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I L S I E U R O P E C O N C I S E M O N O G R A P H S E R I E S

F O O DB I OT E C H N O L O G YAN INTRODUCTION

ILSI Food biotec for pdf 14/04/99 10:54

ILSI Europe

by Dean Madden

FOOD BIOTECHNOLOGYAN INTRODUCTION

ILSI Food biotec for pdf 14/04/99 10:55 Page 3

ILSI EUROPE CONCISE MONOGRAPHS

This booklet is one in a series of ILSI Europe concisemonographs. The concise monographs are writtenfor those with a general background in the lifesciences. However, the style and content of thesemonographs will also appeal to a wider audiencewho seek up-to-date and authoritative reviews on avariety of important topics relating to nutrition,health and food safety.

Concise monographs present an overview of aparticular scientific topic and are usually based onproceedings of scientific meetings. The text of eachconcise monograph is peer reviewed by academicscientists of high standing. The concise monographsmake important results and conclusions available toa wider audience.

The following titles are available in the series:

◆ Starches and Sugars: A Comparison of TheirMetabolism in Man

◆ A Simple Guide to Understanding and Applying theHazard 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: DietaryFat – Some Aspects of Nutrition and Health and ProductDevelopment; The Nutritional and Health Aspects ofSugars; Nutritional Epidemiology: Limits andPossibilities.

The International Life Sciences Institute (ILSI) is aworldwide, non-profit foundation headquartered inWashington, D.C., USA, with branches in Argentina,Australia, Brazil, Europe, Japan, Mexico, NorthAmerica, Southeast Asia, and Thailand, with a focalpoint in China.

ILSI is affiliated with the World Health Organization asa non-governmental organization (NGO) and hasspecialised consultative status with the Food andAgriculture Organization of the United Nations.

ILSI Europe was established in 1986 to provide aneutral forum through which members of industry andexperts from academic, medical and public institutionscan address topics related to health, nutrition and food

safety throughout Europe in order to advance theunderstanding and resolution of scientific issues inthese areas. ILSI Europe is active in the fields ofnutrition and food safety. It sponsors re s e a rc h ,conferences, workshops and publications.

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Copyright © 1995 by the International Life Sciences Institute

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ISBN 0-944398-62-6


FOREWORD

The professor to his cook: You are a little opinionated and Ihave some trouble making you understand that thephenomena which take place in your laboratory [kitchen] arenothing other than the eternal laws of nature, and that certainthings which you do without thinking, and only because youhave seen others do them, derive nonetheless from the highestscientific principles.

Jean Anthelme Brillat-Savarin The Physiology of Taste, 1825

The sentiments expressed by Brillat-Savarin over acentury ago are even more relevant today as thetechnology associated with food production, processingand preparation becomes increasingly complex.Traditional biotechnology — brewing, baking and otherfermentation processes — has been associated with

food for thousands of years. Now modern bio-technology, and genetic modification in particular, isbeginning to make an impact. Given food's deepcultural importance, it is hardly surprising that this hascreated anxieties.

This monograph, which aims to address those concernsand to describe the opportunities presented by modernbiotechnology, is in five sections. In the first two partsthe technology of genetic modification is explained.Numerous examples of its use in food production aredescribed in the third section. Several case studies thenillustrate how enzymes are used in food processing,showing how many modern practices originated fromancient traditions. Finally, some of the social, economicand safety implications of genetic modification areexamined.

Author: Dean MaddenScientific Editors: David A. Jonas, Axel Kahn,

Michael TeuberSeries Editor: Nicholas J. Jardine


CONTENTS

What is biotechnology? ........................................................................................................................................................ 1

Methods used in biotechnology .......................................................................................................................................... 2

Genetics and genetic modification ........................................................................................................................... 2

Other biotechnological methods ............................................................................................................................. . 10

Food production ................................................................................................................................................................... . 12

Microbial production of food, food additives and processing aids ................................................................... 12

Plant biotechnology ................................................................................................................................................... 16

Animal biotechnology ............................................................................................................................................... 23

Examples of the use of enzymes in food processing ....................................................................................................... 26

Cheese manufacture ................................................................................................................................................... 26

Fruit juice production ................................................................................................................................................ 27

Sweetener production ................................................................................................................................................ 29

Regulatory, safety and socioeconomic considerations ..................................................................................................... 31

Food safety ................................................................................................................................................................. . . 31

Social, economic and other effects ........................................................................................................................... 35

The need for public involvement ............................................................................................................................. 36

Bibliography .......................................................................................................................................................................... 37


WHAT IS BIOTECHNOLOGY?

The term "biotechnology" was first coined by aHungarian, Karl Ereky, towards the end of World War I.Ereky used the word to refer to intensive agriculturalmethods. Since that time biotechnology has beenvariously defined, but it has nearly always beenassociated with food production and processing. Inparticular biotechnology has usually encompassed thetraditional manufacture of bread, wine, cheese andother fermented foods. On these grounds, biotech-nology can trace its roots back several thousand years tothe ancient Sumerians, who brewed beer with naturallyoccurring yeasts.

Ancient fermentations were not always successful. Themicrobes that fell into the wine maker's vat could yieldthe finest vintage or transform the entire product tovinegar. In the 1800s Louis Pasteur laid the foundationsof microbiology and identified micro o rganisms —bacteria, fungi, algae and protozoa — as the cause ofboth desirable and undesirable changes in food. Theapplication of Pasteur's research led to safer, morereliable food processing and preservation and helpedensure the consistent high quality of, for example, finewines and cheeses.

Pasteur asserted that fermentation processes wereinextricably linked to the activities of living microbes.Towards the end of the last century it was discoveredthat cell-free extracts from yeast could also bring aboutchemical changes without the intervention of themicrobes from which they were derived. The activecomponents of such extracts were named enzymes(enzyme means "in yeast"). Enzymes are proteins, madeby all living things, that catalyse specific chemicalreactions. Without realizing it, the makers of cheese hadalways used a mixture of natural enzymes — rennet —to transform milk into solid curds and liquid whey.

During the 1940s, large-scale fermentation equipmentwas developed which led to the efficient industrialproduction by microorganisms of pure enzymes andadditives and other valuable compounds (such asvitamins) for use in food.

Just as different breweries have their own carefullymaintained proprietary strains of yeast, enzymemanufacturers culture specially selected strains of theirchosen micro o rganisms. Over many years gre a timprovements have been made to the efficiency ofp roduction and the safety, quality and range ofm i c robial products available. However, much stilldepends on chance occurrence followed by systematicisolation of organisms with desirable characteristics.

With the advent during the 1970s of the ability to makeprecise changes to genetic material, biotechnology wastransformed. The performance of organisms can now be"fine tuned", and biotechnology has now almost becamea synonym for "genetic modification". In 1980, aninfluential British report (the "Spinks Report")attempted to encapsulate nearly half a century ofEuropean and United States thought, defining biotech-nology as "the application of biological organisms, systemsor processes to the manufacturing and service industries".This broad definition suits our purposes, as it includesthe production of food by living organisms, itssubsequent processing with the assistance of microbesor enzymes and the assurance of food quality and safetyusing the tools of molecular biology.

Food Biotechnology 1


METHODS USED INBIOTECHNOLOGY

Genetics and genetic modification

Chromosomes, genes and DNA

Genes, passed from one generation to the next,determine all inherited characteristics. Genes are madefrom DNA (deoxyribonucleic acid), most of which ispackaged, in fungal (including yeast), plant and animalcells, into chromosomes within the nucleus of the cell(Figure 1). Some genes are also found outside thenucleus: in the mitochondria (which release energy forcellular activities) and within the chloroplasts (sites ofphotosynthesis) of plant cells. In bacteria, most genesoccur on a single circular chromosome, although smallrings of DNA called plasmids may also be present.

The double helix of DNA can be likened to a twistedrope ladder. The two intertwined helices are chainsmade from sugar and phosphate molecules linkedtogether alternately. Attached to each sugar molecule isa "base". There are four different bases: adenine (A),thymine (T), cytosine (C) and guanine (G). Weak bondsbetween the bases join the two strands of the doublehelix together like the rungs of a ladder. A always pairswith T, and C always pairs with G. This "base pairing"mechanism ensures identical replication of DNAstrands during cell division (Figure 2).

A particular gene (a stretch of DNA with a particularsequence) determines the structure of all or part of aspecific protein (Figure 3). The sequences of bases in theDNA specify the amino acid residues that are needed tomake proteins. Three bases in a row specify each aminoacid, and the sequence that specifies each — the geneticcode — is the same in all living organisms (see Figure 2).

Also encoded within the DNAare instructions to regulateprotein production. Although all cells of an organism willcontain the same DNA, only certain proteins will be madeat 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, andbecause the genetic code is universal, the possibility israised that genes can be transferred between completelydifferent species. The process of transferring, removing oraltering genetic information by the modification of DNAis commonly called genetic modification (or geneticengineering).

Why alter nature?

In nature, proteins are often made in minute quantitiesand are therefore difficult or impossible to extract andpurify. These proteins include enzymes and a variety ofpharmacologically active compounds such as insulin fordiabetics, interferons for cancer therapy and vaccines tohelp prevent diseases. Often large quantities of suchvaluable proteins are needed. Biotechnologists canachieve this by transferring the relevant genes intomicrobes that can easily be cultivated in large numbers.The same technology, applied to the production of food,could bring significant benefits.

I m p roved varieties for agriculture . Animal and plantbreeders have for centuries selected livestock and plantswith desirable characteristics. Breeding from chosen stockis a very slow process that can be set back by the chancerecombination of genes in the offspring. A breeder mayselect a preferred trait, only to find that it is accompaniedby an equally undesirable one, which then has to bepainstakingly bred out.

Traditional methods of selecting the best plants oranimals from which to breed have been greatly aided bymodern genetic techniques. Furthermore, it is now

2 Concise Monograph Series



FIGURE 1.

The essential structure of bacterial, animal and plantcells

Bacterial cell

chromosome plasmids,small rings of

DNA

cell wall

cell membrane

ribosomessites of protein

synthesis

0,5 µm

cytoplasm

Animal cell

15 µm

nucleus

chromosomes(made from

DNA + protein)

cell membrane

ribosomes

mitochondria,sites of energy

release(have their own

DNA)

cytoplasm

Plant cell

cell membrane

30 µm

cytoplasm

m i t o ch o n d r i o n

vacuole

cell wall ribosomes

chloroplast, site ofphotosynthesis

(has its own DNA)

nucleus,containing

chromosomes

FIGURE 2.

The structure of DNA, the hereditary material.Three bases in a row code for amino acids thata re the "building blocks" of pro t e i n s .

DNA structure

Thegeneticcode

base

sugar

phosphate

hydrogenbondsFirst

positionSecondposition

Thirdposition

nucleotide


possible to make very precise changes to the geneticmaterial. This can help improve the resistance to diseaseand environmental stress amongst crop plants and farmanimals. It can also help boost agricultural productivityand enhance the nutritional status, storage propertiesand ease of processing of food products.

Food additives and processing aids. Enzymes arespecialized proteins that are essential for life. Theycatalyse all biological processes and thus contro lmetabolism in living organisms. Once extracted fromliving organisms, these proteins allow certain processesin food production to be conducted. For thousands ofyears enzymes such as rennet from animals and papainfrom plants have been used to enhance the flavour,


FIGURE 3.

Protein synthesis

DNA determines hereditarycharacteristics by directingthe formation of particularproteins. A stretch of DNAcarrying the instructionsrequired to make a parti-cular protein is called agene.

DNA operates through anintermediary, RNA. Thesequence of bases on theDNA molecule acts as atemplate for the productionof messenger RNA (mRNA).

The mRNA binds to theribosomes, where it directsthe production of theprotein it encodes. ThreemRNA bases in a rowspecify each amino acid tobe added to the protein.

