Part 2 Processing and nutritional quality 8 The nutritional enhancement of plant foods D. Lindsay, CEBAS-CSIC, Spain 8.1 Introduction The question of why it is necessary to improve the nutritional value of plant foods is one that at first hand might seem difficult to justify. What evidence is there that this is a problem? In the developed world there are no overt signs of malnutri- tion even amongst strict vegans. The reasons for this are that many processed plant foods are fortified with essential nutrients. Fortification is utilised to replace nutrients lost in the heat processing of foods and through oxidation. Few vege- tarians are dependent on a single plant source to provide their basic nutritional needs. In addition, vegetarians frequently consume vitamins as supplements and the growth in this industry has been rapid. The fact that people are resorting to the consumption of vitamins as supplements is a reflection of their belief that more of a good thing will result in an improvement in their health. This is a very dubious argument. Nonetheless, it is important to recognise that the recom- mended intakes of nutrients, that have been determined by expert groups of nutri- tionists, are based on the evidence that a specific intake level for a nutrient is required to ensure healthy growth and development. They do not reflect the growing body of evidence that suggests different, and often higher, intakes of these same nutrients are required to optimise health and lead to an active life through the prevention of chronic degenerative diseases associated with ageing. 1,2,3 The critical issue is to determine what intakes are required to optimise health rather than to compromise it. 8.2 The nutritional importance of plants Plants are the staple food for the vast majority of the world’s population. It is known that many staple plant foods are deficient in essential nutrients and, con- sequently, malnutrition is widespread. It has been estimated that over 100 million children worldwide are vitamin A deficient and improving the vitamin A content of their food could prevent as many as two million deaths annually in young chil- dren. 4 This is apart from the deficiencies in iodine intake, resulting in goitre, and in iron-deficient anaemia which are estimated to affect millions in the develop- ing world. There is also an important need to improve the amino acid content of legume proteins that are deficient in essential sulphur amino acids. Nutritional deficiencies can lead to a reduction in immune responsiveness, rather than a spe- cific attributable disorder, making it difficult to establish clearly how many people are suffering from malnutrition. 5 In the developed world all public health authorities are urging consumers to consume more plant-based foods as part of a healthy diet. There is a significant body of evidence to suggest that the traditional Mediterranean diet, rich in plant foods, reduces the risk of many age-related diseases. Epidemiological studies show a strong and consistent inverse relationship between fruit and vegetable intake and the risk of cardiovascular diseases and some cancers. 2 An explosion of interest in trying to define what are the factors in fruit and vegetables which might be responsible for these observations has not yet led to a clear set of expla- nations although many theories abound. Plants contain 17 mineral nutrients, 13 vitamins and numerous phytochemi- cals that have been shown to have potentially beneficial effects on health espe- cially against the initiation or progression of degenerative diseases. Almost all human nutrients can be obtained from plant foods, the exceptions are vitamins B 12 and D. However, the adequacy of a plant diet in delivering a health benefit from a specific component will depend on the amount ingested and its bioavail- ability. Many beneficial plant compounds that are associated with the plant cell wall are not easily bioavailable. Any way in which overall levels can be increased will help overcome this difficulty. 8.3 Strategies for nutritional enhancement There is no single approach to the improvement of the nutritional quality of plant foods since this is affected by a wide variety of factors. Amongst these are: ? The application of traditional breeding methods to select for varieties with an increased level of the bioactive compound. ? A reduction in the content of antinutritional factors. ? The use of genetic manipulation to introduce new traits in plants. ? Improvements in handling, storage and food processing technologies. Each of these approaches has a role to play but genetic manipulation provides a mechanism for the improvement of nutritional quality that overcomes the pro- blem of the absence of a specific biochemical pathway in a staple crop. 196 The nutrition handbook for food processors 8.3.1 Application of ‘traditional’ breeding methods Plant varieties have not been selected to date on the basis of nutritional qualities but there are wide natural variations that can be found in the gene pool of crop plants. Examples of where significant variations in the nutrient content of geno- types have been documented include a: ? 2-fold variation in calcium concentration in beans. 6 ? 4-fold variation in b-carotene concentrations in broccoli. 7 ? 4-fold variation in folates in beetroot. 8 ? 2–3-fold variation in iron and zinc levels in maize. 