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Progress in Biotechnology for Food Applicationswww.esciencecentral.org/ebooks

Edited by Dr. Wing-Fu Lai

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Progress in Biotechnology for Food ApplicationsChapter: Bioengineering in Production of Food Ingredients

Edited by: Wing-Fu Lai

Published Date: June 2014

Published by OMICS Group eBooks

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Progress in Biotechnology for Food ApplicationsEdited by: Wing-Fu Lai

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AbstractBio/Genetic engineering allows genetic material to be transferred between any two

organisms, including between plants and animals. For example, the gene from a fish that lives in very cold seas has been inserted into a strawberry, allowing the fruit to be frost-tolerant. However, this has not yet been done for currently available commercial food crops. Concerns about climatic change may lead to increased development and use of drought tolerant GM food crops. Comparison to the corresponding unmodified organism essentially regulates products of biotechnology on a par with organisms produced by traditional methods. This type of comparison equilibrates the new and old risks by removing new risks commensurate with old, unregulated risks from the regulatory process. However, because it requires a judgment by the manufacturer involving risks that may be unknown and unquantifiable, environmentalists and others skeptical of the new technology may feel that it leaves too many products of that technology unregulated or under regulated. Critics may also think that manufacturers using the new technology have too much discretion to unilaterally decide whether their products meet the required standards.

Keywords: Bioengineering; Food ingredients; Genetically modified foods

IntroductionFood technology is a branch of food science in which modern biotechnological methods

are applied to improve food production or food itself. Various biotechnological processes used to create and improve new food and beverages product include industrial fermentation, plant cultures and genetic engineering. Food biotechnology was advanced in 1871 when Louis Pasteur discovered that heating juices to a certain temperature would destroy bad bacteria which would affect wine and fermentation. This process was then applied to milk production, heating milk to certain temperature to improve food hygiene. Food science and food biotechnology was then progressed to include the discovery of enzymes and their role in fermentation and digestion of foods. With this invention further technological development of enzymes emerged. Typical industrial enzymes used plant and animal extracts. Later this was substituted by microbial enzymes. Foods genetically modified using biotechnology is known as GM foods. Genetic material is altered using non-traditional, laboratory based methods; this is

Omprakash H Nautiyal*Professor of Organic Chemistry/Natural Products Chemistry, Vadodara, Gujarat, India*Corresponding author: Omprakash H Nautiyal, Professor of Organic Chemistry/Natural Products Chemistry, 102, Shubh Building, Shivalik II, Canal Road, Chhani Jakat Naka, Vadodara 390002, Gujarat, India, Tel: +91-8733974519; E-mail: [email protected]

Bioengineering in Production of Food Ingredients

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known as genetic engineering. Individual genes with speficific desirable traits are transferred from one organism to another [1].

An example of this development would be the use of chymosine in the production of cheese which was typically made using the enzyme rennet and was extracted from the stomach lining of the cow. Scientists then started using a recombinant chymosine in order for milk clotting resulting in cheese curds. Food enzyme production using microbial enzymes was the first application of the genetically modified organisms. Food biotechnology has grown to include cloning of plants and animals as well as more developed genetically modified foods in more recent years. In 1946 scientists discovered that DNA can transfer between organisms. The first genetically modified plant was produced in 1983, using an antibiotic resistant tobacco plant. In 1994 the transgenic flavr Savr tomato was approved by the FDA for marketing in the US. The modification allowed the tomato to delay the ripening after picking.

Traditional breeding can achieve similar effects, but works over a much longer time span and is not as targeted as GM. In addition, traditional breeding cannot transfer genes from unrelated species as is possible with GM foods. Genetic modification of food is not new. Humans have been altering food crops and animals through selective breeding for many centuries. However, while genes can be transferred during selective breeding, the scope of exchanging genetic material is much wider using genetic engineering [2].

In theory, genetic engineering allows genetic material to be transferred between any two organisms, including between plants and animals. For example, the gene from a fish that lives in very cold seas has been inserted into a strawberry, allowing the fruit to be frost-tolerant. However, this has not yet been done for currently available commercial food crops. Concerns about climatic change may lead to increased development and use of drought tolerant GM food crops [3,4].

Some foods and fiber crops have been modified to make them resistant to insects and viruses and more able to tolerate herbicides. The major crops that have been modified for these purposes, with approval from the relevant authorities, are [5,6]:

• Maize (corn)

• Wheat

• Rice

• Oilseed rape (canola)

• Chicory

• Squash

• Potato

• Soybean

• Alfalfa

• Cotton

Modified genes are being used in whole foods such as wheat, soybeans, maize and tomatoes. These GM whole foods are not presently available in Australia. GM food ingredients are however present in some Australians food. For example soy flour in bread may have come from imported GM soybeans [7,8].

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Modified genes may have been used in an early stage of the food chain, but may or may not be present in the end product. Nevertheless, gene products for example, phytochemicals (plant chemicals that contain compounds which may prevent disease) may remain in the food chain. The implications for human health are unknown.

Foods certified as Organic or biodynamic should not contain any GM ingredients according to industry guidelines.

Inexpensive, safe and nutritious foods are needed to feed the world’s growing population. Genetic modification may provide:

• Sturdy plants able to withstand weather extremes.

• Drought-tolerant and salt-tolerant crops.

• Better-quality food crops.

• Higher nutritional yields.

• Inexpensive and nutritious food, such as carrots with more antioxidants.

• Foods with a longer shelf life, like tomatoes that taste better and last longer.

• Food with medicinal (nutraceutical) benefits, such as edible vaccines – for example, bananas with bacterial or rotavirus antigens.

• Disease and insect resistant crops that require less pesticide and herbicide – for example, GM canola [9-11].

GM advocates argue that GM foods are better for the environment. By using GM crops that are resistant to attack by pests or disease, farmers can reduce their use of pesticides and herbicides and the residual levels of these chemicals in the environment. However, development of resistance can undermine and even reverse this benefit [12,13].

Genetic engineering can be used to increase amounts of particular nutrients (like vitamins) in food crops. Research into this technique, sometimes called nutritional enhancement is now at an advanced stage.

GM golden rice is a white rice crop modified by the insertion of the vitamin A gene from a daffodil plant. This changes the color and the vitamin level of the rice and is of benefit in countries where vitamin A deficiency is prevalent.

GM researchers are focusing on major health problems like iron deficiency. The removal of the proteins that cause allergies from nuts (such as peanuts and Brazil nuts) is also being studied [14,15].

The Risks of GM CropsConcerns about genetic modification of food raised by scientists, community groups and

members of the public include:

New allergens could be inadvertently created – known allergens could be transferred from traditional foods into GM foods. For instance, during laboratory testing, a gene from the Brazil nut was introduced into soybeans. It was found that people with allergies to Brazil nuts could also be allergic to soybeans that had been genetically modified in this way and so the project was ceased. No allergic effects have been found with currently approved GM foods [16,17].

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Antibiotic resistance may develop – bioengineers sometimes insert a marker gene to help them identify whether a new gene has been successfully introduced to the host DNA. One such marker gene is for resistance to particular antibiotics. If genes coded for antibiotic resistance enter the food chain and are taken up by human gut micro flora, the effectiveness of antibiotics could be reduced and human infectious disease risk increased. Research has shown that the risk is very low; however, there is general agreement that use of these markers should be phased out [18-20].

Cross-breeding – GM crops can cross-breed with surrounding vegetation, including weeds, transferring undesired characteristics. The introduction of glyphosate-resistant soybeans in 1996 has produced glyphosate-tolerant weeds that have driven even greater use of herbicides.

