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GENETIC MANIPULATION IN BREWING PARTICULARLY IN AFRICA - DOES IT HAVE A FUTURE?
Graham G. Stewart The International Centre for Brewing and Distilling, Heriot-Watt University, Riccarton, Edinburgh,
Scotland and The University of Nottingham, Loughborough, England. E-mail: [email protected] ABSTRACT The use of genetic manipulation (GM) during various aspects of brewing (as is the case in the food and beverage industry as a whole) has been controversial for the past 20 years. However, its use is slowly becoming accepted. In 2009, 14 million farmers worldwide grew 134 million hectares of GM crops, an eighty-fold increase since 1996. Specifically, the largest increase is in India where GM accounts for 87% of the cotton crop. The United States is the world’s biggest GM grower, with 64 million hectares of soya, maize, cotton, oil-seed rape, squash, papaya, alfalfa and sugar beet being cultivated. These crops possess enhanced attributes such as drought resistance, reduced use of fertilisers and herbicides, increased yield per acre and elimination of cyst nematodes. In addition, trials with purple tomato with an anti-cancer agent from snapdragons and potatoes containing enhanced quantities of vitamin A are currently ongoing. Many of these developments have direct relevance in Africa. There have been a number of GM developments that have direct application to brewing. These include malting barley varieties with reduced anthocyanogen levels, hull-less barley and varieties with superior agronomic properties. Also, brewer’s yeast strains that produce α-acetolactate decarboxylase for diacetyl management, strains with multiple maltose genes for more rapid wort fermentation and strains that produce amylases for wort dextrin utilisation in light and dry beer production have been developed. In addition, amylases of bacterial origin with enhanced temperature stability for use during starch hydrolysis to more efficiently produce kettle and post-fermentation syrups with specified sugar spectra are being employed. Keywords: brewing syrups, cotton, dextrin utilisation, diacetyl management, genetic manipulation, maltose genes, yeast. INTRODUCTION Genetic engineering (or genetic manipulation GM) is the use of a variety of methods to manipulate the DNA (genetic material) or cells in order to modify hereditary traits in order to produce enhanced and novel biological activity and products. The techniques and methods employed include: • Recombinant DNA – the desired genome is inserted into a host cell and hopefully
expressed (Meaden, 1986). • Monoclonal antibodies – desired antibodies are employed as specific markers (McCafferty
et al, 1996). • Polymerase chain reaction (PCR) – production of considerably more identical DNA
(Bartlett and Stirling, 2003). • DNA fingerprinting – the characterisation of nucleic acid fragmentation patterns
(Walmsley, 1994). • DNA microarrays to detect changes in gene expression levels can accomplish many
genetic tests in parallel (Lashkari et al, 1997, Tang et al, 2009). APPLICATIONS OF GENETIC ENGINEERING Genetic engineering can be applied to a number of areas: • Medicine and therapeutics. • Agriculture. • Environment. • Food and beverage production – plants and microorganisms.
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GENETIC MANIPULATION IN AGRICULTURE AND FOOD/BEVERAGE PRODUCTION There are a number of basic facts that encourage genetic manipulation. The global population is currently 6 billion people and will probably increase to 9 billion people by 2050 (Girling, 2010). In 2009, 14 million farmers worldwide cultivated 134 million hectares of GM crops, this is an eight-fold increase since 1996 (Girling, 2010). The United States of America is the world’s biggest GM grower with 64 million hectares under cultivation with soya, maize, cotton, oil-seed rape, squash, papaya, alfalfa and sugar beet. Rice, maize, soya, tobacco, cotton, tomato and potato (Jacobsen and Schouten, 2008) are easier to manipulate than more complex species such as wheat. Wheat has a large complex genome (much larger than the human genome)(Berg and Mertz, 2010). However, the wheat genome has recently been completely sequenced (Henderson, 2010). In 2009, 93% of the cotton grown in the United States was GM and in China 68% of the cotton was GM. Also, in 2010, 94% of the soya beans grown in the United States was GM (herbicide tolerant). There are many objectives of the genetic manipulation of crops including (Girling, 2010): • Enhanced yield per hectare also increased growth rates. • Fungal, virus and other pest resistant crops. • Insect and weed management. • Improved salt, cold and drought tolerance. • Enhanced nutritional value. • Pharmaceutical crops containing edible vaccines and other drugs. GENETIC MANIPULATION AND WORLD CROP PRODUCTION The question is often asked: will GM crops solve world hunger? The answer is NO, not in isolation. GM is a plant breeding technique not a social or economic system. It needs to be considered in the context of overall world food production. Everyone on this planet could be fed as a result of successful plant breeding and modern agricultural techniques but ensuring that everyone has enough to eat is more about politics and distribution than science! There is increasing demand for land, water and energy. By 2050, because of salt build-up resulting from too much irrigation, over-grazing and desertification (Figure 1), 50% of the world’s arable land may be unusable.
