Shelf Life of Grains
Lipids and lipid-associated components are key factors in the quality of several grains such as maize and oats. The oat and oat products have a relatively high lipid content, a large proportion of unsaturated fatty acids, and a significantly more active lipase than that of either barley or wheat.66 Oats contain antioxidants and oat lipids are stable in mature, undamaged grains67 and in sufficiently heat-treated oat products.68 However, under unfavorable storage conditions or in untreated oat products lipolytic activity will cause rapid release of free fatty acids (FFA), which may then be oxidized and cause rancidity. Oxidative rancidity may also be caused by over-processing.69 Thus, adequate storage conditions70 and appropriate heat treatment for inactivation of lipolytic enzymes before milling of oats68 are essential in achieving stable oat products.71 Because unsaturated FFA in oats are susceptible to oxidation and many form components with undesirable aroma and taste,68 analysis of the amount of FFA may be useful for predicting lipid stability. For oats to be processed into food products, a maximun FFA level of 5% hexane extractable lipids has been suggested.72 FFA in oats enhance formation of bitter compounds.73 An approach to overcome the problems produced by the unsaturated FFA is the genetic engineering of oat crops where the levels of unsaturated FFA are reduced.53,54
Coffee is one of the most commercially important grains in the food industry. Coffee comprises a number of different forms, ranging from coffee cherries (berries); green coffee, which is trade beans that are removed by one of a number of different process sequences after harvesting; roast coffee, which is the green coffee beans that are roasted by a heat process, either domestically or commercially, and which may also be pre-ground; and the coffee beverage, which is the form in which it is actually consumed. It should also be understood that while coffee cherries/beans exist in a number of different botanical species within the corresponding genus, only two are used commercially, Coffea arabica and C. canephora (robusta in the trade), and can reflect different characteristics in storage behavior and in their subsequent coffee products.74
The greatest interest is in the shelf life of roasted (and ground) coffee, since this is the form in which coffee is most familiar, together increasingly with instant coffee to the consumer. Green coffee, roasted coffee, and instant coffee all have two main divisions of their chemical composition; first, the non-volatile matter, some contributing to basic taste sensations of acidity, bitterness, and astringency and the remainder, composed mostly of carbohydrates and proteins of generally neutral flavor characteristics; and second, the volatile substances, present in a very small amounts (ppm levels) but of great significance to overall flavor in the prepared cup of coffee beverages. In all these coffees, environmental factors of temperature, humidity, and oxygen exposure strongly determine storage behavior and therefore shelf life, together with the initial moisture content of the coffee, and its precise composition. The previously mentioned factors determine the condition or quality of roasted coffee after given periods of time. The terms "condition" and "quality", however, are very much subjective and are not amenable to scientific assessment.74 Like all other foodstuffs, roast coffee, and even more rapidly, roast and subsequently ground coffee, deteriorate with time from their initial state of "freshness" (i.e., after roasting in roasted coffee), but the actual deterioration has to be assessed on the cupped beverage, prepared under standardized conditions for all samples being compared, by human senses. Panels of judges are asked to assess changes from "fresh" flavor quality on numerical scales, and at what point the coffee is no longer "acceptable"
(i.e., the end of its shelf life). Such assessment is therefore on the overall flavor quality of the prepared beverage. A separate assessment may be made on the head-space aroma impact from the dry roasted and ground coffee using only the external nostrils of the nose, i.e., by sniffing. In practice an early deterioration may not be so readily marked in actual flavor quality of the beverage. Appearance changes in the dry product are not generally evident, except moisture uptake during storage. Shelf life data are of special relevance for trade purposes.
