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Targeting the gut

Novel milk protein-based microparticles as vehicles for bioactive ingredients

von Michael Betz, Thomas Heidebach, Prof. Dr. Ulrich Kulozik

Michael Betz, Thomas Heidebach, Prof. Dr. Ulrich Kulozik, Chair for Food Process Engineering and Dairy Technology, Technische Universität München, Germany

Realising a healthy and balanced diet in daily life is demanding. The main part of the population of western industrialised countries fails in meeting this challenge resulting in a persistent increase of diet- related diseases. However, a reorientation of eating habits is not foreseeable. For this reason, it is necessary to open new ways in terms of providing people with nutritionally enhanced foods. In this regard, functional foods are a promising possibility due to their high content of bioactive compounds. The aim is to achieve health benefits by the consumption of usual amounts of these foods. Such foods have to be enriched with concentrated bioactive compounds. To ensure theirbioactivity it is necessary to stabilise these mostly sensitive compounds until they reach their desired point of action. The scientists of the TU München have succeeded in stabilising bioactive plant compounds as well as probiotics and in allowing their targeted release by means of microencapsulation in milk protein-based matrices.

Microencapsulation in Food Technology

Microencapsulation is defined as a process for entrapment of substances (core-material) in capsules or matrices, which release their contents depending on specific environmental conditions. This definition induces associations to the pharmaceutical principle of encapsulation. Literally, the pharmaceutical technology is the big brother of microencapsulation in the food sector. However, the prefix “micro” indicates that food-related encapsulation systems are generally smaller than their pharmaceutical analogues.
There is a specific reason to that!
Usually, pharmaceutical formulations are ingested in their pure form whereas food-related encapsulations systems are added to foodstuffs. However, the sensory quality of the foods must not be influenced negatively by the addition. Therefore, in food technology the production of microcapsules is favoured because these are not perceived during ingestion due to their small size. Another important aspect is that materials which are used for the microencapsulation have food-grade status. Hence, the main part of established pharmaceutical encapsulations systems is not applicable for foods because of legal restrictions. Therefore, the scientists at the Chair for Food Process Engineering and Dairy Technology draw on established food material, the milk proteins.

Fig. 1 Whey protein-based microcapsules with 1, 5 and 10 %
encapsulated bioactive plant compounds

Milk proteins for the assembling of microcapsules

Milk proteins can be used in form of two main fractions: the caseins and the whey proteins (cf. Fig. 2).

Fig 2. Main fractions of the milk proteins

The two fractions possess numerous technologically interesting and useful properties. For instance, aqueous solutions of caseins or whey proteins are capable of forming gels, which are a suitable matrix material for microcapsules (cf. infobox). A key component for the thermal gelation of whey proteins is β-lactoglobulin. Aqueous β-lactoglobulin solutions have the ability to form hydrogels even at very acidic conditions below pH 2. For Dipl.-Ing. Michael Betz this is an ideal precondition of encapsulating the bioactive plant compounds, which show their highest stability under these extremely acidic conditions (cf. Fig. 1). Dipl.-Ing. Thomas Heidebach has adapted two enzymatic methods for the generation of casein gels to microencapsulation. The temperature and pH-conditions for both procedures are very gentle, making them ideal for the encapsulation of sensitive probiotic microorganisms. On the one hand he uses the enzyme transglutaminase (Tgase) for the generation of gels from concentrated casein solutions, on the other hand he applies the principle of rennet gelation of milk. For the production of microcapsules the scientists make use of a well known thermodynamic phenomenon: the immiscibility of oil and water.

The emulsification process for the production of microcapsules

Fig. 3 schematically depicts the different steps of the emulsification process. At first, an aqueous matrix solution composed of milk proteins and core-material (and in case of enzymatic gelation the specific enzyme) is emulsified in vegetable oil. A water-in-oil-emulsion is formed. Secondly, the milk protein solution which is dispersed in the oil phase is converted from liquid to solid state by inducing gelation. In addition, the obtained hydrogel microspheres are removed from the oil phase by centrifugation. The size of the generated microcapsules plays an important role for their application. On the one hand they have to be small enough to be imperceivable during ingestion. On the other hand their stabilising and protective effect is improved when the capsule-diameter is increased. Thus, for each field of application the optimum capsule size has to be determined. By variation of the emulsifying intensity during the emulsification process microcapsules in the size range of 20 ?m to 1 mm can be produced and adapted to specific requirements of product and corematerial.

