P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson 24 March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come Part 1: Principles/Food Analysis Crozier A et al 2000 Biosynthesis of hormone and elicitor molecules) In: BB Buchenan, et al (eds.) Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockwell, MD, pp 850–929 Damodaran S et al 2008 Fennema’s Food Chemistry CRC Press, Boca Raton, FL Dangl JL et al 2000 Senescence and programmed cell death) In: BB Buchenan, et al (eds.) Biochemistry and Molecular Biology of Plants American Society of Plant Physiologists, Rockwell, MD, pp 1044–1100 Diehl HA 2008 Physics of color In:C Socaciu (ed.) Food Colorants: Chemical and Functional Properties, CRC Press, Boca Raton, FL, pp 3–21 Duffus CM 1987 Physiological aspects of enzymes during grain development and germination In: Kruger JE, et al (eds.) 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Real-Time PCR: Current Technology and Applications Caister Academic Press, Norfolk P1: SFK/UKS BLBS102-c02 P2: SFK BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come Analytical Techniques in Food Biochemistry Massimo Marcone Protein Analysis Lipid Analysis Carbohydrate Analysis Mineral Analysis Vitamin Analysis Pigment Analysis Antioxidants Gas Chromatography—Mass Spectroscopy References to new limits Gas chromatography (GC), high performance liquid chromatography (HPLC), spectroscopy including near infrared (NIR), and mass spectroscopy (MS) are now considered to be basic laboratory equipment used in food analysis Tandem hybrid analytical equipment such as GC-MS is also gradually becoming common although it is more sophisticated than the single analytical tests PROTEIN ANALYSIS Abstract: Food is a very complex heterogeneous “material” composed of thousands of different nutritive and nonnutritive compounds embedded in a variety of different plant and animal matrices Nondesirable biochemical compounds such as environmental contaminants, microbial and plant toxins, and veterinary drugs are also present with their presence posing a danger to human health In analytical food chemistry, the isolation, identification, and quantification of both desirable and undesirable compounds continue to pose immense challenges to food analysts Methods and tests used to isolate, identify, and quantify must be precise, accurate, be increasing sensitive to satisfy the rigors of investigative and applicable science, have minimal interfering factors, use minimal hazardous chemicals, and produce minimal/no hazardous wastes This chapter is mainly concerned with the analytical methods used to determine the presence, identity, and quantity of all compounds of interest in a food Although it is impossible to address the quantitative analysis of all food components, the major techniques used in food analysis will be addressed in detail The food analyst has a variety of available tests but the test choice is primarily dependent on the goal of the analysis and the use of the final data Many of the traditional analytical biochemical tests such as Kjeldahl digestion for protein determination are still regarded as the gold standard and is still used in many laboratories While useful, traditional analytical methods are continually being challenged by technological and instrumental developments Technology is moving chemical analysis toward the use of more sophisticated instruments (either individually or in tandem) as both instrumental specificity and sensitivity are continually being “pushed” Proteins are a large diverse group of nitrogenous organic compounds that are indispensable constituents in the structure and function of all living cells They contribute to a wide variety of functions within each cell, ranging from structural materials such as chitin in exoskeletons, hair, and nails to mechanical functions such as actin and myosin in muscular tissue Chemically, they influence pH as well as catalyze thousands of critical reactions, producing a variety of essential substances that are involved in functions such as cell-to-cell signaling, immune responses, cell adhesion, cell reproduction, etc Proteins are essentially polymers of 20 l-∞-amino acids bonded together by covalent peptide bonds between adjacent carboxyl and amino functional groups The sequence of these amino acids, and thus the function and structure of the protein, is determined by the base pairs in the gene that encodes it These 20 different amino acids interact with each other within their own chain and/or with other molecules in their external environment, which also greatly influences their final structure and function Proteins are by definition relatively “heavy” organic molecules ranging in weight from approximately 5000 to more than a million Daltons and, over the years, many of these food proteins have been purified, identified, and characterized Chemically speaking, nitrogen is the most distinguishing