P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come 19 Introduction to Food Biochemistry Table 1.16 Selected Enzyme-induced Flavour Reactions Enzyme Alliin lyase (EC 4.4.1.4; garlic, onion) β-Glucosidase (EC 3.2.1.21; strawberry) Catechol oxidase (EC 1.10.3.1; tea) Limonin-d-ring-lactonase (EC 3.1.1.36; lemon and orange seeds) Thioglucosidase (EC 3.2.1.147; cruciferous vegetables) Reaction An S-alkyl-l-cysteine S-oxide → An alkyl sufenate + 2-Aminoacrylate Hydrolysis of terminal non-reducing β-d-glucose residues with release of β-d-glucose (2,5-Dimethyl-4-hydroxy-2H-furan-3-one (DMHF)-glucoside → DMHF) Catechol + O2 → 1,2-Benzoquinone + H2 O Limonoate-d-ring-lactone + H2 O → Limonate A thioglucoside + H2 O → A thiol + A sugar Source: Wong 1989, Eskin 1990, Chin and Lindsay 1994, Orruno et al 2001, IUBMB-NC website (www.iubmb.org) after it is exposed to air due to enzymatic oxidation (discussed earlier) Flavours from cheese fermentation and fresh-fish odour have already been described earlier, and formation of fishy odour will be described later Colour is an intrinsic property of foods, and therefore, a change in colour is often caused by a change in quality Vision is the most important sensory perception in selecting food and appreciating its quality (Diehl 2008) Chlorophylls are the most abundant natural pigments and are responsible for the green colour of plants (Marquez Ursula and Sinnecker 2008) and are the biomolecules responsible for capturing light energy in its transformation into chemical energy during photosynthesis Light is able to be absorbed very efficiently due to the presence of many conjugated double bonds within the large, multi-ring chlorophyll structure Disappearance of chlorophyll during fruit ripening and leaf senescence indicates slowing of photosynthesis The loss of green colour is due to a loss of chlorophyll structure via two main stages: First, various reactions produce greenish chlorophyll derivatives and second, oxidative reactions result in opening of ring structures, thereby causing colourless products (Diehl 2008) Carotenoids are the most widely distributed group of pigments, and although they are not produced by the human body, they are essential to human health Vitamin A/β-carotene are carotenoids critical to a healthy diet Additionally, carotenoids may help reduce the risk of cancer and heart disease (Bertram 1999), are important natural colourants in foods and are used as sources of red, yellow and orange food colouring (Otles and Cagindi 2008) Carotenoids are fat-soluble pigments that provide the colour for many common fruits such as yellow peaches, papayas and mangoes During post-harvest maturation, these fruits show intense yellow to yellowish orange colours due to synthesis of carotenoids from its precursor isopentyl diphosphate, which is derived from (R)-mevalonate Isopentyl diphosphate is a key building block for carotenoids (Croteau et al 2000), and Table 1.17 lists the sequence of reactions in the formation of (R)-mevalonate from acetyl-CoA and from (R)mevalonate to isopentyl diphosphate Flavonoids are not only a group of compounds responsible for various red, blue or violet colours of fruits and vegetables, they are also related to the group of bioactive, anti-plant pathogen compounds called stilbenes Stilbenes have a common precursor of trans-cinnamate branching out into two routes, one that leads to flavonoids and the other leading to stilbenes (Table 1.18) Table 1.18 gives the series of reactions in the biosynthesis of naringenin chalcone, the building block for flavonoid Table 1.17 Mevalonate and Isopentyl Diphosphate Biosyntheses Enzyme Acetyl-CoA C-acetyltransferase (EC 2.3.1.9) Hydroxymethylglutaryl-CoA-synthase (EC 2.3.3.10) Hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) Mevaldate reductase (EC 1.1.1.32) Mevalonate kinase (EC 2.7.1.36) Phosphomevalonate kinase (EC 2.7.4.2) Diphosphomevalonate decarboxylase (EC 4.1.1.33) Source: Croteau et al 2000, IUBMB-NC Enzyme website (www.iubmb.org) Reaction Acetyl-CoA → Acetoacetyl-Co-A + CoA Acetoacetyl-CoA + Acetyl-CoA + H2 O → (S)-3-Hydroxy-3-methylglutaryl CoA + CoA (S)-3-Hydroxy-3-methylglutaryl-CoA + NADPH2 → (R)-Mevalonate + CoA + NADP (R)-Mevalonate + NAD → Mevaldate + NADH2 (R)-Mevalonate + ATP → (R)-5-Phosphomevalonate + ADP (R)-5-Phosphomevalonate + ATP → (R)-5-Diphosphomevalonate + ADP (R)-5-Diphosphomevalonate + ATP → Isopentyl diphosphate + ADP + Pi + CO2 P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm 20 Printer Name: Yet to Come Part 1: Principles/Food Analysis Table 1.18 Naringenin Chalcone Biosynthesis Enzyme Reaction Phenylalanine