The amino acid chain(s)that proteins are formedfrom fold into precise three-dimensional structures,contributing to the protein’sproperties and function.

ribosomemRNA

amino acidchain

bases

gene

DNA

messengerribonucleic acid

(mRNA) amino acids protein

texture and appearance of food. Because of the diversityof microorganisms, it has been possible to find a widerange of microbial enzymes that are active in theconditions encountered in food processing. With geneticmodification a greater range of pure and highly specificenzymes can be produced more eff i c i e n t l y. Theseenzymes can be used to make desirable changes to foodboth rapidly and at relatively low temperatures, with asubsequent reduction in fuel requirements and in thee n v i ronmental impact of food processing. To theconsumer, the direct benefits include better flavour,texture and shelf life of food, often with a reduction inthe need for processing and additives.

aminoacid chains



How genetic modification is done

Cutting and pasting DNA. Special enzymes, obtainedfrom bacteria, are an essential tool of the molecularbiologist. In nature, these enzymes help bacteria fendoff viral attack by precisely dissecting the foreign DNAof invading viruses. In this way, the proliferation of theviruses is restricted. Restriction enzymes (as they areknown) recognize and cut DNA molecules at specificlocations (Figure 4). Many hundreds of re s t r i c t i o nenzymes have been isolated from different microbesand are available commerc i a l l y. With re s t r i c t i o nenzymes almost any section of DNA, and consequentlyany single gene, can be excised at will. The end of oneDNA molecule will readily link to that of another thathas been cut with the same enzyme. To join two DNAmolecules permanently it is necessary to form chemicalbonds along the DNA's sugar-phosphate backbone. Anenzyme called DNA ligase can do this job. The functionof these "cut and paste" enzymes in assembling novelDNA molecules is obvious, but the genetic engineer'stool kit would be incomplete without one or two otherenzymes. To understand their role it is necessary toappreciate how proteins are made.

A genetic intermediary. The genetic information encodedin DNA lies within the nucleus of the cell. However,proteins are not made in the nucleus, but elsewhere, atspecial structures called ribosomes. Before a particularp rotein can be made, a copy of the appro p r i a t einstructions must first be transcribed from the DNAandthen ferried to the ribosomes. The copied instructionsare made from mRNA (messenger ribonucleic acid).This mRNA is virtually a mirror image of the sequenceof bases on one DNA strand, according to the base-pairing rules. Upon arrival at the ribosomes the basesequence within the mRNA directs the construction ofproteins from amino acids. A sequence of three adjacentbases in the mRNA molecule is needed to determineeach amino acid in the protein (see Figure 3).

Cells that are producing a particular protein will havemany identical copies of that protein's mRNA insidethem. It is often easier to search for genes among thesmall mRNA molecules rather than along the entirelength of the cell's DNA. Once a desired length ofmRNA has been isolated, two additional enzymes areneeded.

FIGURE 4.

Restriction enzymes "recognise" and cut DNA molecules at precise locations. The names of these enzymes arederived from those of the organisms that produce them. The sequences of bases that are recognised by threeenzymes (BamHI, HindIII and EcoRI) are shown here.

BamHI HindIII EcoRI



D N A f rom RNA. The enzyme reverse transcriptaseassembles a single strand of complementary DNAalongside a corresponding piece of mRNA. A secondenzyme (DNA polymerase) can then be used toconstruct a double-stranded helix using the first DNAstrand as a template. DNA made in this way is calledcopy or complementary DNA(cDNA). A copy of a genefrom a donor cell is used in genetic modification.

Gene synthesis. By the judicious use of restriction andother enzymes, molecular biologists are able toassemble DNA molecules which contain one or moregenes of interest. Where a particular piece of DNA isdifficult to isolate, it is sometimes possible to make itartificially using a DNA synthesizer. Under computercontrol, these devices string together the biochemicalprecursors needed to make short stretches of DNA. Ofcourse, to programme the synthesizer it is necessary toknow the sequence of bases present in the desired gene;this too can be determined automatically using a DNAsequencer. It is also possible to copy specific genes usingthe polymerase chain reaction (PCR). The PCR has beenlikened to a "genetic photocopier". From a very smallamount of DNA millions of copies of a specific sectionof DNAcan be made quickly. The PCR lies behind manyof the spectacular successes of forensic geneticfingerprinting, where criminals have been identifiedfrom the DNA in just a few drops of blood or even acouple of cells on a cigarette butt.

P l a s m i d s . Once a suitable DNA molecule has beenconstructed, it must be moved into a cell in which it canbe expressed and duplicated so that it passes from onecell division to the next. For microorganisms, one of themost successful methods involves the use of plasmidsas a vehicle for transferring genes. Plasmids are rings ofDNA that are found in some cells. They carry a limitedset of genes and normally constitute only a few percentof a cell's total DNA. During the course of evolution,

plasmids carrying genes that help their microbial hostssurvive have been selected by nature. Some plasmidsconfer on their hosts the ability to degrade substances inthe environment such as nutrients and antibiotics. Manytraditional foods, such as yoghurt, cheese and otherfermented dairy products, contain large numbers ofliving microbes that naturally harbour plasmids.

Like the DNA of chromosomes, that of plasmids can becut with restriction enzymes and additional DNApasted into it. The result is a ring of "recombinant" DNAthat can be put into a bacterium. Specialized plasmidscan be used to ferry genes from bacteria into yeast cellsor even plants (Figure 5).

A limitation of plasmids is that they cannot accom-modate DNAfragments longer than 15 000–20 000 basepairs. However, some harmless, specially tailore dv i ruses can package larger DNA molecules. Suchviruses 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 usedfor a wide variety of plants is the plant tumour-inducingplasmid (Ti-plasmid) found in the soil bacteriumA g robacterium tumefaciens. Through its plasmid,Agrobacterium has the ability to naturally engineer plantcells so that they grow tumours that pro d u c ecompounds which the bacteria need to sustainthemselves. Molecular biologists use disarmed (non-tumour-inducing) versions of this plasmid to introduceforeign genes of their choice into plants. Because everycell carries a complete copy of all the plant's genes in itschromosomes, it is possible to regrow an entire plantfrom a single modified cell (see Cell culture, below).Specially modified Ti-plasmids have now beenproduced which help transfer fairly large genes intoplants. Unfortunately, monocotyledons (including theimportant cereal crops) are resistant to Agrobacterium.



A g ro b a c t e r i u m has proved especially useful whenworking with trees, which, because they are slowg rowing and large, are difficult to improve byconventional breeding. Apricot, plum, apple andwalnut trees have all been genetically modified withAgrobacterium (Figure 6).

Gene ballistics. A procedure called ballistic bombard-ment has achieved success with several crops, includingrice, wheat and soya. With this method, the DNA to beintroduced into the plant cells is first stuck onto minutetungsten or gold particles. The DNA-coated particlesare fired at high velocity into soft plant tissue, usuallycallus (see Cell culture, below). This intro d u c e sfunctional DNA into the plant cells.

Electroporation. DNA can also be introduced into thethin-walled tubes which develop from pollen grains bysubjecting them to microsecond pulses of a stro n gelectric field. This technique, called electro p o r a t i o n ,causes pores to appear momentarily in the pollen tubesthrough which DNA from a surrounding solution canenter. Seeds that develop from ovules fertilized withsuch pollen carry the introduced genes. Electroporationalso works with plant cells from which the cell wall hasbeen removed by enzyme treatment. From these nakedplant cells, whole plants can be regenerated by cellculture (see Cell culture, below). Electroporation has alsobeen used to transfer DNA molecules into a broadspectrum of microorganisms.

FIGURE 5.

Pure chymosin for cheese making can now be obtained from genetically modified yeast

DNACopy of chymosin gene

Sample ofcalf cells

Chymosingene

insertedinto

plasmid

Chymosinfrom

modifiedyeast cells

Calf

Plasmid put intoyeast cells



DNA can be injected into newly fertilized egg cellst h rough a very fine glass pipette. Only a smallproportion of such injected eggs take up the new genes.The injected eggs are transferred into the uterus of asuitable foster mother. This is the only method so far

Remove unwantedgenes from plasmidIsolate desired gene

from donor organism

Insertselected gene

into cutplasmid

Float leaf discs on asuspension of

Agrobacterium

Cultivate leaf discson nutrient medium

Regenerate whole plants,which now contain the

introduced gene

Put novel plasmid intoAgrobacterium tumefaciens

Restriction enzymecuts DNA at specificplaces

DNA ligase joinsDNA fragments Restriction enzyme

cuts DNA at specificplaces

PlasmidBacterialchromosome

Bacteria enter cells at cutedge and introduce novelplasmid into plant tissue

Genetically modified animals. The DNA of animals canalso be modified by genetic engineering. It is necessaryto introduce genes at an early stage of development ifthey are to be present in all of the cells of a matureanimal and be passed on to its offspring.

FIGURE 6.

Agrobacterium naturally infects some types of plants and introduces novel DNA into them. Biologists usespecially tailored forms of Agrobacterium to modify plants genetically.


that works for cows, pigs, sheep and goats.Microinjection can also be used to introduce new genesinto fish eggs, but it is not suitable for the eggs of birds.However, specially modified viruses have been used tointroduce, for example, disease resistance into chickens.The viruses, which are made harmless, are insertedthrough the shell of the egg.

Marker genes and gene probes. Whatever method is used,at best only a small proportion of treated cells take upthe introduced DNA. Screening is therefore necessary todiscover which cells have done so. As mentioned above,plasmids often carry genes which help the microbes thatpossess them break down particular antibiotics. Thesegenes can be used as "markers" to identify those cellswhich have taken up plasmids, for when the cells areplaced in a growth medium which contains anappropriate antibiotic, only those with plasmids willthrive.


Several different types of marker genes exist within theplant kingdom, and these are being developed asalternatives to antibiotic markers. Other methods foridentifying transferred genes include the PCR. Thismethod enables the amplification of transplanted DNAsequences, which can then be detected using "geneprobes". Gene probes are small fragments of single-stranded DNA or RNA which bind to complementarysequences in the DNA that is being sought out(Figure 7). Probes can also be used to detect the DNAofmicroorganisms that might contaminate food. They areso sensitive and specific that it is possible to use probesto differentiate between strains of the same species andto determine whether particular microorganisms arecapable of producing toxins.

Genetic switches. To ensure that the recipient cell'sbiochemical machinery will allow introduced genes tobe expressed, sequences of DNA called control regionsare required. One well-studied control region switches

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 isisolated from

the testorganism, and

amplified ifnecessary

The DNA iscut into

fragments bya restriction

enzyme

The fragments areseparated by size ina slab of agarose gel

DNA fragmentsmigrate towards

the positiveelectrode

DNAfragmentsplaced in

these wells

The DNA istransferred to a

nylon membrane

The probe binds tocomplementary

sequences of DNA

The probe revealsthe presence ofDNA to which it

has bound

cDNA or RNAprobe added

Probe revealed e.g. bychemiluminescence or

autoradiography



on and off a gene for making β-galactosidase. Thisenzyme enables bacteria to metabolise lactose, but it isp roduced only when that sugar is present in thesurrounding medium. The "genetic switch" associatedwith the gene encoding β-galactosidase can be placed infront of other genes, so that the addition of lactose tobroth in which the engineered cells are growing triggersexpression of the new genes. There are several othergenetic switches, some of which can even be thrown bya simple change in temperature.

Other biotechnological methods

Cell culture

Many types of plant cells from a variety of species canbe cultivated in vitro by supplying them with nutrientsand growth substances under strict aseptic conditions(Figure 8). Illumination may also be required. Culturesof individual cells, grown in a fermenter, can be used inpreference to whole plants for producing high-valueproducts such as natural food colourings (for example,betanin, the red colour from beetroot) and flavourings(for example, vanilla and mint oil). Problems with thistechnique arise from the fact that most of thesesubstances are produced only by mature cells that arenot dividing. In culture, the cells tend to be activelygrowing, and so do not yield large quantities of thesedesirable products.

A recent development with considerable promise is thecultivation of "hairy root cultures". By infecting planttissues with Agrobacterium rhizogenes (a relative of A.tumefaciens), the production of root-like structures canbe triggered. These transformed cells can be grownindefinitely on simple solid or liquid media. It ispossible that they could be used to produce natural foodflavourings and colourings without having to rely onplants grown in the open, which are subject to

variations in availability and quality. However, work onhairy root cultures is still at the experimental stage.

From undifferentiated cells (or callus) grown in vitro,whole plants can be regenerated by adjusting thep roportions of various growth factors in thesurrounding medium. This enables the propagation ofmany thousands of plants from just a few cells, greatlyaccelerating the process of plant breeding. Althoughplants produced by cell culture should be geneticallyidentical, the chromosomes in callus cells frequentlyundergo considerable rearrangement. Potato cells areespecially prone to this. Such variation is a rich sourceof new strains, often with desirable characteristics.However, this phenomenon can be a serious drawbackwhen great effort has been devoted to cloning a preciousnew plant variety. Problems of this nature beset a majorproject to grow oil palms in the mid-1980s. Cell cultureis also used for the conservation of those plant varietieswhich cannot be maintained in a normal seed bank.Genetic changes are in this case highly undesirable.

