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Food Biotechnology 2 December 2003
Dr. B. Lee
ENZYME TECHNOLOGY IN FOOD PROCESSING
Word & PowerPoint files available at http://f.rival13.free.fr/enzymes
Lydia ETCHEBEST 260101578
Frédéric RIVAL 260098444
Table of Contents
1.1 Enzymes types and sources
1.2 Reaction conditions
1.3 Commercialization of enzymes processes
2. Immobilization of enzymes
2.1 Carrier Binding
2.1.1 Physical Adsorption mode
2.1.2 Ionic Binding mode
2.1.3 Covalent Binding mode
2.2 Cross Linking
2.3 Entrapping Enzymes
3. Industrial application
3.1 Cheese making
3.2 Meat tenderization
3.3 Bread making
3.4 Production of beverages and fruit juices
3.5 Starch and sugar industries
3.6 The modification of fats and oils
3.7 Other food enzymes from GMOs
4. Legal and safety implications
5. Protein engineering of enzymes
6. Future prospects
The use of enzymes to accomplish specific desirable changes in food has been practiced for centuries. A few examples of the ancient use include the use of malted barley for starch conversions in brewing beer and wrapping the meat in the bruised leaves of the papaya tree to tenderize it. Since then, mankind has tried to improve these processes for fast growing food industry and in order to satisfy consumer demands for healthy and natural, tasty, consistent, convenient and low cost products. Often these processes were passed along from generation to generation by trial and error. Later, more systematic approaches have been used.
An enzyme is a protein that speeds up (catalyses) chemical reactions in physiological processes in the body (e g digestion) and industrial applications for food products (e.g. fermentation of wine and curdling of cheese). An enzyme acts as a catalyst, regulating the rate of chemical reaction taking place without itself being altered at the end of the process. Enzymes are used in food production, rather than other chemical modifications, because enzymatic reactions are carried out under mild temperature and pH conditions and are highly specific. They have also been used as they can help to reduce processing costs, increase yield and improve product handling, shelf-life and sensory characteristics with modifying raw materials and aiding in the processing or cooking stages.
The roles of enzymes include: enhancement of flavour and aroma, removal of unwanted flavours and taints, enhancement of digestibility, modification of texture to aid processing and final product appearance, upgrading raw materials. The main enzyme activity utilised in food processing applications is protease. However, applications utilising lipases and carbohydrate degrading activities are also becoming widespread. The first enzyme to be isolated was diastase(an amylase) from malt in 1833 and the enzyme activity in soy flour was patented as a bleaching agent in 1934. Now there are companies who produce enzyme preparations specifically for baking. Other enzymes which may be used more in the future. There is, however, different legislation covering the use of enzymes in food, in different countries. There are also controversial issues with respect to labeling of products. Should added enzymes be regarded as processing aids, and if their activity is destroyed during processing be omitted from labeling or are they additives which need to be recorded on the labels?
For many years researches were carried out on the actions of enzymes, particularly those that cause food deterioration, such as pectic enzymes that cause destruction and separation of pectic substances in citrus juice and polyphenol oxidase that cause browning, off-flavor development and loss of vitamins in fruits and vegetables. Until recently, chemical and food engineers have gained considerable interest in the potential usefulness of enzymes in processing. Since 1950s, tremendous progress has been made in the development of the submerged fermentation techniques and this has reduced the cost and increased the availability of industrial enzymes.
In the early 1970s, advances in the technology of immobilized enzymes systems became of significance in the processing of foods and the production of food and chemical ingredients. This technique allows for much greater utilization of the relatively expensive enzyme; such as glucose isomerase in the production of high-fructose corn syrup from starch by fixation of the protein onto the column through which a continuous stream of the liquefied starch slurry could be passed (Roller and Goodenough, 1999). In the late 1980s, the food enzyme, chymosin, derived from genetically modified organisms (GMO5) was first introduced in the markets. The large expansion in the application of enzymes in the manufacturing and processing industries has largely been in two areas: the enhancement of traditional processes and the development of totally new uses based on an understanding of enzyme properties (Chaplin and Bucke, 1990). Nowadays, the food industry is the second largest user of enzymes (the detergent industry is the first).