ammonia-lyase (EC 4.3.1.5) Trans-cinnamate 4-monoxygenase (EC 1.14.13.11) 4-Coumarate-CoA ligase (EC 6.2.1.12) Naringinin-chalcone synthase (EC 2.3.1.74) l-phenylalanine → Trans-cinnamate + NH3 Trans-cinnamate + NADPH2 + O2 → 4-hydroxycinnamate + NADP + H2 O 4-Hydroxycinnamate (4-coumarate) + ATP + CoA → 4-Coumaroyl-CoA + AMP + Diphosphate Naringinin chalcone + CoA + CO2 Source: Eskin 1990, Croteau et al 2000, IUBMB-NC Enzyme (www.iubmb.org) biosynthesis Considerable interest has been given to the stilbene trans 3,5,4 -trihydroxystilbene commonly called resveratrol in red grapes and red wine that may have human nutraceutical and/or pharmaceutical applications (Narayanan et al 2009) Table 1.19 summarises various phytochemicals and their sources that are thought to confer health benefits (Gropper 2009) of lipoproteins vary greatly; an excessive level of low-density lipoprotein (LDL) is a critical risk factor for heart disease In contrast, plants produce related molecules called phytosterols, which have various nutraceutical applications (Kritchevsky and Chen 2005) Terpenoids Cholesterol Cholesterol is an important lipid in animal biochemical processes that controls the fluidity of membranes and is the precursor for all steroid hormones Cholesterol’s structure is made up of adjacent carbon rings, making it hydrophobic (fat soluble), and thus it must be transported through the blood complex as a lipoprotein in structures called chylomicrons The densities Terpenoids, a diverse and complex chemical group, are lipidtype molecules important to flavours and aromas of seasonings, herbs and fruits Most terpenoids are multi-cyclic compounds made via successive polymerisation and cyclisation reactions derived from the 5-carbon building block isoprene, which contains two double bonds Terpenoids are components of the flavour profiles of most soft fruit (Maarse 1991) In some fruit species, they are of great importance for the characteristic flavour and aroma Table 1.19 Phytochemicals and Their Sources Phytochemical Anthocyanins Carotenoids Cyanidin β-carotene, α-carotene, lutein, lycopene Flavanols Flavones Flavonols Catechins, epicatechins Apigenin, luteolin Quercetin, kaempferol, myricetin Glucosinolates Gucobrassicin, gluconapin, sinigrin, glucoiberin Genistein, daidzein, equol Allylisothiocyanates, indoles, sulforaphane Isoflavones Isothiocyanates Lignans Organosulfides Phenolic acids Phytosterols Saponins Terpenes Secoisolariciresinol, mataresinol Diallyl sulphide, allyl methyl sulphide, S-allylcysteine Hydroxycinnamic acids (caffeic, ferukic, chlorogenic, curcumin) and hydroxybenzoic acids (ellagic, gallic) β-Sitosterol, campesterol, stigmasterol Panaxadiol, panaxatriol Limonene, carvone Source: Adapted from Gropper et al 2009 Source Berries, cherries, plums, red wine Tomato, pumpkin, squash, carrot, watermelon, papaya, guava Green tea, pear, wine, apple Parsley, some cereals Onions, tea, olive, kale, leaf lettuce, cranberry, tomato, apple, turnip green, endive, Gingko biloba Broccoli, cabbage, Brussels sprouts, mustard, watercress Soy, nuts, milk, cheese, flour, miso, legumes Broccoli, cabbage, Brussels sprouts, mustard, watercress Berries, flaxseed, nuts, rye bran Garlic, onions, leeks, broccoli, cabbage, brussel sprouts, mustard, watercress Blueberry, cherry, pear, apple, orange, grapefruit, white potato, coffee bean, St John’s wort, Echinacea, raspberry, strawberry, grape juice Oils: soy, rapeseed, corn, sunflower Alfalfa sprouts, potato, tomato, ginseng Citrus fruits, cherries, Gingko biloba P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come Introduction to Food Biochemistry (e.g citrus fruits are high in terpenoids as is mango; Aharoni et al 2004) NUCLEIC ACIDS AND FOOD SCIENCE DNA Structure Although nucleic acids are not generally important components of foods, they are perhaps (and ironically) the most important biomolecule class of all for the simple reason that everything we eat was at some point alive, and every process and structure in those organisms were determined via enzymes encoded for by DNA Indeed, the entire purpose of breeding programs is to control the propagation of DNA between successive generations Understanding the basics of DNA (deoxyribonucleic acid) and its biochemical forms is important for food scientists to gain an appreciation of the basis for genetic manipulations of food-related proteins as well as DNA-based food authentication techniques DNA is composed of three main chemical components: a nitrogenous base, a sugar and a phosphate There are four bases: Adenine (A) and guanine (G) are purines, while thymine (T) and cytosine (C) are pyrimidines