Dairy Biotechnology

Module5. Application of biotechnology in dairying

Lesson 21
DAIRY ENZYMES AND WHOLE CELL IMMOBILIZATION

21.1 Introduction

Enzymes are complex proteins which act as catalysts to accelerate the chemical reactions of living cells and bring specific chemical changes for converting specific set of reactants or substrates into specific products, without being changed themselves. These are not permanently modified by their participation in reactions and have a great specificity. In dairy industry, the use of enzymes, particularly exogenous enzymes are not fully exploited and limited to a few major and some minor applications. Enzymes play important role in the preparation of certain dairy products like cheeses, yoghurt etc., by improving texture, flavor and bringing about desirable changes in the product. Lipolytic and proteolytic enzymes can accelerate the production of flavor compounds. Successful use of preparations containing these enzymes is complicated by the need to attain a satisfactory balance among the various enzymes involved in the cheese ripening process.

21.2 Important Enzymes Used in Dairy Industry

The most important enzymes used in dairy industry are rennet, protease, lipases, lactase etc. as the principal constituents of milk are proteins, lipids and lactose.

21.2.1 Rennet

Rennet, an exogenous enzyme, is used as a milk-clotting agent in cheese industry for the manufacture of quality cheeses with good flavor and texture. The action of milk clotting enzyme i.e. rennet in cheese making is splitting of κ-casein which causes destabilization of casein micelles and subsequently leads to the formation of a coagulum.

21.2.2 Proteases

These are enzymes that are added to milk during cheese production for hydrolyzing caseins, specifically κ-casein, which stabilizes micelle formation preventing coagulation. Certain proteinases are used in enhancing the cheese flavor and also in acceleration of ripening process. An extract of B. subtilis, which contains a neutral proteinase, active at higher temperature seems to be quite suitable in this regard. Moreover, it has the advantage of being barely active below 8°C, and even sometimes inactivated at lower temperatures. Hence, over ripening can be prevented. Flavor defects can be kept to a minimum by combining the endopeptidase activity (catalyze the hydrolysis of peptide bonds in the interior of peptide chain or protein molecule) of enzyme preparations with the exopeptidase activity (catalyze the cleavage of the terminal/last or next-to-last peptide bond from a polypeptide or protein, releasing a single amino acid or dipeptide) of extracts of lactic acid bacteria.

21.2.3 Lipases

These are used in the preparation of stronger flavored cheeses, such as Italian, Romano cheese etc., by breaking down milk fats and giving rise to characteristic flavors to cheeses. Lipase acts on Triglycerides and on hydrolysis yields fatty acids, partial glycerides plus glycerol. This process is very critical in natural cheese making and should be controlled as milk fat contains high levels of short chain fatty acids, which when in free form are very volatile and have low flavor threshold.

21.2.4 Beta-galactosidase (lactase)

It helps in the hydrolysis of lactose to glucose and galactose, and hence play a significant role in dairy processing. Lactose intolerance in humans is characterised by the typical symptoms like severe tissue dehydration, diarrhoea, and at times, even death and such condition results due to the lack of ability of individuals to synthesize lactase enzyme which hydrolyses milk sugar i.e. lactose. Hence exogenous lactase is used in preparation of lactase-treated milk and in the manufacture of lactose-free products, particularly milk, for such individuals. Other applications of lactase enzyme include modification of functional properties and preparation of dietetic products, in accelerating the process of cheese ripening.

The other enzymes that have limited applications in dairy industry are catalase, lactoperoxidase, and lysozyme.

21.3 Immobilization of Enzymes

It is always cost-effective to use the enzymes more than once. Most of the enzymes are in solution with the reactants and/or products and hence it is difficult to separate them after completion of chemical reaction. In order to reuse the enzymes again after separation from the products during chemical reaction, it is necessary to employ techniques that are helpful in attaching the enzyme to the reactors. This idea has led to the employment of immobilization techniques for enzymes. The concept of enzyme immobilization was first evolved, when difficulties were experienced during the use of crude enzyme preparations in the production of wine, cheese or in tanning. The phenomenon of immobilization of enzyme on a support, was first reported by J.M. Nelson and E.G. Griffin in 1916. They reported the adsorption (immobilization) of invertase on charcoal/alumina without loss of activity. However, the technique of enzyme immobilization could be established only after a lapse of about 40 years, in 1954- 1961 when many researchers developed relevant procedures and the equipments.