Pesticide-resistant insects – the genetic modification of some crops to produce the natural biopesticide Bacillus thuringiensis (Bt) toxin could encourage the evolution of Bt-resistant insects, rendering the spray ineffective.

Biodiversity – growing GM crops on a large scale may affect the balance of wildlife and the environment. Since bees cannot distinguish GM from non-GM crops, GM crops can affect non-GM and organically–farmed crops through cross-pollination.

Cross-contamination – plants bioengineered to produce pharmaceuticals (such as medicines) may contaminate food crops.

Health effects – minimal research has been conducted into the potential acute or chronic health risks of using GM foods.

Social and Ethical Concerns about GMConcerns about the social and ethical issues surrounding genetic modification include:

The possible monopolization of the world food market by large multinational companies that control the distribution of GM seeds

Concerns related to using genes from animals in plant foods. For example, eating traces of genetic material from pork is problematic for certain religious and cultural groups

Animal welfare could be adversely affected. For example, cows given more potent GM growth hormones could suffer from health problems related to growth or metabolism

New GM organisms could be patented so that life itself could become commercial property [21,22].

Regulation of GM foodsIn Australia, GM foods are regulated by the Food Standards Australia New Zealand (FSANZ)

Code under Standard 1.5.2 – Food produced using Gene Technology. GM foods receive individual pre- market safety assessments prior to use in foods for human consumption.

A GM food will only be approved for sale if it is assessed as being safe and as nutritious as its conventional counterparts. The assessment investigates;

• Nutritional content

• Toxicity (using similar methods to those used for conventional foods)

• Tendency to provoke any allergic reaction

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• Stability of the inserted gene

• Whether there is any nutritional deficit or change in the GM food

• Any other unintended effects of the gene insertion

The safety of GM foods is still being debated, as it is impossible to predict all of the potential effects on human health and the environment. Consequently, some public health experts advocate caution, believing that we do not know whether GM foods are safe [23,24].

GM labelingSince December 2002, Australia law has required that food labels must show if food has

been genetically modified or contains GM ingredients, or whether GM additives or processing aids remain in the final food product. The label on the package must include the statement ‘genetically modified’ in conjunction with the name of the food or ingredient or processing aid.

GM foods are labeled to help consumers make informed decisions about the food they buy, not for safety reasons. Special labels are not required for:

‘Highly refined’ foods that no longer contain the altered DNA or protein (for example, oil from modified corn)

GM food additives or processing aids (unless the new DNA remains in the food to which it is added)

GM flavors constituting less than 0.1 per cent of the food by weight

Food, food ingredients or processing aids unintentionally containing less than one per cent GM material

Food prepared at point of sale (takeaway and restaurant food does not have to be labeled). Labels may be required if:

Genetic modification has altered the food so that its composition or nutritional value is outside the normal range of similar non-GM goods (for example, high omega-3 soybeans)

Food contains toxins which are significantly different to those in similar non-GM foods

The food produced using GM technology contains a new factor which can cause allergic reactions in some people

Genetic modification raises significant ethical, cultural and religious concerns regarding the origin of the genetic material used [25-27].

GM food on the shelvesMany foods on supermarket shelves contain imported GM ingredients. GM foods have also

been approved for production in Australia, including corn, soybeans, potatoes, canola and rice.

Other GM foods are still undergoing field trials approved by the Office of the Gene Technology Regulator, although the moratorium by state governments (lifted in Victoria and NSW in early 2008) stopped some trials. Imported food products are subject to the same regulations as domestically manufactured foods.

Around 20 GM foods, additives, flavorings, growth hormone (bovine somatotropin) and enzymes (like rennet, used to make cheese) are currently approved in Europe. More than 40 GM foods are approved for sale in the USA [28,29].

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The Main Sources of GM Foods in AustraliaSoya imported from the United States – the soya has been genetically modified to be

resistant to herbicide. It can be found in a wide range of foods, such as chocolates, potato chips, margarine, mayonnaise, biscuits and bread.

Cottonseed oil made from GM cotton – this oil, made from cotton resistant to a pesticide, is used in Australia for frying (by the food industry) and in mayonnaise and salad dressings.

Imported GM corn – this is mainly used as cattle feed at present and has not been approved for farming in Australia. However, GM corn may have entered the Australian market through imported foods like breakfast cereal, bread, corn chips and gravy mixes. If so, it is now required to be labeled.

GM ingredients in imported foods including GM potatoes, canola oil, rice, sugar beet, yeast, cauliflower and coffee.

Foods genetically modified using biotechnology is known as GM foods. Genetic material is altered using non-traditional, laboratory-based methods; this is known as genetic engineering. Individual genes with specific desirable traits are transferred from one organism to another [30,31].

Traditional breeding can achieve similar effects, but works over a much longer time span and is not as targeted as GM. In addition, traditional breeding cannot transfer genes from unrelated species as is possible with GM foods.

Genetic modification of plants and animalsGenetic modification of food is not new. Humans have been altering food crops and animals

through selective breeding for many centuries. However, while genes can be transferred during selective breeding, the scope for exchanging genetic material is much wider using genetic engineering.

In theory, genetic engineering allows genetic material to be transferred between any two organisms, including between plants and animals. For example, the gene from a fish that lives in very cold seas has been inserted into a strawberry, allowing the fruit to be frost-tolerant. However, this has not yet been done for currently available commercial food crops. Concerns about climate change may lead to increased development and use of drought-tolerant GM food crops [32,33].

The Society of Toxicology (SOT) is committed to protecting and enhancing human, animal, and environmental health through the sound application of the fundamental principles of the science of toxicology. It is with this goal in mind that the SOT defines here its current consensus position on the safety of foods produced through biotechnology. In this context, biotechnology is taken to mean those processes whereby genes that are not endogenous to the organism (transgenes) are transferred to microorganisms, plants, or animals employed in food production, or where the expression of existing genes is permanently modified, using the techniques of genetic engineering. We intentionally avoid using the term Genetically Modified Organisms (GMOs) or foods in this context, since conventional techniques of plant and animal breeding, which are not considered here, also involve genetic modification. The extent of the genetic changes resulting from such conventional breeding techniques, which is generally undefined, far exceeds that typically produced by transgenic methods. Consequently, it is important to recognize that it is the product, and not the process of modification, that is the

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focus of concern regarding the human or environmental safety of Biotechnology-Derived (BD) foods [34,35].

The principal responsibilities of toxicologists are to define and characterize the potential for natural and manufactured materials to cause adverse health effects and to assess, as accurately as possible, the plausibility and level of risk for human or animal health or for environmental damage under a defined set of circumstances. It is not the task of the Society of Toxicology to determine the overall value of a product or process by balancing health or environmental risks with potential benefits, or to choose between different strategies to manage risk, although toxicological considerations are important in both processes. Our purpose here is rather to identify and consider the primary toxicological issues associated with BD foods. Major areas of concern in the development and application of such foods in agriculture relate to the possibility of deleterious effects on both human health and the environment. We do not consider here some aspects of the possible environmental impact of GM organisms such as gene transfer to non-engineered plants [36,37].

Current techniques of developing organisms used in the production of BD foods typically involve the transfer to the host of the desired gene or genes in combination with a promoter and a gene for a selectable marker trait that allows the efficient isolation of cells or organisms that have been transformed from those that have not. Common selectable markers in plants have included resistance to antibiotics (kanamycin/neomycin or ampicillin) or herbicides [38].

