Application of Immobilized Cells and Enzymes in the Food Industry
Judit Krisch, Erika Beata Kerekes, Miklos Tako and Csaba Vagvolgyi
Immobilization of whole cells, cell debris, purified and crude enzymes, aroma compounds, nutraceuticals, active agents of medicaments or cosmetics and vitamins is an everyday practice in pharmacy, environmental protection, cosmetics, etc. With the aid of nanotechnology, fine delivery systems controlling the release of the immobilized compounds were developed. In food industry, cells and enzymes are usually used directly in food processing and some of them become a part of the food. Therefore, a very careful selection of immobilization materials and techniques must be done. Immobilization offers many advantages compared to traditional processes, and can help design new, healthy foods using environment friendly methods to satisfy the expectations of the consumers. In this chapter, immobilization techniques already used in the food sector or in the developing phase are presented with examples of microbial cells and enzymes suitable for this application.
Immobilization of whole cells or enzymes means fixation to a support, or support- free cross-linking of aggregates. In the food industry, one has to be especially careful with choosing the right support and immobilization technique because of food safety considerations. The support and solvents used cannot be toxic or cause any harm to the consumer. From the industrial point of view, a good support is cheap, renewable and the immobilization is feasible. The support shall protect the immobilized cell or enzyme from the shear forces and harsh environmental circumstances arising during the fermentation process in a tank reactor. It must also be resistant to microbiological degradation.
Support associated immobilization techniques include adsorption, entrapment or encapsulation and covalent binding, the latter being used more for enzymes than cells. In most cases, immobilized cells have to retain their viability for a successful operation but covalent linking can lead to cell wall injuries and limited survival of the cells.
Adsorption is the most simple and cheap immobilization technique where the cells or enzymes attach to inorganic or organic surfaces via dipole-dipole and hydrophobic interactions, Van der Waals forces or hydrogen bonding (Groboillot et al. 1994; Contesini et al. 2013; Rouf et al. 2017). In most cases, preformed, porous materials are used such as cellulose beads or fibers, ceramics and silica materials (Groboillot et al. 1994; Krisch et al. 1995; Szajani et al. 1996). In Figure 8.1 (a and b). the cross section and outer surface of a preformed cellulose bead can be seen. Large cavities within the beads and microspores and rough surface on the outer shell offer sites for adhesion. Immobilization is usually performed in the fermenter by adding the support and cells or enzymes at the same time and after adsorption, the non- adhered cells are washed out (Groboillot et al. 1994; Elakkiya et al. 2016). Adhered cells usually form biofilms on the surface and in the cavities of the support, which results in enhanced resistance against toxic materials and pH changes during fermentation (Krisch and Szajani 1996). In Figure 8.2 (a and b), Acetobacter aceti and Saccharomyces cerevisiae biofilms formed on cellulose beads can be seen. Adsorbed enzymes show enhanced thermal stability (Contesini et al. 2013). Disadvantage of this technique is the easy detachment from the surface of the support on changing environmental conditions (pH, ionic strength, temperature) and wash out from the reactor (Groboillot et al. 1994; Contesini et al. 2013; Rouf et al. 2017). Washed-out cells or enzymes can contaminate the product.
By entrapment, cells and enzymes are packed into a gel matrix usually forming beads with diameters between 1 and 5 mm. Entrapment and gel formation occurs at the same time by adding the cells or enzymes to the liquid gel followed by gelation and beads formation. The gel-forming agent is mostly a polysaccharide and gel formation occurs by temperature changes as in the case of agar, agarose, K-carrageenan and gellan; or by ionic cross-linking with multivalent cations in the case of alginate and chitosan (Groboillot et al. 1994; Elakkiya et al. 2016). These polysaccharides have the advantages of being biocompatible and not degradable by the entrapped microbes. Entrapment results in enhanced mechanical and chemical stability but mass transfer limitations of the substrate into the bead can lead to decreased activity (Rouf et al. 2017). The polysaccharides are used in the concentration of l%-4% to
FIGURE 8.1 Scanning electron microscope images of preformed cellulose beads, (a) Cross section of the bead (35X); (b) surface with micro-pores. Yeast (S. cerevisiae) cells grow in the pores (I000X).
avoid or decrease these diffusion limitations. In the presence of chelating agents or by changing the pH, ionotropic gels will lose integrity and the cells or enzymes will be released from the matrix. Therefore, proper conditions for these gels, viz. continuous presence of the gel-forming cation, are required. Agar and carrageenan gels have less mechanical strength, but formation is simpler and does not need cations in the fermentation broth (Groboillot et al. 1994). The choice of gelation material is based on the fermentation process and the cells or enzymes to be entrapped. Cells usually grow near to the surface of the gel beads and can escape from it. To avoid or reduce cell release from the beads, sometimes a shell is formed on the outer surface by dipping the
FIGURE 8.2 (a) Biofilms on the inner walls of cavities in preformed cellulose beads—Acetobacter aceti (800X); (b) Biofilms on the inner walls of cavities in preformed cellulose beads—S. cerevisiae (700X).
beads in a coating solution containing the same or another polymer (Groboillot et al. 1994). Encapsulation is an alternative technology to entrapment with the advantage of no leakage of the content and higher loading capacity (Kosseva, 2011). Encapsulation is made by emulsification creating a wall around the cell(s) or enzyme(s). Examples for encapsulation are liposomes with one or two layers of phospholipids (Gibbs et al. 1999). Liposomes can range from nanometres to micron size and are non-toxic and acceptable for food applications. Self-assembling proteins like a-lactalbumin of milk can form nanotubes capable of immobilization of enzymes (Weiss et al. 2006; Sozer and Kokini 2009). In this aspect, nanotechnology offers new tools for immobilization.
