encyclopedia of agriculture and food systems || food safety: food analysis technologies/techniques

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Food Safety: Food Analysis Technologies/TechniquesB Jiang, Jiangnan University, Wuxi, Jiangsu, PR ChinaR Tsao, Guelph Food Research Centre, Guelph, Ontario, CanadaY Li and M Miao, Jiangnan University, Wuxi, Jiangsu, PR China

r 2014 Elsevier Inc. All rights reserved.

Introduction

In recent years, consumers have become increasingly con-cerned about the safety and quality of their food supply. Toensure that food supplies meet the highest standards of safetyand nutritional quality, robust and state-of-the-art analyticalmethods are essential for all food products. The rapid growthof novel raw materials and ingredients, and new processes inthe food industry has also brought new challenges to foodscientists. The progresses in food science and technologyhave driven analytical methods toward those that heavily relyon instrumentation and biochemistry, which provide highersensitivity and accuracy. Advances in modern food analysistechnologies have allowed scientists in food safety and qualityto meet such new challenges and demands.

Food analysis is particularly complex due to the intricatefood system, which includes key physicochemical and bio-logical characteristics of raw materials, potential existence ofmicroorganisms, different contaminants and additives, and theprocessing techniques used. For these reasons, various analyticalmethods have been required for the quality control of foodsand selection of appropriate techniques and methodologies arerequired to obtain the best results. In general, the choice of ananalytical technique depends on the purposes of the analysisand the properties of the food. This article attempts to cover therecently developed technologies, as well as traditional instru-mentation, techniques, and applications in food analysis.

Food Composition

Food, a highly complex system, contains essential nutrientssuch as water, carbohydrates, lipids, proteins, vitamins, min-erals, and dietary fibers (Eastwood and Kritchevsky, 2005;Williams, 1995). Recent findings about the beneficial roles ofnonessential micronutrients, such as polyphenols and car-otenoids, to health have led to greater needs for moresophisticated analytical methods. In addition to chemicalcomponents, live microorganisms, such as those in fermentedfoods like probiotic yogurt, can also be a target of the analysis.All these contribute to the increased complexity of the foodsystems that are analyzed. To gain a meaningful insight intothe analysis of these compositions, the aforementionedmacronutrients and micronutrients are briefly discussed in thissection.

Water

Water, the most abundant substance in nearly all food, playsan important role in the food system. Even pure substancessuch as sugar and salt contain a small amount of water

ncyclopedia of Agriculture and Food Systems, Volume 3 doi:10.1016/B978-0-444

absorbed on the surface of the crystal. Moisture content is oneof the quality factors for food products, especially for driedgoods such as milk powder, dehydrated vegetables and fruits,and flours. A reduced moisture content is beneficial for thepacking and transportation of food (Slade et al., 1991), as itinhibits microbial growth. However, certain amount of wateris essential to make a food fresher and tender.

Moreover, water may be present in the food system as freeliquid in which nutrients are dissolved, hydrated, imbibedin gels, or adsorbed on the surfaces of other materials (Levineand Slade, 1991). A method for moisture analysis musttherefore depend upon the form of the water present as well asthe amount in a particular food.

Carbohydrates

Carbohydrates are an important nutrient that supplies abun-dant energy and serves as a source of many physiologicallyactive compounds. In general, carbohydrates are dividedinto three subgroups: monosaccharides, oligosaccharides, andpolysaccharides. Carbohydrate analysis is a key quality assur-ance tool for food processing, not only because it ensuresthat the amount of ingredients added into the food meetthe standards or claims on the label, but it is also a means todetect food deterioration. Moreover, low-caloric sweeteners,such as sugar alcohols and rare sugars, D-psicose and D-tagatose,became increasingly important to consumers and the need forspecific analytical methods for these carbohydrates has alsobeen recognized. Another significant outcome of carbohydrateanalysis is calorie calculation.

Proteins

Like other biological macromolecules such as polysaccharidesand nucleic acids, proteins are vital to the metabolism of allliving organisms. Proteins play an essential role in the tissue oflive animals and carry out various functions, such as actin andmyosin, which are contractile proteins essential for movementand locomotion.

Proteins consist of one or more chains of amino acids, andthey differ from one another primarily in the sequence ofamino acids. Proteins can be hydrolyzed into polypeptides oramino acids by proteases. Amino acids are essential nutrients,and some are supplemented in foods in both natural andsynthetic forms (Ikeda, 2003). Quantitative and qualitativeanalyses of amino acids are important to the food industry ingeneral, but are especially critical for quality assurance in theamino acid industry.

However, for protein molecules their function dependson the specific three-dimensional structures that are easilyaltered by physical, chemical, and biological factors like heat,

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274 Food Safety: Food Analysis Technologies/Techniques

presence of organic solvents, and microorganisms. Functionalproperties of proteins are, therefore, also an important elementof protein analysis (Hall, 1996). Methods commonly used toanalyze protein structures and functions included those basedon site-directed mutagenesis, nuclear magnetic resonance,X-ray crystal diffraction, and mass spectrometry (MS) (Cavanaghet al., 2010; Ladd and Palmer, 2003; Whitford, 2005; Zhang andSmith, 1993). As such, proteins are often purified using a widerange of techniques such as ultracentrifugation, precipitation,chromatography, and electrophoresis (Janson, 2012).