The bases bound to both the sugar and phosphate moieties make up nucleotides and the four building blocks of DNA are deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxycytidine triphosphate (dATP, dGTP, dTTP and dCTP, respectively) These building blocks are typically referred to as ‘bases’ or simply A, T, G and C; however, common usage of these terms are meant to imply nucleotides as opposed to bases alone The nucleotides covalently bond together forming a DNA strand that is synthesised by DNA polymerase Additionally, each nucleotide’s base moiety can bond via hydrogen bonds to other bases; A–T and G–C, termed base pairs and are said to be complementary In fact, DNA exists in two-stranded form, consisting of two complementary strands For example, a strand ATCG would be paired to its complement TAGC 5’-ATCG-3’ 3’-TAGC-5’ Two-stranded DNA spontaneously forms a helix, hence the term double helix, which can contain many thousands of base pairs with molecular weights in the millions, or billions, of Daltons By comparison, the largest known protein is a mere million Daltons Genes are stretches of DNA that encode for the synthesis of proteins; DNA is transcribed, yielding messenger RNA (mRNA), which is then translated at ribosomes, yielding specific sequences of amino acids (proteins) 21 to traditional breeding programs in the search for food plants and animals that have desired traits, such as increased yield, increased pesticide tolerance, lower pesticide requirements/higher pest resistance, longer shelf life post-harvest, etc This alternate strategy is the basis of genetic modification (GM) Critical to the study, transfer and manipulation of genes was the advent of the polymerase chain reaction (PCR), which allows for the easy and accurate copying and, equally important, the amplification of DNA sequences Briefly, PCR works by inducing repeated copying of a given DNA sequence by the enzyme DNA polymerase via repeated temperature cycles such that exponential amplification results, i.e the first round yields only a doubling, but the second round then makes new copies of each of the first round’s copies and the originals; 2, 4, 8, 16, 32, 64, 128, etc After 25–30 PCR rounds, millions of copies result Thus, a gene encoding for a useful gene in organism A (e.g an anti-freeze protein) can be copied, amplified and subsequently transferred to organism B (e.g a fruit) An example of GM is that of the Flavr savrTM tomato, originally available for consumption in 1994 (Martineau 2001) A non-sense gene is a DNA sequence that encodes for complementary mRNA, which base-pair matches and binds to a natural gene transcript, thereby suppressing its translation A ‘non-sense’, gene acting against the polygalacturonase gene, an enzyme responsible for the breakdown of a cell-wall component during ripening, was incorporated into a strain of tomato The result was slowed softening of the texture of the engineered tomato compared to normal tomatoes, thus allowing producers to vineripen the Flavr savrTM , reducing losses (e.g bruising) during subsequent transport to market Superior flavour and appearance relative to natural tomatoes picked green, as well as equivalent micro- and macronutrient content, pH, acidity and sugar content relative to non-transgenic tomatoes resulted In terms of food processing, lactic acid bacteria and yeast have been developed to solve problems in the dairy, baking and brewing industries (Tables 1.20 and 1.21) As with biotechnologyderived food enzymes, the use of genetically modified organisms is governed by laws of nations or regions (e.g the European Union) Safety assessments of GM foods before being released to market are done These comparisons to non-engineered, conventional counterparts include proximate analysis as well as analyses of nutritional components, toxins, toxicants, anti-nutrients and other components relevant to given cases As well, animal feeding trials are conducted to determine if any adverse health effects are observable (Institute of Medicine and National Research Council of the National Academies 2004) Ideally, the reference food for the above comparisons is the isogenic food (i.e non-transformed) from which the GM version was derived Genetic Modification The advances in how to copy DNA, modify its sequence, and transfer genes between organisms efficiently and at low cost has produced a tremendous ability to study the roles of specific proteins in organisms as well as the roles of specific amino acids within proteins This ability has produced an alternate Food Authentication and the Role of DNA Technologies Another area utilising DNA technology