Illustrated feature published in Dairy Industries International, February 2000

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Cultures & coagulants - The genetics game

Rapid development of genetic technology has placed the dairy industry at the dawn of revolution.  Russ Clare reports

Rapid development of genetic technology has placed the dairy industry at the dawn of revolution.  Genes, with their orderly DNA structure - a code for enzymes and other proteins that control living processes - can be isolated, deciphered and used to modify the performance of other organisms.  Already, following one of the earliest applications in the food industry, chymosin (rennet) obtained from genetically modified (GM) microorganisms is used extensively for hard cheese production in Europe.  But the scope and benefits are much broader with many dairy processes open to  improvement by modern biotechnology, while novel horizons beckon in nutrition, food technology and pharmacology.

Designer milk is now a real possibility.  For instance, genetic engineering for secretion of low lactose milk - recently demonstrated - could make whole milk available to the majority of the world's adults currently excluded by lactose intolerance.  Turning to starter cultures, techniques are available to engineer lactic acid bacteria (LAB) to produce health foods - with low cholesterol, for example.  Modification to harness the bacteria's natural antimicrobials could extend shelf-life of dairy foods, offering safe storage in hot countries.  Dairy fermentations also have immense potential to provide enzymes and other products for the food and pharmaceutical industries, while modified LAB may prove effective vehicles for the delivery of vaccines.  Even waste whey is a potential source of fuel alcohol and other chemicals, given its efficient fermentation by bacteria modified with yeast genes.

Although the technology is undoubtedly awe inspiring, opinion on its value and safety remain sharply divided.

Application opportunities
Dramatic advances in understanding the genetics of LAB, notably Lactococcus lactis and Lactobacillus species, have been driven by immediate application opportunities, as Professor Gerald Fitzgerald, Director of Ireland's National Food Biotechnology Centre, at University College Cork, explains: "in the modern dairy industry, tight production schedules and demands for consistently high quality rely on starter cultures with unpredictable behaviour.  Spontaneous genetic change that spreads rapidly can soon shift growth rates, lactic acid production, flavour, and phage resistance."  Historically, the dairy technologist has responded by screening for new strains with desirable traits, but genetic engineering offers a more efficient solution.  "Biotechnologists can construct bacteria with more reliable behaviour, but that quest for the perfect strain must be based on a thorough understanding of how genes control expression of desirable characters," says Fitzgerald.

A major step towards that understanding came in September 1999 at the Sixth International LAB Symposium at Veldhoven, the Netherlands when Dr Dusko Ehrlich and colleagues from Institut National de la Recherche Agronomique, France, announced completion of the entire genome sequence of L. lactis strain IL 1403.  This description of the bacteria's genetic code will not only make the study and engineering of individual genetic traits more efficient, it will also allow multiple gene effects to be investigated - the ordered behaviour of living organisms, including the performance of LAB in dairy fermentations, is fashioned by complex interactions of genes and environment.

Recombinant DNA and gene cloning are techniques central to genetic engineering (Fig.1). Characteristically, genetic information in bacteria is not held solely on the single chromosome.  Smaller, circular DNA plasmids also carry genes.  A gene of interest from an organism - isolated as a DNA fragment - can be recombined into a bacterial plasmid which acts as a carrier or vector for that gene.  Introduced into a bacterial cell, the recombinant gene is cloned when the plasmid and cells replicate.  Thus amplified, the gene can be studied more effectively, and it is available for the modification of other organisms - for instance, a gene that accelerates cheese ripening, recombined on a plasmid vector, could be introduced into starter culture strains.

The European Union (EU) has funded integrated, long-term research on LAB since the mid 1970s, with collaborative work involving 56 laboratories actively disseminating results to nearly 40 companies.  The rewards of this concerted approach are illustrated by work on protein metabolism and cheese flavour at the University of Groningen in The Netherlands, the NFBC at Cork and other key institutions.  Enzyme controlled chemical pathways used by L. lactis to degrade the milk protein, casein have been mapped.  Initial breakdown during fermentation is followed by a cascade of reactions, which continue as the cheese matures.  The reactions' products - a collection of smaller peptide fragments - are responsible for flavour, and some 200 have been identified together with unique genes that code for the controlling peptidase enzymes.  By constructing strains with individual genes added or deleted, respectively to over-express or minimise production of specific enzymes, their contribution to peptide profiles and flavour can be assessed with trial cheeses.  Ultimately, the research should enable commercially useful strains to be constructed that efficiently develop desired flavour.

With similar advances in accelerated cheese ripening, diacetyl synthesis for butter flavour, phage resistance, antimicrobials, and many related fields, Dr Gerard Venema from University of Gronningen and colleagues could confidently assert in a keynote address at the Veldhoven Symposium, "almost all [LAB] modifications to suit the fermentation industry are within reach."

But would foods made with the technology be safe?

The scientists consider such concerns are satisfied if modified LAB retain their original properties.   Self-cloning - the improvement of strains using only genes and plasmid vectors from within the same species - embodies this approach.  As LAB are 'generally recognised as safe' (GRAS) and no foreign DNA is used, the method is deemed intrinsically nonhazardous, as Fitzgerald explains: "food related LAB have a history of safe use stretching back thousands of years, and evidence that they carry genes related to pathogenic (disease causing) mechanisms is lacking."

