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USE OF BIOTECHNOLOGY TECHNIQUE LIKE PCR & RFLP IN FOOD INDUSTRY
B.K.K.K.Jinadasa, Department of Food Science & Technology, University of Sri Jayawardanapura, July-2009
Introduction: ...…………………………………………………………………………………………..1 Polymerase Chain Reaction: .....................................................................................................1 Restriction Fragment Length Polymorphism:........................................................................... 2 Uses of Biotechnology: ................................................................................................................... 3 Agricultural Biotechnology: ....................................................................................................... 4 Biotechnology use in food industry: ......................................................................................... 5 Issues and relevant to food industry:............................................................................................ 8 Conclusion:....................................................................................................................................... 8 References:....................................................................................................................................... 9
1.0 Introduction: According to the Codex statements in March 2000, biotechnology is “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use”. As the some other definition "Any
technological application that uses biological systems, dead organisms, or derivatives thereof, to make or modify products or processes for specific use." A broad definition of biotechnology is "The application of indigenous and/or scientific knowledge to the management of (parts of) microorganisms, or of cells and tissues of higher organisms, so that these supply goods and services of use to the food industry and its consumers. Biotechnology provides powerful tools for the sustainable development of agricultural, fisheries and forestry and food industry. There are different techniques and applications. Biotechnology has included wide range f diverse technologies and they may be applied in many of the different field in food and agricultural sectors. Biotechnology includes technology such as gene modification and transfer, use of the molecular markers, development of recombinant vaccines and DNA based methods like PCR for diseases characterization and diagnostics, in vitro vegetative propagation of plants, embryo transferring technique and other reproductive technologies in animals like fish, DNA typing and cloning of plants and animals. Tissue culture has produced plants that are increasing crop yields by providing farmers with healthier planting material. Rice has been genetically engineered to contain pro-vitamin A (beta carotene) and iron which could improve the health of many low income communities. As well as biotechnology includes a range of technologies used to process the raw food materials produced by crops, fishery and livestock sectors. Biotechnology in the food processing sector targets the selection and improvement of microorganisms with the objectives of improving process control, yield and efficiency as well as quality safety and consistency of bioprocesses products. Microbes are generic terms for the group of living organisms which are microscopic in size. It includes bacteria, yeast and moulds. 1.1. Polymerase Chain Reaction:
In 1983 Kary B. Mullis was driving through California on a moonlight night. He was pondering how to use DNA polymerase with oligonucleotide primers in order to identify a given nucleotide at a given position in a complex DNA molecule, such as the human genome. During this drive he invented or discovered the elegant method of making unlimited DNA copies from a single copy of DNA, and called the method: "Polymerase Chain Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten years after his drive in California, he was awarded the Nobel Prize in Stockholm for his brilliant discovery (Carr, 1993). PCR was first published in 1985 with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published. This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR has become commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple
amplification and detection, and many extensions of the original PCR method have been described. The PCR process was originally developed to amplify short segments of a longer DNA molecule. A typical amplification reaction includes the target DNA, a thermo stable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor of more than a million in a matter of hours. Each cycle of PCR includes steps for template denaturation, primer annealing and primer extension. The initial step denatures the target DNA by heating it to 94°C or higher for 15 seconds to 2 minutes. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermo stable DNA polymerase. In the next step of a cycle, the temperature is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermo stable DNA polymerases, this temperature is in the range of 70–74°C. The extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C for denaturation. Each step of the cycle should be optimized for each template and primer pair combination. If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. After 20–40 cycles, the amplified product may then be analyzed for size, quantity, sequence, etc., or used in further experimental procedures.
Restriction Fragment Length Polymorphism: RFLP (pronounced as a rif-lip) mentioned in the name are sections of a DNA sequence which have been cut by a restriction enzymes. Lengths of the fragments are polymorphic because certain differences in DNA strands such as base substitutions, additions, deletions and other sequence rearrangements. Researchers have developed a method of comparing DNA sample based on this differential cleavage of DNA by restriction enzymes. First step of perform RFLP of extracted DNA from the sample. The DNA must then cut with one or more restriction enzymes. Each resulting fragments is placed into the agarose gel. The electrophoresis separates the restriction fragments by size. The shorter fragments are travel more quickly than larger ones. The final step is visualizing the DNA. When DNA running with Ethilium Bromide, are facilitates to see the bands fluorescence under UV light. Biotechnology facilitates in agricultural fields to produce hybrid seeds, bio fertilizers, bio pesticides, plant extract, plant genetic engineering and plant tissue culture fields also. At the food industrial field biotechnology use in industrial enzymes, bio fuels, polymers, fermented products, microbial strains, biocatalysts and oligonucleotides etc.