For some species, plantlets raised from cloned cells canbe planted directly in farmers' fields. A l t h o u g hexpensive, this method is especially suited to crops suchas bananas, which do not have seeds. More often, plantcell culture is used to generate uniform, viru s - f re eplants of high value (such as ornamental species) or theplants from which seeds are subsequently obtained.Cauliflower seeds, for example, are often obtained fromsuch stock.

Cell culture can also be used to make "artificial seeds" —cultured plant embryos that are subsequently coated bya protective layer of hard gel. The cost of producingsuch artificial seeds currently restricts their use to highvalue crops. However, the process is highly productive;one estimate suggests that from a 10-litre fermentersufficient embryos could be produced to satisfy Frenchfarmers' entire annual demand for carrot seeds.


The walls of plant cells are composed mostly ofcellulose. A cellulase enzyme preparation can be used toremove these walls, leaving spherical pro t o p l a s t sbounded by a thin membrane. The absence of a cell wallmakes protoplasts especially amenable to geneticengineering by electroporation or ballistic impregnation(see page 7).

Animal cells may also be cultured in vitro, although it isnot possible to grow whole animals or anything otherthan sheets of tissue from them. Their main use is inmedical research, where, for instance, they are used tomaintain viruses which cannot be cultured other thanby infecting living cells.

Cell fusion

Plant cell protoplasts from different species can be fusedto create complex "hybrids" with two or more nuclei.Even protoplasts derived from different genera can bejoined; it does not matter that these plants are unable tointerbreed. In theory, hybrid plants with characteristicsfrom both donor cells can be regenerated from fusedp rotoplasts. This could provide a simple route forintroducing, say, the ability to fix atmospheric nitrogenfrom legumes into cereals. However, enthusiasm forthis technique has waned since the first successfulexperiments during the 1970s. Protoplasts of manyspecies, fused or not, have proved difficult orimpossible to culture, let alone regenerate into entireplants.

Cell fusion of a different type, using animal cells, hasbeen more valuable. Antibodies can be produced byhybridomas grown in vitro. A hybridoma is an antibodyproducing cell (which is normally difficult to culture inisolation) fused with a tumour cell (which is virtuallyimmortal in culture). Large quantities of specificantibodies (called monoclonal antibodies because theya re all identical) can be produced by cultivatinghybridomas in a fermenter. These antibodies can be


FIGURE 8.

Plants can be cultured on sterile solid or liquidmedia that contain nutrients and plant growthsubstances ("plant hormones"). Using this method,many thousands of identical plants can be producedfrom a small amount of plant tissue.

Buds Seeds Roots Nodalsegments

Preparation

Surfacesterilization

Consecutives rinsesin sterile distilled water

Incubation Nutrientmedia

containingplant

growthsubstances

LIGHT

Cells in liquidsuspension

culture

AgitationUndifferentiated

callus tissue

Subculture

Callustissue

Nutrient medium

Explants



used in diagnostic tests of great sensitivity. For example,aflatoxins are potentially lethal compounds that areproduced by fungi which contaminate stored food.Antibodies raised against aflatoxins are now usedroutinely in inexpensive and simple-to-use diagnostickits. The same antibody technology is also used todetect the hormones associated with ovulation andp re g n a n c y. Such knowledge brings significantadvantages to animal breeding programmes.

FOOD PRODUCTION

Microbial production of food, foodadditives and processing aids

Fermented foods

Fermented foods are foods in which desirable changeshave been produced by the action of microbes orenzymes. Over 3 500 different traditional fermentedfoods have been catalogued. They include the bread,yoghurt and cheese that are familiar in Europe andNorth America. In Africa, foods made from fermentedstarch crops (yams, cassava, etc.) are more important,whereas in Asia products derived from fermented soyabeans or fish predominate. Fermented beveragesinclude not only the obvious alcoholic drinks, but alsotea, coffee and cocoa (fermentation of the leaves orbeans occurs after tea, coffee or cocoa have beenharvested).

Fermentation can make the food more nutritious, tastieror easier to digest or can enhance food safety.Fermentation also helps preserve food and increase itsshelf life, reducing the need for additives, refrigerationor other energy-intensive preservation methods. Forseveral thousand years, traditional fermentation (suchas brewing) has given people the opportunity tocultivate microbes on a large scale and in a safe manner.

Single-cell protein. In the 1960s, protein from microbialsources (single-cell protein or SCP) was thought to haveconsiderable potential, particularly for Third Wo r l dcountries. Few of the early projects aimed to producefood for humans, aiming instead to provide nutritious,low cost animal feed.

Unfortunately, most of the SCPorganisms (yeasts, fungi,and bacteria) used petrochemical derivatives as a source


of carbon. Even when oil prices were low, the processeswere only marginally economic. Consequently the oilprice rises of the 1970s ended most SCP projects. Onecompany had an efficient large-scale process using thebacterium Methylophilus methylotro p h u s that couldutilize methanol to produce a partially purified proteinfor animal feed. However, the cost of soya or fishmealfor animal feed remained considerably lower than thatof the bacterial protein. Consequently, production of theprotein ceased in the late 1980s.

Of all the SCP projects started in the 1960s, only one hassurvived to become a commercial success. Fungalprotein (in the form of mushrooms and yeasts) has beenaccepted as food for generations. One companypioneered the production of fungal protein from themycelium of Fusarium graminearum, which is cultivatedon glucose made from maize starch. The food ismarketed in the United Kingdom, Ireland, Germany,Switzerland and Belgium. By 1995, annual production isexpected to reach 14 000 tonnes — enough for about 28million meals for a family of four. Unlike soya products,the fungal protein has an excellent texture, attributed toa special linear arrangement of the fungal hyphae whichis similar to that of muscle fibres in meat. It has a highprotein and fibre content, yet contains almost no fat,which accounts for its popularity with health-consciousconsumers. The product has little taste of its own, yetabsorbs other flavours readily and can be combinedwith a wide range of ingredients.

As a form of SCP, algae are of interest in subtropical andtropical regions, where sunlight can be utilized as anenergy source. Often the production of algal protein isassociated with fish farming. Algae of the generaChlorella and Scenedesmus have been used as food inJapan, and Spirulina is being produced commercially inseveral countries, including the United States, Mexicoand Israel. Often the product is sold as a high-value"health food".


I m p roved microbial culture s . All SCP p rocesses usenaturally occurring micro o rganisms that have beencarefully selected from wild populations. Production ofthe fungal protein, for example, uses a strain of Fusariumg r a m i n e a r u m that was isolated from a soil sampleobtained close to a research laboratory. Before that,many thousands of samples from around the globe hadbeen laboriously screened to see whether they containeda fungus with a suitable nutritional profile, pattern ofgrowth and other desirable properties. For the microbialproduction of a substance such as an amino acid, it isoccasionally possible to find a rare natural variant or"mutant" which lacks a critical step in one of itsbiochemical pathways and consequently overproducesthe desired material. Where such mutants are hard tofind, they can sometimes be induced artificially, butsuch techniques are slow and rely heavily on chance.

The goals of genetic modification applied to straini m p rovement are essentially the same as those oftraditional methods, but the techniques are more preciseand far quicker. In addition, genetic modification allowsthe genetic "solutions" developed by nature to betransferred from one species to another, so that thisspecies can also benefit from certain geneticallydetermined traits, for example, disease resistance.

Genetically modified yeasts. In 1990 the United Kingdombecame the first country to permit the use of a live,genetically modified organism in food. This was aspecial strain of baker's yeast engineered to make breaddough rise faster. Existing genes were placed under thec o n t rol of stro n g e r, constitutive promoters, whichhelped the yeast break down sugar maltose faster thanusual.

Ordinary brewer's yeast (Saccharomyces cerevisiae) is ableto utilize a variety of carbohydrates as an energy source.These include glucose, sucrose and maltose.


some of its microbial flora. Starter cultures of only thedesired microbes are then added to the milk in sufficientvolumes and in the appropriate conditions to ensuretheir rapid growth.

When improved starter cultures are developed, it issometimes possible to choose between long-established,conventional techniques and the modern methods ofgenetic modification. However, the organisms that areproduced may be identical, irrespective of the methodchosen. One example where different routes led to thesame result is a new yoghurt that keeps its fresh "homemade" taste for several weeks without the risk ofturning acidic and bitter.

Normally, the starter culture bacteria in yoghurt turnthe milk's sugar, lactose, into lactic acid within days. Allthat is needed to produce a yoghurt that stays mild is aLactobacillus strain that cannot metabolise lactose. Suchvariants already exist in nature. One possibility ist h e re f o re to search for these organisms in nature .Another conventional approach consists of treating thelactobacilli in various ways to increase their mutationrate in the hope that the desired trait will accidentally becreated.

A third option involves modern genetic techniques:identifying the precise gene (or genes) responsible forlactic acid production, isolating and modifying them toinactivate lactic acid production, and then replacingthem in the bacteria.

All three techniques have proved successful and led toidentical mild-tasting yoghurts. In many other cases,however, only the "designer gene" approach can deliverthe desired results. Although this work is still at thelaboratory stage, starter cultures for yoghurt and cheeseproduction have now been altered to provide built-inp rotection against other microbes that cause foodpoisoning. The starter cultures have been modified so


Although sucrose is readily available (as cane or beetsugar), glucose and the other sugars must be preparedby the enzymic breakdown of starch (see Sweetenerp ro d u c t i o n, below). Unlike S. cere v i s i a e, the closelyrelated yeast S. diastaticus is able to grow on starch anddextrins because it makes an extracellular enzyme,glucoamylase, which catalyses the breakdown of starch.Saccharomyces diastaticus cannot be used directly forbrewing because it produces a compound which givesbeer a spicy flavour. Great interest has therefore focusedon transferring the gene for glucoamylase from S .diastaticus into S. cerevisiae. Such a yeast would be betterable to utilize the carbohydrate present in conventionalfeedstocks, which would increase the yield of alcoholand enable the production of a full-strength, low-carbohydrate beer without the use of extra enzymesafter the beer had been brewed. A modified yeast of thissort, produced by a re s e a rch foundation, re c e n t l yreceived approval for use in beer production in theUnited Kingdom.

Food yeasts which have been genetically modified tometabolise a wider range of sugars also help reduce thelevels of polluting waste in effluent. Sugar beetmolasses is widely used as the main raw material in theproduction of baker's yeast. Beet molasses contains, inaddition to sucrose, a small proportion of raffinose. Thissugar is not fully broken down and utilized by theyeast, and the unused part (melibiose) is found in wastewater from factories. The new strains of yeast utilizeraffinose completely, enhancing the yield of baker'syeast and leading to a cleaner effluent.

Improved starter cultures for the dairy industry. Whencheese, yoghurt and similar dairy products are made itis important that only the desired microorganisms beallowed to ferment the milk. Failure to excludeunwanted organisms can lead to poor flavours, lowyields and even food poisoning. Scrupulous hygiene isrequired, and often the milk is heat treated to kill all or


that they produce a compound which breaks down thecell walls of the potentially lethal food poisoningo rganism Listeria monocytogenes. Modified bacteriacould also be put into other foods as a fail-safemechanism in case the food is stored in the wrongconditions. It might also be possible to design foods thatcould be protected against a range of other food-poisoning organisms, such as Salmonella.

Other modified dairy starter cultures underdevelopment produce flavour compounds for better-tasting products and are able to resist attack fromviruses that would otherwise ruin the production ofyoghurt, cheese and similar products.

Food additives and processing aids

For many years, a wide range of food additives,supplements and processing aids have been obtainedfrom microbial sources. These include amino acids,citric acid, vitamins, natural colourings and gums aswell as enzymes. The production microorganisms havebeen selected from nature to ensure that, for example,they produce high yields of a good-quality productand/or are easy to grow or that the product is easy toseparate and purify. Laborious screening and selectionp rocesses are now being augmented by moderngenetics, allowing quicker, more precise selection andimprovement of existing production strains.

