The acceptance and utilization of enzymes by the animal feed industry has become widespread in the last decade. As the understanding of enzymes and their properties has grown, so have both their use and their effectiveness as feed supplements. Why the interest in enzymes by animal nutritionists? The purpose of using enzymes in monogastric animals is to improve availability of nutrients in feedstuffs. The result is improved feed utilization and a reduced impact of anti-nutritional components that can otherwise negatively affect animal production and performance.
How enzymes work
Virtually all chemical reactions in biological systems are catalyzed by macromolecules called enzymes. A catalyst is a substance that increases the rate of a chemical reaction without causing a permanent chemical change; and enzymes exhibit enormous catalytic power. Chemical reactions in vivo rarely proceed at perceptible rates in the absence of enzymes while reaction rates increase as much as a million times when enzymes are present. Nearly all known enzymes are proteins; and they are among the most remarkable biomolecules known.
Enzymes have become big business, with a wide range of industries using commercial enzymes, in addition to the feed industry. The world annual sales of industrial enzymes were recently valued at $1 billion (Bron et al., 1999). Three-quarters of the market is for enzymes involved in the hydrolysis of natural polymers, of which about two-thirds are proteolytic enzymes used in the detergent, dairy and leather industries; and one-third are carbohydrases used in the animal feed, baking, brewing, distilling, starch and textile industries. Detergent manufacturers use 45 percent of all industrial enzymes produced in spot remover and detergent products containing proteases and lipases. This industry is expected to have a 10 percent annual growth rate for the next five years. Food processing enzymes including α-amylases, glucose isomerase and pectinases account for about 45 percent of enzyme usage. The starch processing industry uses half of the enzymes in the food industry, approximately 25 percent are used by the dairy industry and 10 percent by the brewers, fruit juice and wine producers. The textile and paper industries (6 percent) use primarily amylases and hemicellulases, and the leather industry (2 percent) uses proteases. Enzyme supplements for animal feeds account for about 1 percent (Amado, 1993).
Methods for production of commercial enzymes
Industrial enzymes are produced by plants, animals and microbes. By far the largest group exploited for the use of industrial enzymes has been the microbial population. The reason is simple: short generation times and high yields, together with the fact that microorganisms produce extracellular enzymes which are easy to harvest, make microbes the enzyme source of choice. Production of enzymes by microorganisms has also expanded because of the vast amounts of genetic information now available. Several industrially important microbial genomes have been sequenced; and the understanding of gene expression systems in microorganisms is much more advanced when compared to other gene expression systems. This knowledge has made it possible to select a variety of microbial organisms suitable for enzyme production with traditional submerged liquid fermentation (Baily and Ollis, 1986). An alternative fermentation method for enzyme production is solid state fermentation (Mitchell and Lonsane, 1992). How do these processes differ and what are their impacts on feed ingredient needs?
Submerged liquid fermentation
Submerged liquid fermentations are traditionally used in the United States for the production of microbially derived enzymes. Submerged fermentation involves submersion of the microorganism in an aqueous solution containing all the nutrients needed for growth. A research team led by Chaim Weizmann in Great Britain developed a process for production of acetone by submerged liquid fermentation using Clostridium acetobutylicum, which eventually led to the first large-scale aseptic fermentation vessel (Stanbury et al., 1995). The first large-scale aerobic fermenters were used in central Europe in the 1930s for production of compressed yeast (de Becze and Liebmann, 1944). In 1943, the British government decided that solid substrate fermentation was inadequate for the production of penicillin. This decision forced the development of liquid fermenters that are aseptic and contain adequate aeration and agitation. Construction of the first large-scale plant to produce penicillin by liquid fermentation began in 1943 (Callahan, 1944).
Solid substrate cultivation
In addition to submerged liquid fermentation, an ancient fermentation technology known as solid-substrate fermentation is also used to produce enzymes. Solid-substrate fermentations are generally characterized by growth of microorganisms on water-insoluble substrates in the presence of varying amounts of free water (Mitchell and Lonsane, 1992). This process is also referred to as solid state fermentation (SSF). The table shows differences between the SSF and submerged liquid fermentation processes of enzyme production.
The origin of SSF can be traced back to bread-making in ancient Egypt. Solid state fermentations include a number of well-known microbial processes such as soil growth, composting, silage production, wood rotting and mushroom cultivation. In addition, many familiar western foods, such as mold-ripened cheese, bread and sausage, and many Oriental foods including miso, tempeh and soy sauce, are produced using SSF. Beverages derived from SSF processes include ontjom in Indonesia, shao-hsing wine and kaoliang (sorghum) liquor in China and sake in Japan (Mudgett, 1986). The table gives examples of foods that involve an SSF process at some point in production.
In 1896, Takamine produced a digestive enzyme, Takadiastatse, by SSF employing Aspergillus niger on wheat bran (Takamine, 1914). This led to the application of SSF in other food and beverage industries. The most profitable applications of SSF are in the Oriental and African countries where SSF processes have been perfected over long periods. In western countries, traditional applications of SSF are scarce. SSF has been largely neglected since the 1940s and only negligible research and development efforts have been made. The selection of submerged liquid instead over SSF in western countries was not based on economic comparisons of submerged liquid and SSF techniques; the choice was linked to slow growth of the microbial cultivation industries across the world (Ralph, 1976; Hesseltine, 1976).
While commercial use of SSF is not widespread in North America, industrial enzyme production by SSF has occurred for a number of years (Takamine, 1914; Underkofler et al., 1958). After World War II, Underkofler et al. (1947) and Terui et al. (1957) used heaped bed cultures with forced aeration to produce enzymes and citric acid. Tempeh production has been established on a small scale in the United States (Hesseltine, 1987) because it has been accepted as a meat substitute by vegetarians. Mushrooms are cultivated in western countries; and soy sauce production has become highly industrialised and is widely used in both western and oriental countries.
