Final Report Summary - EMULSIFOOD (EMULSION DESIGN TO IMPROVE NUTRITIONAL AND ORGANOLEPTIC QUALITY OF CEREAL BASED FOOD SYSTEMS)
Alarming reports on (the evolution of) the health condition of consumers in the developed world trigger researchers to invest much time and effort in the design of ‘healthier’ foods. Supplementation of food products with bioactive or functional food components is one of the strategies that is currently intensively studied. While bioactive molecules, in this context, are often associated with a health improving effect, the functional food components are often designed to make up for food quality attributes lost during fat, sugar or salt reduction or upon gluten elimination. The mere supplementation of food products with these bioactive or functional components is often not that simple as they are usually not very stable or functional, or are even incompatible with the food matrix. Encapsulation or coating of these bioactive or functional components prior to their supplementation to food products is one of the strategies to overcome these difficulties and challenges or to design controlled release and/or target delivery systems. Structured emulsions and biopolymer-based nanoparticles have already proven promising for enhancement of the nutritional profile and organoleptic quality of ‘liquid’ food systems.
This project aims at the rational design of structured lipid- or biopolymer-based systems as carrier systems for (i) bioactive/functional components and (ii) functionality for ‘semi-solid’ and ‘solid’ foods.
(i) Carrier systems for bioactive or functional components
A distinction has to be made between hydrophobic and hydrophilic bioactive or functional molecules. In this project resveratrol and ascorbyl palmitate, and anthocyanins and potassium iodate were used as model hydrophobic and hydrophilic molecules, respectively. The diameter of the encapsulation systems is preferably as small as possible in order to reduce the chance on detection of the systems during oral processing or to minimise light scattering when applied in transparent food products.
Lipid-based systems, i.e. emulsions, have already proven useful for the encapsulation of hydrophobic bioactive molecules. In this regard, gluten hydrolysates [produced with peptidases with distinct specificity and with varying degree of hydrolysis (DH) (DH 1 to 3 and DH 3 to 10 for trypsin- and alcalase-produced hydrolysates, respectively)] proved to have surface active properties and were used to form and stabilise oil-in-water emulsions. However, the relatively large droplet diameter, the limited stability of the emulsions, the presence of a lipid phase and the difficulties associated with encapsulation of hydrophilic molecules made that the focus of the project was redirected to biopolymer-based systems.
Biopolymer-based systems, i.e. protein nanoparticles, were made by liquid antisolvent precipitation (LAS), a technique that relies on the reduction of the solvent power of the medium in which the proteins are dissolved. This is typically done by adding an antisolvent to the protein solution. Two different protein types were predominantly studied, i.e. water-soluble whey protein isolate (WPI) and water-insoluble gliadin or zein. Water-insoluble proteins were used to encapsulate hydrophobic molecules, while water-soluble proteins were used for the encapsulation of hydrophilic molecules. The versatility in protein properties and interaction possibilities (such as hydrogen bonds, ionic interaction and the hydrophobic effect) makes that protein-based particles are well-suited for the encapsulation and retention of molecules with largely varying properties. However, the encapsulation efficiency and, especially, the retention capacity of the tested particles for hydrophilic molecules were rather limited. Fluorescence quenching experiments were used to shed light on the strength and nature of the interactions and, hence, enable the design of protein particles with tailor-made properties and functionality (encapsulation and release characteristics). Furthermore, protein particle suspensions are very susceptible to changes in pH or ionic strength. Additional stability was achieved by including proteins with flexible conformations (e.g. sodium caseinate) or polysaccharides (e.g. pectin) in the particle structure. Conjugation of dextran to zein e.g. prior to particle production resulted in the formation of particles that were even stable over the entire pH range relevant for food products. The protein particles, however, tended to redissolve once reintroduced in a solvent phase. Tryptophan fluorescence emission intensity and peak location scanning combined with dynamic light scattering revealed that WPI and gliadin particle formation and redissolution largely occur at the same (anti-)solvent concentration and the particle production process seems to be fully reversible. The particles could be stabilised through crosslinking of the biopolymers. Next to glutaraldehyde, a well-known protein crosslinking agent, food-grade alternatives also improved the stability of the particle structure. LAS is a very simple, but versatile and even tuneable process as is shown by a statistical model that was built by exploring the effect of a range of different production parameters on WPI particle properties. The most important production parameters (i.e. protein concentration and solvent:antisolvent ratio for WPI particles) can be used to design particles with a specific particle size (distribution) and predict the properties of particles produced by a set of production parameters.
