Modelo de Valor

For over 5000 years, mankind has, knowingly and unknowingly, made use of spontaneous fermenta- tion of a variety of food items, which include bread, alcoholic beverages, dairy products, vegetable prod- ucts, and meat products. But it was more recently, just in the last century, that scientists realized that the process of fermentation was effected by the ac- tion of microorganisms and that each microorgan- ism responsible for a specific food fermentation could be isolated and identified. Now, with advanced bioengineering techniques, it is possible to charac- terize with high precision important food strains, isolate and improve genes involved in the process of fermentation, and transfer desirable traits between strains or even between different organisms.

ELIMINATION OFCARCINOGENICCOMPOUNDS

Brewer’s yeast (Saccharomyces cerevisiae) is one of the most important and widely used microorganisms in the food industry. This microorganism is cultured not only for the end products it synthesizes during fermentation, but also for the cells and the cell com- ponents (Aldhous 1990). Today, yeast is mainly used in the fermentation of bread and of alcoholic beverages. Recombinant DNA technologies have made it possible to introduce new properties into yeast, as well as eliminate undesirable by-products. One of the undesirable by-products formed during yeast fermentation of foods and beverages is ethyl- carbamate, or urethane, which is a potential carcino- genic substance (Ough 1976). For this reason, the alcoholic beverage industry has dedicated a large amount of its resources to funding research oriented to the reduction of ethylcarbamate in its products (Dequin 2001). Ethylcarbamate is synthesized by the spontaneous reaction between ethanol and urea, which is produced from the degradation of arginine, found in large amounts in grapes. Yeasts, used in wine fermentation, possess the enzyme arginase that catalyzes degradation of arginine. If this enzyme can be blocked, arginine will no longer be degraded into urea, which in turn will not react with ethanol to form ethylcarbamate. In industrial yeast, the gene

CAR1 encodes the enzyme arginase (EC 3.5.3.1) (Dequin 2001). To reduce the formation of urea in sake, Kitamoto et al. (1991) developed a transgenic yeast strain in which the CAR1 gene is inactivated. The researchers constructed the mutant yeast strain by introducing an ineffective CAR1 gene, flanked by a DNA sequence homologous to regions of the arginase gene. Through homologous recombination, the ineffective gene was integrated into the active

CAR1gene in the yeast chromosome, interrupting its function (Fig. 3.12). As a result, urea was eliminated and ethylcarbamate was no long formed during sake fermentation. This same procedure can be used to eliminate ethylcarbamate from other alcoholic bev- erages, including wine (Kitamoto et al. 1991).

INHIBITION OFPATHOGENICBACTERIA

To increase safety, hygiene, and efficiency in the production of fermented foods, the use of starter and protective bacterial cultures is a common practice in the food industry today (Gardner et al. 2001). Starter culture is a liquid consisting of a blend of selected microorganisms, used to start a commercial fermen- tation. The difference between starter and protective cultures is that starter cultures give the food a de- sired aroma or texture, while protective cultures inhibit the growth of undesirable pathogenic micro- organisms, but do not change the food property (Geisen and Holzapfel 1996). For the purpose of practicality during food processing, the same micro- organism should be used for both starter and protec- tive cultures, but unfortunately this is not always possible. Genetic engineering methods help improve available strains of microorganisms used in starter and protective cultures, so that new characteristics can be added and undesirable properties eliminated (Hansen 2002).

Genetic engineering research aimed at optimizing starter cultures is focused on three main goals: (1) to enhance process stability, (2) to increase efficiency, and (3) to improve product safety (Geisen and Hol- zapfel 1996). During the production of some fer- mented food, such as mold-ripened cheese, pH level rises in the culture due to lactic acid degradation by fungal activity. This alkaline media offers an ideal condition for the proliferation of foodborne patho- genic microorganisms such as Listeria monocyto-

genes(Lewus et al. 1991). The safety of food prod- ucts could be greatly improved by the use of starter

cultures that can also serve as protective cultures and inhibit the growth of such harmful microorgan- isms. The enzyme lysozyme (EC 3.2.1.17) can be an effective agent for the inhibition of Listeria in food. Van de Guchte et al. (1992) integrated the gene responsible for lysozyme formation in a strain of the bacterium Lactococcus lactis. After genetic trans- formation, this bacterial strain was able to express and secrete lysozyme at high levels. The researchers cloned lysozyme-encoding genes from E. coli bacte- riophages T4 and lambda in wide-host-range vectors and expressed in L. lactis. Biologically active lysozyme was produced and secreted by the trans- genic L. lactis strains, suggesting that these bacteria can be used as both a starter and a protective culture (Van de Guchte et al. 1992).