A number of investigations have also been performed to determine differences in volatile compounds, as between robusta and arabica; such differences are clearly evident in their respective beverage flavor characteristics. Grosch et al. (cited by Reference 74) used the technique for assessing the important aroma impact compounds by serial dilution techniques to obtain flavor dilution (FD) values in each of arabica and robusta roasted coffee, both from brews and the dry product. Among the differences, they found that 4-vinyl guiacol is especially characteristic in brewed robusta coffee and furaneol in arabica; in the dry product, 3,5-dimethyl-2-ethyl pyrazine appeared with the highest FD-factor in both coffee species. The use of genetic engineering techniques may well be a useful tool to enhance volatile compound production responsible for flavor;3 another approach is the reduction of caffeine content because of consumer demand for decaffeinated coffee.3
The functional role of vegetable and seed proteins in food processing is to provide the required physical properties to the food material either during processing or in the final product. The physical properties of both the starting materials (protein extracts, isolates, concentrates, or flours) and products are determined by the level of protein present, the proportions of different protein types, and the presence of nonprotein components, and such properties are likely to manifest themselves in different ways, depending on the processing procedures used.63 The types of functional properties sought in proteins are many and include those responsible for emulsification, foam formation, and stabilization, and also for texturing.63 65 Whereas many functional properties rely on maintenance of the native configuration of the proteins, several others arise through complete or partial denaturation of the proteins, followed by rearrangements of the polypeptide chains and formation of new intramolecular and intermolecular bonds. In all cases, however, the behavior of a particular protein type depends ultimately on its intrinsic primary structure (amino acid sequence) encoded by the genes. Thus, the proportions and functional properties of certain seed proteins could be manipulated by genetic engineering to suit particular applications in food processing.63-65
One example of the importance of protein functional properties is in baking quality. Payne and Rhodes (cited by Reference 63) have described a number of different baked products and the different qualities of wheat grain and wheat protein required for each. An important part of the basis of baking quality lies in the composition of the gluten protein fraction in wheat flour. Gluten is the water-insoluble viscoelastic protein mass left after soluble proteins, starch, and other nonprotein materials have been washed out from the flour. Gluten is composed mainly of hydrated forms of the two major wheat protein fractions, namely, gliadins (wheat prolamins) and glutenins (wheat glutelins).63 75 These proteins contribute in different ways to the properties of the flour during processing and in the final product. Thus, gliadins provide viscosity and extensibility to bread dough, whereas glutenins provide the elasticity that is all-important in dough stability and in the structure and texture of bread. Differences in proportions and properties of these two protein fractions determine whether a particular wheat variety has good bread-making properties or is more suitable for the production of other products, such as pasta or biscuits. A molecular basis for these properties has been proposed, based on the characteristics of the purified components, their amino acid composition and the primary structures of certain wheat proteins.75 The components responsible for good bread-making quality have been tentatively identified and ascribed to the glutenin fraction, notably to the high molecular weight (HMW) glutenin proteins (95,000 to 150,000 apparent MW).75 76 Payne et al. (cited by Reference 63) have shown a positive correlation between the molecular weight of native glutenin and the amount of HMW glutenin subunits, and have suggested that interactions of these subunits with other polypep-tides are important in stabilizing the glutenin structure. Furthermore, it has been concluded that allelic variation does correlate with good or poor baking quality, although other factors, possibly other wheat proteins, may be involved.63 Certain of the gliadin polypeptides have also been implicated in baking quality and dough strength. The positive identification and cloning of the genes encoding polypeptides that contribute to good bread-making quality could potentially allow the transfer of this trait to poorer-quality wheats carrying other desirable attributes.63 Additionally, it might be possible to manipulate the functional properties of the gluten for purposes other than bread making by transferring multiple copies of certain genes, thereby altering the proportion of the HMW glutenins.77
There are 12 genes for HMW glutenin proteins in hexaploid bread wheat, four coming from each of the three progenitor species although two genes are inactive in all varieties.75 The genes are of two types, Glu-1-1 and Glu-1-2, which give rise to X and Y HMW glutenin subunits, respectively. Glu-1-1 and Glu-1-2 loci are very closely linked on the long arms of the chromosomes of the homologous group 1. Thus, there are six pairs of loci each of which carries an X and Y gene.78 Series of genotypes have been assayed, possessing new combinations of X and Y subunit genes at the Glu-1D-1 and Glu-1D-2 loci.79 The comparisons have enabled the separate contributions of subunits associated with poor bread-making quality [2(X) and 12(Y)] and subunits associated with good bread-making quality [5(X) and 10(Y)]. Flavell et al.75 have showed that in these seeds the major variation was contributed by the Y subunits, with subunit 12 conferring poorer dough quality than subunit 10. They compared the amino acid sequences of these closely related proteins and found that differ in their central regions, which consist of an array of repeating hexamers and nonamer amino acid units; HMW glutenin 10 has a higher proportion of repeats of the consensus type than glutenin 12 and they postulated that this produces a more regular pattern of repetitive p turns in the protein, contributing to dough elasticity. A cysteine residue, likely to become involved in intra- or intermolecular linkages, may also be in a different configuration in the two subunits. There are also other protein components, such as low molecular weight glutenin subunits8081 and gliadin proteins that have been found to be associated with quality.8283 Branlard and Dardevet84 used the French wheat cultivar Darius, which has very good bread-making quality, even though it possesses the HMW glutenin subunit combination 2, 7 and 12, associated with poor quality, and a null allele at the Gli-D1 locus. The absence of the Gli-D1 encoded m-gliadins was associated significantly with higher dough tenacity and strength. These results demonstrated that using only one locus breeders can improve particular quality traits.84 In this way, genetic engineering could be used to aid in the construction of wheat and other cereal varieties with predetermined functional properties designed for a precise processing purpose, such as bread-making, breakfast foods, meat analogs, and hydrolyzed products, as well as for nonfood applications.63