Figure 1: Overgrazed land became a desert.
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SOME REACTIONS TO GENETIC MANIPULATION IN AGRICULTURE AND FOOD It is often said that GM is interfering with nature. GM will NOT solve world hunger. It is a breeding method not a social or economic system. GM is development and evolution in a long line of plant and microbial breeding techniques which were not so precise. Contemporary GM techniques are methods that permit genetic changes that would never occur with conventional methods. GM crops are currently grown in 23 countries on 114 million hectares and research trials are ongoing on six continents. Food and drinks prepared from GM raw materials are not necessarily different from food and drink that has been developed employing traditional methods of selective breeding. GENETIC MANIPULATION IN AFRICA Africa (similar in many ways to Europe) has been slow to adopt GM techniques. This has been because of a lack of resources (human and physical) and a reluctance to adopt the “unknown”. Nevertheless, changes are occurring and GM techniques are being adopted in many African countries. For example: • Uganda has approved a National Biotechnology and Biosafety Policy. • Tanzania is developing a drought tolerant maize. The project involves both public and
private institutions. Similar projects are ongoing in Kenya, South Africa and Uganda. • The North Africa Biosciences Network is supporting a project to improve barley
production. This project is funded (in part) by the Canadian International Development Agency (CIDA). This project has an agronomic focus.
• Drought tolerant maize varieties are being developed in various parts of Africa. • Pesticide resistant cowpea varieties are being developed for use primarily amongst
African smallholders. What are the Advantages of Genetic Manipulation in Food and Beverages? • Higher raw material yields are probably the primary objective. • Products with a greater nutrition characteristic are often sought. • Crops that can be grown in harsh environments. • Crops that are more resistant to pests thus eliminating the use of potentially hazardous
pesticides. • More efficient production processes. • Undesirable characteristics can be removed. • Food and beverages that possess a “better” flavour and longer shelf life. • Crops can be used on a less expensive source of medicines (for example, Girling, 2010). What are the Disadvantages of Genetic Manipulation in Food and Beverages? • A gene for herbicide resistance may spread to other crops and create some form of
“superweed”. • Genetic modification that is passed on through pollination might possess a hazard to the
ecosystem. • Unusual gene expression to lead to crops causing more allergic reactions in the
population. • Trials with purple tomatoes containing an anti-cancer agent from snapdragons and
potatoes containing enhanced quantities of vitamin A are currently ongoing. • A virus can be used as a vector to insert the gene inside the host organism and it is feared
that resilient factors may have been enhanced. • Finally, a gene could land in a spot in the genome where it is not wanted and cause harm
due to expression in unusual ways. COTTON Cotton is a shrub (Figure 2) native to tropical and subtropical regions in the Americas, Africa, China and the Indian subcontinent (Yafa, 2004). Its cultivation requires long frost-free periods, plenty of sunshine and moderate rainfall. GM cotton was developed to reduce reliance on
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pesticides. The gene for the Bacillus thuringiensis insecticide toxin has been inserted into cotton in order to produce this toxin. This gene has also been cloned into potato and maize plants (Demont and Tollens, 2004).
Figure 2: Cotton is a shrub.