(ii) Carrier systems for functionality
When fat is taken out of a food product, the functionality (i.e. rheology, mouthfeel, visual properties and machinability) of the fat droplets is also lost. The lost functionality can be (partially) replaced by adding other structures to the food system. Empty capsules of a similar size as the oil droplets e.g. are theoretically very promising novel structures to come up to the lost functionality. Starch granules were swollen in the presence of sodium stearoyl lactylate and subsequently coated with WPI (and calcium chloride) and pectin after which the pectin layer was crosslinked by laccase. Subsequent hydrolysis of the starch leads to the production of empty capsules which can be used to mimic the rheological behaviour of oil droplets in a fat reduced product. The coating procedure can be repeated to produce multilayer structures and eventually empty capsules with a thicker shell.
Some of the above systems have been tested in ‘model’ foods or conditions relevant for food processing:
• Zein and gliadin based nanoparticles encapsulating anthocyanins produced by LAS were incorporated in a citric acid system pH 3.5 containing sodium benzoate and ascorbic acid, a system that closely matches a soft drink (liquid food system). Ascorbic acid is known to speed up the colour loss of anthocyanins. Preliminary experiments showed that encapsulating anthocyanins in protein particles slowed this deterioration down.
• Potassium iodate was encapsulated in WPI particles in order to release its oxidising power later in the breadmaking procedure (‘semi-solid’ or ‘solid’ food system). Pure potassium iodate surprisingly increased the dough development time as determined by mixograph experiments. The same, but slightly less outspoken, result was found for the flour sample to which potassium iodate encapsulated in WPI particles were added. Full breadmaking experiments using the same additives should be conducted to verify these observations and the effect on bread quality.
• Crosslinked WPI nanoparticles were subjected to an in vitro digestion test in which stomach conditions were mimicked. Particle suspensions were supplemented with pepsin and brought to pH 3.0. The digestion process was followed up by monitoring the amount of hydrochloric acid that had to be added in order to keep the pH constant. Further research is necessary to increase the reproducibility of this test.
• Suspensions of starch granules coated with WPI and pectin were subjected to rheological tests at a constant shear rate of 10 s-1. This shear rate is believed to, to some extent, correlate with the perception of viscosity of ‘semi-solid’ food products in the mouth. The tests were also performed in the presence of amylase. Rheological tests pointed to shear-thinning and limited thixotropic behaviour of the (coated) starch suspensions and a potential reduced accessibility of the starch for amylases upon coating. Oscillatory tests pointed to a predominant elastic behaviour of the starch suspensions.
In conclusion, different encapsulation strategies and systems have been explored. All of them hold promise to be used in real food products, be it as encapsulation system for water-soluble bio-active molecules to be incorporated in ‘semi-solid’ and ‘solid’ food products or to come up to the functionality loss in e.g. fat-reduced products. However, more research is needed to add these systems in a real-world system. The identification of lipid- and biopolymer-based delivery systems suitable for the encapsulation of bioactive and quality improving components and with the desired functionality in food products will come to the benefit of the design of healthier and tastier food products and can contribute to the longstanding quest to reduce diet-related diseases in the Western World.
This project aims at the rational design of structured lipid- or biopolymer-based systems as carrier systems for (i) bioactive/functional components and (ii) functionality for ‘semi-solid’ and ‘solid’ foods.
(i) Carrier systems for bioactive or functional components
A distinction has to be made between hydrophobic and hydrophilic bioactive or functional molecules. In this project resveratrol and ascorbyl palmitate, and anthocyanins and potassium iodate were used as model hydrophobic and hydrophilic molecules, respectively. The diameter of the encapsulation systems is preferably as small as possible in order to reduce the chance on detection of the systems during oral processing or to minimise light scattering when applied in transparent food products.
Lipid-based systems, i.e. emulsions, have already proven useful for the encapsulation of hydrophobic bioactive molecules. In this regard, gluten hydrolysates [produced with peptidases with distinct specificity and with varying degree of hydrolysis (DH) (DH 1 to 3 and DH 3 to 10 for trypsin- and alcalase-produced hydrolysates, respectively)] proved to have surface active properties and were used to form and stabilise oil-in-water emulsions. However, the relatively large droplet diameter, the limited stability of the emulsions, the presence of a lipid phase and the difficulties associated with encapsulation of hydrophilic molecules made that the focus of the project was redirected to biopolymer-based systems.