Fig. 3 Microencapsulation of bilberry extract and probiotics by means of the
emulsification process

Microencapsulation of probiotics

To ensure that probiotics unfold their full potential inside the human body they have to reach their target point of action in an active state and in sufficient amounts. Microorganisms, in particular, have to overcome several obstacles. Hitherto, probiotics are added to foods like yoghurts in a dried or frozen form. However, when the bestbefore- date of the products is reached the concentration of contained microorganisms has strongly decreased. Acids contained in the yoghurt harm the living microorganisms during storage, and after ingestion the gastric acid poses a threat for the probiotics. For this reason, only a fraction of the microorganisms which were initially added to the yoghurt reach their target location, the gut. This problem can be solved by microencapsulation. By means of entrapment in milk protein gels, the stability and survival rate of the probiotics under detrimental acidic conditions can significantly be improved, as fig. 4 depicts [1,2].

Fig. 4
Comparison of survival rate of microencapsulated and
non-encapsulated probiotics in simulated gastric fluid

Microencapsulation of bioactive plant compounds

While microorganisms are retained within the relatively close-meshed milk protein gels due to their size, most bioactive plant compounds are of low molecular weight and are therefore able to migrate through the gel network. Fig. 5 shows the consequence, namely the diffusive release of the core-material in aqueous environment [3].

Fig. 5 Release of anthocyanins from the whey protein-based microcapsules
in aqueous environment

This seems to be a challenge, when such low- molecular compounds are to be encapsulated. Or is it a chance rather than a problem? To answer this question it is necessary to take a closer look at the properties of the bioactive plant compounds, which in the present case is bilberry extract. Bilberries contain large amounts of secondary plant metabolites like anthocyanins (AC). Their high AC-content accounts for the high antioxidative potential and thus bioactivity of the bilberries. To be effective the resorption of a sufficient amount of AC from the gastrointestinal tract has to be ensured. Hitherto it is known, that just a small amount of ingested AC is resorbed and hence bioactive. In this context it is not yet fully understood at which point and by which resorption mechanisms various forms of AC are resorbed in the gastrointestinal tract.

Cluster project of the food industry

The microencapsulation of the bilberry extract is intended to help answering these questions. Within the scope of the interdisciplinary cluster project “Bioactive compounds from microstructured multicapsule- systems” the food technologists of the TUM collaborate with scientists throughout Germany coordinated by the University of Karlsruhe. The objective is to develop encapsulation systems which allow the stabilisation and targeted release of the bilberry extract within the human gut. By this means the mechanisms of resorption and action can be examined. Another goal is to establish a basis for the future development of functional foods [4]. Let us go back to the question whether the diffusive release of the AC from the microcapsules has to be seen as a problem or a chance. “It depends”, is the scientists’ usual answer. On the one hand, the microcapsules are intended for application in aqueous foods and must not release their load until their target location is reached. For this purpose a shell-capsule is ideal. On the other hand, a time-dependent release at the target location can be advantageous in regard to stabilising the unreleased fraction within the protective environment of a matrix-capsule.

In vitro, ex vivo, in vivo

The resistance of the microcapsules against degradation by gastrointestinal digestive fluids is the precondition for the realisation of a time-dependent release in the gut. To get an impression of the stability of the capsules in the human gastrointestinal tract (in vivo) laboratory investigations with simulated digestive media (in vitro) are carried out. Project partners at the Martin-Luther Universität Halle-Wittenberg were able to show that the milk protein capsules were stable at simulated gastric and intestinal conditions. Neither in diluted hydrochloric acid with pH 1.2 (stomach) nor in buffer media with pH 6.8 and additional digestive enzymes has a significant capsule degradation been observed. Eventually, project partners at the TU Kaiserslautern incubate ileostomy fluid (content of the small intestine) with the bilberry extract-loaded microcapsules (ex vivo). Free extract was incubated as a reference sample. While the non-encapsulated extract was degraded continuously, the timedependent release from the microcapsules counteracted a decrease of the AC concentration within the first hour of incubation.

Acknowledgement

These research projects are financially supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn); Projects AiF 15611 N and AiF 15327 N.

[1] Heidebach et al. (2009) Food Hydrocolloids 23, 1670-1677
[2] Heidebach et al. (2009) Int. Dairy J. 19, 77-84
[3] Betz, M. et al. (2009) Proceedings of the 5th ISFRS, Zurich, 188-191
[4] http://www.fei-bonn.de/projekte/projektdatenbank.html/fv_cluster_1/