GM cotton has also been produced with resistance to the herbicide glyphosate (also called Roundup). The gene for glyphosate resistance has also been cloned into soya, maize, sorghum and alfafa – wheat is still under development. GM cotton acreage in India has increased from 50,000 hectares in 2002 to 8.4 million hectares in 2009. This is 87% of India’s cotton cultivation area. India is currently the largest area of GM cotton cultivation in the world with China employing 3.7 million hectares for cotton cultivation (68% of the total crop) in 2009. The U.S. cotton crop was 93% GM in 2010. Although cotton is grown in Africa (Moseley and Gray, 2008) (for example in West Africa and Egypt), GM varieties are only being cultivated on an experimental basis (Bolt, 2007). GENETIC MANIPULATION IN BREWING Interest in genetic manipulation during various aspects of the brewing process began in the 1970s (Sofie et al, 2010). Although considerable effort and expense was devoted to this objective no genetically manipulated example of any part of the processes has been employed in the brewing process except for the use of GM amylases with enhanced thermostability for the production of syrups with defined sugar and dextrin spectra (Stewart, 2006a). Other examples of experiments with raw materials employing GM techniques include: • Barley varieties with reduced anthocyanogen levels for beer with enhanced physical
stability (Jende-Strid, 1997). However, there were agronomic difficulties with the low anthocyanogen barley varieties.
• Hull-less barley in order to improve sweet wort runoff employing a hammer mill and a mash filter for wort separation (Singh and Taino, 1977).
• As already discussed, maize able to be grown in the presence of reduced pesticides are being developed worldwide including Africa.
In addition, the genetic manipulation of brewer’s yeast strains has received considerable attention during the past 30 years, although no genetically manipulated strain has
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knowingly been employed on a production basis. A comprehensive review on this subject has recently been published (Sofie et al, 2010).
Examples of early GM studies with brewer’s yeast strains (Hammond, 1998) that will be discussed in some detail are:
• Yeast with α-acetolactate decarboxylase (ALDC) for controlled diacetyl management (Yamano et al, 1994). It is beyond the scope of this paper to discuss in detail conventional techniques for diacetyl management. Suffice to say, diacetyl is found during wort fermentation as a result of the spontaneous conversion of extracellular α-acetolactate to diacetyl (Figure 3). The diacetyl is subsequently re-adsorbed by yeast at the end of fermentation and during maturation (Figure 4) (Wainwright, 1973). A novel alternative approach was to clone the gene alpha acetolactate decarboxylase (ALDC) into a brewing yeast strain. The ALDC gene was obtained from a strain of the Gram-negative bacterium Acetobacter aceti.
Pyruvate
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butyrate
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Figure 3: Mechanism of diacetyl production by yeast.
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Figure 4: Mechanism of diacetyl reduction by yeast.
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Wort fermentation trials with the ALDC yeast were conducted and compared to the same uncloned strain as control. The diacetyl production and reduction profile were profoundly different when compared to the uncloned control culture (Figure 5). Because of the presence of ALDC, the α-acetolactic acid was not spontaneously converted to diacetyl but to acetoin instead. Acetoin does not have the same flavour impact as the butterscotch aroma from diacetyl. The overall fermentation performance of some cloned yeast strains can be adversely affected when compared to the uncloned control strain (Tada et al, 1995). This was not the situation with the ALDC yeast (Figure 6).
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Figure 5: Effect of alpha acetolactate decarboxylase (ALDC) expression in a
brewing yeast strain on diacetyl metabolism during wort fermentation.
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Figure 6: Effects of alpha acetolactate decarboxylase (ALDC) expression in a brewing yeast strain on overall fermentation rate during wort fermentation.
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• Maltose genes for rapid wort fermentation. Wort contains five fermentable sugars:
glucose, fructose, sucrose, maltose and maltotriose and unfermentable dextrins. These sugars are taken up by yeast in a distinct order (Figure 7)(Stewart, 2006b). Although maltose is the largest concentration sugar in most worts it is not taken up until approximately 60% of the glucose has been metabolised (glucose repression). The metabolism of maltose in Saccharomyces sp. is controlled by the MAL gene cassette which consists of three component genes: - A maltose permease system that controls the active transport of the disaccharide. - α-Glucosidase that hydrolyses the maltose into two glucose molecules once the
maltose is inside the cell. - A system that regulates permease and α-glucosidase activity.
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Figure 7: Order of uptake of wort sugars by yeast.