Several key issues have been raised with respect to the potential toxicity associated with BD foods, including the inherent toxicity of the transgenes and their products, and unintended (pleiotropic or mutagenic) effects resulting from the insertion of the new genetic material into the host genome. Unintended effects of gene insertion might include an over-expression by the host of inherently toxic or pharmacologically active substances, silencing of normal host genes, or alterations in host metabolic pathways. It is important to recognize that, with the exception of the introduction of marker genes, the process of genetic engineering does not, in itself, create new types of risk. Most of the hazards listed above are also inherent in conventional breeding methods [39].

It has been 17 years since the groundbreaking 1975 meeting at A silo mar where scientists discussed the emerging technology of molecular biology, its vast potential and the possible risks that could result from the ability to transfer DNA from one organism to another.1 Since then, a number of biotechnology-derived pharmaceutical products have already gone on the market,2 and the first food and agricultural products have been approved or are close to approval.3 Many more such products are under development, and there have been no adverse impacts on human health or the environment. Rather, reputable scientific and medical sources stress the potential of biotechnology to improve human health and nutrition, and to ameliorate the adverse impacts of traditional agricultural practices on the environment. Despite the abundance of data indicating the beneficial potential of biotechnology and the absence of harmful incidents, genetic engineering has aroused considerable public suspicion and from some quarters a demand for government oversight out of proportion to the demonstrated risks. The negative perception and resulting regulatory response threatens to adversely affect the development and competitiveness of this fledgling industry, and may also delay or even block the introduction of beneficial products [40].

This Comment examines issues in the regulatory oversight of the production and consumption of bioengineered food. As an introduction, the Comment first examines the

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range of products under development and their potential benefits and risks. It next considers the recent controversy over the use of genetically engineered bovine growth hormone, which illustrates many of the issues in this area. The next section presents biotechnology’s possible risks and then discusses the advantages of a comparative approach to risk regulation of bioengineered food. Next, the Comment examines the current government regulation of this technology. Since the potential risks involve both the genetically engineered food products and the environmental release of the organisms that produce them, regulation in these areas is analyzed. This regulation has been implemented through the “coordinated framework,” an adaptation of existing statutes to the oversight of biotechnology. Although regulation under the coordinated framework has the advantage of not singling out biotechnology for special oversight, the Comment examines some of the criticisms that stem from adapting existing statutes to address biotechnology. The Comment next considers, in light of the benefits of a comparative approach, some recently proposed approaches to regulation. Finally, the Comment contemplates what modifications in government policy and regulation might improve the public perception of biotechnology, strengthen the industry, and foster the generation of products that are not merely profitable, but also truly beneficial to both human health and the environment [41].

This Comment examines issues in the regulatory oversight of the production and consumption of bioengineered food. As an introduction, the Comment first examines the range of products under development and their potential benefits and risks. It next considers the recent controversy over the use of genetically engineered bovine growth hormone, which illustrates many of the issues in this area. The next section presents biotechnology’s possible risks and then discusses the advantages of a comparative approach to risk regulation of bioengineered food. Next, the Comment examines the current government regulation of this technology. Since the potential risks involve both the genetically engineered food products and the environmental release of the organisms that produce them, regulation in these areas is analyzed. This regulation has been implemented through the “coordinated framework,” an adaptation of existing statutes to the oversight of biotechnology. Although regulation under the coordinated framework has the advantage of not singling out biotechnology for special oversight, the Comment examines some of the criticisms that stem from adapting existing statutes to address biotechnology. The Comment next considers, in light of the benefits of a comparative approach, some recently proposed approaches to regulation. Finally, the Comment contemplates what modifications in government policy and regulation might improve the public perception of biotechnology, strengthen the industry, and foster the generation of products that are not merely profitable, but also truly beneficial to both human health and the environment [42].

Biotechnology used in the Production of FoodBiotechnology as applied to the production of food (Figure1) has the potential to greatly

benefit the public, as well as to improve agricultural productivity. The Council on Scientific Affairs of the American Medical Association has stated that agricultural biotechnology has the potential to “meet the needs of a rapidly growing population and minimize the toxic influences of traditional farming practices on the environment.” However, due to agricultural economics as well as scientific complexity, the function of many of the first genetically engineered food products is to improve agricultural efficiency and productivity. Since the technology for producing foreign proteins in genetically engineered bacteria is more established than the

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technology for transforming entire plants or animals, some of the first products are proteins that can be inexpensively produced to replace or augment the same naturally occurring protein. Thus, the first genetically engineered food ingredient approved by the Food and Drug Administration (FDA) is chymosin, an enzyme traditionally obtained from the stomach of calves and used in the production of cheese. Since the genetically engineered chymosin is identical to the enzyme obtained from the traditional preparation and contains no ingredients that are not Generally Recognized As Safe (GRAS), the FDA has concluded that this product, like the traditional product, is GRAS.8 Bovine somatotropin, a hormone used to increase milk production, has been produced in genetically engineered bacteria and is virtually identical to the naturally occurring protein. It is expected to be approved in 1992 [43].

Figure 1: Food production (http://www.ied.edu.hk/biotech/eng/classrm/class_food1.html).

Genetic engineering techniques have also been applied to commercial food crops. The “anti-sense” tomato, one of the bioengineered products closest to FDA approval, has been modified to retard the softening and subsequent spoilage that accompanies ripening. This modification improves (Figure 2) the farmer’s ability to machine-pick ripe tomatoes without bruising them, thus producing a tasty but easily harvested tomato. This type of modification improves the efficiency of the agricultural industry and can result in increased supply and lower food prices for consumers. Genetically-engineered herbicide-resistant plants that survive the application of herbicides during weed eradication may also improve the efficiency of farming.12 Such plants will be used in conjunction with recently developed herbicides that are rapidly biodegraded and are of low toxicity.13 Finally, many crops, including tomato, tobacco, potato, alfalfa, cucumber, corn, and soybeans have been genetically engineered to resist plant viruses that might otherwise devastate these plants. This should improve both crop yield and quality, since harvested plants would have far less viral contamination than is present in unmodified plants [44].

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Figure 2: Activation of silent genes. (http://www.naro.affrc.go.jp/org/nfri/english/organization/bio/kouso.html National Food Research Institute).

The state of agricultural biotechnology today is that a variety of items are in development or close to marketing. Those that are farthest along are of the type that will increase agricultural efficiency or productivity, but the current technology has the potential for improving nutritional quality and reducing the use of chemical pesticides. Moreover, there have been no hazardous incidents that should create a fear of this technology and lead to tight regulatory oversight. To the contrary, the Council on Scientific Affairs of the American Medical Association has recommended that physicians play a role in educating the public that “genetic manipulation is not inherently hazardous and that the health and economic benefits of recombinant DNA technology greatly exceed any risk posed to society.”Yet public perception of biotechnology is one of suspicion, leading to calls for tighter regulation. The following examination of the

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recent controversy over bovine somatotropin may illuminate some of these conflicting views on biotechnology [45].