element in proteins, varying in amounts from 13% to 19% due to the variations in the specific amino acid composition of proteins (Chang 1998) Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 26 P1: SFK/UKS BLBS102-c02 P2: SFK BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come Analytical Techniques in Food Biochemistry For the past several decades, protein analysis from food products has been performed by determining the nitrogen content after complete acid hydrolysis and digestion by the Kjeldahl method followed by an analytical step in which the resulting ammonium ion is quantified by titrimetry, colorimetry, or by the use of an ion-specific electrode The result is then multiplied by a pre-established protein conversion factor that determines the final protein content of the sample (Chang 1998, Dierckx and Huyghebaert 2000) While this “wet” analytical technique is still the gold standard in protein analysis, it is time-consuming and involves the use of many dangerous chemicals both to the analyst and to the environment Its main advantage is that the food sample used in this analytical procedure is considered large enough to be a genuine representative of the entire product On the other hand, Dumas combustion is a more recent and faster “dry” analytical instrumental method of determining the protein content in foods and is based on the combustion of a very small sample at 900◦ C in the presence of oxygen The resulting liberated nitrogen gas is analyzed in three minutes by the equipment through built-in programmed processes with the resultant value also multiplied by pre-determined conversion factors, requiring no further analysis or the use of dangerous chemicals Both of these methods assume that all the nitrogenous compounds in the sample are proteins, but other organic molecules such as nucleotides, nucleic acids, some vitamins, and pigments (e.g., chlorophyll) also contains nitrogen, overestimating the actual protein content of the sample Both techniques measure crude protein content, not actual protein content While the Kjeldahl method is the internationally accepted method of protein determination for legal purposes, Dumas combustion is slowly becoming more acceptable as its accuracy and repeatability will soon be superior to that of the Kjeldahl method (Schmitter and Rihs 1989, Simonne et al 1997) As far back as the turn of the century, colorimetric methods for protein determination became available with procedures such as the Biuret, Lowry (original and modified), bicinchoninic acid (BCA), Bradford, and ultraviolet (UV) absorption at 280 nm (Bradford 1976) These colorimetric methods exploit the properties of specific proteins, the presence of specific amino acid functional groups, or the presence of peptide bonds All require the extraction, isolation, and sometimes purification of the protein molecule of interest to attain an accurate absorbance reading Considering that the nutrients in foods exist in complex matrices, these colorimetric methods are not practical for food analysis Additionally, only a few dye-binding methods (official methods 967–12 and 975–17) have been approved for the direct determination of protein in milk (AOAC 1995) The Biuret procedure measures the development of a purplish color produced when cupric salts in the reagent complex react with two or more peptide bonds in a protein molecule under alkaline conditions The resultant color absorbance is measured spectroscopically at 540 nm with the color intensity (absorbance) being proportional to the protein content (Chang 1998) and with a sensitivity of 1–10 mg protein/mL This method “measures” the peptide bonds that are common to cellular proteins, not just the presence of specific side-groups While it is less sensitive compared to other UV methods, it is considered to be a good 27 general protein assay for which yield is not an important issue Likewise, the presence of interfering agents during absorbance measurement is not an issue as these substances usually absorb at lower wavelengths While the color is stable, it should be measured within 10 minutes for best results The main disadvantage of this method is that it consumes more material as well as it requires a 20-minute incubation period Over the years, further modifications to the colorimetric measurement of protein content have been made with the development of the Lowry method (Lowry et al 1951, Peterson 1979), which combines both the Biuret reaction with the reduction of the Folin-Ciocalteu (F-C) phenol reagent (phosphomolybdic–phosphotungstic acid) The divalent cupric cations form a complex with the peptide bonds in the protein molecules, which cause them to be reduced to monovalent cations The radical side groups of tyrosine, tryptophan, and cysteine then react with the Folin reagent, producing an unstable molybdenum/tungsten blue color when reduced under alkaline conditions The resulting bluish