9 In the case of the pro-vitamin A carotenoids, plants provide highly variable amounts depending on their colour. Varieties of sweet potato may contain levels varying from 0.13 mg to 11.3 mg g -1 dry weight b-carotene. 10 Similar variations in levels can be found in carrots and cassava. In the case of the tomato, genes have been identified that are associated with high and low lycopene content. Incorpo- ration of genes that increase lycopene content and/or elimination of genes that decrease the lycopene content can be achieved by pedigree selection and back- cross programmes. Such techniques have produced hybrids with a three- or four- fold increase in content of lycopene in tomato fruits. 11 8.3.2 Reduction in antinutritional factors The interest in reducing antinutritional factors in plants has been predominantly focused around improving the nutritional value of feedstuffs. Phytates are present in many plant seeds and limit phosphorus uptake as well as other elements. The potential for introducing a phytase gene into feedstuffs has been explored. 12 However, there are other strategies that seem to be of greater overall value in human nutrition. Thioredoxin is thought to be an activator of the germination process in seeds. 13 It is able to activate proteins to degradation by proteolysis and results in improved digestibility. 14 It also has the potential advantage of being able to reduce allergenicity, presumably because of its capacity to break disul- phide bonds by the action of the reduced thiol groups in the molecule and ensure the tertiary structure of the protein is accessible to degradation by proteases. 14 The insertion of the wheat thioredoxin gene into barley has produced a transgenic plant where thioredoxin accounts for 7% of the total protein content in the barley and is a good source of sulphur amino acids. 15 8.3.3 The application of genetic manipulation Genetic engineering is being applied to enhance levels of functional compounds in food crops. Indeed, for some purposes it will be the only approach feasible especially where there are widespread deficiency diseases and the population is dependent on staple crops which are not sources of the nutrient required. There are many examples where the technology has been applied with success although there are no products which have yet reached the marketing stage where nutri- tional benefits have been the main focus. The nutritional enhancement of plant foods 197 Potential strategies for the enhancement of specific metabolites could target on: 1. Over-expression of enzymes that control the final steps in the biosynthesis of a metabolite. 2. Over-expression of rate-limiting enzymes. 3. Silencing of genes whose expression causes the metabolite to be degraded. 4. Increased expression of genes that are not subject to metabolic feedback control. 5. Increasing the number of plastids in a plant. 6. Increasing metabolic flux into the pathway of interest. 7. Expression in storage organs using site-specific promoters. The strategy that has had the greatest success at present is the first one, espe- cially in conjunction with the last strategy. In practice, if a substantial increase in the concentration of a metabolite is required, the use of specific promoters directing the synthesis to a particular organelle normally used for storage pur- poses, or where the plant normally synthesises the metabolite, is essential. Failure to use these approaches could cause toxicity in the plant by interfering with the production or function of other essential metabolites. However, this strategy pre- supposes the metabolite of interest is the final one in a particular pathway. Few strategies have yet been applied where multiple gene insertions are nec- essary to produce the metabolite, although these are progressing rapidly, and none where plastid numbers have been increased. However, the accumulation of sequence data of both chromosomal DNA and expressed sequence tags of plants and other species is providing rapid advances in knowledge of the genetic make- up and functions of several plants, and it is expected that these other possibili- ties will soon be feasible. 8.4 The priorities for nutritional enhancement 8.4.1 For the developed world Although it is known that the distribution and processing of food can lead to a significant loss in nutritional quality, there are few instances where present evi- dence suggests there is a need to change current practices. There is very little evi- dence for nutritional deficiencies. In those cases where public health authorities have thought there is a potential problem, food supplementation with nutrients is a commonly adopted policy. The use of nutritional supplements is widespread. Whilst the focus of current interest is on the need to consider nutrients and other phytochemicals as protective against the development of disease in later life, the levels of intake that may be necessary to optimise protection are far from resolved at the present time. The only plant-derived food product on the market where nutritional health benefits are claimed (as opposed to implied) is the enrichment of margarines with plant sterol and stanol esters for the reduction in plasma cholesterol levels (Fig. 198 The nutrition handbook for food processors 8.1) These products do not require the development of specifically bred plants since it is possible to extract stanols and sterols from existing plants (albeit in the case of the stanols from the bark of a tree) for use in their manufacture. Experiments with plant stanol esters were shown to lower serum cholesterol consistently by about 10–15% and LDL-cholesterol by about 20% in patients with high serum cholesterol levels as well as in normal individuals. 