In simple terms "immobilized" means unable to move or stationary. An immobilized enzyme is an enzyme which is attached to an inert, insoluble material such as calcium alginate over which a substrate is passed and converted to product. This technique has revolutionized the prospects of enzyme application in industry.

Immobilization is defined as the imprisonment of a biocatalyst in a distinct phase that allows exchange with, but is separated from, bulk phase in which substrate, effector, inhibitor molecules are dispersed and monitored.

Immobilized enzyme is physically entrapped or covalently bonded by chemical means to an inert and usually insoluble matrix, where it can act upon its natural substrate. The matrix is usually a high molecular weight polymer such as polyacrylamide, carrageenan, chitan, cellulose, starch, glass beads, etc.

The use of insoluble form of an enzyme in a process offers a number of advantages as given below:

• Immobilization allows separation of enzymes from the products after completion of chemical reaction and thus can be reused or recycled.

• Immobilized enzymes have ability to bind to a matrix, by which it typically possess greater resistance to change in pH and temperature and have operational stability than the soluble form of the enzyme.

• Reaction mixture or products specifically contain only solvent and reaction products and so more or less do not require complex purification as the processed product is not contaminated with the enzyme.

• Immobilization improves the efficacy and efficiency of an enzyme.

• Certain manipulations of chemical reactions are better possible with immobilized enzymes

21.3.1 Methods of immobilization

It is very important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemical nature or reactive groups in the binding site or active site of the enzyme during its immobilization to a surface. The selection of immobilization method depends upon the application as well as the resources available.

The Traditional Methods of Enzyme Immobilization can be classified as Follows

21.3.1.1 Carrier binding

In this method, the enzymes are bound to water in-soluble carrier molecules. Based on the technique of binding, this is further divided into

a. Physical adsorption

This is the immobilization of an enzyme on the surface of water-insoluble carriers.

b. Ionic binding

This process involves ionic binding of the enzyme to water-insoluble carriers containing ion-exchange residues as indicated in Fig 21.1. e.g. Polysaccharides and synthetic polymers having ion-exchange centers

Fig. 21.1 Ionic binding

c. Covalent binding

This is based on the binding of enzymes and water-insoluble carriers by the formation of covalent bonds between the enzyme and the support matrix (Fig 21.2) e.g. Glutaraldehyde

Fig. 21.2 Covalent binding

21.3.1.2 Cross linking

This is the process of intermolecular cross-linking of enzymes by bi-functional or multi-functional reagents (Fig 21.3).

Fig. 21.3 Cross linking

21.3.1.3 Entrapment

Here enzyme is trapped in insoluble beads or microspheres i.e. incorporating enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a semi-permeable polymer membrane (Fig 21.4).

Fig. 21.4 Entrapment

21.3.2 Application of immobilized enzymes

Application of immoblized enzymes is increasing day by day and the Table 21.2 given below elucidates the use this technology in various food applications.

Table 21.1 Immobilized enzymes and their uses

21.4 Whole Cell Immobilization

The term “immobilized cells” refers to keeping the cell in one place. Generally in a reaction environment, cells are floating around in nutrient liquid whereas in cell immobilization, the cells are trapped or stuck to a sticky surface while nutrient flows over them. This method is more appropriate and useful when there is a need for using or when a series of enzymes are required in the process.

Whole cell immobilization was defined as "the physical confinement or localization of intact cells to a certain region of a space with preservation of some desired catalytic activity", and is a process that often mimics what occurs naturally when cells grow on surfaces or within natural structures.

Full-scale systems using immobilized higher cells came into use in the eighties. A well known example is the plant of Bayer, USA, for the production of factor VIII that plays a key role in blood clotting. Here, the cells are not immobilized on a carrier but retained by a membrane in a continuously operated system.

21.4.1 Advantages of whole-cell immobilization

• Enzyme isolation and purification steps are not required

• Show the higher stability in the catalytic power and enzyme activity,

• Have multivariate enzyme applications, eliminating the need for immobilization of multiple enzymes

• Intracellular enzymes need not be extracted prior to the reaction; they may be used directly

• The end products can be recovered in a simple manner.