color is read at both 500 nm and 750 nm wavelengths, which are highly sensitive to both high and low protein concentrations with a sensitivity of 20–100 ug, respectively The modified Lowry method requires the absorbance measurement within 10 minutes, whereas the original Lowry method needs precise timing due to color instability Additionally, the modified Lowry method is more sensitive to protein than the original method but less sensitive to interfering agents The BCA protein assay is used to determine the total protein content in a solution being similar to the Biuret, Lowry, and Bradford colorimetric protein assays The peptide bonds in the protein molecules reduce the cupric cations in the BCA in the reagent solution to cuprous cations, a reaction that is dependent upon temperature Afterwards, two molecules of BCA chelate the curprous ions, changing the solution color from green to purple, which strongly absorbs at 562 nm The amount of cupric ions reduced is dependent upon the amount of protein present, which can be measured by comparing the results with protein solutions with known concentrations Incubating the BCA assay at temperatures of 37–60◦ C and for longer time periods increases the assay’s sensitivity as the cuprous cations complex with the cysteine, cystine, tyrosine, and tryptophan side-chains in the amino acid residues, while minimizing the variances caused by unequal amino acid composition (Olsen and Markwell 2007) Other methods exploit the tendency of proteins to absorb strongly in the UV spectrum, that is, 280 nm primarily due to the presence of tryptophan and tyrosine amino acid residues Since tryptophan and tyrosine content in proteins are generally constant, the absorbance at 280 nm has been used to estimate the concentration of proteins using Beer’s law As each protein has a unique aromatic amino acid composition, the extinction coefficient (E280 ) must be determined for each individual protein for protein content estimation Although these methods are appropriate for quantifying the actual amounts of protein, they have the ability to differentiate and quantify the actual types of proteins within a mixture The most currently used methods to detect and/or quantify specific protein components belong to the field of spectrometry, P1: SFK/UKS BLBS102-c02 P2: SFK BLBS102-Simpson 28 March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come Part 1: Principles/Food Analysis chromatography, electrophoresis, or immunology or a combination of these methods (VanCamp and Huyghebaert 1996) Electrophoresis is defined as the migration of ions (electrically charged molecules) in a solution through an electrical field (Smith 1998) Although several forms of this technique exist, zonal electrophoresis is perhaps the most common Proteins are separated from a complex mixture into bands by migrating in aqueous buffers through a polyacrylamide gel with a pre-determined pore size (i.e., a solid polymer matrix) In nondenaturing/native electrophoresis, proteins are separated based on their charge, size, and hydrodynamic shape, while in denaturing polyacrylamide gel electrophoresis (PAGE), an anionic detergent sodium dodecyl sulfate (SDS) is used to separate protein subunits by size (Smith 1998) This method was used to determine the existence of differences in the protein subunits between control coffee beans and those digested by an Asian palm civet as well as between digested coffee beans from both the Asian and African civets (Marcone 2004) These protein subunits differences lead to differences in the final Maillard browning products during roasting and therefore flavor and aroma profiles During the analysis of white and red bird’s nests, Marcone determined that SDS–PAGE might be a useful analytical technique for differentiating between the more expensive red bird nest and the less expensive white bird (Marcone 2005) Additionally, this technique could possibly be used to determine if the red nest is adulterated with the less expensive white nest Using SDS-PAGE analysis, Marcone was the first to report the presence of a 77 kDa ovotransferrin-like protein in both red and white nests, being similar in both weight and properties to ovotransferrin in chicken eggs In isoelectric focusing, a modification of electrophoresis, proteins are separated by charge in an electrophoretic field on a gel matrix in which a pH gradient has been generated using ampholytes (molecules with both acidic and basic groups, that is, amphoteric, existing as zwitterions in specific pH ranges) The proteins migrate to the location in the pH gradient that equals the isoelectric point (pI) of the protein Resolution is among the highest of any protein separation technique and can separate proteins with pI differences as small as 0.02 pH units (Chang 1998, Smith 1998) More recently, with the advent of capillary electrophoresis, proteins can be separated on the basis of charge or size