16, 17 Similar effects have been seen with plant sterol esters but at least 1 g/day of plant sterols need to be consumed. 18 Consequently, they require extraction and addition to foods. Plant sterols can be in the free form or predominantly esterified with long chain fatty acids or with phenolic acids as in rice-bran oil (ferulate) and shea butter (cinnamates). Sterol esters are better absorbed than the free sterols and most sterol esters are hydrolysed to the free sterols in the intestine. As campesterol esters are better absorbed than sitosterol esters, serum levels of campesterol could rise to those levels that are found in the very few people who suffer toxic symptoms from phytosterolemia. Thus there may be a benefit in increasing the sitosterol to campesterol ratio in plants. The nutritional enhancement of plant foods 199 Fig. 8.1 Structure of plant sterol and stanol esters. The ideal situation would be for sufficient sterols to be present in our diets to ensure that plasma cholesterol levels are kept reasonably low without the need to buy a specific functional food, and that they would be in a fat-soluble form for effective uptake. The evidence favours, in increasing order of preference, the use of: 1. Plant sterol esters with low campesterol contents. 2. Sterol esters from tall oil (derived from pine wood) which have a higher stanol content than edible oils. 3. Plant stanol esters. A vegetable oil rich in plant stanols, especially in sitostanol esterified with polyunsaturated fatty acids, would also have the benefit of being less susceptible to oxidation at frying temperatures than the sterols. The potential health benefits of this class of bioactive compounds are unlikely to be met by the use of classi- cal plant breeding methods but genetic engineering could make these targets feasible. 8.4.2 For the developing world The world-wide deficiency of vitamin A is being tackled both through conven- tional plant breeding and by genetic manipulation. However, the use of conven- tional plant breeding to deliver adequate intakes is dependent on the availability of carotenoid-rich staple foods. Often these are available for very restricted times of the year in some societies. In those countries where rice is a dietary staple the problem is particularly severe and the deficiency is likely to be corrected only by the introduction of rice that has been genetically manipulated to produce b- carotene. However, yellow rice is produced and this may give rise to problems of acceptability to consumers used to white rice. Manipulation of the carotenoid pathway in rice The nature of the challenges faced in manipulating plant secondary metabolites is well illustrated through the attempts that have been made to produce carotenoids in rice plants. A simplified version of the pathways leading to the synthesis of the carotenoids principally found in food plants is shown in Figure 8.2. Immature rice endosperm is capable of synthesising the early pathway inter- mediate geranylgeranyl diphosphate (GP). Four plant genes corresponding to the enzymes phytoene synthase (psy) (1), phytoene desaturase (2), zeta carotene desaturase (3) and lycopene cyclase (crt) (4) are required. Enzyme (1) was obtained from the daffodil (narcissus pseudonarcissus), (2) from a bacterium Erwinia uredovora – which is capable of achieving steps (2) and (3) from the single enzyme, and (4) from the daffodil. The genes need to be expressed in a tissue-specific manner through the inser- tion of specific promoters. This has been achieved in rice through the use of the daffodil psy gene. 19 In rice the daffodil psy cDNA insertion is under the control 200 The nutrition handbook for food processors of an endosperm-specific promoter. The choice of promoter will very much affect the timing and tissue-specific expression of a gene. Surprisingly, seeds that expressed psy and crt did not accumulate lycopene. Instead they contained b-carotene and other xanthophylls. Thus it would seem that the enzymes required to make these metabolites are either normally expressed in rice endosperm or are induced if lycopene is formed. The maximum level of carotenoids in the endosperm of plants that were heterozygous for the transgenes was 1.6 mg kg -1 which is likely to help to meet the nutritional needs of people consuming rice as a staple. Interestingly, good progress is being made in adding a gene coding for ferritin – the iron storage protein found in mammals and plants – to rice. 20 It is likely that this would, in addition, help improve the iron deficiency also seen in these communities if it is shown to be bioavailable. The controversy over the use of advanced technologies for producing sus- tainable food in the developing world has been addressed by the developers of modified rice. They have in effect waived all intellectual property rights for exploitation of the technologies in the developing world and are actively involved in assisting the International Rice Research Institute to breed stable and agro- nomically successful lines for use in vitamin A-deficient areas. 