Bovine somatotropinBovine Somatotropin (bST), (Figure 3) also called bovine growth hormone, was the first

major product of recombinant DNA technology available for use in agriculture. bST occurs naturally in cows, but when additional bST produced by genetically engineered bacteria is administered to dairy cows, their milk production is expected to increase an average of 12% without a commensurate increase in feed consumed. The use of bST has been heralded as the technological advance that will have the most dramatic effect on the efficiency of milk production in this decade. Moreover, numerous studies have shown that milk produced by bST-dosed cows is safe for human consumption. The use of bST is also expected to lower environmental pollution, because the decreased intake of feed relative to milk output will decrease the production of manure, urine, and methane-a gas with a strong greenhouse effect. Simply put, fewer cows are required to produce the same amount of milk. Despite these benefits, bST has elicited considerable public outcry. Consumer groups are threatening to boycott the milk from bST supplemented cows, grocery chains and food processing companies refused the milk that was approved by the FDA for sale during the investigation period, and states have considered or taken action either banning the use of bST or requiring labeling of products derived from its use [46].

Figure 3: Production of bST and recombinant of DNA technology. (http://mmg-233-2013-genetics-genomics.wikia.com/wiki/Bovine_somatotropin).

Though ten states introduced bills restricting the use of bST and two states actually enacted such laws, only a few individuals generated the original controversy. Samuel Epstein,

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a professor at the University of Illinois, joined with genetic engineering critic Jeremy Rifkin to attack the use of bST. In an evaluation of other scientific studies, Epstein questioned the safety of bST-produced milk for human consumption; in addition, he concluded that bST adversely affects the health of cows. Rifkin’s group, Foundation on Economic Trends (F.E.T.), demanded the release of environmental assessment records relating to bST. F.E.T. also petitioned the FDA for an environmental impact statement prior to field testing bST. The petition asserted that use of bST would (1) significantly affect agricultural land use in milk-producing regions of the United States; (2) adversely affect the internal environment of cattle injected with [bST]; and (3) have adverse economic and social impacts on the dairy industry [47].

The bST controversy illustrates some of the difficulties that may be encountered during the introduction of bioengineered food products. Since biotechnology products are already suspect in the public eye, they are easily attacked by a vocal minority. Even if they meet the current regulatory standards, they are especially vulnerable to criticisms that they have adverse environmental, economic, or social impacts that may result from modern dairy or agricultural practices as a whole rather than just from biotechnology. State or local agencies may impose additional regulation that could impede the development of the biotechnology industry and delay advances that might actually be environmentally, economically, or socially beneficial. The question, then, is what regulatory balance should be struck between the potential or perceived risks of biotechnology and its unknown but almost certain benefits [48].

Risks and regulatory issues concerning bioengineered foodAlthough new and unknown technologies are often viewed with suspicion, some features

of biotechnology make it particularly susceptible to an exaggerated perception of risk. Public concern may stem from scientists themselves, who initiated a moratorium on some aspects of genetic engineering in 1974. While scientists have since grown comfortable with the technology, the public perception of unreasonable risks lingers on. A recent survey found that 52% of the public “believes that genetically engineered products are at least somewhat likely to represent a serious danger to people or the environment.” Biotechnology often suggests the “Frankenstein image.” While the current technology generally changes but a single gene, producing a relatively small modification, many people may believe that any interspecies exchange of genetic information results in a dramatic change. Perhaps such views underlie the finding that 24% of a group aware of biotechnology felt that creation of hybrid plants and animals through genetic engineering is morally wrong. Another aspect of biotechnology that invites public concern is the ability of living things to reproduce; thus any deleterious effects of genetically engineered organisms have the ability to escape human control and self-perpetuate [48,49].

This leads to a fear that although a deleterious result is unlikely, if it occurs, the outcome could be a problem of substantial magnitude. Such fears of an unlikely but potentially disastrous outcome could greatly hinder the progress of biotechnology. A majority of the public would object to the use of genetically engineered organisms if the risk were unknown.46 Food products of biotechnology generate their own specific concerns. Production of bioengineered food usually involves not only a consideration of the safety of the food for human consumption, but also the safety of environmental release of the altered plant. The public’s perception of potential danger from food biotechnology is enhanced by its heightened awareness of environmental damage from the introduction of exotic species and of health problems that are manifested only decades after exposure to the causative agent. Yet many similar risks from food stem from traditional agricultural and plant breeding practices that are essential to provide sufficient food to the growing population or to assure the taste, quality, and convenience that consumers and farmers have come to expect. Thus, society accepts environmental risks

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of pesticide use and dispersal of domesticated plants and animals within certain limits and tolerates low levels of pesticide residues in food. Other risks from food are inherent in the food itself. Food contains many naturally occurring toxicants and carcinogens that are nearly unavoidable in the ordinary diet [48,49].

Biotechnology presents few risks beyond those already accepted in traditional foods. As to their environmental risks, “crops modified by molecular and cellular methods should pose risks no different from those modified by classical genetic methods for similar traits.” Bioengineered organisms’ potential for dispersal and environmental disruption is generally similar to their traditional counterparts. Society has long accepted the fact that traditional plant and animal breeding practices may change the nutrient or toxicant levels in the food or alter an organism’s potential for environmental dispersal. Although traditional methods usually enhance the safety of the food, they have occasionally increased the level of a deleterious component. The use of antibiotic resistance marker genes in the production of bioengineered food has raised some questions, but most experts agree that the genes should cause no health or safety problem. Bioengineering as an extension of traditional breeding practices, should pose no greater concern over the safety of the food consumed; it should actually be safer since the recombinant techniques are more specific and thus less likely to produce unwanted side effects such as increased levels of toxicants or weediness. Indeed, as considered above, bioengineering may lower both the environmental and food consumption risks [48,49].

Thus, to foster technological advance and its resultant benefits, Huber argues that a comparative system of regulation of old and new risks, one that permits new technologies functionally similar to established technologies and of no greater risk, should be implemented. The comparative approach, allowing a new risk, is justified when the old, risky product is one that society accepts either because it is essential or desirable. “Excessively strict regulation of the safer-than-average products will drive consumption toward the more hazardous ones.” Comparative regulations, on the other hand, would favor the safer product, particularly because modern technology usually replaces an old outmoded source of risk rather than adding to it [48,49].

Huber suggests a four-step process for implementing comparative regulation:

1) The agency must define a risk market comprising products that are functional substitutes for each other.

2) It next must identify typically risky products already allowed to compete in that market.

3) The agency must then compare the risks of the new substitute with those of products not in fixed supply and already in the market. Only the less safe substitutes must be excluded or otherwise regulated.

4) If a new product offers exceptional price or other advantages over existing, more hazardous products, introduction of the safer product could conceivably increase net risk by increasing total consumption. As a final step in comparative regulation, an agency must therefore consider whether a candidate for regulation is this type of risk.

The regulatory framework for bioengineered food is in transition from an approach that, by focusing on the process used to produce genetically engineered food, did not always accurately assess the risk of the product. The Bush Administration sought to cure this problem by adopting a policy similar to the comparative regulatory approach discussed above. The federal regulatory agencies are currently implementing this policy. This policy approach has the advantage of removing unjustified oversight of biotechnology, but it may have the disadvantage of under regulating the field, especially because it relies on existing statutory authority not directed at biotechnology. Moreover, this policy approach does not address the non-risk based

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social and economic concerns that contribute to the public’s objections to biotechnology. It may, therefore, fuel the demand for state and local regulation of biotechnology [48,49].

The regulatory frameworkRegulation of bioengineered food falls under the general regulatory scheme that has been

established for biotechnology as a whole.