Amino acids. In foods, amino acids are used to enhanceflavours and to act as seasonings, nutritional additivesand improvers (in flour). They are used in both humanfood and animal feed. Bacteria or fungi which havebeen specially selected to overproduce specific aminoacids are grown in large fermenters. The acids aresecreted into the fermentation medium and harvested.The most important commercial products are glutamicacid, which is used as monosodium glutamate, aflavour enhancer; lysine, cysteine and methionine,

which are used as supplements in animal feeds that areusually deficient in these essential amino acids; andphenylalanine, which is used in animal feed and in them a n u f a c t u re of the sweetener aspartame (seeSweeteners, below).

Most of the 20 amino acids needed to make proteins areproduced by fermentation in hundred- or thousand-tonne quantities. In 1990, a company made a batch oftryptophane that was subsequently implicated in a raredegenerative disord e r. Concern arose because thetryptophane had been produced by a geneticallyengineered strain of Bacillus. However, it is thought thatthe illness was caused by insufficient purification of thetryptophane and not by the genetic modification itself.

Gums. Several gums produced by microorganisms andplants are used widely in the food industry asthickeners, emulsifiers and fillers. Recently a processwas developed to turn relatively cheap guar gum(obtained from seeds) into something akin to the moreexpensive locust bean gum. An enzyme (α-galactosidase) from the guar seeds is responsible for thistransformation. The gene encoding the enzyme wasinserted into baker's yeast, and α-galactosidase can nowbe produced in quantity. Production and processing ofα-galactosidase by modified organisms brings severalother benefits (Table 1). Bacterial polysaccharidescurrently occupy only a small fraction of the foodingredients market, but genetically modified cells couldproduce a wide range of novel gums with improvedproperties.

Sweeteners. Aspartame, a peptide which is 160 timessweeter than sucrose, is now used in an increasing rangeof foods and beverages. Aspartame's sweetness was(allegedly) discovered by accident when JamesSchlatter, working in the United States, licked hisfingers to separate a stack of papers. He had previouslyspilt some aspartame on his hands while working in the



Plant biotechnology

Plant breeding techniques

Since the origins of agriculture some 10 000 years ago,farmers have been trying to improve their cro p s .Initially, people simply replanted some of the seedsf rom their best plants each year. Crosses betweenchosen individuals of the same species led to gradualimprovements in the stock. Ancient South Americancivilizations relied on sweet corn (maize), the product ofa cross between two dissimilar wild plants, for much oftheir diet. For thousands of years this species has beentotally dependent on human intervention because it hasno natural mechanism for dispersing its closely boundseeds. Around the turn of the century the scientific


laboratory. Chemists at another laboratory in the UnitedKingdom had independently synthesized aspartamesome years before but had failed to notice its sweetness.

The original manufacturing process linked the twoamino acids phenylalanine and aspartic acid (bothproducts of fermentation) by chemical means. Today amore efficient enzymatic method has been developed.

Other food additives from microbes. Citric, acetic, lactic andascorbic acids are produced in large volumes for thefood industry by microbial fermentation.

Enzymes. A wide range of microbial enzymes are usedby the food industry. Some of the more importantapplications are shown in Table 2.

A comparison of production and downstream processing requirements of α-galactosidaseproduced by standard and modified yeasts

TABLE 1

Standard yeast Modified yeast

ProductionYeast required 236 tonnes 10 tonnesWaste water 2 000 tonnes 90 tonnesWaste yeast 400 tonnes 12 tonnes

Downstream processingAmmonium sulphate 1 100 tonnes 25 tonnesPotassium sulphate 25 tonnes 1 tonneAluminium gel 1 tonne NoneFiltration aid 133 tonnes 5 tonnesSolid waste 540 tonnes 18 tonnesLiquid waste 1 125 tonnes 26 tonnesDemineralized water 3 700 m3 50 m3

Iced water 52 000 m3 2 000 m 3

Energy 44 500 kW 9 000 kWSteam 220 tonnes 50 tonnes



Uses of enzymes in the food industry

TABLE 2

Enzyme Product Use

α-Amylase Sweeteners Liquefaction of starchBeer Removal of starch hazeBread, cakes and biscuits Flour supplementation

Amyloglucosidase Sweeteners SaccharificationLow-carbohydrate beer SaccharificationWine and fruit juice Starch removalBread manufacture Improved crust colour

β-Galactosidase (lactase) Whey syrup Greater sweetnessLactose-reduced milk and dairy Removal of lactose for those who

products are lactose intolerantIce cream Prevention of "sandy" texture

caused by lactose crystals

Chymosin (rennin) Cheese Coagulation of milk proteins

Glucose isomerase High-fructose syrup Conversion of glucose to fructose

Glucose oxidase Fruit juices Removal of oxygen

Invertase Soft-centred sweets Liquefaction of sucroseSugar syrups

Lipases Cheese Flavour developmentAccelerated ripening

Flavourings Ester synthesis

Papain Beer Removal of protein

Pectinases Wine and fruit juice Increased yield, clarificationCoffee Extraction of the bean

Proteases (various) Dairy products Modification of milk proteinsCaviar Viscosity reduction of "stickwater"Bread, cakes and biscuits Gluten weakeningMeat Tenderisation

Removal of meat from bones


principles which govern inheritance started to beunderstood, and plant breeding began to be conductedon a more systematic basis.

Artificially induced mutation and selection, like thatapplied to microbial strains, has yielded moreproductive varieties of the world's major cereal crops.Genetic mapping methods similar to those used topinpoint human genes have enabled scientists toidentify precisely those plants which carry specificdesirable genes. Such techniques have and will continueto lead to major improvements in yield, quality andresistance to disease. Despite these refinements to thec rude hybridisation methods of former centuries,several problems remain. For instance, although plantbreeders would like to introduce specific genes intocrops, conventional breeding permits the recombinationonly of whole sets of chromosomes. Undesirable traitsa re there f o re likely to be inherited alongside thedesirable ones. Artificially induced mutationoccasionally gives rise to improvements, but more oftenthe mutations have a deleterious effect. Traditionalplant breeding remains a tediously slow pro c e s s ,governed mainly by the time it takes plants to grow andset seed. Genetic modification, while it will likely neverreplace traditional methods, complements them ando ffers the opportunity to overcome some of theirlimitations.

About 80% of contemporary re s e a rch in plantbiotechnology is directed towards the improvement offood plants; the remaining 20% is concerned withnonfood products such as cotton, tobacco, ornamentalplants and medicines. The initial emphasis in plantbiotechnology has generally been directed towards theimprovement of agronomic qualities. The second andthird generations of genetically modified food plantswill bring direct advantages to the consumer andcommercial food processor.

Pest resistance

The main emphasis in this area has been thedevelopment of crops that produce a bacterial proteinfrom Bacillus thuringiensis (Bt). Proteins from differentstrains of Bt act on specific pests such as beetles, mothsand soil nematodes but do not affect mammals. Overmany years, more than 2 000 tonnes of Bt spores orp roteins derived from them have been used asbiological alternatives to conventional pesticides.However, Bt pesticides are expensive to produce, andbecause they break down quickly frequent reapplicationis necessary. Several genes encoding Bt proteins havebeen inserted into a variety of food plants, includingcabbage, mustard, oilseed rape, maize, potato andtomato. The hope is that crops with such built-inresistance will reduce the reliance on conventionalpesticides.

Herbicide tolerance

The production of genetically engineered plants that aretolerant of or even resistant to herbicides has generatedconsiderable interest and much controversy in recentyears. Critics fear that the availability of herbicide-tolerant plants will lead to an increase in the use ofherbicides. The true picture is more complex.

Farmers currently apply herbicides as a ro u t i n epreventative measure, usually before weed infestationhas progressed too far. It is argued that with the newcrops farmers will need to apply chemicals only whenthey are really needed, confident that this treatment willbe effective. This should lead to a reduction in the use ofherbicides, which would seem to be against the interestsof the agrochemical companies. However, many of themodified plants now under test are tolerant of the safestherbicides available, so that farmers will be able toreplace older chemicals with ecologically morefavourable ones. A g rochemical companies areinterested in selling these more advanced products.




Tolerance of the herbicide glyphosate, for example, hasbeen introduced into soya, maize, oilseed rape andsugar beet. Glyphosate is effective at low concen-trations, is not toxic to humans or other mammals andis rapidly degraded by soil microorganisms. Unfort-unately this herbicide cannot be used on conventionalcrops because it would kill them. When the modifiedcrops become available farmers could use glyphosate inpreference to more hazardous chemicals.

Disease resistance

Viral diseases. Plant viruses cause major reductions inyield and adversely affect the quality of many crops. Atpresent there is no effective chemical control for viral

diseases of plants. The conventional response has beento select varieties (using classical plant bre e d i n gmethods) that are naturally resistant to viral attack. Themechanism of natural resistance is not pro p e r l yunderstood, and very few of the genes responsible haveso far been identified. However, this does not mean thatit is impossible to create virus resistant plants.

For example, hybrids have been made between virusresistant and virus susceptible potato species. Hybridscannot usually be created between different species.H o w e v e r, in this case protoplasts from two potatospecies were fused together, and the resultant cells werecultured on a nutrient medium to regenerate wholeplants. These plants were resistant to three major virusdiseases. Field trials of this type of potato plant havebeen carried out in several countries since the mid-1980s.

Antiviral "vaccines". The insertion of genes for viralproteins into a plant inhibits subsequent infection by thatvirus or a closely related one. Potatoes, melons, tomatoesand cucumbers have now been modified to resist virusesin this way.

Antiviral antisense. Another route to engineering virusresistant plants is the suppression of viral genes usingantisense technology (see box and Figure 9). Hereantisense viral genes are inserted into the plant's DNA.The plant then produces antisense RNAto block the RNAof the invading virus (98% of plant viruses have RNArather than DNAas their genetic material). This approachis still experimental, but initial results show promise.

Genes fight fungi — and locusts! Fungal diseases,particularly of soft fruits such as tomatoes, are beingcombated by the transfer of chitinase genes. Unlike thecell walls of plants, those of fungi contain chitin.Therefore the novel enzyme allows the plant to fend offfungal infection without damaging its own cells.

Antisense technology

The sequence of bases along only one strand of theDNAdouble helix directs the production of proteins.This strand is called the "sense" strand.

When proteins are made, an mRNA copy of thea p p ropriate instructions is transcribed from theDNA sense strand and ferried to the ribosomes.There, the mRNA directs the production of proteinsfrom amino acids.

Antisense RNA is an RNA sequence transcribedf rom the wrong (antisense) strand of the DNAmolecule. Antisense and sense RNA c o m b i n e ,preventing the production of proteins encoded byspecific genes. For the method to work, anappropriate piece of inverted DNA is inserted intothe sense strand of the host cell's DNA. AntisenseRNAmolecules are formed from the inverted sectionwhen the DNA is transcribed (see Figure 9).



exo-polygalacturonasebreaks links between

galacturonic acid residues atthe ends of chains

FIGURE 9.

Pectin is an essential component of many fruit. It is naturally broken down by polygalacturonase enzymes.Antisense technology can be used to "neutralise" the production of polygalacturonases, delaying the softening of fruits once they have been harvested.

endo-polygalacturonasebreaks bonds between

galacturonic acid residueswithin the chain

galacturonicacid residue

DNA "sense" strand encoding polygalacturonase

messenger RNA strand

anti-messenger RNA strand

RNA strands "neutralise" each other

DNA "anti-sense" strand — complementary to polygalacturonase


R e s e a rchers in the United Kingdom, working withBrazilian scientists, recently isolated a chitinase from atropical cereal that can degrade fungal cell walls. Anunusual feature of this protein is that it also inhibitscertain digestive enzymes of locusts. This was the firstdemonstration of a protein that acts as both an enzymeand an enzyme inhibitor. If the gene encoding thisprotein could be put into major cereal crops, it mightoffer an attractive means of combating damage of storedgrain.

Improved food quality: The antisense route

A diverse range of food crops with qualities to benefit thefood pro c e s s o r, retailer and consumer are curre n t l yundergoing field trials. Many of these plants have beenmodified so that their fruits reach the consumer in peakcondition after transportation and storage. Antisensetechnology has been used to limit or neutralise the actionof undesirable genes. The first food plants to be altered inthis way were tomatoes.