8.5 Relationship of structure to nutritional quality (bioavailability) The overall content of a given nutrient in a food is not always a useful indicator of its nutritional value because not all of the nutrient present is absorbed. Nutri- tionists must concern themselves with understanding the proportion of an avail- The nutritional enhancement of plant foods 201 Fig. 8.2 The carotenoid biosynthetic pathway (simplified). able nutrient that is digested, absorbed, and ultimately utilised. In the case of nutrients or phytochemicals, whose beneficial effects are directed towards inhibit- ing degenerative diseases, it is important to know whether or not the nutrient is reaching the particular target organ and in a form which is active. Otherwise the claims for the health benefits of that chemical would not be justified, especially as it is difficult to demonstrate benefits from long-term human studies. Diet plays an important role in the uptake of specific nutrients and phyto- chemicals. Those that are lipophilic are absorbed much more readily from a lipid- rich diet. Frying tomatoes in oil dramatically improves the uptake of lycopene compared with the consumption of fresh tomatoes. 21 Raw carrots, which have high levels of pro-vitamin A carotenoids, are poorer sources of b-carotene than gently cooked carrot. 22 The bioavailability of certain trace elements is increased on cooking or processing; for example, the bioavailability of iron is increased in canned spinach. 23 The chemical form of the phytochemical present in food is very important in determining uptake through the gastro-intestinal tract. Quercitin-b-glucoside is more easily absorbed than the aglycone quercitin. Isorhamnetin-b-glucoside, which is chemically similar to quercitin, differing only by a single methoxyl group, is more readily absorbed. Flavonoid rutinosides (rhamnosyl 1–6 gluco- sides) are less easily absorbed. 24 Thus whilst some phenols might be better antiox- idants than others when tested in in vitro systems, this is of little significance in terms of health relevance. What matters is whether the compounds are easily absorbed, are not quickly degraded in tissues, and are able to reach the target sites. Flavonoids that are not absorbed undergo extensive degradation by gut microorganisms, and may play only a limited role in preventing oxidative damage in the colon. 8.6 Nutritional enhancement versus food fortification The importance of enhancing the levels of a natural protective constituent in plant foods is well illustrated in the case of the folates. There is a good chance that folate status even in affluent countries is not optimal. 25,26 The most important sources of folates in the diet are liver, products derived from yeast, eggs, green vegetables, legumes and certain fruits. Plant foods (vegetables, fruits and pota- toes) are by far the single largest contributor to the overall folate intake of adults. 27,28,29 Some 40% of the total folate intake is from fruit and vegetable consumption in these countries even when the average consumption is not very high. Folates have the effect of reducing the levels of plasma homocysteine which is a sensitive biomarker of folate status. A variety of studies have suggested that increased plasma homocysteine levels are a risk factor for cardiovascular disease and stroke. 30 Human studies have shown that if individuals consume a supple- ment of 100 g/day of folic acid their plasma homocysteine is reduced to a level 202 The nutrition handbook for food processors of about 7.0mM/l. Increasing the intake of folic acid beyond that level has no further effect. However, the bulk of the population have homocysteine levels in excess of 7.0mM/l. Folic acid is not the natural form of folate that is found in plants where natural folate consists of ten different polyglutamate complexes. Folic acid is, however, the form of folate that is used in the fortification of food as it is more stable. It is also found to be more bioavailable. Natural folates show only 50% or less of the bioavailability of folic acid. 31, 32 There is good evidence that to achieve the ideal level of plasma homocysteine dietary levels of folate (as opposed to folic acid) would have to increase from the current average of 200mg/day to 600mg/day. 33 This increased intake is also likely to have an important impact on the reduction of Neural Tube Defects (NTDs). Women with a low folate status (about 150mg/l red cell folate) have a 0.7% risk of NTDs in their offspring, whereas supplementation with folic acid at doses of between 100 and 200 g/day resulted in red blood cell folate levels that have been associated with an optimal reduction in NTD incidence. Since average intakes of natural folates are about 100mg/day from the diet it would require at least 500 mg of natural folate to be consumed (preferably 600 mg/day) to ensure that the incidence of NTD in the population was kept to a minimum. The fact that supplemental folic acid can achieve these same effects whilst being more stable and bioavailable would imply that there was little purpose in supplementing natural levels of folate. This ignores the intrinsic difference between the cellular metabolism of synthetic pro-vitamin folic acid compared to the natural folates. The mucosa converts all of the natural forms of folate into 5-methylenetetrahydrofolate monoglutamate. This reaction also occurs when folic acid is consumed but the difference is that for folic acid the process can be saturated at around 300mg. Intakes in excess of this cause un-metabolised folic acid to enter the circulation. 