The “Coordinated Framework for the Regulation of Biotechnology” (coordinated framework), introduced by the Office of Science and Technology Policy (OSTP) in 1985-86, describes the policies for federal regulation of biotechnology. Under the coordinated framework, regulation of biotechnology relies on existing federal statutes, with each agency maintaining jurisdiction over biotechnology applications within its traditional domain. Oversight of each product is within a single agency, but where more than one agency is involved, one is designated the lead agency. Agencies rely upon existing statutory authority to provide immediate health and safety protection, as well as to eliminate any regulatory delay or uncertainty that might hurt the new biotechnology industry. Underlying this decision was the premise that genetic engineering techniques are basically extensions of the traditional techniques of selective breeding and hybridization, and thus the laws that governed products of those techniques could also apply to biotechnology [48,49].

The Biotechnology Science Coordinating Committee (BSCC), established by the OSTP in 1985, had broad authority for promoting cooperation between the agencies and establishing consistent scientific policy and reviews. It was composed of senior policy officials from the United States Department of Agriculture (USDA), the FDA, the National Institutes of Health (NIH), the Environmental Protection Agency (EPA), and the National Science Foundation (NSF). In late 1990, the BSCC was replaced by the Biotechnology Research Subcommittee (BRS) of the interagency Committee on Health and Life Sciences. The BRS is said to have responsibilities similar to the BSCC. Although the BSCC, in its evaluation of the issues, could “develop generic scientific recommendations that could be applied to similar, recurring applications,” it did not re-evaluate agency decisions and thereby delay that agency’s response. Two important facets of BSCC’s initial mission were to ensure that its constituent agencies regulate biotechnology using scientific reviews of similar stringency, and to establish consistency as to which genetically engineered organisms were subject to regulatory oversight [48,49].

The FDA has followed a policy, consistent with that stated in the coordinated framework, that oversight of biotechnology products under its jurisdiction requires no new procedures or requirements. The FDA is responsible for assuring the safety and quality of both plant and animal bioengineered food products. New animal drugs, including those produced by biotechnology, require pre market approval by the FDA. Moreover, the FDA must approve for human consumption the edible portions of animals that have been administered a new drug. To approve the use, the FDA must confirm the safety of the food product for human consumption; the drug must not accumulate as unsafe residues in the edible portions of the animal. Finally, the efficacy of the drug and its safety for both the animals and the environment must be established. When a new drug produced by genetic engineering is virtually identical to an approved substance produced by conventional technology, the showing for approval is reduced and only a supplemental application to the FDA is necessary. Regulation of biotechnology-derived foods from plants will depend largely on the use of the food. Generally, regulations differ for “whole foods” such as fruits, vegetables, or grains; for substances unintentionally added to foods; and for food additives. The FDA does not require pre market approval for

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whole foods, but the burden is on the producer or manufacturer to assure that such foods are safe. However, the FDA can regulate whole foods, including those that are products of biotechnology, under section 402(a)(1) of the FDCA, which sets different safety standards for inherent natural constituents of the food and unintentionally added substances that are poisonous or deleterious. Naturally occurring constituents posing safety problems, such as elevated levels of solanine in a new potato variety or poisons in a toxic mushroom, make the food legally adulterated only “if the quantity of such substance[s] ordinarily renders[s] it injurious to health.” Unintentionally added substances on the other hand, are contaminants and are subject to a more rigorous standard. The contaminants may be chemicals introduced accidentally by human activities (e.g., Polychlorobiphenyls (PCBs), mercury, and lead) or they may be naturally occurring contaminants (e.g., aflatoxin). “Added substance[s]” causes a food to be legally adulterated if they “may render it injurious to health.” Adulterated food is subject to an enforcement action if it enters into interstate commerce [48,49].

Food additives are subject to pre market clearance by the FDA, unless the additive is Generally Recognized As Safe (GRAS). A substance is GRAS either if its safety is known from common use in foods consumed by a significant number of consumers prior to January 1, 1958, or if its safety is determined by well-controlled scientific studies. A company may market a product, believing it to be GRAS, but it runs the risk that the FDA may decide that it is not GRAS and force it off the market. A company wishing to clarify the matter at the outset may obtain the FDA’s opinion on the substance by filing a GRAS affirmation petition. If a substance added to food is not GRAS, it is a food additive and under section 409 of the FDCA a company must submit a food additive petition for FDA approval. Thus, if a bioengineered product is a food additive, it requires submission of scientific data showing that it is safe under the conditions for which it will be used. Moreover, under the requirements of NEPA, the manufacturer must prepare an environmental assessment or an impact statement if the manufacturing process or the use of the food additive will significantly affect the environment [48,49].

Regulation under the coordinated framework: Not surprisingly, regulation of bioengineered food products, and biotechnology in general, is often viewed as too stringent by biotechnology companies, as too lax by environmentalists, and as lacking a sound scientific basis by academicians. Biotechnology companies often cite regulatory uncertainty as a substantial concern in developing new products. In recent years, the main regulatory hurdle that food biotechnology companies have had to face has involved the release of genetically engineered organisms [50].

The companies acknowledge that APHIS’ handling of small field tests has worked well and that many delays have been due to suits or to local restrictions on release. Thus, at the federal level, industry concerns for the future stem from uncertainty over regulation of large scale release of genetically engineered crops during commercialization; the adequacy of coordination between the EPA, the USDA, and the FDA when a single product requires oversight by all three agencies; and over how the FDA will handle food products. Industry may actually welcome a case-by-case analysis of the first bioengineered foods, because FDA approval will give an assurance of safety that will boost public confidence in the products. In the long run, however, industry representatives feel that bioengineered foods should require no more screening than traditionally produced foods. Since under current law this would mean that genetically engineered foods classified as whole foods would require no pre market approval, the International Food Biotechnology Council, an industry association, recommends that the FDA establish a voluntary pre market notification system. However, industry’s greatest

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regulatory concern may not be with federal regulations but with the increasing patchwork of state and local regulations. This concern, which will be considered further below, has led industry to lobby for more explicit federal regulation [50].

Environmental groups have faulted the coordinated framework for incompletely regulating biotechnology through existing statutes not directed at genetic engineering and for not keeping environmental considerations paramount. Environmentalists have criticized the use of the Federal Plant Pest Act to regulate environmental release of genetically engineered agricultural plants and animals because it covers only plant pests. Although use of the Ti plasmid as a vector has brought most plant genetic engineering under the authority of the regulations, environmentalists are concerned that increased use of other means of introducing foreign DNA will leave many bioengineered plants unregulated. Moreover, environmentalists and university researchers are concerned that USDA’s statutory authority is inadequate to cover genetically engineered animals. Finally, environmentalists argue that the EPA, rather than the USDA, should be the lead agency in charge of environmental release of genetically engineered organisms, since EPA’s mandate is to protect the environment as a whole, whereas USDA’s interest is to promote agriculture [50].

Specific policy changes reflecting this philosophy have been proposed both for the regulation of the release of bioengineered food producing organisms and the assessment of food safety and quality. The new policy concerning the release of food producing organisms mandates equivalent, risk-based regulation of traditional and genetically engineered organisms, and thus should lower industries’ burden of federal regulation. The new policy no longer specifies that “inter generic organisms” or “pathogenic” species require oversight. The policy broadly covers all types of genetic modifications, including those resulting from traditional methods, by stating that “a determination to exercise oversight should not turn on the fact that an organism has been modified by a particular process or technique.” Rather, all organisms should be regulated according to the risk of introducing them into a particular environment. Federal agencies should not exercise oversight of such introductions unless the risk is unreasonable. However, federal agencies need not choose between imposing or not imposing oversight. Agencies have a range of options, such as “issuance of suggested industry practices, development of guidelines for certain introductions, and requirements for notification, labeling, prior review or approval of certain introductions.” In determining what level of oversight should be applied, the policy adopts a comparative approach similar to Huber’s. Thus, “an introduction should be subject to no greater degree of oversight than was a comparable organism or product previously used in past safe introductions in a comparable target environment.” In short, this policy suggests a comparative approach to regulation by applying comparable oversight to comparable organisms in similar environments. Such an analysis would apply whether the new organism is, for example, a genetically engineered variety or a newly introduced exotic species [50,51].