“The challenge to food technologists is to recognize the potential of biotechnology to fulfill the food requirements of today’s society” (Sanders, 1991).
Biologically active enzymes may be extracted from any living organism: animal, plant and microbiological sources. Most organisms have certain ‘core’ enzymes in common. For instance, enzymes of the Embden-Meyerhof pathway can be found in microbes, plants and animals. Similarly, amylase activity is found widely in human saliva, in plant seedling and in many microbes that use starch as an energy source. For enzymes like these, there are many potential sources. Other enzymes are specific to an organism or even provide that organism its characteristic features. Examples are the specialized enzyme systems in nitrogen-fixing bacteria and enzyme alliinase in onion and related plants, which catalyses the breakdown of peptide precursor to liberate sulphur-containing volatiles that defines the characteristic aroma. In cases like these, the source is limited as well as obvious. Although some animal and plant enzymes such as rennet and papain, are still in use, the large majority of bulk enzymes today are manufactured using fungal or bacterial fermentation. The advantages of using sources from fungi and bacteria have is that they can be easily grown and are usually not difficult to scale up a production process. In addition, the sources are not subject to seasonal or other factors, e.g. chymosin (rennin) that is extracted from the fourth stomachs of young calves for cheese production. The majority of enzymes that have so far been used are hydrolytic enzymes and many of these are produced extracelluarly by fungi. In general, animals and plants are poor sources of enzymes as they are slow growing and expensive. Extraction of enzymes from animal tissues or plants cells can also be difficult and time consuming, further increases the production cost of the enzymes. The possibility of producing larger quantities of enzyme from animal and plant sources by use of tissue culture methods is now being explored. Some proteins, such as vaccines, are already being produced in tissue culture.
With microbial enzymes it is often possible to increase the yields by changes in the growth conditions, addition of inducers, or strain selection, including increasing the number of gene copies by genetic engineering (Price, 1999). With enzymes from animal and plant sources, the yields may be increased by the introduction of the appropriate genes and their promoter regions into the more rapidly growing microorganisms. This has removed the technical problems of securing adequate sources of raw materials. However, there are often problems, such as the formation of inclusion bodies through incorrect folding, the lack of glycosylation, or degradation of the recombinant protein, which have to be overcome before a satisfactory product is obtained. There are also strict controls on the use of recombinant protein in the food industry.
Types of enzyme
The process of traditional products like cheese is due to enzymes that are endogenous; that is they occur naturally in the tissues of plant or animal or in the micro-organism. The endogenous enzymes can be manipulated to some extent to improve product quality but there are limitations. The idea of adding enzymes from other sources (exogenous enzymes), to improve existing reactions or to initiate new reactions, dates from the start of this century (Wolnak, 1980). Early work in the USA led to development of enzymes for the leather industry and started the commercial production of papain for use in the beer industry.
Enzymes are often referred to as endogenous or exogenous in foodstuff. Exogenous enzymes are added during processing to involve a wide range of effects. Among these are the control of texture, appearance and nutritive value, as well as the generation of desirable flavors and aromas or their precursors. The applications of these enzymes to generate a desirable end product are often a blanching act in which the degree of enzymatic modification of foodstuff must be carefully controlled. For example, the use of proteases in hydrolyzing proteins, such as that from soybean can cause production of bitter peptides if hydrolysis proceeds too far. One way of eliminating such bitter peptides is by treatment with peptidases to yield a protein-like material plastein.
Endogenous enzymes may cause desirable or deleterious effects in texture, aroma, flavor and appearance just as exogenous enzymes. For example, natural food flavors such as terpenes, hydrocarbons, alcohols, aldehydes, ketones, esters, lactones, amines, and sulfur-containing compounds are enzymatically produced in fruits and vegetables. The major difference is that endogenous enzymes are already in the foodstuff and control may be more difficult.