34 The control of how much natural folate is taken up and retained by cells is regulated by the enzyme methionine synthase which acts on 5-methyltetrahydrofolate to conjugate it into a polyglutamate which is then retained in the cell. Folic acid does not pass through the methion- ine synthase pathway and can be conjugated directly, retained and metabolised. If folic acid is present in excess of the mucosal capacity to metabolise it can bring about DNA biosynthesis in vitamin B 12 -deficient cells in cases of pernicious anaemia via the DNA cycle. This causes a haematological response with the risk that the anaemic state is masked and the associated neuropathy is not avoided. Natural folates on the other hand will be poorly metabolised by vitamin B 12 -deficient cells enabling the anaemia to be detected at an earlier stage. Another concern that has been raised against increasing population levels of folate is that the increased capacity to cause DNA biosynthesis could promote tumour growth. This would be expected to be more of a problem with folic acid than natural folates because of folic acid’s less controlled uptake into cells. The nutritional enhancement of plant foods 203 8.7 Constraints on innovation The potential to exploit fully GM technologies is severely limited by constraints on the use of the technology itself, as well as in satisfying the legislation that exists on the pre-market approval of foods that have been produced by the tech- nology, or are in some way novel. These constraints are so severe in Europe that in very few cases will any producer see a return on their investment if nutritional improvement is their goal. This market is also affected by the widespread addi- tion of specific nutrients as additives to certain processed foods. Enhancement of any component considered to be beneficial is likely to be of market value only if positive claims can be made. Whenever possible ‘conventional’ plant breeding will be used. 8.7.1 Genetic manipulation There has been a de facto European Union moratorium on the approval of GMO products since October 1998. Eighteen products have already been approved under the general EU Directive (90/220/CEE) whilst 14 are pending approval. Five Member States have temporarily banned already approved GM products, which is permitted under the Directive. Two new EU labelling regulations have been drafted but have not been implemented because of a lack of testing method- ologies, certifying labels and inspection procedures. The ultimate intention is to ensure that products can be labelled GM free to enable consumers to make an informed choice. It has been argued that products labelled as containing products derived from GM will convey negative messages to consumers. This is likely to be so in the absence of benefits that are clearly seen by consumers. This will occur if plants are used as factories for the production of vaccines and pharma- ceutical products. However, it is unclear at present where these benefits will lie in the nutritional field other than for the developing world. The lack of public confidence in the European food safety system is already causing harm to markets in the US and in developing nations where the technol- ogy is already embraced. This is likely to lead to major problems in international trade unless it is resolved. 8.7.2 Safety No scientific development in food can ignore the very strict regulatory controls that exist before any new or ‘novel’ product or process can be applied in its pro- duction. Food plants produced by ‘conventional’ plant breeding techniques in general are not subject to any regulatory controls. In some countries voluntary codes of practice have been developed within the plant breeding sector when it was discovered that varieties of potatoes with good agronomic characteristics were found to contain high levels of toxic glycoalkaloids. 35 At the present time, genetically-modified (GM) foods are regulated applying the concept of ‘substantial equivalence’. 36 This concept is applied as the basis 204 The nutrition handbook for food processors from which to determine the extent of the requirements for food safety assess- ment. If a genetically modified food can be characterised as substantially equiv- alent, it can be assumed to pose no new health risks over its conventional counterpart and can be marketed without the need to undertake extensive toxi- cological and nutritional studies to determine its safety-in-use. The principle of substantial equivalence was adopted into the EU Regulation on Novel Foods and Novel Food Ingredients. 