To view the following tables, please click on the links: Comparison of solid-substrate fermentation and submerged liquid cultures; Foods produced by solid substrate fermentation; Comparison of enzyme activities.
Future potential for feed savings
Solid state fermentation technology could offer an opportunity to produce a unique enzyme complex that can be utilized to improve performance and save feeding costs. SSF may also be the key to the conversion of fiber into protein. Various high fiber materials could be utilized as feed ingredients if the digestibility could be improved. One potential way to increase digestibility is by creating unique enzyme complexes with SSF technology. The high fiber material can be used as a substrate in SSF, thus producing an enzyme complex that is designed specifically to digest the material. By utilizing this concept, the raw material can be used as a feed ingredient as well as a substrate in SSF.
The single most important feature of SSF is the low moisture content of the medium, which makes SSF very different from submerged liquid cultures. Water is essential for microbial growth; and the limited water in SSF has several consequences. It is adsorbed and to some extent held tightly; and there may even be some free water in the interior and on the surface. Water activity can be below 0.99 in SSF, where free water is virtually absent. These conditions favor filamentous fungi, many of which grow well between water activities of 0.93 and 0.98 (Corry, 1973). Bacteria and yeast grow above a water activity of 0.99.
Heat transfer is restricted in SSF, which can lead to overheating problems in large-scale fermentations (Laukevics et al, 1984). Evaporative cooling is the most effective cooling method, although this will reduce water availability (Trevelyan, 1974). Proper temperature conditions during the fermentation are a balance between the need for heat removal and the necessity of keeping the substrate sufficiently moist to support growth.
Solid substrates used in SSF are composite and heterogeneous products from agriculture or by-products of agro-industries. For many processes, substrates are chosen because they are readily available and therefore inexpensive. All substrates have a common macromolecular structure. The macromolecular portion can provide a structural component for the substrate as well as serve as the carbon and energy source (e.g. cellulose) for the microorganism. If the macromolecule serves as a structural source only, the carbon and energy source is provided by a non-structural macromolecule such as starch or a smaller, soluble compound (e.g. soluble sugar).
Differences in enzymes produced
Evidence has been accumulating to support the view that SSF processes are qualitatively different than submerged liquid fermentations. The data suggest that microbial physiology and regulation within the cell are influenced by the fermentation environment (Viniegra-Gonzalez, 1997). Ayeres et al. (1952) reported that pectinases produced by SSF had noticeable biochemical differences from those produced by submerged fermentation. A glucosidase produced Aspergillus phoenicis in SSF was more thermotolerant than when produced in submerged liquid fermentation (Deschamps and Huet, 1984). Alazard and Raimbault (1981) showed that amylases produced by A. niger using SSF were more resistant to heat denaturation than those produced in submerged liquid fermentation by the same strain. Other differences have also been reported (Romero et al., 1993; Villegas et al., 1993) and reinforced by the observation that the induction and repression patterns of pectinase production by A. niger are different for each fermentation technique (Solis-Pereira et al., 1993).
Exoenzyme production results in increased amounts of some enzymatic activities not produced by cultures in liquid fermentation. In a comparison of aphytases produced by SSF and a commercially-available phytase produced using submerged liquid fermentation, carbohydrase and protease side activities were found in the SSF, but not the liquid fermentation product (see table). The complex nature of feedstuffs means these side activities should be analyzed for their potential benefits to the animal industry (Classen, 1996). In vitro comparisons have shown increased rates of reducing sugar and amino nitrogen, and an associated increase in phophate release by an SSF phytase product (Filer et al., 1999).
Advantages and disadvantages
Solid-state fermentation systems have features that may be advantageous and are worth considering when choosing an enzyme production process for animal feed formulation (Cannel and Moo-Young, 1980; Mudgett, 1986):
The medium is often simple, consisting of unrefined agricultural product, which may contain all the nutrients for microbial growth. Examples of substrates are cereal grains, wheat bran and wheat straw.
Substrates require less pretreatment compared to liquid fermentation. Pretreatment for SSF must increase the accessibility of nutrients, while pretreatment for liquid fermentation must achieve extraction of the nutrients into the bulk liquid phase.
The restricted availability of water helps to select against undesirable contaminants, especially bacteria and yeast.
Forced aeration is often easier in solid-state cultures than in liquid cultures because the inter-particle spaces allow transfer of fresh air to thin films of water at the substrate surfaces.
Downstream processing and waste disposal is often simplified or minimized. If drying is required, less water is present to be removed.
But solid state fermentation as an enzyme production technique is not without difficulties that must be overcome. A reduced moisture level and difficulties in heat removal can be drawbacks, depending on the application or needs.
A number of disadvantages must be addressed to make a successful product (Cannel and Moo-Young, 1980; Mudgett, 1986):
The process is restricted to microorganisms that grow at reduced moisture levels. The majority of commercially profitable processes involve fungi, however, and this problem is avoided.
Removal of metabolic heat generated can be a problem in large-scale fermentations. Depending on the organism, heat can drastically influence end-product production. This problem can be lessened by using organisms that are heat tolerant.
The solid nature of the substrate presents problems in monitoring process parameters. Changes in pH are not easily identified and controlled in SSF; and the control of moisture content and substrate concentrations is extremely difficult. Heat production, oxygen consumption and carbon dioxide are parameters that can be measured.
Many important basic scientific and engineering aspects are poorly understood. Little is known about the mode of growth of fungi within substrate masses composed of irregularly shaped solid particles.
Cultivation times are often longer.