Enzymes have a number of distinct advantages over conventional chemical catalysts. Primarily are their specificity and selectivity, not only for particular reactions, but also in their discrimination between similar parts of molecules (regiospecificity) or between optical isomers (stereospecificity). This means that the chosen reaction can be catalyzed to the exclusion of side reactions, eliminating undesirable by-products. Thus, higher productivities may be achieved, reducing material costs. As a bonus, the product is generated in an uncontaminated state, so reducing purification costs and the downstream environmental burden. Enzymes work under generally mild processing conditions of temperature, pressure and PH.
Classical enzyme studies, are carried out in dilute aqueous solutions under optimal conditions with only substrate, enzyme, buffer and necessary co-factor present. The efficiency of the reaction is measured by the enzyme activity, which was defined by the International Union of Biochemistry in the 1 960s in an attempt to produce a standard system. One Unit (U) is defined as “the amount of enzyme that catalyses the transformation of 1 of substrate per minute under defined conditions”. The defined conditions normally refer to 25°C and optimal substrate concentration and pH; however, these conditions are rarely found when enzymes are used in the food industry (Fullbrook, 1983) and it is difficult to predict activity and therefore the amount of enzyme that is required. Fullbrook also raises difficult questions about how a mole of industrial substrate, e.g. corn starch, can be defined when the molecular weight varies and the fact that enzyme activity in industry may be measured, not in terms of µmoles of substrate transformed but in terms of reduced viscosity or a related chemical value, e.g. a color standard. Although there is a great deal of published information about enzymes, the application of these data to the industrial context is not always straightforward.
Other problems in applying pure biochemical criteria to the food situation are associated with substrate concentration, which is rarely optimal and normally governed by other factors such as solubility. The optimum temperature of commercial enzymes (typically around 50 to 100°C) is also far removed from the standard temperature of 25°C and the pH optimum may be temperature dependent (Fullbrook, 1983). Physical factors also affect the enzymes and there are certainly differences between reaction rates in aqueous solution and when enzymes are membrane bound. When enzymes are immobilized or encapsulated for convenience in food processing, the properties of the enzymes will also change, Reactions at low water activity or in fat/water mixtures (e.g. in the modifications of lipids where the lipid/water interface is important) are also outside the classical enzyme studies. Application of these reactions bas been hindered by a lack of understanding of the basic chemistry, although the enzymatic modification of lipids has considerable commercial potential (Critchley, 1987).
There are many food enzymes available that originate from different sources and therefore have different pH and temperature characteristics. It is worth testing a number of these to see if there are significant differences in performance or not. In the case of proteases, there is a wide range available with p11 optima from 2.5 to 9 although they do have different affmities for certain amino-acid bonds. Other types of enzymes generally have narrower ranges of optimum pH.
Recent advances in genetic engineering have provided the means for improving the stability of enzymes; this is achieved by altering the structure at vulnerable points by substitution of a different amino acid.
Another factor that may limit the usefulness of an enzyme in the industrial context is product inhibition. In normal metabolism, this property is useful, as it helps regulate metabolic pathways, but if the enzyme is required for the complete conversion of a substrate, the product needs to be removed to increase the percentage conversion. Enzyme processes need to be designed so that the desired changes can occur. The product may be removed to increase conversion and the design of enzyme reactors is critical.
When enzymes are used over relatively long periods and at elevated temperature, there is a decline in enzyme activity. In some applications, this is welcome, as active enzyme may be unacceptable in the final food product. In other applications, it leads to decreased conversion rate and loss of efficiency. Again, design of the process can overcome these problems so that a constant degree of conversion is achieved.