2.0 Uses of Biotechnology: 2.1 Agricultural Biotechnology: World population is increasing very fast; concern about that the quality of food, bio agricultural technique should developed to fulfil that needs. Normally agricultural farmers wish to take more return than investment and also increase productivity by using these techniques. The challenge of producing more food grains to feed the ever increasing world population. Plant breeding has been enhanced considerably by in vitro development of improved verities which are better adapted to a specific environment. Using the techniques of modern biotechnology, one or two genes (Smartstax from Monsanto will use 8, starting in 2010) may be transferred to a highly developed crop variety to impart a new character that would increase its yield. However, while increases in crop yield are the most obvious applications of modern biotechnology in agriculture, it is also the most difficult one. Current genetic
engineering techniques work best for effects that are controlled by a single gene. Many of the genetic characteristics associated with yield (e.g., enhanced growth) are controlled by a large number of genes, each of which has a minimal effect on the overall yield. There is, therefore, much scientific work to be done in this area. The application of tissue cultural has some advantages like rapid reproduction and multiplication, availability of seed materials throughout the year. Tissue cultural technique very popular technique, because do not need very high tech instruments, and knowledge. As an example tissue cultural technique use to take banana seed in Sri Lanka. As well as biotechnology help to reduce the use of agrochemicals. It will help to enhance the productivity, reduction in toxic elements in final product and environment also. Biotechnology use many ways to increase yield; for example by increasing flowering capacity and increasing photosynthesis. The plant cloning can help reduce the work in harvesting period. It can use to take more uniform character in plant like growing speed, ripening at same time. As well as scientist have been able to produce insect resistant plant verities such as BT corn. Food shortages would not exist in many countries if the problems of post harvest losses could be solved. In the future genetic engineer may be used to remove plant components that cause early deterioration of the harvest. Recently the genetic engineer had used to take box type water lemon, it is reduced the storage space when transport. Other example is reduce the presence of normal tomato enzymes involved in the softening of ripe tomato fruit has been patented and would be found very useful for enhance the shelf life of crops varies verities. When we consider livestock and poultry fields; several growth enhancing novel genes have been introducing into pigs. By using this several qualities enhance the meat; example meat is lean and tenderer. Many modifications to milk have been done by gene manipulation technology; as an example Newzealand developed GM cows that produce milk with increased levels of casein protein. Use of that protein increases the efficiency of cheese production. Other application of genetic modification in animal production in the early stages of research and development include improvement of disease resistance, increased birth rates of sheep, altered sex ration in poultry, increased egg production in poultry by creating two
active ovaries, and improved feed conversion in the pig. When we consider aquaculture field, enhanced the growth of Atlantic salmon containing a growth hormone gene from Chinook salmon is likely to be first GM animal on the food market. These fish grow 3-5 times faster than their non transgenic variety. At least eight other farmed fish species have been genetically modified for growth enhancement. One of above species has introduced in Sri Lanka few years ago call GIFT Tilapia (genetically improved farm Tilapia). 2.2 Biotechnology use in food industry: Biotechnology in food processing sector targets the selection and improvement of microorganisms with the objectives of improving process control, yields and efficiency as well as the quality, safety and consistency of bio-processed products. Fermentation is one of the oldest forms of food preservation method which is conversion of organic substance by microorganisms or enzyme of microbial, plant or animal origin. Bread, cheese, wine, yogurt, sausages, soy sauce, cider, cocoa, coffee, kefir, miso, salami, sauerkraut are the typical example for that. Food fermentation is important to food safety, nutritional value and food security. Even though fermentation technology has been used many years, the technology is very simple as well as not developed comparing with time. Some bacteria used in food fermentation produce compounds that kill other food poisoning and spoilage bacteria. Microorganisms have been essential in food industry as a source of many food additives and processing aids. Many food additives are substances nature has provide microbial origin, such as xanthan gum, guar gum. Many of amino acid supplements, flavours, flavour enhancers, vitamins added to breakfast cereals are produced by microbial fermentation. The enzymes produced by microbial fermentations play essential roles as processing aids in the food industry. Before the bio technique, the enzymes like rennin had are extracted from stomach of calves, lambs and bay goats, but it now produced by microorganisms that were given the gene for this enzymes. Modern biotechnology can be used to slow down the process of spoilage so that fruit can ripen longer on the plant and then be transported to the consumer with a still reasonable shelf life. This alters the taste, texture and appearance of the fruit. More importantly, it could
expand the market for farmers in developing countries due to the reduction in spoilage. However, there is sometimes a lack of understanding by researchers in developed countries about the actual needs of prospective beneficiaries in developing countries. For example, engineering soybeans to resist spoilage makes them less suitable for producing tempe which is a significant source of protein that depends on fermentation. The use of modified soybeans results in a lumpy texture that is less palatable and less convenient when cooking. The first genetically modified food product was a tomato which was transformed to delay its ripening. Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are currently working on delayed-ripening papaya in collaboration with the University of Nottingham and Zeneca. Biotechnology in cheese production; enzymes produced by micro-organisms provide an alternative to animal rennet – a cheese coagulant - and an alternative supply for cheese makers. This also eliminates possible public concerns with animal-derived material, although there are currently no plans to develop synthetic milk, thus making this argument less compelling. Enzymes offer an animal-friendly alternative to animal rennet. While providing comparable quality, they are theoretically also less expensive. About 85 million tons of wheat flour is used every year to bake bread. By adding an enzyme called maltogenic amylase to the flour, bread stays fresher longer. Assuming that 10-15% of bread is thrown away as stale, if it could be made to stay fresh another 5–7 days then perhaps 2 million tons of flour per year would be saved. Other enzymes can cause bread to expand to make a lighter loaf, or alter the loaf in a range of ways. When we consider health factors, plant scientist have able to make healthier cooking oils which is decreased in total amount of saturated fatty acids in certain vegetable oils. Other vice normal hydrogenation process of oil form trans-fatty acids. But using biotechnology heat stability of oil has improved. Produce high protein potato verity using amaranth gene transfer, canola oil with high amount of vitamin A, increase the ellagic acid like cancer protective agent in strawberry, are the other example for health and nutrional and health benefits. Most of food allergens also can remove from food by using biotechnology.