Support-free aggregates of yeast cells can be formed by natural flocculation, which is an important process in separating yeast cells from beer (Genisheva et al. 2014). Aggregation, especially in the case of enzymes, can be forced by using cross-linking agents such as glutaraldehyde and bis-isodiacetamide (Abdelmajeed et al. 2012; Rouf et al. 2017). The large three-dimensional structure of enzyme aggregates multiplies the active area for a faster reaction. The criterion for successful cross-linking or covalent binding of an enzyme to a support is that sites not involved in the reaction are chemically bound. Covalent binding can enhance the rigidity and thus the stability of the enzyme structure but on the other hand, this rigidity can inhibit the free movement of the enzyme resulting in decreased activity (Rouf et al. 2017). A special type of enzyme immobilization is when non-viable whole cells are immobilized. In this case, multiple- step reactions can be implemented where more enzymes are needed at the same time, like in aspartate production for the sweetener aspartame (Johnson-Green 2002).
Bioreactors Used for Immobilized Systems
Immobilization allows continuous production without loss of the biocatalyst. The type of bioreactor used for immobilized systems depends on the mass transfer requirements and easy removal of product. Stirred tank reactors can be used for batch or continuous production; in the latter case, often more reactors are connected to each other to enhance productivity. Shear forces in this reactor can damage the support material, leading to loss of the biocatalyst. The packed bed reactors are commonly used in the industry. In these reactors, the biocatalysts are packed into a column and the fermentation broth flows through the bed. Anaerobic conditions can easily be achieved, for example, for ethanol fermentation. Packed bed reactors are cheap and simple to operate, but some problems with gas bubble formation, product inhibition at upper layers, and compaction of the bed can occur (Groboillot et al. 1994; Kosseva 2011; Rouf et al. 2017). Fluidized bed reactors are used for processes where oxygenation is needed. The added gas moves the biocatalyst and mixes the fermentation broth. In this reactor, substrate inhibition is minimal owing to the good mixing, and heat transfer is excellent (Rouf et al. 2017). Different variations of these bioreactors were designed to solve the problems arising during operation.
Properties of Immobilized Cells and Enzymes
In many cases immobilization brings higher productivity, shorter fermentation time, and higher product yield. Immobilized cells and enzymes are physically protected by the support from the changing conditions in the environment. Immobilized biocatalysts tolerate temperature and pH changes better, and are less sensitive to substrate or product inhibition (Krisch and Szajani 1996, 1997, Mitropoulou et al. 2013). Advantages of immobilized systems over traditional fermentation using free cells or enzymes cannot be explained only with the protective effect of the support matrix. Changes in the physiology of immobilized cells (Willaert 2011; Zur et al. 2016) as well as changes in the conformation of immobilized enzymes (Flores-Maltos et al. 2011) also contribute to the enhanced activity. Adsorbed cells usually form biofilms on the surface of the support, which is a multi-step process. The first adhesion of cells is the result of different interactions between the surface of the cell and the support causing reversible attachment followed by the reinforcement of the bonding resulting in irreversible connection. Attached cells began to form micro-colonies and produce extracellular matrix that connects the cells to each other and the surface (Kerekes et al. 2015). During biofilm maturation, a three-dimensional structure will be formed in which different layers can be found causing physiological, structural and mass transfer heterogeneity (Sutherland 2001; Zur et al. 2016). In the matured biofilm, cells from the top layer can detach and go into the fermentation broth.
In a biofilm, cell to cell or cell to surface interactions depend on the expression of special proteins such as the Flo protein family in S. cerevisiae. Regulation of the FLO genes contribute to the switch from non-adhesive planktonic cells to adhesive cells (Bojsen et al. 2012).
The extracellular matrix called EPS (extracellular polymeric substances) consists of polysaccharides, proteins, nucleic acids and lipids. This EPS plays a crucial role in the adhesion of biofilms, adsorbs water and nutrients from the environment, and represents a protective barrier against antimicrobial agents. Increased resistance of the cells in the biofilm to inhibitors may be the consequence of horizontal gene transfer in this EPS. Compositional changes or reorganization of cell wall or membrane (e.g. higher rate of unsaturated lipids) are also responsible for enhanced stress tolerance (Zur et al. 2016).
Entrapped cells in a gel are surrounded by the matrix and have no direct connection to the environment unlike adsorbed cells. Cells within the matrix can enjoy stronger protection than adsorbed cells (Krisch and Szajani 1997). In the gel matrix, oxygen and nutrient diffusion rate decreases gradually, and due to the phenomenon, the cells migrate close to the outer surface of the bead and form large colonies (Groboillot et al. 1994). Owing to the diffusion limitations, cells grow slowly in the beads, which in turn leads to higher plasmid stability (Willaert 2011).
As can be seen from the aforementioned examples, the protective effect of the support and changes in the cell physiology contribute to the better performance of immobilized biocatalysts over the free ones.