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ilsi europe concise monographs this booklet is one in a series of ilsi europe concise monographs the concise monographs are written for those with a general background in the life sciences however the style and content of these monographs will also appeal to a wider audience who seek up-to-date and authoritative reviews on a variety of important topics relating to nutrition health and food safety concise monographs present an overview of a particular scientific topic and are usually based on proceedings of scientific meetings the text of each concise monograph is peer reviewed by academic scientists of high standing the concise monographs make important results and conclusions available to a wider audience the following titles are available in the series starches and sugars a comparison of their metabolism in man a simple guide to understanding and applying the hazard analysis critical control point concept food allergy and other adverse reactions to food dietary fibre oxidants antioxidants and disease prevention caries preventive strategies sweetness the biological behavioural and social aspects concise monographs in preparation include dietary fat some aspects of nutrition and health and product development the nutritional and health aspects of sugars nutritional epidemiology limits and possibilities international life sciences institute and ilsi europe the international life sciences institute ilsi is a worldwide non-profit foundation headquartered in washington d.c usa with branches in argentina australia brazil europe japan mexico north america southeast asia and thailand with a focal point in china ilsi is affiliated with the world health organization as a non-governmental organization ngo and has specialised consultative status with the food and agriculture organization of the united nations ilsi europe was established in 1986 to provide a neutral forum through which members of industry and experts from academic medical and public institutions can address topics related to health nutrition and food safety throughout europe in order to advance the understanding and resolution of scientific issues in these areas ilsi europe is active in the fields of nutrition and food safety it sponsors research conferences workshops and publications for more information about its programmes and activities please contact ilsi europe avenue e mounier 83 box 6 b-1200 brussels belgium telephone 32 2 771.00.14 telefax 32 2 762.00.44[close]
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the use of trade names and commercial sources in this document is for purposes of identification only and does not imply endorsement by the international life sciences institute ilsi in addition the views expressed herein are those of the individual authors and/or their organisations and do not necessarily reflect those of ilsi copyright © 1995 by the international life sciences institute all rights reserved no part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior written permission of the copyright holder ilsi press 1126 sixteenth street n.w washington d.c 20036 usa telephone 1 202 659-0074 telefax 1 202 659-8654 ilsi europe avenue e mounier 83 box 6 b-1200 brussels belgium telephone 32 2 771.00.14 telefax 32 2 762.00.44 printed in belgium isbn 0-944398-62-6[close]
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foreword the professor to his cook you are a little opinionated and i have some trouble making you understand that the phenomena which take place in your laboratory [kitchen are nothing other than the eternal laws of nature and that certain things which you do without thinking and only because you have seen others do them derive nonetheless from the highest scientific principles jean anthelme brillat-savarin the physiology of taste 1825 the sentiments expressed by brillat-savarin over a century ago are even more relevant today as the technology associated with food production processing and preparation becomes increasingly complex traditional biotechnology brewing baking and other fermentation processes has been associated with food for thousands of years now modern biotechnology and genetic modification in particular is beginning to make an impact given food s deep cultural importance it is hardly surprising that this has created anxieties this monograph which aims to address those concerns and to describe the opportunities presented by modern biotechnology is in five sections in the first two parts the technology of genetic modification is explained numerous examples of its use in food production are described in the third section several case studies then illustrate how enzymes are used in food processing showing how many modern practices originated from ancient traditions finally some of the social economic and safety implications of genetic modification are examined author dean madden scientific editors david a jonas axel kahn michael teuber series editor nicholas j jardine[close]
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contents what is biotechnology methods used in biotechnology genetics and genetic modification other biotechnological methods food production microbial production of food food additives and processing aids plant biotechnology animal biotechnology examples of the use of enzymes in food processing cheese manufacture fruit juice production sweetener production regulatory safety and socioeconomic considerations food safety social economic and other effects the need for public involvement bibliography 1 2 2 10 12 12 16 23 26 26 27 29 31 31 35 36 37[close]