is food authentication Analysing processed food and ingredients for the presence of fraudulent or foreign components by DNA technologies can be P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm 22 Printer Name: Yet to Come Part 1: Principles/Food Analysis Table 1.20 Selected Commercial Biotechnology-derived Food Enzymes Enzyme Application Acetolactate decarboxylase (EC 4.1.1.5) α-Amylase (EC 3.2.1.1) Amylo-1,6-glucosidase (EC 3.2.1.33) Chymosin (EC 3.4.23.4) Lactase (EC 3.2.1.108) Glucan-1,4-α-maltogenic α-amylase (EC 3.2.1.133) Beer aging and diacetyl reduction High-fructose corn syrup production High-fructose corn syrup production Milk clotting in cheese manufacturing Lactose hydrolysis Anti-stalling in bread Source: Roller and Goodenough 1999, Anonymous 2004, IUBMB-NC website (www.iubmb.org) very complex due to degradation of genetic material as the result of processing conditions Detecting misrepresentation of food components is a task well-suited for DNA-based detection in the case of meat species of origin, especially when the meat product is processed such that appearance and physical traits cannot easily cue experts as to product identity Gene sequences within mitochondrial DNA are usually used as the PCR targets for such purposes (Wiseman 2009) For example, different tuna species can be differentiated (Michelini et al 2007) Likewise, the presence of meat in cattle feed can be assayed (Rensen 2005) Such tests can be done for high-heat-processed samples at a low cost, and such tests can be highly automated for steps post sample collection Lastly, another example of a food authentication application for PCR is the case of detecting the presence and/or the quantity of genetically modified ingredients in a food For the purposes of quantification, real-time PCR must be used (Wiseman 2009), the most modern type of PCR technology, because it is capable of highly precise, quantitative determinations This capability is due to real-time PCR’s ability to very quickly and accurately modulate PCR reaction temperature via the use of low-volume reactions Jurisdictions that have Table 1.21 Selected Genetically Modified Microorganisms Useful in Food Processing Microorganism Lactobacillus lactis Saccharomyces cerevisiae (Baker’s yeast, Brewer’s yeast) GM labelling laws (e.g the European Union) can thus verify the correct labelling of incoming ingredients In the authentication process, DNA within foods can also be characterised and differentiated by use of DNA probes, an earlier food authentication technology that dates back to at least 1990, where it was used to distinguish between heat-processed ruminant (goat, sheep and beef), chicken and pig meat (Chicuni et al 1990) DNA probes are short pieces of DNA (Rensen et al 2005) that contain a detectable feature (usually fluorescence) and that are complementary to a DNA sequence of interest Thus, a signal is produced only if the probed sequence is present in the food, since the probe will bind only to its complement in a manner that yields a signal In order to probe a food product, genetic material is isolated and immobilised on a piece of nitrocellulose (blotting), which is then ‘probed’ with a DNA complementary probe The information from the above types of DNA-based authentication tests can aid in processing quality control to ensure both authenticity and safety at relatively cheap cost NATURAL TOXICANTS The contamination of various foods by toxicants may occur as a result of microbial production, crop plant production or ingestion by animals for human consumption Microbial sources of toxins are mycotoxin-producing fungi and toxin-producing bacteria Notable examples of microbially derived toxins are botulinum toxin produced by Clostridium botulinum and the Staphylococcus aureus toxin These toxins are produced in the food itself and result in food poisoning Both toxins are heatlabile; however, the extreme toxicity of the botulinum toxin, potent at 10−9 g per kg body weight, makes it of particular concern for food processing of anaerobically stored foods Mycotoxins are extremely toxic compounds produced by certain filamentous fungi in many crop plants (Richard 2007) Ingestion of mycotoxins can be harmful to humans via contaminated foods or feed animals via their feed, particularly in maize, wheat, barley, rye and most oilseeds Mycotoxins produce symptoms that include nervous system disorders, limb loss and death Aflatoxins are a well-studied type of mycotoxin that are known carcinogens An example of a mycotoxin structure, aflatoxin B1, is shown in Figure 1.6 Mycotoxin poisoning can take place either directly or indirectly, through