Approximately 1% of the enzymes so far identified are used commercially as technical enzymes. The largest volume (35%) is proteases for use in detergent manufacture. In food processing, technical enzymes are used to reduce processing cost, to increase yields of extracts from raw materials or improve handling of materials, and to improve the self-life and sensory characteristics of foods.
Enzymes are active at low concentrations and the rates of reaction are easily controlled by adjustment of incubation conditions. However, the cost of enzymes is high, and in some products, enzymes must be inactivated or removed after processing, which adds to the cost of the product. Like other proteins, enzymes may cause allergic responses in some people, and they are usually coated or immobilized on carrier materials to reduce the risk of inhalation of enzyme dust by operations.
Theory
Microbial enzymes have optimum activity under similar conditions to the optimum growth conditions for the microorganism concerned. Enzymes from closely related microbial species have optimum activity under similar conditions, whereas those from unrelated species have widely differing properties. Microbial enzymes are either extra-cellular (secreted by the cells into the surrounding medium) or intracellular (retain within the cell). Extra-cellular enzyme production occurs in either the logarithmic phase or the stationary phase of growth, whereas intracellular enzymes are produced during logarithmic growth but are only released into the medium when cells undergo lysis in the stationary or decline phase.
Enzyme production from microorganisms
The requirements of commercial enzyme production from microorganisms are as follows:
1. Microorganisms must grow well on an inexpensive substrate,
2. They should produce a constant high yield of enzyme in a short time,
3. Methods for enzyme recovery should be simple and inexpensive and
4. The enzyme preparation should be stable
These requirements are met by constitutive mutant strains of microorganism, which permanently retain the required characteristics.
Enzymes are produced by either surface culture on solid substrate (for example rice hulls, fruit peels, soybean meal, wheat flour and peanut meal) or by submerged culture using liquid substrates. Submerged cultures have lower handling costs and a lower risk of contamination and are more suited to automation than are solid substrates. The substrate should contain carbon and energy source and a source of nitrogen for cell growth. In addition, specific nutrients may be required for cell growth and specific minerals may be necessary for enzyme production. In submerged culture, a seed inoculum is produced using similar incubation conditions to those used for production. The substrate (for example molasses starch hydrolysate or corn steep liquor) is low cost and readily available in adequate quantities, with a uniform quality. In batch methods, the inoculum is added to sterile substrate at 3-10% of the substrate volume. Fermenter capacities range from 1000 to 100.000 l. Cells are grown under controlled conditions for 30-150 h. Microprocessors are used to control pH, dissolved oxygen, carbon dioxide and temperature automatically.
Enzyme recovery
Extra-cellular enzymes are recovered from the fermentation medium by centrifugation, filtration, fractional precipitation, chromatographic separation, electrophoresis, membrane separation, freeze drying or a combination of these methods (Skinner, 1975). Intracellular enzymes are extracted by disruption of cells in a homogenizer or mill. Recovery is more difficult and the yield is lower than for extra-cellular enzymes, because some enzymes are retain within the cell mass. If required, the specific activity of the enzyme is increased by precipitation using acetone, alcohols, or ammonium sulfate or by ultra-filtration. The success of commercial enzyme production depends on maximizing the activity of the microorganism and minimizing the costs of the costs and incubation and recovery procedures.
Application of enzymes to foods
In batch operation the enzyme is mixed with food and, after completion of activity, is either retained within the food or inactivated by heat. This method is widely used when the cost of the enzyme is low.
In continuous operation, enzymes are immobilized on support materials by:
1. Micro-encapsulation in polymeric membranes, which retain the enzyme but permit passage of substrates and products,
2. Attachment by electrostatic attraction to ion exchange resins,
3. Cross-linking with for example glutaraldehyde,
4. Adsorption onto colloidal silica and cross-linking with glutaraldehyde,
5. Covalent bonding of non-essential residues on the enzyme to organic polymers, (the most permanent form of attachment),
6. Entrapment in polymer fibers (for example cellulose triacetate and starches)
7. Copolymerization with maleic anhydride and
8. Adsorption onto charcoal, polyacrylamide, or glass.
In adsorption onto charcoal, polyacrylamide, or glass, porous carriers have a high surface area and hence permit higher enzymic activities than non-porous carriers do. They also give protection to the enzyme against variations in the pH or temperature of the substrate but are more difficult to regenerate.
The main advantages of enzyme immobilization are:
1. Enzymes are re-used without the cost of recovery from a food,
2. Continuous processing, and
3. Closer control of pH and temperature to achieve optimum activity.
Immobilization is at present used when an enzyme is difficult to isolate or expensive to prepare. However, because of these advantages, the technique is expanding into areas of processing. The main limitations are:
1. The higher cost of carriers, equipment and process control,
2. Changes to the pH profiles and reaction kinetics of enzymes,
3. Loss of activity (25-60% loss) and
4. Risk of microbial contamination
In operation, either immobilized enzymes are mixed with a liquid substrate and then removed by centrifugation on filtration and reused, or the feed liquor is passed over an immobilized bed of enzyme fixed into a reactor. Immobilized enzymes should have the following characteristics:
1. Short residence times for a reaction,
2. Stability to variations in temperature and other operating conditions over a period of time (for example glucose isomerase is used for 1000 h at 60-650C).
3. Suitability for regeneration and
4. High mass transfer rates between the carrier material and the substrate.