• The technique is cost effective

21.4.2 Disadvantages

• Low productivity

• Lower resistance

• Limitation of mass transfer

• Problems with degradation of product

• Byproducts are formed due to lysis of cells or toxic metabolites

21.4.3 Methods of whole cell immobilization

Most of the cell immobilization techniques are modifications of the techniques developed for enzymes. The techniques used for whole cell immobilization are briefly described below:

21.4.3.1 Binding

These methods are further classified based on the chemical forces used in the binding process of microbial cells with carrier molecules.

a. Weak bonds

Flocculation: The microbial cells are encouraged to aggregate to form clumps and thus resulting in floc formation under the influence of the weak chemical forces such as Van Der Waal forces, hydrogen bonds etc. However, such weak chemical forces are strongly influenced by the pH, ionic strength and reagents that influence flocculation.

Adsorption: Living microbial cells are weakly adsorbed onto the non-charged surfaces with Van Der Waal forces, hydrogen bonds and hydrophobic interactions, so that the cells can be easily shed from the surface under the influence of shear forces of flow. However, the microbial cells will be replenished due to continuous multiplication with the passage of time.

Ionic binding: This is mainly due to the ionic charge interactions between positively charges anionic ionic exchangers made of cellulose or sephadex and negatively charged microbial cells.

b. Strong bonds

Covalent binding: Covalent binding process involves the immobilization of whole cells on inert, stable carrier by covalent binding e.g. silanized or derivatized porous glass. The method is not very successful for immobilization of whole cells because of general toxicity of reagents used and low loading capacity of the carrier molecule, even though it is very effective and efficient in immobilization of enzymes.

Cross linking: Microorganisms are crossliked by chemical substances, e.g., by glutardialdehyde. The surfaces (especially the proteins) of microorganisms are liknked with the surfaces of other microorganisms by aldehyde groups of glutardialdehyde

21.4.3.2 Physical retention

The physical methods of whole cell immobilizations are broadly categorized as entrapment and membrane retention.

a. Entrapment

The method depends on the entrapment of microbial cells in the pores of polymer material. Microbial cells are entrapped by gels that permit the diffusion of small molecules, both substrate and product, at rates that are adequate for the cells’ viability and functioning. Entrapment can be carried out using by introducing microbial cultures into polymerization medium i.e. cells are included in polymerizing monomer or into polymer solution which is then used to form flat, biologically active membrane. The variations in the entrapment procedures are as follows:

Ionotropic gelation: Ionotropic gelation is based on the ability of polyelectrolytes to crosslink in the presence of counter ions to form hydrogels. Entrapment by ionic gelation, especially in the form of alginate beads, is the most widely used method. Alginate is a polysaccharide that forms a stable gel in the presence of cations, such as calcium. Beads of alginate-containing cells, are formed by dripping a cell suspension-sodium alginate solution mixture into a stirred calcium chloride solution.

Polymerization: Gel entrapment by polymerization is most commonly carried out by using polyacrylamide. However, toxicity of cross-linking agents used in polymerization may cause a loss of cell viability. Among the polymerization agents available, k-carrageenan is reported to be more advantageous.

Microencapsulation: Microencapsulation is a process by which very tiny droplets or particles of liquids or solid materials or microbial cell suspensions are surrounded or coated with a continuous film of polymeric material. A prevalent objective is to protect the cells from degradation by reducing their reactivity to environmental conditions. Biopolymers, such as alginate, gellan-gum, xanthan, κ-carrageenan and more recently proteins are used as matrix materials are being widely used for microencapsulation

b. Membrane Retention

Semipermeable membranes with porous structures are used for the retention of microbial cells. The porous membranes offer the advantage of allowing the passage of substrates and products during reaction processing. The methods include dialysis tubing (a type of semi- or partially permeable membrane tubing made from cellulose or cellophane which selectively allows some molecules to pass through the membrane through passive transportation or active transportation), ultrafiltration units (hollow-fiber anisotropic membranes most commonly used for downstream processing of conventional fermentation).

21.4.4 Biomass production

Methods are used to support cell release from gel beads that occurs suddenly as microcolonies form near the surface of the biocatalysts. The release of cells growing in the peripheral layer of highly colonized gel beads can be used to efficiently produce biomass in the bulk liquid medium. This cell release activity can be used for producing single or mixed strain cultures and to continuously inoculate food liquids to process fermented foods such as fermented milk products.

21.4.4.1 Production of bulk lactic starter cultures

The LAB are largely used in single and mixed cultures for the production of fermented milks like yoghurts and cheeses. The Immobilized Cell (IC) technology can be explored to produce mixed lactic starters in continuous fermentation. The high IC concentration results in higher productivity and decreases contamination risks, due to the high dilution and inoculation rates provided by cell release from beads. Immobilisation also improves plasmid stability in the starters to express desirable functions optimally during pilot production of fermented dairy foods.