Ripening research. In the mid-1970s researchers at theUniversity of Nottingham in the United Kingdomstarted to investigate the complex processes involved inthe ripening of fruit. The tomato was selected for thiswork because it has a relatively small set of genes and iscomparatively easy to work with (for example, it canreadily be grown by tissue culture). The aim was not toproduce a better tomato but to understand the ripeningprocess more fully so that the knowledge acquiredcould be applied to fruit and vegetables in general. Thismight allow farmers in tropical countries to benefit fromthe demand for exotic fruit and out-of-season temperatecrops, because the fruit could be transported withoutrefrigeration yet retain its texture and flavour. Foodprocessors might also benefit because they would beable to produce high-quality fruit juices and purees thatrequired less processing and fewer additives.

Pectinase key. Many genes are involved in thedevelopment of colour, flavour and texture in fruit.After several years the key to changing texture duringtomato ripening was identified, an enzyme calledpolygalacturonase (PG). As tomatoes ripen, PG breaksdown the pectin which holds cell walls together, causingthe fruit to soften. A g roup of industry scientists,working with the Nottingham group, discovered a wayto inhibit the PG gene without interfering with otheraspects of the tomato's natural ripening mechanism.This meant that the softening process could be sloweddown while the tomatoes continued to develop thedesirable colour and flavour.

Antisense activity. The PG gene was "neutralised" by theintroduction of an antisense gene into the tomato plant(see Figure 9). Although in early experiments some 10%of the PG remained active — sufficient to break downpectin and lead to mushy fruit — the antisense gene wasinherited stably, so that cross-pollination between plantswith the lowest levels of PG activity produced offspringwhich had just 1% of the usual enzyme activity.Researchers in the United States made similar use of anantisense PG gene. Their modified tomato is called FlavrSavr™, and has gained approval for sale to U.S.consumers. Pending regulatory approval, Euro p e a nshoppers will have to wait a little longer for ahomegrown alternative to watery, flavourless tomatoes.

Other slow-ripening fruit. The gas ethylene is produced byf ruit as part of the natural ripening process. A na t m o s p h e re of ethylene can be used to ripen fru i tartificially. Antisense technology has been used to inhibitthe production of ethylene and thereby impede ripening.Among the crops to have benefited from this type ofmodification are broccoli, raspberries, tomatoes and the"Euromelon" — the result of a joint project amongFrench, Spanish, British and Greek scientists. All of thesefruits can be picked when unripe and will remain in thatcondition until exposed to an atmosphere of ethylene.



Examples of other improvements to food quality

Potatoes pass the acid test. Human beings synthesize onlyhalf of the 20 different amino acids that are needed tomake proteins. A healthy diet must therefore containadequate amounts of the remaining 10 amino acids. Therisk of malnutrition is enhanced where there is relianceon a single staple food. The proteins in some crops (forexample, many of the legumes that are cultivated inAfrica and South America) contain only small amountsof certain essential amino acids. These deficienciescould be overcome by inserting into affected cropsgenes from other organisms which have substantialamounts of these amino acids. Genetically modifiedpotatoes with an improved amino acid content havealready been developed in Israel. This work couldprove highly beneficial, since potatoes are the world'sfourth most important crop and an important source ofnutrition in some countries.

Potato production could also be enhanced following theisolation, by Australian researchers, of a gene whichdoubles the yield of potato tubers from plants into whichit has been inserted. Tests of the nutritional status andcooking properties of the potatoes are now underway.Other plant breeders have introduced a bacterial geneinto potato plants that increases the proportion of starchin the tubers while reducing their water content. Thismeans that they absorb less fat on frying.

A l t e red oils. A combination of conventional plantbreeding, genetic modification and protoplast fusion hasbeen used by numerous companies and research groupsto alter the properties of oil from oilseed rape. Theresults range from plants that produce a gre a t e rp roportion of saturated fats (suitable for marg a r i n eproduction) to others which yield a high-temperaturefrying oil that contains a low proportion of saturated fat.

Sweeter fruit — with no added sugar. Sweeter crops (forexample, lettuces and tomatoes) have been produced by

transplanting into them the genes for two naturalprotein sweeteners, monellin and thaumatin. The genesfor both proteins came originally from tropical plants.Thaumatin is 3 000 times sweeter than sugar.

Removal of antinutritional factors. Many plants producechemicals to fend off attack by pests and pathogens.U n f o r t u n a t e l y, these natural chemicals can act asantinutritional compounds. Most legume seeds, forexample, contain chemicals which inhibit the action ofdigestive enzymes. Many legumes contain relativelyhigh concentrations of lectins, which, if they are notremoved by soaking or destroyed during cooking, cancause severe nausea, vomiting and diarrhoea. In cassavaand some legumes which form the staples of severalAfrican countries, levels of cyanide-generatingcompounds can lead to death or chronic neurologicaldisease if these foods are not cooked properly. In somecountries, all new varieties of potatoes are screened forlevels of the toxin solanine. Genetic modification couldbe used to produce plant varieties with low levels ofthese antinutritional factors.

P rocessed products from genetically modified plants. InJanuary 1995 the United Kingdom governmentannounced the food safety clearance of severalp rocessed products obtained from three geneticallymodified crops: oil derived from oilseed rape that hadbeen genetically modified to confer male sterility,thereby preventing self-fertilization and allowing thep roduction of vigorous high-yielding hybrids;processed products (oil, meal and protein fractions)f rom soya plants modified to be resistant to theherbicide glyphosate; and tomato paste from tomatoesmodified to slow down the process of softening. Unlikethe antisense tomatoes described above, these tomatoeshad been modified using "sense" technology. Theycontain a truncated polygalacturonase gene in the"sense" orientation, which, for reasons that are note n t i rely clear, has the same effect as the antisensepolygalacturonase gene.



Long-term goals

N i t rogen fixation. The capacity to fix atmosphericnitrogen into a form that can be taken up by plants inthe same way they use nitrogen in fertilizers — anability possessed by Rhizobium bacteria that live in closeassociation with leguminous plants — has been a majorp reoccupation of re s e a rchers. Despite decades ofintensive study, the goal of a self-fertilizing cereal is stillelusive. Doubts have now arisen as to whether this aimis realistic and should be a high priority, for tworeasons. The first is that to receive ammonia from itsassociated bacteria, the plant has to provide the bacteriawith "food" so that they can survive in small nodules onits roots. This food is provided at the expense of theplant's productivity. Legumes have had millions ofyears to evolve a mutually beneficial physiology withtheir Rhizobia, and it seems unlikely that suchrelationships could be artificially established withc e reals; indeed there is evidence to suggest thatn i t rogen-fixing cereals could have decre a s e dp ro d u c t i v i t y. The second reason concerns the ro o tnodules: these complex structures would have to beprovided on the plant's roots. This is likely to involvethe manipulation of very many genes.

A better approach might be to transfer nitrogen-fixingabilities into Agrobacterium, which infects a wider rangeof plants. Unfortunately this does not include cereals,which form the majority of food crops. For theforeseeable future it would probably be more profitableto try to improve existing nitrogen-fixing species,especially tropical legumes and their associatedbacterial strains. Other readily available options includethe traditional improvement of crop management, suchas rotation between legumes and cereals.

Drought resistance. Drought-resistant crops would bringobvious advantages to farmers in areas of low rainfall.N u m e rous organisms are able to withstanddehydration without harm and can be resuscitated

simply by adding water; dried yeast is a familiarexample. A desert plant, appropriately named there s u r rection plant, is similarly able to withstandprolonged periods of drought. Both organisms rely forthis remarkable ability on a sugar in their cells calledtrehalose. In 1993, the first successful attempt was madeto place the genes involved in the production oftrehalose into potato and tomato plants. The results ofthis work have yet to be evaluated.

Animal biotechnology

Animal nutrition

Probiotics. For many years low levels of antibiotics havebeen added to animal feed, with the result that theanimals gain extra weight. This is thought to be becausethe antibiotics kill detrimental bacteria in the animal'sgut. An alternative approach is to add live, nonpatho-genic bacteria to the animal feed. These organisms(termed probiotics) are thought to compete with thedetrimental species, leading to a similar weight gain.Genetically modified bacteria might one day be used asprobiotics to improve the health and efficiency of feed toweight conversion of farm animals.

Rumen bacteria. The nutrition of ruminant animals (forexample, sheep and cattle) is highly dependent on thebacteria which live in their stomachs, enabling them todigest plant material effectively. Genetically modifiedbacteria could be introduced into the rumen by addingthem to animal feed, enabling the animals to makebetter use of a wider range of food plants.

Silage. Silage is grass which has been fermented bynaturally occurring bacteria to enhance its nutritionalvalue. Microbial inoculants are now used to improve theformation of silage, but there is a possibility thatgenetically modified species could be used to furtherimprove the process.



Bovine growth hormone. Bovine somatotropin (BST) is ap rotein produced by cows that has a variety ofphysiological effects, including the regulation of milkyield. BST is naturally present in all cow's milk. Thegene for BST has been cloned into bacterial cells so thatit can be produced in bulk. Injections of BST can be usedto enhance milk yield. The hormone also improvesgrowth rate and protein-to-fat ratios in meat. There hasbeen a moratorium on the general use of BST incountries of the European Union for several years,although it is approved elsewhere, including the UnitedStates. BST is probably one of the most severely testedsubstances in the history of the U.S. Food and DrugAdministration, but its use has been opposed in somequarters. Such opposition is based partly onuncertainties over its effects on animal health, and onworries that the hormone might contaminate meat ormilk. There has also been opposition on political andeconomic grounds, as a surplus of milk is alreadyproduced in many developed countries. The advantageof BST is that it would allow the same volume of milk tobe produced by fewer cattle.

Animal breeding and health

Classical animal breeding has done much to improvethe productivity and well-being of farmed livestock.Changes in characteristics such as maturity, fecundityand the distribution of muscle tissue are noticeable inmany modern breeds compared with their wildancestors and old domestic breeds. The achievements oftraditional breeding are now being augmented bymodern genetic analysis.

The majority of the desirable features in livestock seemto be controlled by many genes, each with a small effect,working in concert. The modification of animals bygenetic engineering is still in its infancy, so the genesthat should be altered to improve animal productivityor health are still difficult to predict. Much of the workso far has concentrated on simple changes, such as the

introduction of growth hormone genes. The results,some of which are described below, were not completelyforeseen.

Most of the transgenic animals created to date are micewhich are used in medical research. This lies outside thescope of the current discussion. However, this work islikely to benefit human and animal health.

A g r i c u l t u re in Europe and North America alre a d yproduces sufficient food for the indigenous population.The real benefits from improved animal productionmight be seen in the Third World. For example, it mayone day be possible to introduce disease resistance intootherwise vulnerable animals. There are well-advancedp o rcine, ovine and bovine genome projects, whichparallel similar efforts to map and sequence the entirehuman genome. The Bovine Genome Project couldresult in, for instance, resistance to trypanosomiasisbeing introduced into more productive breeds of cattlef rom their naturally resistant African counterparts.However, in the immediate future more benefit is likelyto come from the development of new diagnosticagents, vaccines and therapeutic agents than bymodifying the animals themselves.

Genetically modified pigs. Genetically modified pigs werefirst produced in the United States in 1988. Pig embryoswere injected with a gene encoding growth hormone.Genetic "switches" were included so that a change in theanimals' diet would greatly increase the levels ofhormone circulating in the blood. Contrary to somereports, the result was not giant pigs, but animals withvery lean muscle tissue. Unfortunately, the high levelsof growth hormone led to gastric ulcers, arthritis,kidney disease and other undesirable conditions.Insufficient attention had been paid to what was alreadyknown about growth hormone, namely, that it isgenerally produced only for limited periods during ananimal's life, not continuously.



Classical breeding methods supplemented by moderngenetic mapping have produced more desirable results.For example, the genes which control leanness in pigsare linked to those that may cause sudden death duringa period of stress. Humans also carry similar genes andmay, for example, die inexplicably when undergoingroutine surgery, although such a genetic predispositionis very rare in humans. Selection for leanness in pigsover many generations has also increased the frequencyof the stre s s - related genes, but modern bre e d i n gmethods are allowing such genes to be bred out.

Herman the transgenic bull. The world's first transgenicbull was bred in the Netherlands. Herman, the animalin question, became famous in 1992 when the Dutchparliament had to decide whether or not he would beallowed to mate. Scientists at the company that bredHerman wished to reduce cows' susceptibility tomastitis. To do this, they transplanted an extra copy ofthe gene for a milk protein called lactoferrin into severalbovine embryos. Lactoferrin is part of the mammalian(including the human) defence against bacterialinfection. Unfortunately, the only embryo to take up thetransplanted gene produced not a cow, but a bull.Undeterred, the company hoped that Herman wouldpass on the gene to his daughters and that they wouldbe less likely to suffer from mastitis. The Dutchparliament eventually gave its consent to the requestfrom the company. Herman has sired several offspring,at least one of which carries the extra lactoferrin gene.