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food biotechnology 1 what is biotechnology the term biotechnology was first coined by a hungarian karl ereky towards the end of world war i ereky used the word to refer to intensive agricultural methods since that time biotechnology has been variously defined but it has nearly always been associated with food production and processing in particular biotechnology has usually encompassed the traditional manufacture of bread wine cheese and other fermented foods on these grounds biotechnology can trace its roots back several thousand years to the ancient sumerians who brewed beer with naturally occurring yeasts ancient fermentations were not always successful the microbes that fell into the wine maker s vat could yield the finest vintage or transform the entire product to vinegar in the 1800s louis pasteur laid the foundations of microbiology and identified microorganisms bacteria fungi algae and protozoa as the cause of both desirable and undesirable changes in food the application of pasteur s research led to safer more reliable food processing and preservation and helped ensure the consistent high quality of for example fine wines and cheeses pasteur asserted that fermentation processes were inextricably linked to the activities of living microbes towards the end of the last century it was discovered that cell-free extracts from yeast could also bring about chemical changes without the intervention of the microbes from which they were derived the active components of such extracts were named enzymes enzyme means in yeast enzymes are proteins made by all living things that catalyse specific chemical reactions without realizing it the makers of cheese had always used a mixture of natural enzymes rennet to transform milk into solid curds and liquid whey during the 1940s large-scale fermentation equipment was developed which led to the efficient industrial production by microorganisms of pure enzymes and additives and other valuable compounds such as vitamins for use in food just as different breweries have their own carefully maintained proprietary strains of yeast enzyme manufacturers culture specially selected strains of their chosen microorganisms over many years great improvements have been made to the efficiency of production and the safety quality and range of microbial products available however much still depends on chance occurrence followed by systematic isolation of organisms with desirable characteristics with the advent during the 1970s of the ability to make precise changes to genetic material biotechnology was transformed the performance of organisms can now be fine tuned and biotechnology has now almost became a synonym for genetic modification in 1980 an influential british report the spinks report attempted to encapsulate nearly half a century of european and united states thought defining biotechnology as the application of biological organisms systems or processes to the manufacturing and service industries this broad definition suits our purposes as it includes the production of food by living organisms its subsequent processing with the assistance of microbes or enzymes and the assurance of food quality and safety using the tools of molecular biology.[close]
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2 concise monograph series methods used in biotechnology genetics and genetic modification chromosomes genes and dna genes passed from one generation to the next determine all inherited characteristics genes are made from dna deoxyribonucleic acid most of which is packaged in fungal including yeast plant and animal cells into chromosomes within the nucleus of the cell figure 1 some genes are also found outside the nucleus in the mitochondria which release energy for cellular activities and within the chloroplasts sites of photosynthesis of plant cells in bacteria most genes occur on a single circular chromosome although small rings of dna called plasmids may also be present the double helix of dna can be likened to a twisted rope ladder the two intertwined helices are chains made from sugar and phosphate molecules linked together alternately attached to each sugar molecule is a base there are four different bases adenine a thymine t cytosine c and guanine g weak bonds between the bases join the two strands of the double helix together like the rungs of a ladder a always pairs with t and c always pairs with g this base pairing mechanism ensures identical replication of dna strands during cell division figure 2 a particular gene a stretch of dna with a particular sequence determines the structure of all or part of a specific protein figure 3 the sequences of bases in the dna specify the amino acid residues that are needed to make proteins three bases in a row specify each amino acid and the sequence that specifies each the genetic code is the same in all living organisms see figure 2 also encoded within the dnaare instructions to regulate protein production although all cells of an organism will contain the same dna only certain proteins will be made at any one time or in any particular type of cell that is only certain genes will be expressed dna has an identical structure in all living things and because the genetic code is universal the possibility is raised that genes can be transferred between completely different species the process of transferring removing or altering genetic information