One feature of this assessment, in addition to qualitative and analytical evaluation, is a comparative approach. Acceptable and unacceptable levels of certain food constituents are determined by reference to the host plant when that plant has a history of safe use. By following FDA’s guidelines, a manufacturer is expected to determine whether the product is safe, requires FDA consultation because of questionable safety, or is unsafe. Thus, the FDA suggests that the level of toxicants in the new variety should be within the range of toxicant levels in the host variety, and that “the concentration and bioavailability of important nutrients in the new variety should be within the range ordinarily seen in the host species.” If toxicant levels present a safety concern, the food is unacceptable; if nutrient levels are outside of the

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normal range, the manufacturer must consult the FDA to determine its course of action. The primary concern raised by the donor species is the potential transfer of allergens or toxicants to the host. The manufacturer must consult the FDA if it is possible that allergens have been transferred from the donor to the host plant. The assessment of food safety may entail qualitative, as well as quantitative comparisons. Thus, a manufacturer must consult the FDA if the introduced protein, carbohydrate, fat, or oil is likely to be a major component of the diet and is not derived from an edible source, or differs substantially from that in the edible source [50,51].

Comparison to the corresponding unmodified organism essentially regulates products of biotechnology on a par with organisms produced by traditional methods. This type of comparison equilibrates the new and old risks by removing new risks commensurate with old, unregulated risks from the regulatory process. However, because it requires a judgment by the manufacturer involving risks that may be unknown and unquantifiable, environmentalists and others skeptical of the new technology may feel that it leaves too many products of that technology unregulated or under regulated. Critics may also think that manufacturers using the new technology have too much discretion to unilaterally decide whether their products meet the required standards. Moreover, it is difficult to see how agencies can apply a risk-based policy to certain organisms (e.g., transgenic fish) perceived to be immune to oversight due to gaps in statutory authority. Nor does the policy address ideological deficiencies that result from using existing statutory and regulatory authority. Thus, while the policy may ease regulatory burdens at the federal level, it may create a backlash from the public and from state and local bodies that perceive greater risks from biotechnology and wish to regulate it more stringently [50,51].

Horizontal Gene TransferHorizontal gene transfer (HGT) is the process by which an organism transfer genetic

material to another organism other than its offspring and which is followed by integration and expression of the genetic material. This process is common among bacteria and other prokaryotes. Speculation that HGT could occur between ingested bioengineered food and enteric bacteria present in the human mouth, stomach, and gut has been expressed. Of special concern are bioengineered foods made from transgenic plants that express antibiotic-resistance markers (ARMs), which are employed during the development of the transgenic plant to select for those that have incorporated the transgenes. When humans ingest food derived from plants that express an ARM, it is theoretically possible that the ARM could be taken up and stably integrated into enteric bacteria through HGT, resulting in bacteria that are resistant to specific antibiotics. This situation has never been reported, although studies point to its possibility. The epsps transgene, which confers resistance to a common herbicide, survives intact through the small intestine of humans when bioengineered food made with Roundup Ready soybeans (resistant to the herbicide glyphosate, commonly called Roundup®) is consumed. Also, M13 bacteriophage DNA has been shown to survive transiently in the gastrointestinal tract of mice and is able to enter the bloodstream. However, these studies demonstrate only the ability of certain DNA molecules to resist degradation by salivary and gastric enzymes; no studies to date have demonstrated the ability of the DNA molecules to become stably integrated into the bacterial genome by HGT [50,51].

Some consumers have reported concerns that consumption of bioengineered foods means that humans will ingest the “foreign” DNA present in transgenes. A DNA sequence of particular concern is the cauliflower mosaic virus 35S promoter, commonly used to direct expression of plant transgenes. This promoter is efficient and functional in a variety of organisms, and

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it has been suggested that it might lead to inappropriate over expression of genes in species into which it is transferred and promote HGT, or recombine with dormant endogenous viruses present in humans, leading to new infectious viruses. However, almost all genomes of human endogenous retroviruses contain lethal mutations that prevent replication and production of viral particles. Also, the cauliflower mosaic virus is present naturally in approximately 10% of cabbages and cauliflowers, and so is regularly ingested by humans. No adverse consequences from the consumption of this virus have been reported [50,51].

Food-grade micro-organismsThe use of micro-organisms (Table 1) in food production is accepted when they have a long

history of safe use. However it is not scientifically defined what a long history is and what a safe use is. Moreover, a recent draft document suggests that only micro-organisms that have a long and safe history of use in food and that also have a qualified presumption of safety status (see below) should be applied in the food industry. These strains could be used as recipients for genetic modification. The use of pathogenic micro-organisms should not be allowed. However, the history of safe use in food should not be an everlasting guarantee that the strain could always be applied in food fermentation processes. If new research shows that strains with a long history of safe use are producing toxic components in levels that may harm human health, these strains should not be accepted anymore for use in food fermentations. For instance, certain LAB has an unblemished history of safety in food fermentation, but, as was discovered later, may produce unfavorable amines under some conditions. Evidently these strains should not be used in preparation of foods without a profound safety assessment, which, especially in this case, also investigates the actual concentrations of the harmful compound to which the consumer will be exposed [50,51].

Host strain Donor strain Gene involved Intended effect Modification technique

Reference

Lb. bulgaricus Not applicable lacZ limited lactose fermentation

IS mediated deletion Mollet and Delley, 1990

L. lactis Not applicable ldh and others increased carbon dioxide production

spontaneous and induced random mutagenesis

El Attar et al. 2000

L. lactis Not applicable ldh and others increased acetoin and diacetyl production

NNG induced random mutagenesis

Boumerdassi et al. 1997

L. lactis Not applicable aldB increased diacetyl production

NNG induced random mutagenesis,

Monnet et al. 2000

L. lactis Not applicable aldB increased diacetyl production

Spontaneous random mutagenesis

Goupil et al. 1996

L. lactis Not applicable ribC increased riboflavin production

induced random mutagenesis

Burgess et al. 2003

S. thermophilus Not applicable gal operon fermentation of galactose spontaneous random mutagenesis

Vaughan et al. 2001

L. lactis Not applicable glk, eIIman/glc no glucose fermenting capacity

spontaneous random mutagenesis

Thompson et al. 1985.