21.4.4.2 Production of mass Probiotic cultures

IC technology can be used to continuously and stably produce mixed-strain starters containing fastidious and non-competitive micro-organisms, such as bifidobacteria, with a high volumetric productivity and high biomass concentrations in the outflow of the continuous fermentation, even at high dilution rates.

21.4.4.3 Pre-fermentation of milk

Starter culture preparation is of paramount importance in the manufacture of fermented dairy products. The continuous inoculation-prefermentation of milk for yoghurt production in a stirred tank reactor by separately entrapped cells of Lactobacillus delbruekii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus in Ca-alginate gel beads was the first studied dairy application of IC. This technology allowed a reduction in fermentation time by approximately 50 and 20% compared with freeze-dried strains and a liquid yoghurt culture respectively. The inoculation-prefermentation of milk for fresh cheese production with a mixed culture entrapped in κ-carrageenan/LBG gel beads has also been extensively studied. Fermentation time to produce the fresh cheese curd was considerably reduced by more than 50% compared with the traditional industrial process.

21.4.4.4 Metabolite production

The ultrafiltration of cheese whey yields large volumes of low-value whey permeate, which has limited uses. The immobilized cell technology can be used for production of different metabolites and functional ingredients from LAB using this low value whey permeate containing high lactose and mineral contents, used as a culture medium for the production of lactic starter cultures or metabolites.

21.4.4.5 Lactic acid production

Lactic acid is widely used as an acidulant and preservative agent in foods and as a precursor for production of emulsifiers, such as stearoyl-2-lactylates, in baking industry for the baking. High lactic acid productivities and long-term stability have been obtained during continuous IC fermentation of yeast extract-supplemented whey permeate by Lactobacillus helveticus immobilized in κ-carrageenan/locust bean gums gel beads, but with limited conversin of lactose.

21.4.4.6 Exopolysaccharide production

Very little research has been done on cell immobilization for Extracellular polysaccharide (EPS) production. Mucoid properties of L. Rhamnosus RW 9595M were explored for cell immobilization by adsorption on solid porous supports (ImmobaSil®). The production of EPS was investigated during pH-controlled IC repeated-batch cultures in SWP. IC technology results in changes in cell morphology and physiology, and the formation of very large aggregates containing very high cell and insoluble EPS concentrations. The high potential of the strain, L. rhamnosus RW9595M, and of IC technology has been judiciously exploited for the production of EPS as a functional food ingredient. In addition, the production of insoluble EPS allows an easy recovery of the product and the aggregates containing high EPS and viable cell concentrations could have interesting applications as symbiotic product, combining both probiotic and prebiotic activities.

21.4.4.7 Bacteriocin production

Cell immobilization has been used to increase cell density for bacteriocin production in supplemented whey permeate medium. A very high nisin Z production was demonstrated in the broth after repeated-cycle pH-controlled batch (RCB) cultures of Lactococcus lactis ssp. lactis biovar diacetylactis UL719 immobilized in κ-carrageenan/LBG gel beads in Supplemented Whey permeate (SWP).

21.4.4.8 Other applications in food industry

Novel technologies were developed based on immobilized cells in gel beads, which are principally involved in fermentation of liquid substrates, and the immobilized cells are not found in the product. Thus the most important industrial exploitation of immobilized cell technology is found mainly in beer and wine making.

Some potential applications of IC technology in the food industry have been given in Table 21.2

Table 21.2 ICT Applications in the food industry

References

Hartmier, W., Immobilized Biocatalysts; An Introduction, Springer Verlag, Berlin, 1988.

Mattiasson, B. (ed.), Immobilized Cells and Organelles, CRC Press, Boca Raton, FL, 1983, vol. 1–2.

Tampion, J. and Tampion, M.D. Immobilized Cells: Principles and Applications, Cambridge University Press, Cambridge, 1987.

Nelson J.M and E.G. Griffin (1916) J. Am. Chem. Soc.38, 1109–1115

Liliana Gianfreda, Palma Parascandola, and Vincenzo Scardi (1980) European J. Appl. Microbiol. Biotechnol. 11, 6-7

Chenn, P. Microorganisms and Biotechnology (1997) John Murray, London. ISBN O719575095

Simpkins, J. & Williams, J.I. Advanced Biology (1997) Collins Education Ltd. ISBN 0748744673

http://www.iisc.ernet.in/currsci/jul10/articles17.htm