When enzymes are considered for use in a food process, it is essential to ensure that they will confer some commercial benefit. There are several ways of defining this latter parameter. Enzymes may improve the conversion of a raw material to its constituent parts as in the hydrolysis of starch to glucose. Acid hydrolysis gives limited conversion, whereas enzymes can improve the yield. This example of starch hydrolysis also illustrates another beneficial effect of using enzymes, namely that the effluent from enzyme hydrolysis is less toxic and therefore cheaper in terms of waste disposal.
In the brewing industry, savings on raw material costs can be achieved by the use of enzymes in the mashing process. The traditional mash process relies on the enzyme activities in the malt constituent to hydrolyze the macromolecules of malt and barley into fermentable substrate. Malt is an expensive commodity, however, and it is also variable in terms of enzyme activity. Since brewing is a complex process, the complete replacement of malt enzymes by commercial enzymes may have other effects on the quality of the final product.
Rather than total replacement of malt enzymes, commercial enzymes are often used in conjunction with the malt enzymes, so that brewers can standardize the processes and produce consistent quality beer, regardless of raw material fluctuations. Thus the commercial benefits of using enzymes may be expressed in different ways as:
Given the fact that food is biological in nature and that food processing involves some type of conversion of raw materials to processed foods, it is surprising that enzymes are so little used in the industry. In their book Food Biotechnology, Angold et al. (1989) presented several reasons why biotechnology (which includes enzyme technology) has not found greater use in the food industry. They first differentiate between small-scale and large-scale biotechnology.
The pharmaceutical industry is typical example of the small-scale operation where the high costs of research and development can be recouped by charging (relative) high prices for drugs. Indeed, it could be argued that pharmaceutical research and development creates markets, as without research into diseases and ailments, no cures could be found. In contrast, the food industry can be described as “large-scale, commodity transformation characterized by a low margin operation” (Angold et al., 1989). Since food is a basic commodity, consumers expect it to be available at a reasonable cost. Moreover, it is difficult to improve food significantly so that is might attract a premium price. People in the Western World will pay a little extra for improved quality but, apart from specialties like caviar or truffles, food generally is cheap. In addition, the population in developed countries already consumes a sufficient variety and quantity of food to satisfy their nutritional requirements and over-consumption is now recognized as undesirable. There is therefore a limit to the market size and expansion can only be achieved by increasing the market share of a particular company.
Food is a traditional, craft-based industry and consumers are already suspicious about scientists “messing about” with their food. The public has seen so many contradictory statements by food experts, that the credibility of science as a whole has decreased. Improvements in the processing of food are more likely to be achieved through optimization of existing processes or through advances in engineering to allow efficient production of novel products (e.g. co extrusion machines).
The economic system of the consumer and producer is normally allowed to find its own balanced in the so-called free market, but food is such an important strategic commodity, that there is sometimes political intervention. The exogenous enzymes play a major role in industries such as food & beverages, starch sugar & alcohol, animal feed, brewing, textiles, paper & pulp, leather, detergents and health care. Many companies produce enzymes for food application e.g. biocone or biocatalyst. The groups of these different enzymes are:
However, each group has several enzymes and the names depend on the company. Each enzyme has a specificity to improve the process. The most important advantage is the regularity of production thanks to exogenous enzymes. The quality of endogenous enzymes is very variable. The tables show the different amylases for baking products that the company: Biocone produce.
2. Methods of Immobilization
When immobilizing an enzyme to a surface, it is most 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 of the enzyme. In other words, attach the enzyme but do as little damage as possible. It is desired to avoid reaction with the essential binding site group of the enzyme. Alternatively, an active site can be protected during attachment as long as the protective groups can be removed later on without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate or a competitive inhibitor of the enzyme.
The surface on which the enzyme is immobilized is responsible for retaining the structure in the enzyme through hydrogen bonding or the formation of electron transition complexes. These links will prevent vibration of the enzyme and thus increase thermal stability. The micro environment of surface and enzyme has a charged nature that can cause a shift in the optimum pH of the enzyme of up to 2 pH units. This may be accompanied by a general broadening of the pH region in which the enzyme can work effectively, allowing enzymes that normally do not have similar pH regions to work together.
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