeses show a high diversity in flavour and textures that are the result of using different organisms for the fermentation.

New food-grade strains to create new flavours are continuously isolated from nature. Interestingly, these strains do not have to be dairy-based. We at Nizo have shown that a food-grade bacterium from a plant can be evolved to grow on milk within a period of months. This can lead to an even larger variety of flavours that are the result of fermentation.

It is exciting to see that the technology, long used in the dairy industry, is finally translated to the coffee and chocolate industry. What I see as the next step is to use yeast isolated from coffee beans for cacao fermentation and vice versa. Although this may seem challenging at first from a scientific point of view, this is a much smaller step than the previously successfully conducted conversion of a plant bacterium to a dairy bacterium. This can lead to an even greater diversity of potential new flavours in the coffee and chocolate industry.

The biggest challenge in this will be to find out what type of flavours are of interest to a consumer, which is what marketing departments are looking for. To study thousands of new fermented products at Nizo we have developed a high throughput platform that screens thousands of fermentations simultaneously at a 200mg scale. In addition to the miniaturised process being an almost perfect copy of the industrial scale process, using cheese as a model in this case, it also proven to be able to detect specific flavour molecules and link this to the desired flavours.

It is clear that the food industry is at the verge of a breakthrough in finally leveraging the large natural diversity of bacteria and yeasts in nature. This will results in new flavours and therefore completely new products with unexpected, original and unique characteristics. I myself cannot think of a more exciting and brighter future.

By Kevin van Koerten, expert from our Processing Department

Food and beverage manufacturers are presented with several challenges. Organisations strive to minimise production and supply-chain costs, realise a perfect delivery efficiency and meet a multitude of regulatory needs merely to stay reasonably competitive. Processing efficiency in both waste and energy management is a key component in reducing production costs and creating more value from ingredients. However, increasing efficiency is almost never a single straightforward solution, but a delicate trade-off between cost, energy and product quality. As such, to make the most appropriate decisions, a deeper understanding is needed of the effects of processing parameters on both product quality and capacity.


The processing temperature is one of the most important factors in multiple processing steps. Higher processing temperatures will result in larger heat flows, thus increasing capacity. Additionally, in pasteurisation processes, higher temperatures will more effectively destroy harmful microorganisms, ensuring product safety. On the other hand, lower processing temperatures generally decrease energy consumption and decrease the heat load on the product. The latter is especially important in protein-rich formulations, since proteins can denature at temperatures as low as 40°C. Not only will proteins lose their functionality when denatured, potentially negating their use in specific products, they also tend to aggregate with each other and equipment surfaces. The consequent fouling has a large effect on runtime and the amount of cleaning required. It is therefore important to take all these consequences into account when determining appropriate processing temperatures.

Water content

Another important factor in processing efficiency is the water content of the product, . Removing water from product streams is one of the main sources of energy consumption in the food processing industry. Some production processes revolve around the addition of water followed by removal of that same water (e.g. starch production). Less water usage in processes is therefore a direct improvement in energy efficiency. On the other hand there is a difference in energy usage for different water removal units:

  • Membrane concentration generally requires 20-40 MJ/tonns of water removed
  • Falling film evaporators, depending on number of stages and steam supply, require around 55-500 MJ /tonnes of water removed
  • Spray drying requires 3500-4500 MJ/tonnes of water removed.

It is clear from these numbers that if water removal can be moved from more energy intensive drying processes to less intensive drying, up to 99% energy savings can be achieved. Naturally there are limits to the amount of dry matter in a processing stream that certain equipment can handle, but even minimal changes will have a significant effect with the large throughputs generally used in food processing plants.


A final factor that stronlgy affects processing efficiency is the humidity or moisture content of the process air used for drying. The maximum moisture content of air at atmospheric pressure differs with temperature, from close to zero moisture per amount of dry air at temperatures below 0 °C up to an infinite amount at 100 °C (pure steam). As such, the capacity of spray dryers is highly dependent on the outlet temperature of the air. However, this outlet temperature is usually bound by the moisture content of the outgoing powder. Therefore, the only ways to improve capacity is to build a bigger spray dryer with more air or to decrease the humidity of the ingoing air. Naturally, the latter is the only option for optimization of an existing spray dryer. Dehumidification treatments with silica or zeolites are options for decreasing and/or standardizing the humidity of the drying air. Another option, which requires much less investment, is to match the dryer capacity to the outside air humidity, i.e. increase ingoing product flow when the outside humidity becomes lower. This can have a large impact on capacity as the daily variation in humidity can be quite significant.

The effect of product-process interactions on efficiency

Besides processing conditions, product-process interactions can also determine the process efficiency, as already mentioned in the form of protein denaturation. Another important interaction is the relation between product powder stickiness and drying air humidity in a spray dryer. The equilibrium between the moisture content of dry matter and air is described by a sorption isotherm. This captures the behaviour of moisture becoming harder to evaporate at higher dry matter content due to binding and adsorption energies. The stickiness of powders depends on moisture content and temperature, becoming more sticky at higher moisture contents and at higher temperatures. Since spray dryers approach ideal mixing, the outgoing powder is in near-equilibrium with the outgoing process air. Therefore, the stickiness of the powder depends on outgoing air humidity and temperature. If the powder becomes too sticky during spray drying, fouling will occur, leading to shorter runtimes and longer cleaning times. By modelling the drying process, the sorption isotherm and the dependence of powder stickiness on moisture and temperature, the optimal conditions for the spray dryer can be set to meet the requirements for non-sticky conditions, optimal capacity and final product moisture content.