Cloned sheep. Breeders of sheep in the United Kingdomand New Zealand are trying to disentangle the role ofgenes and the environment in an effort to improve thequality of their livestock. They intend to do this with thehelp of identical sheep placed in several flocks. Thesheep are produced by splitting an embryo when itconsists of just eight cells. This happens naturally withthe production of identical offspring, such as twins.Each of the eight cells is implanted in a foster mother.

Differences between the identical sheep will be duesolely to environmental influences (such as diet orexercise). The hope is that this comparison will enablesheep breeders to conduct their work on a more rationalbasis.

Chickens. Because viral infections are often a problem inintensive poultry production, a lot is known about theviruses which affect chickens. This knowledge has beenuseful to genetic engineers, because modified versionsof the viruses themselves can be used to combat disease.Birds have been given immunity to fatal viruses thatproliferate in crowded broiler houses by adding genesfor viral proteins to the chicken genome.

Transgenic fish. Compared to mammals and birds, fisha re still relatively wild, with little history ofi m p rovement through selective breeding. There arevery many fish species and consequently a large poolf rom which genes can be drawn for bre e d i n gprogrammes. Fish, especially when farmed, are prone toinfection. Recent genetic re s e a rch on fish hasconcentrated on inserting genes that impart resistanceto disease. Successful modifications have also beenmade to genes that influence growth, which could leadto more productive fish for aquaculture. Otherpossibilities are the introduction of "antifreeze" genes toextend the range of Atlantic salmon into colder watersand to delay the breeding season of fish so that theygain weight. Concerns have been raised about thewisdom of increasing the geographical range of apredator at the top of a food web. Work in this area mustp roceed with careful consideration of its possibleecological consequences.



EXAMPLES OF THE USE OFENZYMES IN FOODPROCESSING

Cheese manufacture

Ancient tradition

In ancient times the nomadic tribes which rangedt h rough eastern Europe and western Asia carriedliquids in bags made from animals' stomachs. Milkstored in these containers, warmed by the sun, souredby naturally occurring bacteria and laced with enzymesfrom the stomach lining, would have been transformedinto solid curds and liquid whey. Thus, almost byaccident the first cheeses were made.

The Romans were the first Europeans to describe cheesemaking in detail. To coagulate the milk protein, an enzymepreparation from goat, lamb or even hare stomachs wasmixed with sheep's or goat's milk (cow's milk was notproduced on a large scale before the 13th century). Afterthe whey had been drained from the coagulated milk, thec u rds that remained were salted and stored forconsumption later in the year. Vegetarian cheeses werealso produced using the juices of plants which possessedmilk-clotting properties, such as lady's bedstraw (Galiumverum) or butterwort (Pinguicula vulgaris).

First commercial rennet

Early cheese makers either placed strips of the kid, lambor calf stomach directly into warm milk or prepared acrude rennet extract by soaking the strips in salt water.By 1837, some farms were selling rennet extracts insmall quantities for the convenience of domestic cheesemanufacturers. In 1874, the Danish chemist ChristianHansen founded a laboratory in Copenhagen andstarted the first industrial production of calf rennet

extract. This was obtained from the stomachs ofunweaned calves that were slaughtered for vealproduction and not specifically to obtain the enzyme.World production of rennet now exceeds 25 millionlitres per year.

Rennet substitutes

In the 1960s the Food and Agriculture Organization ofthe United Nations predicted a severe shortage of calfrennet. It was anticipated that an increased demand formeat would lead to more calves being re a red tomaturity, so that less rennet would be available. Overthe last 30 years several substitutes for calf rennet havebeen developed, allowing the supply of enzymes tokeep pace with cheese production. Today there are sixmajor sources of protease for coagulating milk (that is,chymosins), three from animals (veal calves, adult cowsand pigs) and three from fungi (Rhizomucor miehei[formerly Mucor miehei], Endothia parasitica a n dRhizomucor pus i l l u s [formerly Mucor pusillus]). Inaddition, there are now chymosins derived fro mgenetically modified microbes (Escherichia coli,K l u y v e romyces lactis and A s p e rgillus niger). See Figure 5.

Purity and safety of recombinant Chymosin

Chymosin obtained from recombinant organisms hasbeen subjected to rigorous tests to ensure its purity.Biochemical, toxicological and microbiological testshave also been done. Today at least 50% of cheese in theUnited States is made with chymosin from geneticallymodified microbes.

Other enzymes for cheese making

In some cheeses fat-degrading enzymes (lipases) may beadded to promote the formation of piquant flavours asthe cheese ripens. Many traditional Italian cheesesbenefit from such additions to augment the activity ofnaturally occurring lipolytic microbes. Recently, lipase



f rom genetically modified microbes has beenintroduced to accelerate the ripening of other cheeses.Cheddar cheese takes up to 9 months to mature fully.However, with the addition of a suitable lipase, thisperiod can be reduced to a mere 6 weeks. In somecountries food additive regulations based on traditionalpractices prohibit the use of enzymes in cheese otherthan those used to coagulate milk. These may be revisedin the light of current technological developments.

Immobilised enzymes for whey processing

In several countries methods have been developed forthe enzymatic treatment of surplus whey from cheesemaking. The enzyme lactase (β-galactosidase) isimmobilised (for example, on porous beads) inside a tallcolumn. Lactase is expensive and immobilisationpermits it to be reused many times. Whey is passed overthe beads in the column, emerging some 10 minuteslater with 80–90% of its lactose sugar split into a mixtureof sweeter-tasting glucose and galactose. After enzymetreatment the pH of the whey is adjusted and salt (fromthe cheese-making process) is removed. Finally thep roduct is concentrated by evaporation to give anopaque, honey-like, protein-rich syrup.

Whey syrup finds a wide range of applications in theconfectionery industry, particularly in the manufactureof soft toffee or caramel, where it substitutes forsweetened condensed milk. However, for thisapplication it may be necessary to replace the caseinthat was precipitated when the whey was formed, sincetoffee made without this protein may be too fluid.Balanced against this, toffee made with whey syrup ishygroscopic, that is, it absorbs moisture. It is thusespecially useful in the manufacture of wafer biscuits,since moisture is absorbed by the toffee, leaving the wafercrisp and extending the product's shelf life.

This important quality is also conferred on cakes madewith whey syrup, since it allows manufacturers to reduce

or omit humectants such as sorbitol or glycerol from theirrecipes. Because of its protein content, whey syrup alsoacts as an emulsifier, helping bind the cake ingredientsand replacing some of the egg protein found in traditionalrecipes.

New uses for whey syrup are still being developed andmay include products as diverse as mousse (in which thesyrup's foaming, "mouth feel" and stabilising propertiesa re important) to fermented meat products such assausages. It may also be possible to use whey syrup as asubstrate in microbial fermentations.

Fruit juice productionTo appreciate the action of enzymes in fruit juiceproduction, it is necessary to understand something of thestructure of fruits and the changes that occur as theyripen.

Changes during ripening

As fruit ripens it becomes pro g ressively softer. Muchresearch has been done to discover exactly which enzymesare responsible for this and other changes in maturingfruit. One hope is that by monitoring the levels of theseenzymes, growers might be able to harvest their crops atjust the right time or to select varieties with desirablecharacteristics, such as better storage properties or greateryields and improved quality of juice. Naturally occurringcellulases, acting on the cell walls, play a part in thedevelopment of softer fruits such as peaches and tomatoes.Pectin is also broken down as fruits ripen. In unripe fruit,the pectin is bound firmly to the cell walls. Such pectin isinsoluble and the liquid within the cells remains fluid,conferring rigidity on them. During ripening, the pectin isaltered by enzymes in the fruit. As a result the pectinbecomes more soluble, its grip on the surrounding cellwalls is loosened and the plant tissue softens.



Dissolved pectin makes juice more viscous and difficult topress from fruit. Pectin also helps retain important colourand flavour compounds within fruit so that juice pressedfrom it is of inferior quality. Juice may also be difficult toclarify and filter because of suspended pectin particles. Inthe fruit juice industry pectinases obtained from fungi areused to help overcome all of these problems.

Use of pectinases in the fruit juice industry

Pectinases were first utilized in the commerc i a lpreparation of wines and fruit juices in 1930. Before this,juices had been prepared by mechanical means, simply bypressing fruit and filtering the liquid which emerged. Theearly methods using pectinase were developed by trialand error. Only in the 1960s did the chemical structure ofplant tissues become known, and with this knowledgefood technologists began to use a greater range ofenzymes more effectively.

Enzymes are used to help extract, clarify and modifyjuices from many plants, including berries, stone andcitrus fruits, grapes, apples, pears and even vegetables(Table 3). Where a cloudy juice or nectar is preferred (forexample, with oranges, apricots, pineapples or carrots)

there is no need to clarify the liquid, and enzymes are usedinstead to enhance extraction or perform othermodifications.

Future developments

The enzyme commonly referred to as "pectinase" is not asingle substance but a complex cocktail of enzymes able toattack a variety of bonds in correspondingly diverse pectinmolecules. The pectolytic enzymes used to date have beenc rude preparations, but greater knowledge of thesubstances they act on has stimulated the demand for purerenzyme preparations with specific, well-defined activities.

Although improvements in enzyme purification processesare important, the focus has turned to gene technology top rovide, for example, pectinases with impro v e dt e m p e r a t u re stability and lower pH optima. Theavailability of a range of specific enzymes from geneticallymodified, food-grade organisms will also lead to newapplications for such enzymes. For example, a pectatelyase from the bacterium which causes soft rot in carrotsand potatoes (Erwinia carotovora) has recently been clonedin Escherichia coli. This enzyme could be used in tea leafextraction in the manufacture of instant tea.


Enzymes and their applications in the fruit and vegetable juice industry

TABLE 3

Amylase Removal or prevention of "starch haze" in juice concentrates

Arabanase Removal of araban haze from juice concentrates (especially pear juice)

Glucose oxidase Removal of excess oxygen from juices

Naringinase Reduction in bitterness of citrus juices

Pectinase Increasing yield of juice from fruit

Clarification of juice

"Limoninase" Reduction in bitterness of citrus juices


Sweetener production

Sugars from starch

During the Napoleonic wars Europe was isolated fromits major sources of cane sugar in the tropics. In 1811German chemists succeeded in producing sugar bybreaking down starch with acid, and this process wasadopted in several countries until the introduction ofsugar beet into European farming. In World War II agentler enzymatic method of converting starch tosugars was developed which had the advantage ofyielding sugar that lacked the bitter compoundscharacteristic of the acid treatment.

Enzymatic treatments are a major way of producingsweeteners today, including syrups derived fro msucrose or starch that contain mixtures of glucose,maltose, fructose and other sugars. High-fructose syrup(HFS) from corn (maize) starch has now eclipsedsucrose as the major sweetener used in the U.S. foodindustry. More than 8 million tonnes of HFS are soldannually, although the production and use of HFS in thecountries of the European Union has been limited byquotas intended to protect European sugar beetgrowers.

HFS is an excellent alternative to sucrose or invert sugarin food processing. It is used in many pro d u c t s ,including soft drinks, jam, ice cream, cakes, cannedfruit, pickles and sauces. Unlike sucrose, HFS remainsstable in chilled, frozen and acidic foods withoutforming crystals or undergoing conversion to othersugars.

High-fructose syrup

HFS is made from a low-cost raw material, starch. Thestarch is converted into syrup by several enzymes,which are used in three distinct stages:

Liquefaction. Starch is obtained as a by-product aftervaluable oil and protein have been extracted fro mmaize. The starch solution is boiled and treated with α-amylase, an enzyme from B a c i l l u s. This tre a t m e n tgelatinizes and dissolves the starch and starts to break itdown. The partly degraded starch molecules are knownas dextrins.

S a c c h a r i f i c a t i o n . Depending on the carbohydratecomposition required in the finished product, a cocktailof various fungal enzymes is then added to the dextrins.For syrups with a high glucose content a mixture of aamylase or pullulanase with amyloglucosidase is used.Over 1–3 days these enzymes break down the dextrinsprogressively to glucose. Evaporation of water yields aviscous glucose syrup.