L. lactis L. lactis aldB increased diacetyl production

double crossover homologous recombination

Swindell et al. 1996

L. lactis Lb. helveticus pepN, pepX, pepC, pepI

modulation of proteolytic system for enhancement cheese ripening

food grade vector cloning

Joutsjoki et al. 2002

L. lactis L. delbrueckii pepI, pepL, pepW, pepG

modulation of proteolytic system for enhancement cheese ripening

NICE System Wegmann et al. 1999

http://www.ejbiotechnology.info/content/vol9/issue4/full/12/index.html#80

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L. lactis Peptostreptococcus asaccharolyticus

Gdh increased production of alpha-ketoglutarate

Vector cloning Rijnen et al. 2000

L. lactis lytic phage phi31 phage inducible promoter

expression of lethal three-gene restriction cassette LlaIR+,

Vector cloning Djordjevic et al.1997

L. lactis lytic phage phi31 anti sense phage RNA

silencing of phage genes Vector cloning Walker and Klaenhammer, 1998

L. lactis lytic phage anti sense phage RNA

silencing of phage genes encoding structural genes

Vector cloning Kim et al. 1992

L. lactis Not applicable pip inactivation of phage infection protein


Monteville et al. 1994

L. lactis strains L. lactis strains lacticin encoding genes

lacticin production Conjugation O’Sullivan et al. 2003

L. lactis strains L. lactis strains lacticin encoding gene

lacticin production conjugation (plasmid stacking)

Mills et al. 2002

L. lactis S. thermophilus abiA, abiG abortion of cells upon phage induction

Vector cloning Tangney and Fitzgerald, 2002

L. lactis and others

Pediococcus acidilactici and others

lcnC, lcnD lantibiotic production Vector cloning Horn et al. 1999

L. lactis phage lytA, lytH production of lysin and holin

NICE de Ruyter et al. 1997

L. lactis S. thermophilus Sfi6 EPS gene cluster altered EPS production Vector cloning Stingele et al. 1999L. lactis S. thermophilus

Sfi39EPS gene cluster altered EPS production Vector cloning Germond et al. 2001

Lb. gasseri L. lactis folate gene cluster introduction folate biosynthesis pathway

Vector cloning Wegkamp et al. 2004

S. thermophilus Not applicable pgmA, gal U inactivation of phosphoglucomutase

double crossover homologous recombination and vector cloning

Levander et al. 2002

L. lactis B. sphaericus ldh, alaD, alr rerouting of pyruvate to L-alanine

double crossover homologous recombination, vector cloning

Hols et al. 1999

L. lactis L. lactis riboflavin gene cluster

overexpression riboflavin biosynthesis pathway

Vector cloning Burgess et al. 2003

L. lactis L. lactis folate gene cluster overexpression folate biosynthesis pathway

Vector cloning Sybesma et al. 2003b

L. lactis L. lactis glk, pfnABCD, pfcBA, genes lactose-PTS and tagatose-6P

inactivation of glucose fermenting system and introduction of lactose fermentation

double crossover homologous recombination, vector cloning

Pool et al. 2003

L. lactis L. lactis galA, aga introduction a-galactosidase activity

food grade vector cloning

Boucher et al. 2002

Lb. plantarum B. subtilis phyC introduction phytase activity

Vector cloning Kerovuo and Tynkkynen, 2000

Lb. plantarum L. amylovorus amyA introduction a-amylase activity

chromosomal integration

Fitzsimons et al. 1994

Lb. plantarum B. stearothermophilus, C. thermocellum

α-amylase gene, celA

introduction a-amylase and cellulase activity

single homologous recombination

Scheirlinck et al. 1989

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S. thermophilus S. thermophilus glyA increased acetaldehyde production

Vector cloning Chaves et al. 2002

Lb. fermentum Not applicable ldhD, ldhL increased mannitol production


Aarnikunnas et al. 2003

Lb. helveticus Not applicable ldhD production of pure L-(+) lactic acid


Kyla-Nikkila et al. 2000

Lb. delbrueckii Not applicable EPS genes altered EPS production Chemically induced random mutagenesis

Welman et al. 2003

S. thermophilus S. thermophilus bacteriophage

anti sense phage RNA, helicase gene

silencing of phage genes Vector cloning Sturino and Klaenhammer, 2002

Streptococcus mutans

Zymomonas mobilis ldh, adh prevention of dental caries double crossover homologous recombination, gene replacement

Hillman, 2002

Lb. delbrueckii Not applicable β-galactosidase gene

Increased b-galactosidase activity

EMS or NNG induced random mutagenesis

Ibrahim and O'Sullivan, 2000

L. lactis Eimeria tenella, L. plantarum

M1Pase gene, MtlD

increased mannitol production

NICE Wisselink et al. 2005

L. casei ATCC 334

L. casei LC202 dhic increased alpha-keto acid dehydrogenase activity

Vector cloning Broadbent et al. 2004

L. plantarum Lactobacillus sake promotors and regulatory genes

increased gene expression

Vector cloning Mathiesen et al. 2004

L. lactis L. lactis Prt+- and Lac+-derivatives of L. lactis MG1363

increased proteolytic and acidifying activity

conjugation Picon et al. 2005

L. lactis S. simulans Lss production of lysostaphin NICE Mierau et al. 2005L. lactis P. stipitis XYL1 production of xylitol Vector cloning Nyyssölä et al. 2005L. lactis Not applicable unknown increased oxidative-stress

tolerancespontaneous mutations (natural selection)

Rochat et al. 2005

L. lactis L. lactis nisRK, nisFEG increased nisn Z production

Vector cloning Cheigh et al. 2005

L. lactis Z. mobilis, L. lactis pdc, nox increased acetaldehyde production

NICE Bongers et al. 2005

L. lactis G. stearothermophilus, L. Brevis, L. lactis

sgsE, slpA, usp production of excreted S-layer protein

NICE Novotny et al.2005

L. lactis E. coli gshA, gshB increased glutathion production

NICE Li et al.2005

L. lactis, L. paracasei

L. paracasei groEL increased stress tolerance NICE Desmond et al. 2004

L. lactis Not applicable unknown increased L(+)-actate production

random mutagenesis Bai et al. 2004

S. thermophilus Not applicable deoB, gst, rggC, and unknown

increased oxidative-stress tolerance

random insertional mutagenesis

Fernandez et al. 2004

EMS: ethyl methanesulfonate; NNG: N-methyl-N'-nitro-N-nitrosoguanidine

Table 1: Overview of lactic acid bacteria with controlled or uncontrolled genetic alterations.

Controlled genetic alteration of LAB (Lactic Acid Bacteria)Relation of a specific aspect to the application of vectors in industrial strain improvement is




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the use of selection markers. The use of antibiotic resistance markers might result in transfer of antibiotic resistance from one organism to another. As a consequence, the practical value of antibiotics that are used for human health or in veterinary practice will be severely reduced. Hence, food-grade resistance markers are preferred. Currently there are many food-grade selection markers for vector cloning in LAB. For instance, transfer of the α-galactosidase gene (aga) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (galR) of Lactococcus raffinolactis ATCC 43920 into L. lactis and Pediococcus acidilactici strains modifies the sugar fermentation profile from Melibiose negative (Mel(-)) to Melibiose positive (Mel(+)). A similar food-grade vector is based on complementation of the lactose operon in L. lactis NZ3600 or L. casei by introduction of lacF, or lacG, respectively, enabling growth on lactose. An alternative system is based on a suppressor tRNA allowing growth in milk of a purine auxotrophic strain. A newly developed food-grade marker is characterized by the requirement of D-alanine in the medium to enable growth of the micro-organisms. An overview of general strategies for constructing food-grade markers has previously been reported. As mentioned before, the current use of these food-grade markers if constructed via self-cloning techniques is restricted to applications with contained use [50,51].

Other targeted modifications of the genetic content of DNA may occur via conjugation and transduction. These processes are considered natural events. According to the current legislation, bacteria that are changed by using these transfer systems are not considered as GMOs (Table 2).