by the modification of dna is commonly called genetic modification or genetic engineering why alter nature in nature proteins are often made in minute quantities and are therefore difficult or impossible to extract and purify these proteins include enzymes and a variety of pharmacologically active compounds such as insulin for diabetics interferons for cancer therapy and vaccines to help prevent diseases often large quantities of such valuable proteins are needed biotechnologists can achieve this by transferring the relevant genes into microbes that can easily be cultivated in large numbers the same technology applied to the production of food could bring significant benefits improved varieties for agriculture animal and plant breeders have for centuries selected livestock and plants with desirable characteristics breeding from chosen stock is a very slow process that can be set back by the chance recombination of genes in the offspring a breeder may select a preferred trait only to find that it is accompanied by an equally undesirable one which then has to be painstakingly bred out traditional methods of selecting the best plants or animals from which to breed have been greatly aided by modern genetic techniques furthermore it is now[close]
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food biotechnology 3 figure 1 the essential structure of bacterial animal and plant cells chromosome figure 2 the structure of dna the hereditary material three bases in a row code for amino acids that are the building blocks of proteins bacterial cell plasmids small rings of dna dna structure cytoplasm ribosomes sites of protein synthesis cell wall 0,5 µm cell membrane nucleotide animal cell nucleus cell membrane chromosomes made from dna protein cytoplasm 15 µm mitochondria sites of energy release have their own dna first position second position third position hydrogen bonds base sugar phosphate ribosomes plant cell chloroplast site of photosynthesis has its own dna cell membrane the genetic code mitochondrion cytoplasm vacuole nucleus containing chromosomes 30 µm cell wall ribosomes[close]
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4 concise monograph series figure 3 protein synthesis dna messenger ribonucleic acid mrna amino acids amino acid chain protein gene bases mrna ribosome amino acid chains dna determines hereditary characteristics by directing the formation of particular proteins a stretch of dna carrying the instructions required to make a particular protein is called a gene dna operates through an intermediary rna the sequence of bases on the dna molecule acts as a template for the production of messenger rna mrna the mrna binds to the ribosomes where it directs the production of the protein it encodes three mrna bases in a ro w specify each amino acid to be added to the protein the amino acid chains that proteins are formed from fold into precise threedimensional structures contributing to the protein s properties and function possible to make very precise changes to the genetic material this can help improve the resistance to disease and environmental stress amongst crop plants and farm animals it can also help boost agricultural productivity and enhance the nutritional status storage properties and ease of processing of food products food additives and processing aids enzymes are specialized proteins that are essential for life they catalyse all biological processes and thus control metabolism in living organisms once extracted from living organisms these proteins allow certain processes in food production to be conducted for thousands of years enzymes such as rennet from animals and papain from plants have been used to enhance the flavour texture and appearance of food because of the diversity of microorganisms it has been possible to find a wide range of microbial enzymes that are active in the conditions encountered in food processing with genetic modification a greater range of pure and highly specific enzymes can be produced more efficiently these enzymes can be used to make desirable changes to food both rapidly and at relatively low temperatures with a subsequent reduction in fuel requirements and in the environmental impact of food processing to the consumer the direct benefits include better flavour texture and shelf life of food often with a reduction in the need for processing and additives.[close]
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food biotechnology 5 figure 4 restriction enzymes recognise and cut dna molecules at precise locations the names of these enzymes are derived from those of the organisms that produce them the sequences of bases that are recognised by three enzymes bamhi hindiii and ecori are shown here bamhi hindiii ecori how genetic modification is done cutting and pasting dna special enzymes obtained from bacteria are an essential tool of the molecular biologist in nature these enzymes help bacteria fend off viral attack by precisely dissecting the foreign dna of invading viruses in this way the proliferation of the viruses is restricted restriction enzymes as they are known recognize and cut dna molecules at specific locations figure 4 many hundreds of restriction enzymes have been isolated from different microbes and are available commercially with restriction enzymes almost any section of dna and consequently any single gene can be excised at will the end of one dna molecule will readily link to that of another that has been cut with the same enzyme to join two dna molecules permanently