An argument for the safety of modification using foreign genes can also be made where the donor species is another GRAS organism, and the inserted DNA is integrated into the host's chromosome.  Many of the processes essential to dairy fermentations are controlled by plasmid genes - lactic acid production, for instance.  That has helped the geneticist since plasmid genes are easier to manipulate.  But for the dairy technologist such genes are a source of unreliability because, in continued culture, bacteria can loose plasmids along with their desirable traits.  Safety concerns arise because recombinant DNA lost in this way has the potential to modify other bacteria with unintended consequences.  Chromosomal DNA is not vulnerable to such instability, and now, biotechnologists can integrate plasmid cloned genes into the chromosome.

Food grade genetic modification also requires a different approach to selectable markers which are used to isolate successfully engineered bacteria.  In common research practise, genes are cloned on a plasmid vector alongside a gene for antibiotic resistance.  The recombinant bacteria are selected by their ability to grow on medium containing the antibiotic.  Given the possible transfer of antibiotic resistance genes to other bacteria, there is absolute agreement on their absence from food grade applications.

Several alternatives have been investigated, including the mechanism based on lactose metabolism in L. lactis developed by Professor Willem de Vos and colleagues at NIZO Food Research, The Netherlands.

The lacF gene, located in the lac operon - a collection of genes controlling lactose metabolism - codes for an enzyme essential in lactose breakdown (Fig. 2).  De Vos explains: "Natural mutant strains of L. lactis deficient in the enzyme are unable to grow in lactose medium.  We can also construct similar strains artificially by deleting the lacF gene.  In these strains, with the lac operon integrated into the chromosome, a food grade clone that grows on lactose can be constructed by introducing a plasmid carrying the lacF gene alongside the gene of interest." The patented system uses no foreign DNA so is ideal for genetic improvement of L. lactis by self-cloning.  It has immediate application in contained use of modified bacteria for production of enzymes and other metabolites for the food industry, and also potential application in GM fermented foods.

Substantial equivalence
Approval of GM foods within the EU is regulated by 1997 legislation on novel foods and ingredients which employs the substantial equivalence principle.  The concept, endorsed by the World Health Organisation and adopted by many other countries,  focuses on unintended effects of genetic modification - approval follows only when the novel food is shown to be as safe as the existing food it is meant to replace.  Safety assessments are made by each member state.  In the UK, independent experts appointed to the Government's Advisory Committee on Novel Foods and Processes (ACNFP) judge applications on a case by case basis - the only clear policy being the rejection of antibiotic resistance markers in foods with living GM microorganisms.  Among the issues considered are the structure, origins and stability of inserted DNA, and the techniques used.  For foods containing GM microrganisms, the health effects of any past exposure to the conventional species are also assessed.

Consensus within the scientific community maintains GM foods are safe, given proper regulation.  In the UK, the Government's Chief Medical Officer, Professor Liam Donaldson, and Chief Scientific Adviser, Sir Brian May reported in May 1999, "there is no current evidence to suggest that GM technology is inherently harmful."  Nonetheless, there is widespread resistance to GM foods in Europe - unsurprisingly, given the erosion of consumer confidence in food safety following health crises, such as BSE.  Genetic technologies and their attendant risks are also poorly understood, while an ethical dimension arises from an intuitive sense that 'tinkering with the secret of life' breaks previously accepted boundaries of science.

Despite this negative climate, biotechnology's advocates can take some encouragement from research showing a pragmatic view among consumers.

Psychologist Lynne Frewer and colleagues at the UK's Institute of Food Research are investigating public concern about genetic engineering.  They have shown a majority of consumers, given a simple option, would choose a conventional cheese over one involving genetic modification.  However, when asked to consider advantages of modification, a majority stated they would choose GM cheese if it offered tangible benefits. "But that trade-off only applied to consumer benefits - for instance, improved nutrition or flavour," says Frewer.  "Consumers were not swayed by benefits only to the producer - like a faster production time - although price reductions were moderately attractive."

Meanwhile, in 1997, food market research group Seymour-Cooke Ltd reported widespread lack of support for biotechnology among major European dairies.  "Many dairies see little point in investing in production processes which are not acceptable to their core markets," says Director, Tim Cooke.  But, without such immediate consumer acceptance it may be difficult to maintain the momentum of research.  "We are at the beginning of a huge biotechnological revolution," says Fitzgerald.  "The technology is progressing well, but we have to be careful not to stunt development.  In the next 10 to 20 years many more applications will be possible, especially in nutrition and health management.  But if we don't get over the hurdle of acceptance, we may be unable to deliver them - research funders will say, 'look, people are not interested'."

The arguments for dairy biotechnology are compelling, and given greater attention to  consumers' understanding of the science perhaps they will prevail.  Time will tell.

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[A copy of reference material for this article may be obtained from the editorial secretary at DII, or from the author.]

Article published in Dairy Industries International, February 2000, and reproduced here with permission from the publisher: Wilmington Publishers Ltd

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