consumption of a contaminated food or by a food ingredient that may be contaminated and subsequently eaten as part of a final product Application Phage resistance, lactose metabolism, proteolytic activity, bacteriocin production Gas (carbon dioxide) production in sweet, high-sugar dough Manufacture of low-calorie beer (starch degradation) Source: Hill and Ross 1999, Roller and Goodenough 1999, Anonymous 2004 O O O O O O Figure 1.6 The structure of aflatoxin B1, produced by Aspergillus flavus and A parasiticus P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come Introduction to Food Biochemistry H N H O O N H N N H H N H N N H O O H H Figure 1.7 The structure of saxitoxin responsible for paralytic shellfish poisoning An important example of food-related mycotoxins is ‘ergot’, the common name for fungi of the genus Claviceps Ergot species produce ergot alkaloids, which are derivatives of lysergic acid, isolysergic acid or dimethylergoline When ingested, the various alkaloids produce devastating symptoms such as vasoconstriction, convulsion, gastrointestinal upset and central nervous system effects (Peraica et al 1999) Another type of natural toxins is that of shellfish toxins (phycotoxins), which are produced by certain species of marine algae and cyanobacteria (blue-green algae) The best known algal toxins are saxitoxins, responsible for paralytic shellfish poisoning (PSP; see Figure 1.7 for chemical structure) Saxitoxins block voltage-gated sodium channels of nerve cells Thus, this group of 20 toxins induces extreme symptoms, including numbness, tingling and burning of the lips and skin, giddiness, ataxia and fever; severe poisoning may lead to muscular incoordination, respiratory distress or failure (Garthwaite 2000) The poisoning results from bioaccumulation of the algal toxins in algae-eating organisms and toxins can be step-wise passed up the food chain, e.g from marine algae to shellfish to crabs to humans CONCLUSION In 1939, the newly formed IFT was the world’s first organisation to organise and coordinate those working in food processing, chemistry, engineering, microbiology and other sub-disciplines in order to better understand food systems Food science began mainly within commodity departments, such as animal science, dairy science, horticulture, cereal science, poultry science and fisheries Now, most of these programs have evolved into food science, or food science and human nutrition, departments Many food science departments with a food biochemistry emphasis are now available all over the world In addition, food science departments developed not only applied research programs, but also basic research programs that seek to understand foods at the atomic, molecular and/or cellular levels The ‘cook ‘n’ look, approach is not food science Food science is a respected part of academic programmes worldwide, whose researchers often form collaborations with physicists, chemists, parasitologists, etc Over the past several decades, food biochemistry research has led to industry applications, e.g lactase supplements, lactose-free dairy products, BeanoTM , application of transglu- 23 taminase to control seafood protein restructuring, protease-based meat marinades for home, production of high-fructose syrups, rapid pathogen detection tests, massively diversified natural and artificial flavourings and many more Thus, food biochemistry will continue to play critical roles in developments in food microbiology, packaging, product development, processing, crop science, nutrition and nutraceuticals REFERENCES Aharoni A et al 2004 Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species Plant Cell 16: 3110–3131 Institute of Medicine and National Research Council of the National Academies 2004 Methods for Predicting and Assessing Unintended Effects on Human Health In: Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects National Academies Press, Washington, DC, pp 127– 174 BeMiller JN, Whistler RL 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Carotenoids and gene regulation Nutr Rev 57: 182 –191 Besler M et al 19 97 Allergenicity of hen’s egg-white proteins: IgE-binding of native and deglycosylated ovomucoid Food Agric Immunol 9: 2 27? ??3 28 Bewley... poultry science and fisheries Now, most of these programs have evolved into food science, or food science and human nutrition, departments Many food science departments with a food biochemistry emphasis... 2012 11 :8 Trim: 276 mm X 219mm Printer Name: Yet to Come Introduction to Food Biochemistry (e.g citrus fruits are high in terpenoids as is mango; Aharoni et al 2004) NUCLEIC ACIDS AND FOOD SCIENCE