it is necessary to form chemical bonds along the dna s sugar-phosphate backbone an enzyme called dna ligase can do this job the function of these cut and paste enzymes in assembling novel dna molecules is obvious but the genetic engineer s tool kit would be incomplete without one or two other enzymes to understand their role it is necessary to appreciate how proteins are made a genetic intermediary the genetic information encoded in dna lies within the nucleus of the cell however proteins are not made in the nucleus but elsewhere at special structures called ribosomes before a particular protein can be made a copy of the appropriate instructions must first be transcribed from the dnaand then ferried to the ribosomes the copied instructions are made from mrna messenger ribonucleic acid this mrna is virtually a mirror image of the sequence of bases on one dna strand according to the basepairing rules upon arrival at the ribosomes the base sequence within the mrna directs the construction of proteins from amino acids a sequence of three adjacent bases in the mrna molecule is needed to determine each amino acid in the protein see figure 3 cells that are producing a particular protein will have many identical copies of that protein s mrna inside them it is often easier to search for genes among the small mrna molecules rather than along the entire length of the cell s dna once a desired length of mrna has been isolated two additional enzymes are needed.[close]
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6 concise monograph series dna from rna the enzyme reverse transcriptase assembles a single strand of complementary dna alongside a corresponding piece of mrna a second enzyme dna polymerase can then be used to construct a double-stranded helix using the first dna strand as a template dna made in this way is called copy or complementary dnacdna a copy of a gene from a donor cell is used in genetic modification gene synthesis by the judicious use of restriction and other enzymes molecular biologists are able to assemble dna molecules which contain one or more genes of interest where a particular piece of dna is difficult to isolate it is sometimes possible to make it artificially using a dna synthesizer under computer control these devices string together the biochemical precursors needed to make short stretches of dna of course to programme the synthesizer it is necessary to know the sequence of bases present in the desired gene this too can be determined automatically using a dna sequencer it is also possible to copy specific genes using the polymerase chain reaction pcr the pcr has been likened to a genetic photocopier from a very small amount of dna millions of copies of a specific section of dnacan be made quickly the pcr lies behind many of the spectacular successes of forensic genetic fingerprinting where criminals have been identified from the dna in just a few drops of blood or even a couple of cells on a cigarette butt plasmids once a suitable dna molecule has been constructed it must be moved into a cell in which it can be expressed and duplicated so that it passes from one cell division to the next for microorganisms one of the most successful methods involves the use of plasmids as a vehicle for transferring genes plasmids are rings of dna that are found in some cells they carry a limited set of genes and normally constitute only a few percent of a cell s total dna during the course of evolution plasmids carrying genes that help their microbial hosts survive have been selected by nature some plasmids confer on their hosts the ability to degrade substances in the environment such as nutrients and antibiotics many traditional foods such as yoghurt cheese and other fermented dairy products contain large numbers of living microbes that naturally harbour plasmids like the dna of chromosomes that of plasmids can be cut with restriction enzymes and additional dna pasted into it the result is a ring of recombinant dna that can be put into a bacterium specialized plasmids can be used to ferry genes from bacteria into yeast cells or even plants figure 5 a limitation of plasmids is that they cannot accommodate dnafragments longer than 15 00020 000 base pairs however some harmless specially tailored viruses can package larger dna molecules such viruses have been used to transfer genes into microbes plants and animals and even to treat human disease genetically modified plants a vector system that is used for a wide variety of plants is the plant tumour-inducing plasmid ti-plasmid found in the soil bacterium agrobacterium tumefaciens through its plasmid agrobacterium has the ability to naturally engineer plant cells so that they grow tumours that produce compounds which the bacteria need to sustain themselves molecular biologists use disarmed nontumour-inducing versions of this plasmid to introduce foreign genes of their choice into plants because every cell carries a complete copy of all the plant s genes in its chromosomes it is possible to regrow an entire plant from a single modified cell see cell culture below specially modified ti-plasmids have now been produced which help transfer fairly large genes into plants unfortunately monocotyledons including the important cereal crops are resistant to agrobacterium.[close]