Biotechnology helps to improve product quality, especially in raw materials quality. Increase the shelf life of fresh fruits and vegetables using this technique, improving the crispness of carrots, peppers and celery, certain seedless verities of grapes and melons, improving the flavour of tomato, lettuce, pepper, peas and potatoes and creating caffeine free coffee and tea. Recently the Japanese scientists have identified the gene which is produce enzymes that make s cry when we slice onion. By using biotechnology, they are able to make the tearless onion. The normal potato with high water content absorbed the high amount of oil when frying process, it is not healthier. Now scientist able to produce high starch potato verity, which are absorbed less oils as well as less cost to handle. Biotechnology is helping many ways to enhance food safety. It is providing many tools to detect microorganisms and the toxins they produce. Monoclonal antibody tests, biosensors, PCR, and DNA probes are being developed to determine the presence of harmful bacteria such as Listeria and Clostridium, E. coli 0157:H7. Biotechnology based detection method have been developed now to detect toxin like aflatoxin. Unhygienic processing and handling help to add food borne pathogens into the food, it will critical for ensuring the safety to consumers. Traditional detection method takes need 3-4 days, but biotechnological method need maximum 36 hrs and it also specific, sensitive and faster than normal conventional method. As well as PCR based method are also used for the detection of ingredients in food product such as soy in meat products, whet in non whet products or allergens in diverse food. 3.0. Issues and relevant to food industry: Application of biotechnology to food processing in developing countries is in issues for a long time. A Socio economic and cultural factor is one of issues. Traditional food fermentation processes are low input with minimal investment requirement. These product and process normally uncontrolled, unhygienic and inefficient as well as product variable quality, short shelf life. But fermented food play major role in food security and nutrition in developing countries.
Infrastructural and logistic factors are the next important one. Physical infrastructural requirement need for the manufacture, distributor and storage of generally these technologies. Other issues are the consumer education, intellectual property rights should be considering. 4.0. Conclusion: DNA-based methods are increasingly used for the detection of foreign food constituents, such as microbial pathogens, or the presence of genetically modified crop material. Furthermore, the detection of plant and animal species as well as allergens, certain ingredients or contaminants in the final food products has been shown to be feasible with DNA-based methods. The methods are in general fast, very specific and provide a sensitive tool for the detection of specific food constituents. The choice of the analytical method applied is however, mainly dependent on the food concerned (availability of specific PCR primers) and on the history of processing involved during food production (degradation or even removal of DNA). However, it has to be considered that PCR-based assays detect only the presence of DNA from a living entity. However, this is not always the compound of concern. PCR-based assays do not detect for example the allergen or mycotoxin itself. Therefore, the PCR-based result cannot be tied to actual allergenic exposure or an actual contamination of the sample with mycotoxins. Nevertheless, PCR-based detection methods provide an excellent alternative to more traditional methodologies in the quality and safety assurance of food. References: U.S. Department of State International Information Programs, “Frequently Asked Questions about biotechnology”, USIS online available from: http://usinfor.state.gov/ei/economic_issues/biotechnology/biotech_faq.html. Bio-2008: biotechnology industry organization, Washington DC, www.bio.org Molecular biology training manuals-2007: Anfaco - Cecopesca, Spain. Executive summary of food biotechnology in Asia, 2008: Asian food information centre (AFIC), www.afic.org.
Modern food biotechnology, human health and development an evidence based study, 2005: World health organization, www.who.int.foodsafety
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