Biopolymer-based systems, i.e. protein nanoparticles, were made by liquid antisolvent precipitation (LAS), a technique that relies on the reduction of the solvent power of the medium in which the proteins are dissolved. This is typically done by adding an antisolvent to the protein solution. Two different protein types were predominantly studied, i.e. water-soluble whey protein isolate (WPI) and water-insoluble gliadin or zein. Water-insoluble proteins were used to encapsulate hydrophobic molecules, while water-soluble proteins were used for the encapsulation of hydrophilic molecules. The versatility in protein properties and interaction possibilities (such as hydrogen bonds, ionic interaction and the hydrophobic effect) makes that protein-based particles are well-suited for the encapsulation and retention of molecules with largely varying properties. However, the encapsulation efficiency and, especially, the retention capacity of the tested particles for hydrophilic molecules were rather limited. Fluorescence quenching experiments were used to shed light on the strength and nature of the interactions and, hence, enable the design of protein particles with tailor-made properties and functionality (encapsulation and release characteristics). Furthermore, protein particle suspensions are very susceptible to changes in pH or ionic strength. Additional stability was achieved by including proteins with flexible conformations (e.g. sodium caseinate) or polysaccharides (e.g. pectin) in the particle structure. Conjugation of dextran to zein e.g. prior to particle production resulted in the formation of particles that were even stable over the entire pH range relevant for food products. The protein particles, however, tended to redissolve once reintroduced in a solvent phase. Tryptophan fluorescence emission intensity and peak location scanning combined with dynamic light scattering revealed that WPI and gliadin particle formation and redissolution largely occur at the same (anti-)solvent concentration and the particle production process seems to be fully reversible. The particles could be stabilised through crosslinking of the biopolymers. Next to glutaraldehyde, a well-known protein crosslinking agent, food-grade alternatives also improved the stability of the particle structure. LAS is a very simple, but versatile and even tuneable process as is shown by a statistical model that was built by exploring the effect of a range of different production parameters on WPI particle properties. The most important production parameters (i.e. protein concentration and solvent:antisolvent ratio for WPI particles) can be used to design particles with a specific particle size (distribution) and predict the properties of particles produced by a set of production parameters.
(ii) Carrier systems for functionality
When fat is taken out of a food product, the functionality (i.e. rheology, mouthfeel, visual properties and machinability) of the fat droplets is also lost. The lost functionality can be (partially) replaced by adding other structures to the food system. Empty capsules of a similar size as the oil droplets e.g. are theoretically very promising novel structures to come up to the lost functionality. Starch granules were swollen in the presence of sodium stearoyl lactylate and subsequently coated with WPI (and calcium chloride) and pectin after which the pectin layer was crosslinked by laccase. Subsequent hydrolysis of the starch leads to the production of empty capsules which can be used to mimic the rheological behaviour of oil droplets in a fat reduced product. The coating procedure can be repeated to produce multilayer structures and eventually empty capsules with a thicker shell.
Some of the above systems have been tested in ‘model’ foods or conditions relevant for food processing:
• Zein and gliadin based nanoparticles encapsulating anthocyanins produced by LAS were incorporated in a citric acid system pH 3.5 containing sodium benzoate and ascorbic acid, a system that closely matches a soft drink (liquid food system). Ascorbic acid is known to speed up the colour loss of anthocyanins. Preliminary experiments showed that encapsulating anthocyanins in protein particles slowed this deterioration down.
• Potassium iodate was encapsulated in WPI particles in order to release its oxidising power later in the breadmaking procedure (‘semi-solid’ or ‘solid’ food system). Pure potassium iodate surprisingly increased the dough development time as determined by mixograph experiments. The same, but slightly less outspoken, result was found for the flour sample to which potassium iodate encapsulated in WPI particles were added. Full breadmaking experiments using the same additives should be conducted to verify these observations and the effect on bread quality.
• Crosslinked WPI nanoparticles were subjected to an in vitro digestion test in which stomach conditions were mimicked. Particle suspensions were supplemented with pepsin and brought to pH 3.0. The digestion process was followed up by monitoring the amount of hydrochloric acid that had to be added in order to keep the pH constant. Further research is necessary to increase the reproducibility of this test.
• Suspensions of starch granules coated with WPI and pectin were subjected to rheological tests at a constant shear rate of 10 s-1. This shear rate is believed to, to some extent, correlate with the perception of viscosity of ‘semi-solid’ food products in the mouth. The tests were also performed in the presence of amylase. Rheological tests pointed to shear-thinning and limited thixotropic behaviour of the (coated) starch suspensions and a potential reduced accessibility of the starch for amylases upon coating. Oscillatory tests pointed to a predominant elastic behaviour of the starch suspensions.
In conclusion, different encapsulation strategies and systems have been explored. All of them hold promise to be used in real food products, be it as encapsulation system for water-soluble bio-active molecules to be incorporated in ‘semi-solid’ and ‘solid’ food products or to come up to the functionality loss in e.g. fat-reduced products. However, more research is needed to add these systems in a real-world system. The identification of lipid- and biopolymer-based delivery systems suitable for the encapsulation of bioactive and quality improving components and with the desired functionality in food products will come to the benefit of the design of healthier and tastier food products and can contribute to the longstanding quest to reduce diet-related diseases in the Western World.