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food biotechnology 7 figure 5 pure chymosin for cheese making can now be obtained from genetically modified yeast dna copy of chymosin gene chymosin from modified yeast cells sample of calf cells chymosin gene inserted into plasmid calf plasmid put into yeast cells agrobacterium has proved especially useful when working with trees which because they are slow growing and large are difficult to improve by conventional breeding apricot plum apple and walnut trees have all been genetically modified with agrobacterium figure 6 gene ballistics a procedure called ballistic bombardment has achieved success with several crops including rice wheat and soya with this method the dna to be introduced into the plant cells is first stuck onto minute tungsten or gold particles the dna-coated particles are fired at high velocity into soft plant tissue usually callus see cell culture below this introduces functional dna into the plant cells electroporation dna can also be introduced into the thin-walled tubes which develop from pollen grains by subjecting them to microsecond pulses of a strong electric field this technique called electroporation causes pores to appear momentarily in the pollen tubes through which dna from a surrounding solution can enter seeds that develop from ovules fertilized with such pollen carry the introduced genes electroporation also works with plant cells from which the cell wall has been removed by enzyme treatment from these naked plant cells whole plants can be regenerated by cell culture see cell culture below electroporation has also been used to transfer dna molecules into a broad spectrum of microorganisms.[close]
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8 concise monograph series figure 6 agrobacterium naturally infects some types of plants and introduces novel dna into them biologists use specially tailored forms of agrobacterium to modify plants genetically isolate desired gene from donor organism remove unwanted genes from plasmid restriction enzyme cuts dna at specific places dna ligase joins dna fragments restriction enzyme cuts dna at specific places insert selected gene into cut plasmid float leaf discs on a suspension of agrobacterium plasmid bacterial chromosome put novel plasmid into agrobacterium tumefaciens bacteria enter cells at cut edge and introduce novel plasmid into plant tissue cultivate leaf discs on nutrient medium regenerate whole plants which now contain the introduced gene genetically modified animals the dna of animals can also be modified by genetic engineering it is necessary to introduce genes at an early stage of development if they are to be present in all of the cells of a mature animal and be passed on to its offspring dna can be injected into newly fertilized egg cells through a very fine glass pipette only a small proportion of such injected eggs take up the new genes the injected eggs are transferred into the uterus of a suitable foster mother this is the only method so far[close]
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food biotechnology 9 figure 7 dna probes can be used to detect particular genes or fragments of dna that have been isolated from organisms this allows the presence of genetic characteristics to be confirmed rapidly without the need to for example grow a plant to maturity dna fragments placed in these wells cdna or rna probe added probe revealed e.g by chemiluminescence or autoradiography dna is isolated from the test organism and amplified if necessary the dna is cut into fragments by a restriction enzyme dna fragments migrate towards the positive electrode the fragments are separated by size in a slab of agarose gel the dna is transferred to a nylon membrane the probe binds to complementary sequences of dna the probe reveals the presence of dna to which it has bound that works for cows pigs sheep and goats microinjection can also be used to introduce new genes into fish eggs but it is not suitable for the eggs of birds however specially modified viruses have been used to introduce for example disease resistance into chickens the viruses which are made harmless are inserted through the shell of the egg marker genes and gene probes whatever method is used at best only a small proportion of treated cells take up the introduced dna screening is therefore necessary to discover which cells have done so as mentioned above plasmids often carry genes which help the microbes that possess them break down particular antibiotics these genes can be used as markers to identify those cells which have taken up plasmids for when the cells are placed in a growth medium which contains an appropriate antibiotic only those with plasmids will thrive several different types of marker genes exist within the plant kingdom and these are being developed as alternatives to antibiotic markers other methods for identifying transferred genes include the pcr this method enables the amplification of transplanted dna sequences which can then be detected using gene probes gene probes are small fragments of singlestranded dna or rna which bind to complementary sequences in the dna that is being sought out figure 7 probes can also be used to detect the dnaof microorganisms that might contaminate food they are so sensitive and specific that it is possible to use probes to differentiate between strains of the same species and to determine whether particular microorganisms are capable of producing toxins genetic switches to ensure that the recipient cell s biochemical machinery will allow introduced genes to be expressed sequences of dna called control regions are required one well-studied control region switches[close]
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