Protein Structures as Delivery Vehicles in Foods - American Chemical

Chapter 5

Protein Structures as Delivery Vehicles in Foods 1,2

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Downloaded by UNIV OF SOUTHERN CALIFORNIA on January 26, 2016 | http://pubs.acs.org Publication Date: March 3, 2009 | doi: 10.1021/bk-2009-1007.ch005

Paul Smith and Mark Plunkett 1

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YKI Institute for Surface Chemistry, Box 5607, SE 114 86, Stockholm, Sweden Current address: Cargill R&D Centre Europe, Havenstraat 84, B1800 Vilvoorde, Belgium

Protein structures are important for providing many properties of foods. Hydrophobins have recently been extracted from filamentous fungi. These are amphiphilic molecules with unusual surface-active properties. These can be used in order to make unique nano-structures in foods and also to give different behaviour in other applications. There are a great many opportunities and challenges for food scientists. Structures could be used for incorporation of ingredients or to create novel textures and products.

© 2009 American Chemical Society

In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Qingrong, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Introduction Proteins have many different roles in foods. They are very important nutritional components of the diet. Certain proteins are surface active. They are widely used in food systems where a surface-active component is required. Obvious examples are in milk and dairy products. These are generally low-tech foods and a great deal of specific functionality is hot needed. It is the other ingredients that people have looked for in developing newer foods. However specific active proteins exist that can be developed and applied to give new functionalities and opportunities. One such proteins are the hydrophobins. Hydrophobins are proteins that exist on the outside of filamentous fungi. They were initially discovered because they were stable to boiling during extraction (7). This is very unusual for such a complicated material. The structure of the proteins has been well characterized (2) and reviews of the literature have been published. The hydrophobins tend to be found on the outside of the fungi. The proteins are all about lOkDa in size and contain a large proportion of hydrophobic amino acids. The main unifying feature is the presence of 8 Cys residues. Study has shown that different hydrophobins are formed and expressed at different stages of the fungi's life. They are expressed all through the life of fungi. The properties and biological roles are fully described in different reviews (5-5). However it can be seen that they have two main roles. They help fungi survive and adapt to the environment and they also have various structural roles. The 3D structure of a hydrophobin has been discovered relatively recently. The relationship between the structure and the properties is very interesting. The structure is illustrated in Figure 1. It can be seen that the molecule is amphiphilic with one hydrophobic and one hydrophilic part. This means that when the molecules are placed together they can form different self-assembled structures. This work will describe the properties of the different structures and suggest ways in which they can be incorporated and used in foods.

Experimental The experimental work was performed using a variety of different pieces of equipment. Hydrophobin II was extracted and manufactured at V T T Biotechnology, Espoo, Finland. It was supplied as a gift. Sodium caseinate was purchased from Aria Foods, Stockholm, Sweden. Lipids were obtained from Sigma Chemicals.



Figure L The structure of a hydrophobin 2 molecule (after réf. 1).


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92 Solutions of different quantities of hydrophobin in milli Q water were prepared and degassed. They were used as fresh. Varying concentrations were used up to 5 mg/ml. Foam stability was measured using a Turbiscan Classic, Formulaction, Toulouse, France. A i r was bubbled into the solutions and the density of the foam was measured for up to 17 days. Contact angle measurements on the solutions were performed using the pendant drop technique. Surface rheology measurements were performed using a surface rheometer from K S V Instruments, Helsinki, Finland. A Langmuir-Blodgett trough was obtained from the same source.

Results On investigation of the samples by Mastersizer we saw very clear evidence of structuring within the system (Figure 2). This was also apparent after extensive degassing of the system. This was seen repeatedly. If we consider the size of the structures then we see that they appear to be of a scale for small vesicles, which can agglomerate in the system. The larger structures are presumably agglomerations. Contact angle measurements showed that the hydrophobins were surface active, with surface activity increasing with concentration (Figure 3). Despite this, attempts at using the materials for emulsification were unsuccessful. A n extremely large amount of work was given over to the attempt to manufacture emulsions, either water in oil or oil in water, but this was ultimately unsuccessful. In conjunction with other surface-active proteins such as sodium caseinate, emulsification was possible. These emulsions did not differ significantly in character from sodium caseinate stabilized systems and so presumably it is the sodium caseinate that is responsible for the effect. However it was found that the hydrophobins had very strong foaming effects. Foams made at 5 mg/ml of additive were stable for over a month. Decay was measured with a turbiscan and over 17 days only relatively small changes were seen with the 5 mg/ml solution. Even at lower concentrations, very good and stable foams were seen that lasted for several days at 0.01 mg/ml. This indicates that the hydrophobins have very strong interactions and a profanity for the air / water interface. Because of this strong foam building ability and the poor emulsification properties it was decided to use surface rheology in order to study the behavior. On performing these measurements it was found that extremely low concentrations of hydrophobin in water were necessary in order to be able to achieve any measurements at all. In fact concentrations lower than lxl0" mg/ml were needed. 6



Figure 2. Particle Sizing Distribution of a Hydrophobin in water. Continued on next page.


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97 At these low concentrations, a two dimensional gel was formed between the particles. These were relatively strong interactions between the individual molecules. These have resulted in the interactions that are seen. As can be deduced from Figure 4 these films are very strong and elastic gel.


Conclusions We can see that the behavior of the hydrophobins is very interesting. Although the properties that have been studied are bulk ones it is in fact the behavior at the nanoscale that is having a significant effect. The evidence is that small structures of hydrophobins are forming. These can then partition to the air water interface where the amphiphilic nature of the molecule causes them to interact to form strong elastic films. There is clearly little partition to any oil/ water interface. This indicates that the specific nano-scale interactions of the particles are important. In order to further establish the use and applicability of these systems further work is needed. The results also reveal the complexity and functionality of the hydrophobin molecules and their applicability in fungi. It seems that the film building functionality is extremely critical.

Acknowledgements We would like to thank Rauni Seppanen, Per Claesson and Eva Blomberg for interesting discussions. Hans Ringblom, Annika Dahlman and Anne-Marie Hârdin provided experimental help. We are grateful to V I N N O V A and T E K E S for financial support.

References 1. 2. 3. 4. 5.

Wessels J.G., de Vries O . M . , Asgeirsdottir S.A., Springer J. J. Gen. Microbiol. 1991, 137, 2349-2345. Linder M . B . , Szilvay G.R., N a k a r i - S e t ä l äT, Pentilä M . E . , FEMS Microbiol. Rev. 2005, 29, 879-896. Wösten Η.Α., van Wetter W.A., Lugones L . G . , van deer Mei H.C., Burscher H.J., Wessels J.G., Curr. Biol., 1999, 9, 85-88. Talbot N.J., Nature, 1999, 398, 295-296. Scholtmeijer K . , Wessels J.G., Wösten H.A., Appl. Microbiol. Biotechnol, 2001, 56, 1-8.


Protein Structures as Delivery Vehicles in Foods - American Chemical

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