NATURALSWEETENERPRODUCED BYMICROORGANISMS

Techniques to enhance flavor in food have been known for a long time, but only recently has it been

recognized that microorganisms can also be used in flavor production and enhancement. Today, many of the techniques for flavoring food and beverages make use of synthetic chemicals (Vanderhaegen et al. 2003). With increased public concern about the danger of using synthetic chemicals, flavors pro- duced by biological methods, also called bioflavors, are becoming more popular with consumers (Arm- strong andYamazaki 1986, Cheetham 1993). The fla- vor and fragrance industry is estimated worldwide at $10 billion per year; and although thousands of natu- ral volatile and synthetic fragrances are known, only a few hundred are regularly used and manufactured on an industrial scale (Somogyi 1996). There are sev- eral methods for the production of bioflavors includ- ing (1) product extraction from plant materials and (2) the use of specific bioengineered microorganisms for their biosynthesis. Biotechnological production of bioflavors using microorganisms has certain advantages such as large-scale production with low cost, nondependence on plant material, and preserva- tion of natural resources (Krings and Berger 1998).

Xylitol, also called wood sugar, is made from xylose, which is found in the cell walls of most land plants (Nigam and Singh 1995). Pure xylitol is a white crystalline substance that looks and tastes like sugar, making it important for the food industry as a sweetener. One of the main advantages of xylitol over other sweeteners is that it can be used by dia- betic patients, since its utilization is not dependent on insulin (Pepper and Olinger 1988). Xylitol is be- lieved to reduce tooth decay rates by inhibiting

Streptococcus mutans, the main bacteria responsible for cavities. Because xylitol is slowly absorbed and only partially utilized in the human body, it contains 40% fewer calories than regular sugar and other car- bohydrates. In the United States, xylitol has been used since the 1960s, and it is approved as an addi- tive for foods with special dietary purposes (Emodi 1978). Yeast (S. cerevisiae) is considered the ideal microorganism for commercial production of xylitol from xylose because of its well-established use in the fermentation industry. South African researchers (Govinden et al. 2001) isolated a xylose reductase (EC 1.1.1.21) gene (XYL1) from Candida shehatae and introduced it into S. cerevisiae. The XYL1 gene from Candida was cloned into the yeast expression vector pJC1, behind the PGK1 promoter, and the construct was transformed into yeast by electropo- ration. Xylitol production from xylose by the trans- formant was evaluated in the presence of different cosubstrates including glucose, galactose, and malt- ose. The highest xylitol yield (15 g/L from 50 g/L of xylose) was obtained with glucose as cosubstrate.

PRODUCTION OFCAROTENOIDS IN MICROORGANISMS

Carotenoids are structurally diverse pigments found in microorganisms and plants. These pigments have a variety of biological functions, such as coloration, photo protection, light harvesting, and hormone pro- duction (Campbell and Reece 2002). Carotenoids are used as food colorants, animal feed supplements, and more recently, as nutraceuticals in the pharmaceutical industry. Recent studies have suggested many health benefits from the consumption of carotenoids. Carot- enoids such as astaxanthin,-carotene, and lycopene have high antioxidant properties, which may protect against many types of cancers, enhance the immune system, and help relieve the pain and inflammation of arthritis (Miki 1991, Jyocouchi et al. 1991, Giovan- nucci et al. 1995). There is an increased interest in

extracting large amounts of carotenoids from natural sources. The 1999 world market for carotenoids was $800 million, with projections for $1 billion in 2005 (Business Communications Co. 2000). Although re- searchers have found certain microalgae such as

Haematococcus pluvialisthat produce high amounts of the carotenoid astaxanthin, extraction of carot- enoids from these microalgae is difficult because of their thick cell wall. For this reason, genetic engineer- ing methods have been applied to produce carotenoids in other microorganisms. The edible yeast Candida

utilisis a good candidate for commercial carotenoid production. It is a “generally recognized as safe” (GRAS) organism, and large-scale production of peptides, such as glutathione, has already being suc- cessfully achieved in C. utilis (Boze et al. 1992).

In microorganisms and plants, carotenoids are syn- thesized from the precursor farnesyl pyrophosphate (FPP) (Fig. 3.13). Miura et al. (1998) developed a

Figure 3.13. Biosynthetic pathway of the carotenoids lycopene, -carotene, and astaxanthin.

de novo biosynthesis of the carotenoids lycopene, - carotene, and astaxanthin in C. utilis using cloned bacterial genes that encode enzymes of the biosyn- thetic pathway. They used four genes (crtE, crtB,

crtI, crtY) from Erwinia uredovora and two genes (crtZ, crtW) from Agrobacterium aurantiacum for construction of four different plasmids. The plas- mid pCLR1EBI-3 contained crtE, crtI, and crtB, required for the production of lycopene (Fig. 3.14a). For synthesis of -carotene, the plasmid pCRAL- 10EBIY-3 contained the genes crtY, crtI, crtE, and

crtB (Fig. 3.14b). A dual plasmid system with pCLEIZ1 containing crtE, crtI, and crtZ and pCLBWY1 containing crtW, crtY, and crtB was used to produce astaxanthin (Fig. 3.14c,d). In order to integrate these genes into the yeast chromosome, the plasmids were linearized by restriction digest and transformed into C. utilis by electroporation. The resultant transgenic yeast produced significant amounts of lycopene (1.1 mg/g dry weight), - carotene (0.4 mg/g dry weight), and astaxanthin (0.4 mg/g dry weight), in quantities similar to amounts found in microorganisms that naturally produce these carotenoids (Miura et al. 1998). These results indicate that C. utilis has a great potential for use in large-scale production of commercially important carotenoids.

DETECTION METHODS IN FOOD