Isomerization. Glucose shares its chemical compositionwith fructose but has a different molecular structure.This makes glucose about half as sweet as fructose. Theenzyme glucose isomerase converts glucose to fructose,t h e reby increasing the sweetness of the syru p .Immobilised glucose isomerase is packed into a column,and glucose syrup heated to 60˚C is passedcontinuously over it. At this temperature, the glucoses y rup has a low viscosity, microbial spoilage isprevented and conversion occurs swiftly. Typical HFShas a dissolved-sugar composition of 42% fructose and53% glucose (the remainder being other sugars). Shouldsyrups with a greater fructose content be required,glucose can be separated from the liquid leaving thecolumn. This sugar can be recycled over the enzymecolumn to achieve a greater overall conversion rate.

Better enzymes from nature

Over the last 25 years much research has been directedtowards finding better enzymes for HFS production. In1974 a Danish company introduced a bacterial α-amylase from Bacillus licheniformis that catalysed thebreakdown of starch at 100˚C or more. This led to



significant improvements in the initial liquefactionprocess. A diverse range of dextrin-degrading enzymeshas also become available to satisfy the demand forspecialised sugar syrups, for example, for baby food, fordiabetic confectionery and for use in brewing and winemaking. These developments have resulted fro mcareful selection of microorganisms that produce theenzymes. Finding the ideal production strain takesmany years, however, and is largely a matter of luck. Amicrobe with one attribute in its favour will likely lackanother equally important characteristic. Modernmolecular biology has started to reduce thisdependence on trial and error.

Modified microbe

Bacillus stearothermophilus produces an α-amylase whichis well suited to sugar syrup production. Unfortunately,this species makes only small amounts of the desirableenzyme. Several copies of the appropriate gene weretransferred into a closely related species, Bacillus subtilis,enabling commercial production of the superiorenzyme. After extensive safety tests, this amylasebecame the second enzyme in the world (afterchymosin) from a genetically modified organism to beapproved for food processing in the United States.

A designer enzyme

At the relatively high temperatures (60˚C) used inimmobilised enzyme columns, glucose isomeraserapidly becomes inactive. Typically the enzyme'sactivity halves every 55 days, so that after severalmonths the expensive enzyme has to be replaced. In1986, a company in the Netherlands in cooperation witha Belgian company initiated an ambitious re s e a rc hp rogramme to improve the stability of glucoseisomerase. The research team first sought to understandthe reasons for the decline in enzyme activity.

S t ronger links. C a reful examination of crystals of theenzyme revealed its structure. Glucose isomerase wasfound to consist of four identical subunits joined togetherby fragile bonds. At raised temperatures the protein brokeapart at these bonds and linked instead to glucosemolecules in the syrup as they passed through thecolumn. This explained the inactivation of the enzyme.

Further investigations showed that of the manyhundreds of amino acid residues making up glucoseisomerase, just two were responsible for the weak links.By substituting these amino acids with others thatbound more tightly to their neighbours, the "proteinengineers" were able to produce a more stable enzyme.This was done by subtly altering a small section of theDNA that coded for glucose isomerase, so that one outof the 20 lysine residues in each subunit of the proteinwas replaced by an arginine residue.

Production processes. The remaining technical hurdles offermentation, product recovery and immobilisationwere gradually overcome, and by 1993 a new productwas created. The improved glucose isomerase has a halflife that is roughly double that of the original form,resulting in a doubled productivity in the enzymecolumn. A further benefit could come from the newenzyme's greater thermostability. With standard glucoseisomerase there is a limit to the proportion of fructosethat can be produced in the syrup leaving the column.At higher temperatures, a greater proportion of fructoseis formed; the new enzyme may therefore permit one-step production of HFS at very high temperatures.

Product registration. Before the new enzyme can be soldto HFS producers, both it and the production processmust undergo extensive testing and gain the approval ofregulatory bodies. The U.S. Food and Dru gAdministration has already been consulted, and it hasaccepted the description, purpose and conditions of usefor this glucose isomerase.



REGULATORY, SAFETY ANDSOCIOECONOMICCONSIDERATIONS

To date, relatively few products of genetic modificationhave reached the supermarket shelves. Geneticmodification of farm animals is in its infancy, and workwith the major crop plants, especially cereals, hasproved more difficult than was at one time anticipated.Outside the laboratory, modified plants and microbeshave so far been restricted to closely monitored fieldtrials. Nevertheless, during the coming century theimpact of food biotechnology is likely to be bothprofound and far reaching.

For this reason, there has been much debate about thepotential social and economic implications ofbiotechnology. The safety of new developments hasbeen examined with rigour, and legislation has beendrafted to protect the interests of the public.

Food safetyIn addition to issues of environmental safety, theimplications of modern biotechnology for food safetyneed to be considered. Recognising the numerous benefitsto health, nutrition, food preservation and foodp roduction that biotechnology could bring, theO rganization for Economic Cooperation andDevelopment (OECD) established a working group ofnational experts to consider the safety implications ofmodern food biotechnology. The intention was toexchange ideas, data and information among experts toenhance international cooperation in the field. Theworking group considered numerous examples of howthe safety of novel foods and food components had beenevaluated in the past and established some concepts andprinciples that underpin the safety evaluation of foodsderived by modern biotechnology. These principles have

been widely accepted and are similar to recommendationsmade by other influential groups such as the World HealthOrganization and the Food and Agriculture Organizationof the United Nations. The major conclusions of the OECDreport are summarised below.

Food is considered safe if there is reasonable certainty thatno harm will result from its consumption underanticipated conditions. Historically, food prepared andused in traditional ways is considered safe on the basis oflong-term experience, even though it may naturallycontain harmful substances. In principle, food is presumedto be safe unless a significant hazard has been identified.

Modern biotechnology broadens the range of geneticchanges that can be made to food and widens the rangeof possible sources of food, although this does noti n h e rently lead to food that is less safe than thatdeveloped by conventional techniques. Therefore theevaluation of foods derived from modern biotechnologydoes not require a fundamental change in establishedprinciples of food safety, nor does it require a differentstandard of safety.

Furthermore, modern biotechnology provides precisetechniques for the direct and focused assessment ofsafety (for example, by the detection of minute amountsof contaminating material), which can be usefullyapplied to foods derived from both modern andtraditional methods.

Substantial equivalence

The OECD report said that the most practical method toestablish food safety was to consider whether a novelfood (or food component) was substantially equivalent toan analogous conventional food product, where oneexisted. Account should be taken of the processing (suchas cooking) that the food might undergo, as well as howmuch food was to be consumed, by whom and thedietary pattern.



To demonstrate substantial equivalence, a number offactors have to be considered, such as:

• the characteristics and composition of theconventional food to which the new one is to becompared

• knowledge of the component parts of the newproduct or organism, such as any introducedgenes, the method used to introduce the newgenetic material and how that new geneticmaterial is expressed

• the characteristics and composition of the newproduct or organism compared with the existingfood or food component.

If the novel food is judged to be substantially equivalentto an existing food, it is treated in the same manner asits conventional counterpart.

Where new classes of foods or food components areintroduced it is more difficult to apply the concept ofsubstantial equivalence. Here experience gained in theevaluation of similar materials is taken into account.Where a product is thought not to be substantiallyequivalent to an existing one, further investigationsfocusing on the identified diff e rences are re q u i re d .Totally new foods, where no similar materials have everbeen consumed, must be evaluated solely on the basisof their own composition and properties.

Genetically modified potatoes

As an example of the application of the principle ofsubstantial equivalence, potatoes are an established partof the human diet. They can contain toxic alkaloids, butpeople generally know how to prepare them and avoideating green potatoes, which contain significantamounts of alkaloids. Potatoes are often infected bynaturally occurring viruses, but these do no harm tohumans and have a long history of humanconsumption. If a potato were genetically modified

with one of these viruses so that it produced viralprotein at levels comparable to those from naturallyinfested potatoes, it would be considered to besubstantially equivalent to the infected potatoes thathave a long history of safe use and consumption. Thistheoretical analysis applies only to viral proteins in theparts of the potato plant that are traditionallyconsumed. It also assumes that the insertion of the viralcoat protein gene does not lead to secondary effectsthrough, for example, interruption of coding sequenceswithin the plant's genome.

Chymosin from genetically modified bacteria

As described in the section on cheese manufacture, theenzyme chymosin can be obtained from geneticallymodified micro o rganisms. Chymosin from thebacterium Escherichia coli is obtained from a completelydifferent organism and by a method that is completelydifferent from its traditional counterpart, which comesfrom an animal. Thus the types of potential impuritiesdiffer and the characteristics of the enzymes may differ,too. To determine whether the two preparations weresubstantially equivalent, the U.S. Food and Dru gAdministration compared the activities of the twoenzymes and evaluated whether impurities in theproduct from the modified organism affected its safeuse. After a battery of tests, both enzymes were found tobe substantially equivalent in safety and function.

Safety implications of marker genes

The current generation of genetically modified organismsfrequently contains marker genes, some of which encoderesistance to antibiotics. In such cases it is important toconsider whether food derived from an organism withsuch a gene would be substantially equivalent to aconventional product. Among the questions that must beasked are whether the marker gene encodes a proteinsuch as an enzyme which catalyses the breakdown of anantibiotic and, if so, what levels of that protein (if any)



would be expected in the food. Does the gene encoderesistance to a clinically useful antibiotic, and if so,would ingestion of the food while someone was beingtreated with that antibiotic interfere with the drug'seffectiveness? Finally, is it at all likely that the gene couldbe transferred to other organisms such as microbes in thefood or in the intestine of the consumer? For foodenzymes produced by genetically modified organisms,the Organisation for Economic Cooperation andDevelopment (OECD) considered that levels of markergenes or their products in food would almost always bebiologically insignficant. Would the same be true ofplants with marker genes?

With these concerns in mind, the Food Safety Unit of theWorld Health Organization (WHO) organised aworkshop. WHO recognised that there was a need formarker genes, which may have no function in the finalproduct, but that at present it was impractical for suchgenes to be removed from modified organisms afterthey had fulfilled their function. The presence of markergenes per se in food was not thought to constitute asafety concern. It was also noted that the number ofmarker genes in plant varieties appro a c h i n gc o m m e rcialisation was restricted to two antibioticresistance markers and to a few herbicide tolerancemarkers. The workshop concluded that there is norecorded evidence for the transfer of genes from plantsto microorganisms in the gut. This is highly unlikely tooccur for theoretical reasons, since the mechanism forcontrolling gene expression is fundamentally differentin bacteria and plants. Unless the gene transferred intothe plant were under the control of a bacterial system(and therefore unable to be expressed in the plant), therewould be no mechanism for expression of the gene,even if it were transferred to the gut bacteria.

Regulatory approval

European Union. Within the countries of the EuropeanUnion, regulations have been proposed to cover the useof novel foods or food ingredients. In particular, theregulations will apply to modified or new molecules, toany products that have not previously been eaten byhumans to a significant degree, to genetically modifiedorganisms and their products, and to novel processingmethods. This proposal is still under discussion by theEuropean Union Council of Ministers.

United States. In the United States, foods are regulatedby three bodies. The Food and Drug Administration(FDA) is concerned with food safety relating to newplant varieties, dairy products, seafood, food additivesand processing aids, whereas the Department ofAgriculture regulates meat and poultry products andfield tests all genetically modified plants. TheE n v i ronmental Protection Agency is responsible forpesticide chemicals, and it may there f o re have toapprove new plant varieties resistant to attack by pests.Of the three agencies, the policy of the FDAwith respectto new plant varieties is most clearly defined at present.

The FDA regards the key factors in reviewing safety tobe the characteristics of the food and its intended use,rather than the fact that new methods have been used inits production. Novel food products are not subject toregulatory approval if the constituents of the food arethe same or substantially similar to substances currentlyfound in other foods (such as proteins, fats, oils andcarbohydrates). For example, if a gene from a bananaw e re transferred to a tomato, approval would notordinarily be required before that food was placed onthe market. However, if a sweetening agent that hadnever been an ingredient of any other food were addedto a variety of grapefruit, the novel food would needregulatory approval. The sweetener would be regardedas a food additive and therefore be subject to other, morestringent regulations.



Deliberate release of modified organisms

The conduct of laboratory-based genetic modification ofm i c ro o rganisms, while requiring care, presents fewhazards other than those normally associated with theuse of similar microorganisms. Such risks are readilycontained by well-established and tested laboratoryprocedures. However, when a proposal is made to use amodified organism (microbe or plant) beyond theconfines of the laboratory, particularly a ro b u s torganism capable of withstanding the rigours of thenatural environment, a different set of safety questionsarises.