There are five MAL gene cassettes – MAL1, MAL2, MAL3, MAL4 and MAL6. If any one of these gene systems is present in the genome it will confer upon a strain the ability to metabolise maltose. However, brewing yeast strains are thought to contain more than one MAL gene. In order to investigate this area further, a strain with two MAL2 and two MAL4 gene copies was constructed and its wort fermentation rate compared to a strain containing only one copy of MAL2. As expected, the overall fermentable rate with the strain containing multiple MAL genes was considerably faster than the strain containing the single copy MAL2 (Figure 8). The principal reason for this faster fermentation rate was due to an increased rate of maltose uptake and subsequent metabolism compared to the yeast strain containing the single MAL2 copy (Figure 9). (Stewart, 1981; Kodama et al, 1995)
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MAL2/MAL2;MAL4/MAL4MAL2/mal2;mal4/mal4
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Figure 8: Fermentation profile of a 16°Plato wort with a diploid yeast strain containing multiple maltose (MAL) genes*.
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Figure 9: Uptake of maltose from 16°Plato wort by a diploid yeast strain containing multiple maltose (MAL) genes*.
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• Production of low dextrin beer. It has already been discussed in this paper that wort contains unfermentable dextrins (maltotetraose and larger molecular weight dextrins)(Figure 7). This dextrin remains in the finished beer and consequently gives it mouth feel and contributes to its calorific value. In order to produce a low calorie beer, its dextrin level must be reduced. There are a number of techniques to reduce dextrins (Erratt and Stewart, 1978). One method would be to employ a yeast strain that has an ability to metabolise wort dextrins. There is a grouping of yeast – Saccharomyces cerevisiae var. diastaticus – that is taxonomically closely related to brewer’s yeast strains. These strains contain the genetic ability to produce an extracellular glucoamylase that can hydrolyse the dextrins to glucose which will be taken up by the yeast during wort fermentation. These genes have been identified as: STA1/DEX1, STA2/DEX2 and STA3/DEX3) (Erratt and Stewart, 1978; Perry and Meaden, 1988; Russell et al, 1983). A strain incorporating these genes was constructed and its fermentation characteristics assessed during a wort fermentation. The amylolytic yeast exhibited a faster fermentation rate and to a lower final wort degree Plato than the control yeast that was unable to metabolise wort dextrins (Figure 10).
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Figure 10: Effects of glucoamylase in a brewing yeast strain during wort fermentation.
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Figure 11: Production of glucose in pasteurised beer during storage at 21°C.
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The extrcellular glucoamylase produced by this group of yeast is thermotolerant probably because it is heavily glycosylated (it is a mannoprotein). As a consequence of this, the glucoamylase was not inactivated during pasteurization of the low dextrin beer. This resulted in beer produced with an amolytic yeast contained increasing concentrations of glucose and became sweeter and sweeter. Nevertheless, a low dextrin beer produced with a glucoamylase-containing yeast has been employed on a semi-production scale – Nutfield Lyte – as part of a collaborative project between the Brewery Research Foundation (now Campden BRI, Brewing Division) and Heriot-Watt University (Figure 12). This strain has been approved for use on a production basis by the United Kingdom’s Novel Food Products and Processes (Baxter, 1995). However, it is not being employed on a production basis for brewing.
Figure. 12: Brewing Research Foundation International – Nutfield Lyte.
SUMMARY The use of GM techniques employed on raw materials used in the brewing process (as is the case in many aspects of the food and beverage industry) has been controversial for the past two decades. However, its adoption is being very gradually accepted. In 2009, 14 million farmers grew 134 million hectares of GM crops including: soya, maize, cotton, oilseed rape, squash, papaya, alfalfa and sugar beet. GM wheat is not so far advanced because it possesses a very complex genome. As its genome has recently been sequenced, GM developments with this cereal are expected soon. A number of GM developments have potentially direct application to brewing including: low anthocyanogen barley, hull-less barley and yeast strains which support diacetyl management, containing multiple maltose genes and producing extracellular glucoamylase.
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ACKNOWLEDGMENTS Gratitude is due to Bettie Lodolo and Alastair Kennedy for providing information regarding genetic manipulation developments in Africa. Thanks are also due to Anne Anstruther for her assistance with the development of the written version and also the oral presentation of this paper. REFERENCES Bartlett, J.M., and Stirling, D. (2003). A short history of the polymerase chain reaction. In: Methods in Molecular Biology 226: 3-6.
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