37 The Regulation excludes from its controls foods and food ingredients obtained through traditional propagating or breeding practices and which have a history of safe use. GM plants are consid- ered as ‘novel’ under the terms of the Regulation. However, the detailed safety evaluation provisions of the Regulation do not apply to foods produced by genetic manipulation ‘if on the basis of the scientific evidence available they are sub- stantially equivalent to existing foods with regard to their composition, nutritional value, metabolism, intended use, and the level of undesirable substances present’. The Regulation regards food as ‘novel’ if the characteristics of the food differ from the conventional food regarding the accepted limits of natural variation of such characteristics. It is clear that most nutritionally enhanced plants would be caught under the definition of a ‘novel’ food. The principle of substantial equivalence is vague and difficult to define in many cases. Consequently the whole issue of regulation of GM foods is under intensive debate. Meanwhile the EU has applied a de facto moratorium on GM plant introductions. The US attitude to regulation has so far been to regard safety as an issue that relates to the characteristic of the food and not to the process(es) that lead to it. Novel food products, of which products produced by GM are included in the definition, are not subject to any specific approval on safety ground if the constituents of the food are the same, or substantially similar, to substances currently found in other foods. It is clear that it is never going to be possible to argue that a GM plant is safe any more than it is possible to argue that a plant produced by conventional plant breeding is safe. The very concept can be addressed only in the context of a history of safe use as a human food. Clearly, the overwhelming evidence supports the view that health benefits arise as a consequence of the regular consumption of a variety of fruits and vegetables, few if any of which have any close compositional relationship to the wild types from which they were bred. Similarly their production, storage and distribution has depended on the use of a wide range of chemical fertilisers and pesticides. These chemicals are extensively tested for safety before approval is given for their marketing and use but this has not removed the widely held view amongst consumers that ‘organic products’ are better for your health. There is no evidence to support this view and any adverse health effects that there might be as a consequence of the use of pesticides appear to be outweighed by the beneficial effects from the consumption of fruit and vegetables. What determines ‘safety’ is the overall effect of consumption over a period, not the effects of a specific chemical that might be present. The issue of ‘safety’ in the context of the ability to market foods which are ‘novel’ is emotionally charged and without a solid scientific base. Consequently The nutritional enhancement of plant foods 205 it is unlikely that any industry would want to take on these issues unless they had a product with a potentially large market. 8.8 Future trends It is clear that the developing world will adopt whatever approach is technically feasible for them to meet the food and nutritional needs of their populations. Genet- ically modified crops will be used if there are clear benefits. In terms of resistance to disease and adaptation to harsh environments the technology has clear poten- tial. Improvements in nutritional quality can be added to the list of benefits. In Europe and other developed countries the impetus for improving the nutri- tional value of foods will occur only if there are clear health benefits in doing so. As there is growing evidence that nutritional needs will vary according to age and genetic susceptibility, it will be hard to convey a consistent message since intakes that benefit one sector of society might not benefit another. The priority is to demonstrate clearly what are the functional effects of nutrients, or benefi- cial phytochemicals, at the physiological level. The information is generally rudi- mentary. In those cases where the function is clearer the relationships between dose and effect are not known. When it comes to marketing foods that have been genetically manipulated the benefits will have to be very great indeed if current consumer resistance to their use is to be overcome. 8.9 Further information Sources of further information about the potential for nutritional enhancement can be found in reviews by Willis, Lencki and Marangoni (1998), 38 Grusak and DellaPenna (1999), 39 Dixon et al., (1996), 40 Yamauchi and Minamikawa (1998). 41 An overview of the subject is contained in Lindsay, D. G. (2000) ‘Maximising the functional benefits of plant foods’, in: Functional Foods. Chapter 8, edited by Williams, C. M. and Gibson, G. R., Woodhead Publishing Ltd. Cambridge, England. pp. 183–208. ISBN 1 85573 503 2. A account of the issues to be addressed in tackling the nutritional enhance- ment of plants (including increasing levels of bioactive secondary metabolites) can be found in published reviews commissioned under the EU’s concerted action project ‘The Nutritional Enhancement of Plant Foods in European Trade (NEODIET)’, 2000, J Sci Food & Agric. 80(7): 793–1137. Some of the issues raised in relation to the use of plant biotechnology in food and feed produc- tion are discussed in papers contained in a special edition of Science (Plant Biotechnology: Food & Feed, 1999, Science 285: 367–389). 8.10 References 1 buring j e and smith c v (1997), Antioxidant vitamins and cardiovascular disease Nutrn Rev, 55(1), S53–S58 206 The nutrition handbook for food processors 2 steinmetz k a and potter j d (1996), Vegetables, fruit and cancer prevention: A review. 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