What might the risks be? The deliberate planting ordispersal of genetically modified organisms outside thelaboratory could cause unforeseen effects if, forexample, modified plants or microbes were able to:

• avoid limiting factors that regulate naturallyoccurring populations and thereby change theusual balance between populations (for example,an increased tolerance of drought, high temp-eratures or high salt concentrations could achievethis)

• p roduce new compounds, (for example,insecticides, herbicides)

• transfer their newly inserted genetic material toother plants.

Note that these changes, should they occur, would notnecessarily be harmful; indeed some, such as theproduction of toxins or improved nitrification, might bethe purpose of the modification in the first place. Thenature of the potential environmental impact falls intotwo broad categories: effects which result from theactivities of the modified organism itself and thosewhich might arise should genetic material betransferred into other organisms.

Making pre d i c t i o n s . P redicting the influence of anyorganism (genetically modified or not) introduced into anew habitat is difficult. However, it is important torecognise that nearly all agricultural species areintroductions which have been cultivated by humans,often for many thousands of years. In this time theyhave been subjected to enormous random geneticchange — selection by nature and by humanintervention. It is unlikely that the deliberate andtargeted genetic modification of such species wouldresult in hazards any greater than those that havealready occurred.

Field trials. M o re than a thousand field trials ofgenetically modified crops have been conductedworldwide. Extensive research with the specific aim ofdetermining the ecological effects of modified crops hasbeen carried out in the United Kingdom. The PlannedRelease of Selected and Modified Organisms researchtook 3 years, and was funded by European industry andthe United Kingdom government. Independent publicsector ecologists conducted the work as they saw fit. Theresults suggested that the genetically modified plants(potatoes, maize, oilseed rape and sugar beet) presentedno intrinsic environmental threat. Genes were notreadily transferred from the modified plants to theirwild relatives, and there was no increase in the ability ofthe modified crops to invade natural habitats. Theecologists warned that their findings should not besimply extrapolated to other crops, but suggested that acautious step-by-step approach to planned releases hasmuch to commend it. Similar work involving more than200 field trials and more than 500 locations andtransgenic lines has been carried out in France.

E u ropean regulations governing deliberate re l e a s e s . A l lEuropean Union nations are party to a European Councildirective governing the deliberate release of geneticallymodified organisms (GMOs) into the environment. Thisincludes provisions for marketing food pro d u c t scontaining or made from GMOs. Under this directive all



of the member states have agreed to put into place lawsand regulations to ensure that common policies onGMOs are adopted throughout the union. Theregulations do not apply to organisms obtained byconventional breeding techniques (for example,mutagenesis) that have been applied over a long time.

The directive requires that proposed releases should beconsidered individually. People intending to make arelease must first seek permission from the appropriatenational authority. The application must contain atechnical dossier including information about thepersonnel concerned and their training and a fulle n v i ronmental risk assessment with details ofappropriate safety and emergency responses.

In certain cases interest groups or the public may beconsulted about a proposed release. Action has beentaken in several countries to ensure that there is publicinterest representation on the relevant advisory bodiesand that the public has access to information aboutreleases. Companies or others may ask for someinformation to be kept confidential on grounds ofcommercial competitiveness, but in such cases theymust give verifiable justification. Following thedestruction of some field trials in the Netherlands, therea re now provisions in Dutch law allowing for thelocation of field test sites to be kept secret, but normallythis is not done.

A general principle is that releases should be conducteda step at a time and that the scale of release should beincreased gradually only as it becomes apparent that it issafe to do so. Once a release has been made, a report onit must be sent to the appropriate authorities, withparticular reference to any risk to human health or theenvironment. The directive makes provisions for theexchange of information on releases between memberstates and for the publication of details.

It should be emphasised that the European directivediscussed above is based on a precautionary approach.As more experience has been gained, the risks presentedby genetically modified plants appear to be smaller thanwas first anticipated. International research in this areais leading increasingly to the conclusion that there is nog reater risk from introducing genetically modifiedorganisms into the environment than is presented bysimilar introductions of organisms produced byconventional breeding techniques.

Social, economic and other effects

Biotechnology could bring considerable benefits toworld agriculture, but there could be accompanyingdisadvantages. Insufficient space permits only anindication of some of the main issues here.

Many of the consequences of modern biotechnologycould be similar to those produced by existing trends,such as the shift towards larger farms and more capitalintensive farming systems. This tends to favour wealthyfarmers in the Northern Hemisphere who can invest innew technologies rather than those in the impoverishedSouth. Biotechnology may reduce developed countries'dependency on products imported from developingcountries, with adverse effects in the producer nations.

Concerns in the Western world about overproductionare unlikely to be shared by countries whose populationgrowth far outstrips the capacity of farmers to provides u fficient food. However, if biotechnology bringsgreater agricultural productivity to developing nations,there could be major shifts in employment patterns,with adverse effects on the countries' stability.

Some people also question whether developmentsprovided by the new biotechnology are really needed— the so-called fourth hurdle (after the safety, efficacyand quality of a new product have been demonstrated).



However, the only place where the market need for aproduct can be assessed properly is the marketplace.The requirement to demonstrate need in advance couldseverely restrict innovation.

Biodiversity

Biotechnological methods (including the geneticmodification of plants) have already led to plants withgreatly improved characteristics compared to the oldcultivars produced by conventional methods. Excessiveplanting of a limited number of cultivars would lead toi n c reased genetic uniformity. This might beaccompanied by a greater risk of widespread epidemicsof plant diseases, and it could lead to a reduction inbiodiversity (in crop plants, weed species, insects andmicroflora in the fields in question).

C o n v e r s e l y, biotechnology might encourage t h ep roduction of a far wider variety of new cro p s ,increasing biodiversity. In the developed nations, the"green revolution" of the 1960s and 1970s has been sosuccessful in increasing the yield of food that farmers inthe United States and Europe are paid to take land outof cultivation, again increasing diversity. Thebiotechnological revolution may further accelerate thistrend.

On a wider scale, biotechnologists depend on thegenetic resources of the world for their raw materials,and thus have a vested interest in maintainingb i o d i v e r s i t y. The techniques of plant tissue culturemight be used to help maintain rare and endangeredspecies. Some argue in favour of patents on livingorganisms, as they might allow Third World countriesto obtain payment for the use of their genetic resources.

The need for public involvement

Although the potential of genetic engineering in thefood sector is often exaggerated, many improvementsare possible in the areas of food quality, variety andsafety. The economic effects of such developments couldbe considerable, and there are opportunities to reducethe environmental impact of food processing andproduction, making a positive contribution towardsmore sustainable development. Much will depend onthe attitudes of consumers. Some of the new foods willbe indistinguishable, at least on the supermarket shelf,f rom those currently available. In other pro d u c t s ,genetic engineering will lead to significant andnoticeable improvements, such as better flavour. Thep rovision of appropriate information and publicunderstanding are therefore a crucial concern. Althoughconcerns about some aspects of biotechnology arejustifiable, it is important that these be judged againstthe enormous benefit that technologies such as geneticengineering could bring.

In a democratic society public involvement is essential.The strong case for the involvement of the informed laypublic on decision-making bodies is reflected in thelegislative framework for biotechnology devised inseveral countries. Some sectors of the food industry arewilling to search for applications of genetic modificationthat are mutually acceptable to consumers, legislators,food producers and processors. It is to be hoped thatthis monograph will contribute to that discussion.



BIBLIOGRAPHY

Scientific and technical background

Fincham, J.R.S., and Ravetz, J.R. (1991) G e n e t i c a l l yEngineered Organisms: Benefits and Risks.(ISBN 0 471 93217 5). Council for Science andSociety/Open University Press, Milton Keynes Organization for Economic Cooperation and Develop-ment (1992) Biotechnology, Agriculture and Food(ISBN 92 64 13725 4). OECD, Paris

Food safety

Department of Health/Advisory Committee on NovelFoods and Processes (1991) Guidelines on the Assessmentof Novel Foods and Processes. Her Majesty's StationeryOffice, LondonOrganization for Economic Cooperation and Develop-ment (1993) Safety Evaluation of Foods Derived by ModernBiotechnology — Concepts and Principles (ISBN 92 64 13859 5). OECD, Paris World Health Organization (1991) Strategies forAssessing the Safety of Foods Produced by Biotechnology(ISBN 92 4 156145 9). WHO, Geneva

Ethical issues

Report of the Committee on the Ethics of GeneticModification and Foods Use (1993) Her Majesty'sStationery Office, LondonStraughan, R. (1992) Ethics, Morality and Crop Biotech-nology. Zeneca Seeds, HaslemereStraughan, R. (1989) The Genetic Manipulation of Plants,Animals and Microbes: The Social and Ethical Issues forConsumers: A Discussion Paper. National ConsumerCouncil, London



Dietary Starches and Sugars in Man: A Comparison1989. Edited by J. DobbingILSI Human Nutrition Reviews SeriesAvailable from ILSI Press (originally published bySpringer-Verlag). ISBN 3-540-19560-2

Re-evaluation of Current Methodology of ToxicityTesting Including Gross Nutrients1990. Edited by R. Kroes & R.M. HicksFood and Chemical Toxicology, Vol. 28 No. 11.Published by Pergamon Press. ISSN 0278-6915

Recommended Daily Amounts of Vitamins &Minerals in Europe1990. Nutrition Abstracts and Reviews, Series A, Vol. 60No. 10. Published by C.A.B. International.

Diet & Health in Europe - the Evidence1991. Edited by G.B. BrubacherAnnals of Nutrition & Metabolism, Vol. 35, supplement 1.Published by Karger. ISBN 3-8055-5400-1

Food Packaging: a Burden or an Achievement?(2 volumes)1991. Published by Symposia Paris. ISBN 2-9506203-0-2

Monitoring Dietary Intakes1991. Edited by I. MacdonaldILSI MonographsAvailable from ILSI Press (originally published bySpringer-Verlag). ISBN 3-540-19645-5

Food Policy Trends in Europe: nutrition, technology,analysis and safety1991. Edited by H. Deelstra, M. Fondu, W. Ooghe & R.van HavereEllis Horwood Series in Food Science and TechnologyPublished by Ellis Horwood. ISBN 0-7476-0075-9

Modern Lifestyles, Lower Energy Intake andMicronutrient Status1991. Edited by K. PietrzikILSI Human Nutrition Reviews SeriesAvailable from ILSI Press (originally published bySpringer-Verlag). ISBN 3-540-19629-3

Dietary Fibre – A Component of Food: NutritionalFunction in Health and Disease1992. Edited by T.F. Schweizer and C.A. EdwardsILSI Human Nutrition Reviews SeriesAvailable from ILSI Press (originally published bySpringer-Verlag). ISBN 3-540-19718-4

Health Issues Related to Alcohol Consumption1993. Paulus M. Verschuren, Executive EditorPublished by ILSI Press. ISBN 0-944398-17-0

ILSI Europe Workshop on Nutrition and PhysicalPerformance1993. Edited by P.O. AstrandInternational Journal of Sports Medicine, Vol. 14 No. 5Published by Thieme. ISSN 0172-4622

Scientific Evaluation of the Safety Factor for theAcceptable Daily Intake1993. Edited by R. Kroes, I. Munro and E. PoulsenFood Additives and Contaminants, Vol. 10 No. 3Published by Taylor & Francis. ISSN 0265-203X

High Temperature Testing of Food Contact Materials1993. Edited by J. GilbertFood Additives and Contaminants, Vol.10 No. 6Published by Taylor & Francis. ISSN 0265-203X

Nutritional Appraisal of Novel Foods1993. Edited by Å. Bruce, N. Binns and A. JacksonInternational Journal of Food Science and Nutrition, Vol.44, Suppl. 1, S1-S100Published by The Macmillan Press. ISSN 0963-7486

European Food Packaging and Migration ResearchDirectory1994. Edited by J. GilbertPublished by PIRA International. ISBN 1-85802-069-7

Light Foods – An Assessment of Their Psychological,Sociocultural, Physiological, Nutritional and SafetyAspects1995. Edited by P.D. Leathwood, J. Louis-Sylvestre andJ.-P. MareschiPublished by ILSI Press. ISBN 0-944398-44-8

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