LAB has a long history of use by man for food production and food preservation. LAB is Gram-positive, non-spore forming bacteria and naturally present in raw food material and in the human gastro-intestinal tract. The heterogeneous group of LAB includes the rod-shaped bacteria like lactobacilli, and cocci such as streptococci, lactococci, pediococci and leuconostocs. LABS are widely used as starter cultures for fermentation in the dairy, meat and other food industries. Their properties have been used to manufacture products like cheese, yoghurts, fermented milk products, beverages, sausages, and olives. These food-grade bacteria can also improve the safety, shelf life, nutritional value, flavor and quality of the product. Moreover, LAB can be used as cell factories for the production of food additives and aroma compounds. It is further assumed that LAB may function as probiotics and contribute to the general health of the consumer upon consumption. The use of probiotics falls currently within a grey area between food and medicine and many health claims assigned to probiotics are not yet scientifically proven. Another application - the use of LAB in the production of proteins for application in health care or for development of new vaccines is more related to Pharma than to food. In the future it is predicted that knowledge about the interaction between LAB and the human host will open new avenues for developing LAB which support human health [50-53].

Modification of DNA Directed genetic alteration

Un-controlled genetic alteration

Acceptance of contained use, 90/219/EC

Acceptance of deliberate release, 2001/18/EC

Spontaneous mutations - + + non-GMO + non-GMOInduced mutations - + + non-GMO + non-GMOMutations via insertion elements - + + non-GMO + non-GMOConjugation + - + non-GMO + non-GMOTransduction + - + non-GMO + non-GMOSelf-cloning + - + non-GMO - GMONon-self-cloning + - - GMO - GMO

Table 2: Types of DNA modification methods and the acceptability to be used in food production.

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ConclusionsClaims by a manufacturer that a food or its ingredients, including foods such as raw

agricultural commodities, is not bioengineered should be able to substantiate that the claim is truthful and not misleading. Available validated testing is the most reliable way to identify bioengineered foods or food ingredients. For many foods, however, particularly for highly processed foods such as oils, it may be difficult to differentiate by validated analytical methods between bioengineered foods and food ingredients and those obtained using traditional breeding methods. Where tests have been validated and shown to be reliable they may be used. However, if validated test methods are not available or reliable because of the way foods are produced or processed, it may be important to document the source of such foods differently. Also, special handling may be appropriate to maintain segregation of bioengineered and non-bioengineered foods. In addition, manufacturers should consider appropriate recordkeeping to document the segregation procedures to ensure that the food’s labeling is not false or misleading. In some situations, certifications or affidavits from farmers, processors, and others in the food production and distribution chain may be adequate to document that foods are obtained from the use of traditional methods. A statement that a food is “free” of bioengineered material may be difficult to substantiate without testing. Because appropriately validated testing methods are not currently available for many foods, it is likely that it would be easier to document handling practices and procedures to substantiate a claim about how the food was processed than to substantiate a “free” claim.

References1. Arshad SH, Malmberg E, Krapf K, Hide DW (1991) Clinical and immunological characteristics of Brazil nut allergy. Clin Exp Allergy

21: 373-376.

2. Astwood JD, Leach JN, Fuchs RL (1996) Stability of food allergens to digestion in vitro. Nat Biotechnol 14: 1269-1273.

3. Berberich SA, Ream JE, Jackson TL, Wood R, Stipanovic R, et al. (1996) The composition of insect-protected cottonseed is equivalent to that of conventional cottonseed. J. Agric. Food Chem 44: 365–371.

4. Betz FS, Hammond BG, Fuchs RL (2000) Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regul Toxicol Pharmacol 32: 156-173.

5. Carver JD (1999) Dietary nucleotides: Effects on the immune and gastrointestinal systems. Acta Paed 88: 83–88.

6. CDC (2001) Investigation of human health effects associated with potential exposure to genetically modified corn. A Report to the U.S. Food and Drug Administration from the Centers for Disease Control and Prevention.

7. Conner AJ, Jacobs JM (1999) Genetic engineering of crops as potential source of genetic hazard in the human diet. Mutat Res 443: 223-234.

8. Doerfler W (1991) Patterns of DNA methylation--evolutionary vestiges of foreign DNA inactivation as a host defense mechanism. A proposal. Biol Chem Hoppe Seyler 372: 557-564.

9. Doerfler W (2000) Foreign DNA in Mammalian Systems. Wiley-VCH, Weinheim.

10. FAO/WHO (2000) Safety aspects of genetically modified foods of plant origin: Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agriculture Organization of the United Nations and World Health Organization. WHO, Geneva, Switzerland.

11. FAO/WHO (2001) Safety assessments of foods derived from genetically modified microorganisms. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agriculture Organization of the United Nations and World Health Organization. WHO, Geneva, Switzerland.

12. FDA (1992) Statement of policy: Foods derived from new plant varieties. Food and Drug Administration. Fed. Reg. 57:22984–23002.

13. Flavell RB, Dart E, Fuchs RL, Fraley RT (1992) Selectable marker genes: safe for plants? Biotechnology (N Y) 10: 141-144.

14. Gatehouse AM, Ferry N, Raemaekers RJ (2002) The case of the monarch butterfly: a verdict is returned. Trends Genet 18: 249-251.

http://www.ncbi.nlm.nih.gov/pubmed/1863901



http://pubs.acs.org/doi/abs/10.1021/jf950304i

http://pubs.acs.org/doi/abs/10.1021/jf950304i




http://www.cdc.gov/nceh/ehhe/cry9creport/pdfs/cry9creport.pdf

http://www.cdc.gov/nceh/ehhe/cry9creport/pdfs/cry9creport.pdf





http://onlinelibrary.wiley.com/book/10.1002/9783527613465

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http://www.who.int/foodsafety/publications/biotech/en/ec_sept2001.pdf



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15. Goldsbrough AP, Tong Y, Yoder JI (1996) Lc as a non-destructive visual reporter and transposition excision marker gene for tomato. Plant J. 9:927–933.

16. Hammond BG, Vicini JL, Hartnell GF, Naylor MW, Knight CD, et al. (1996) The feeding value of soybeans fed to rats, chickens, catfish and dairy cattle is not altered by genetic incorporation of glyphosate tolerance. J Nutr 126: 717-727.

17. Kimber I, Dearman RJ (2001) Can animal models predict food allergenicity? Nutr. Bull 26:127–131.

18. Koprek T, McElroy D, Louwerse J, Williams-Carrier R, Lemaux PG (2000) An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function. Plant J 24: 253-263.

19. Kuiper HA, Kleter GA, Noteborn HP, Kok EJ (2001) Assessment of the food safety issues related to genetically modified foods. Plant J 27: 503-528.

20. Lemaux PG, Frey P (2002) Biotechnology Information.

21. Losey JE, Rayor LS, Carter ME (1999) Transgenic pollen harms monarch larvae. Nature 399: 214.

22. Lozzia GC, Furlanis C, Manachini B, Rigamonti IE (1998) Effects of Bt corn on Rhopalosiphum padi L. (Rhynchota, Aphididae) and on its predator Chrysoperla carnea Stephen (Neuroptera chrysopidae). Boll. Zool. Agrar. Bachicolt 30:153–164.

23. MacKenzie D (1999) Unpalatable truths. New Sci 162:18–19.

24. Maryanski JH (1995) Food and Drug Administration policy for foods developed by biotechnology. In Genetically Modified Foods: Safety Issues (K.-H. Engel, G. R. Takeoka, and R. Teranishi, Eds.), pp.12–22. ACS Symposium Series No. 605. American Chemical Society, Washington, DC.

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