The technology of producing beer involves the processes of malting and brewing. In malting, the barley grain is germinated under conditions leading to enzymatic hydrolysis or modification of starch and protein reserves and the production of flavor compounds, whereas brewing involves the fermentation of sugars to produce alcohol by yeast.85 Different varieties differ in regard to their suitability for malting, and in general barley suitable for malting should have a low protein content.86 It is thought that storage proteins released from protein bodies during germination surround starch grains, and also reduce access to amylolytic enzymes and delay sugar release, and this leads to poor fermentation.63 Malting quality may be affected by the hordein fractions,63,87 particularly B hordeins and especially disulfide-linked aggregates that may be less easily degraded when adhering to starch. However, malting quality is a complex character, and hordeins may affect other stages of brewing, such as filterability, foaming, and haze formation,88 but with further knowledge it should be possible to identify specific proteins (e.g., disulfide-linked components) that could be manipulated or their expression reduced such that malting quality will be improved. Other aspects that could be manipulated include identification and removal of genes involved in polyphenol (proanthyocyanidins) production to prevent the haze formed by interaction of polyphenols and protein.8689 The copy numbers of a-amy-lase genes and those of other hydrolytic enzymes (e.g., p-glucanases) can be increased to speed up breakdown of seed reserves during malting. The wheat a-amy-lase gene, the major enzyme of starch degradation, has already been cloned, and it has been expressed in modified yeast cells. The a-amylase has also been used for production of low-calorie beer.90
Improving Nutritional Properties Seed Storage Proteins
The nutritional quality of grains very much depends on the amino acid composition of the storage proteins, and being macromolecules directly coded by specific genes, unlike storage carbohydrates, they should be easier to manipulate. In terms of total storage protein production for cereals, wheat, maize, rice, and barley, it is much more significant than that produced by legumes, except for soybean.63 64 Lysine is the first limiting amino acid in wheat, barley, maize, sorghum, and triticale; threonine (barley, sorghum) or tryptophan (maize) is the second. Thus, in a pure cereal diet in which lysine is limiting, the quality will be poor because the grain protein will not be metabolized efficiently by humans and animals.91
The discovery that the maize opaque2 (o2) mutation dramatically increases the lysine content of the grain92 led to the development of high lysine corn.93 However, the soft, starchy endosperm of this mutant, which causes the kernel to be susceptible to pests and mechanical damage,94 prevented significant utilization of the mutation. After the initial characterization of o2, genes that alter the mutant phenotype were identified, giving it a normal appearance. These genes designated o2 modifiers95 were subsequently used by plant breeders at the Centro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT)96 to develop o2 varieties with normal kernel hardness and protein content, as well as an enhanced percentage of lysine. These modified o2 mutants are called quality protein maize (QPM).97-99
The major storage fraction of most cereals are prolamins, which are given trivial names such as gliadin (wheat), zein (maize), hordein (barley), and secalin (rye). In order to increase the limiting amino acids in cereals, two approaches have been suggested:100 (1) insertion of extra codons for lysine, threonine, or tryptophan into cloned storage protein genomic DNA, followed by reintroduction of the gene into the plant; and (2) modification of the expression of existing genes so that proteins rich in limiting amino acids are preferentially synthesized. A specific problem is that prolamins are coded by multigene families (e.g., for zein, possibly up to 150 closely related genes), so replacement of a single modified copy would have little effect. Some likely approaches to circumvent this problem may include the following: (1) introduction of a modified gene into a recipient that has a deletion lacking part of the gene family; (2) inactivation of normal gene expression (without deletion), with expression of introduced modified genes; (3) insertion of a modified gene with a strong promoter such that it is transcribed more frequently than natural genes; and (4) insertion of multiple copies of the modified gene, perhaps combined with approaches 1 through 3. Eggum et al.101 have developed several rice mutants for prolamin and glutelin in order to improve the nutritional properties of rice protein. They obtained mutants with a higher lysine content and a higher net protein utilization (NPU).
It is also possible to improve the nutritional quality of cereals by increasing specific soluble amino acid levels; there has been some success in producing mutants with feedback-insensitive regulatory pathways, particularly those of lysine biosynthesis.91 The amino acids lysine, threonine, methionine, and isoleucine are derived from aspartic acid, and it is known for barley that there is a negative feedback to three isozymes of aspartate kinase, the first enzyme in the pathway, by the end products lysine, threonine, and S-adenosyl methionine. Thus, cloning of the genes that encode for these isozymes is being developed in order to increase the production of such amino acids.63
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