in an electric field within a very short period of time The primary difference between capillary electrophoresis and conventional electrophoresis (described above) is that a capillary tube is used in place of a polyacrylamide gel The capillary tube can be used over and over again unlike a gel, which must be made and cast each time Electrophoresis flow within the capillary also can influence separation of the proteins in capillary electrophoresis (Smith 1998) HPLC is another extremely fast analytical technique having excellent precision and specificity as well as the proven ability to separate protein mixtures into individual components Many different kinds of HPLC techniques exist depending on the nature of the column characteristics (chain length, porosity, etc.) and elution characteristics such as mobile phase, pH, organic modifiers, etc In principle, proteins can be analyzed on the basis of their polarity, solubility, or size of their constituent components Reversed-phase chromatography was introduced in the 1950s (Howard and Martin 1950, Dierckx and Huyghebaert 2000) and has become a widely applied HPLC method for the analysis of both proteins and a wide variety of other biological compounds Reversed-phase chromatography is generally achieved on an inert packed column, typically covalently bonded with a high density of hydrophobic functional groups such as linear hydrocarbons 4, 8, or 18 residues in length or with the relatively more polar phenyl group Reversed-phase HPLC has proven itself useful and indispensable in the field of varietal identification It has been shown that the processing quality of various grains depends on their physical and chemical characteristics, which are at least partially genetic in origin, and a wide range of qualities within varieties of each species exist (Osborne 1996) The selection of the appropriate cultivar is an important decision for a farmer, since it largely influences the return he receives on his investment (Dierckx and Huyghebaert 2000) Size-exclusion chromatography separates protein molecules on the basis of their size or, more precisely, their hydrodynamic volume, and has in recent years become a very useful separation technique Size-exclusion chromatography utilizes uniform rigid particles whose pre-determined pore size determines which protein molecules can enter and travel through the pores Large molecules not enter the pores of the column particles and are excluded, that is, they are eluted in the void volume of the column (i.e., elute first), whereas smaller molecules enter the column pores and therefore take longer to elute from the column An application example of size-exclusion chromatography is the separation of soybean proteins (Oomah et al 1994) In one study, nine peaks were eluted for soybean, corresponding to different protein size fraction, with one peak showing a high variability for the relative peak area and could serve as a possible differentiation among different cultivars Differences, qualitatively and quantitatively, in peanut seed protein composition were detected by size-exclusion chromatography and contributed to genetic differences, processing conditions, and seed maturity In 1990, Basha demonstrated that size-exclusion chromatography was an excellent indicator of seed maturity in peanuts as the area of one particular component (peak) was inversely proportional to increasing peanut seed maturity, which also remained unchanged toward later stages of seed maturity (Basha 1990) The peak was present in all studied cultivars, all showing a mature seed protein profile with respect to this particular protein, which was subsequently named Maturin LIPID ANALYSIS Compared to most other food components, lipids are a group of relatively small, naturally occurring molecules containing carbon, hydrogen, and oxygen atoms, but with much less oxygen than carbohydrates This large group of organic molecules includes fats, waxes, cholesterol, sterols, glycerides, phospholipids, etc The most simplistic definition of a lipid is based on its solubility, that is, it is soluble in organic solvents (e.g., alcohol) but insoluble in water Lipid molecules are hydrophobic, but this generality is sometimes not totally correct as some lipids are amphiphilic, that is, partially soluble in water and ... Springer, New York, pp 145–1 58 Mine Y, Yang M 20 08 Recent advances in the understanding of egg allergens: basic, industrial, and clinical perspectives J Agric Food Chem 56(13): 487 4:4900 Murphy OJ 1999... HA 2004 Update on food allergy J Allergy Clin Immunol 113: 80 5? ?81 9 Sampson HA, Cooke SK 1990 Food allergy and the potential allerginicity–antigenicity of microparticulated egg and cow’s milk proteins... Branen (ed.) Food Additives Marcel Dekker New York, pp 543–561 Schormuller J 19 68 The chemistry and biochemistry of cheese ripening Adv Food Res 16: 231–334 Sicherer SH, Sampson HA 2006 Food allergy