Nutritive value of a protein also needs to be evaluated apartfrom amino acid composition, protein content, and proteinstructure and function. The most commonly evaluated par-ameters are the amino acid score (AAS), net protein utilization(NPU), true digestibility (TD), and biological value (BV)(Owusu-Apenten, 2002).

Lipids

Lipids are essential to living organisms because they contributeto vital metabolic processes in cells. Fats such as butter andlard are derived from animal products such as milk and meat,whereas oils such as soybean oil, peanut oil, and olive oil arederived from plants. Lipids can be categorized into two classes:saturated and unsaturated, which are dependent on the fattyacid backbone of the triacylglyceride. Melting points of satur-ated fatty acids are higher than those of unsaturated fatty acids.Therefore, most animal fats with a high content of saturatedfatty acids are in the solid form at room temperature, whereaslipids from plants such as olive oil are liquid on account of thehigher content of unsaturated fatty acids.

The taste, flavor, and nutritional value of food, as well asthe health of humans, are affected by the variety and quantityof the lipids, because of the potentially toxic oxidationbyproducts of lipids (Kubow, 1992). In addition, some mono-and polyunsaturated fatty acids can help reduce the risk ofhuman chronic diseases beyond their basic nutritional func-tions (Jew et al., 2009). An accurate and precise analysis oftotal and individual lipids is essential for the development ofsafe and high-quality foods and functional foods.

Minerals

What makes minerals different from organic nutrients is thatminerals cannot be synthesized or decomposed in a biologicalsystem like the human body. According to the requirement ofminerals by living organisms, they can be categorized intomacrominerals such as calcium, potassium, phosphorus, andtrace minerals like iron, iodine, and zinc (Faller et al., 2004).Although the requirement of trace minerals is low, they arevery important for metabolism. Minerals are obtained throughfood, and those necessary in maintaining healthy metabolismare called essential microelements. In some cases, fortificationis allowed in the diet. Mineral analysis is of great significancebut highly challenging for evaluating the nutritional value,potential toxicity, and food safety, especially for the traceelements in food. Modern instrumentation plays an importantrole in obtaining accurate, precise, and sensitive results inmineral analysis.

Vitamins

A vitamin, including fat-soluble and water-soluble vitamins, isdefined as an organic compound present in minute amountsin natural foodstuffs that are required by humans or any otherorganisms as a vital nutrient at low concentrations (McDowell,2008). Insufficient levels of vitamins result in hypovitaminosisor avitaminosis.

Many vitamins are lost or decomposed during food pro-cessing. Monitoring the stability and reducing the loss ofvitamins in raw and prepared foods or food supplementsthus requires accurate and sensitive analytical methods. Suchmethods are also important for assessing dietary adequacy.

Dietary Fibers

Dietary fibers, including soluble and insoluble ones, havespecial physiological functions such as protecting against coloncancer, lowering the cholesterol absorbed, reducing the risk ofobesity, and cardiovascular diseases. Research has shown thatdietary fibers changed the microbial contents of the gastro-intestinal tract in favor of the growth of beneficial bacteriaagainst the pathogenic ones (Eastwood and Kritchevsky,2005).

Physicochemical and Biological Properties

Energy

Food energy is defined as the energy released from carbo-hydrates, fats, proteins, and other organic compounds. Whenthe three major calorigenic nutrients (carbohydrates, fats, andproteins) in a food are burnt entirely with sufficient amountsof oxygen, it releases energy or food calories that are expressedin kilojoules (kJ) or kilocalories (kcal). Food energy is usuallymeasured by a bomb calorimeter based on the heat of com-bustion (Insel et al., 2012).

Energy released by a particular food is a critical parameterin nutrition. Several chronic diseases such as obesity, diabetes,and cardiovascular disease have been considered to be causedby excess energy intake. All food manufacturers are now re-quired to label the energy of their products to help consumerscontrol their energy intake. Fats have the greatest amount offood energy per mass, up to 9 kcal g−1. Most of the carbo-hydrates and proteins have approximately 4 kcal g−1, whereasfibers have less due to its low digestibility and absorbance inhuman bodies (Insel et al., 2012). Restricting long-term con-sumption of high-energy foods rich in fats and sugar canreduce the incidence of obesity and other diseases.

pH and Acidity

There are two indicators of food flavor and quality: pH andacidity. Most common organic acids in foods are just partiallyionized. In food processing, optimization of flavor and taste,and improvement of formulations are often done by control-ling the pH and the acidity, which are also important foreffectively inhibiting the activities of enzymes and the growthof microorganisms, ultimately improving the food quality and

Food Safety: Food Analysis Technologies/Techniques 275

prolonging the shelf-life of the product (Brown, 2010; Vaclavikand Christian, 2003).

Texture

The texture of the food is its physical and chemical interactionin the mouth and can directly correlate to food rheology.Food texture encompasses hardness, smoothness, thickness,and other mouth-feel characteristics (Lawless and Heymann,2010). Food is constantly evaluated, from the initial im-pression when the product touches the palate, until the after-taste. Physical properties that contribute to the texture of afood are now widely used to represent the state and taste ofthat respective food. The collective expression of food texturethrough such properties at present is measured using mech-anics and by visual and auditory methods.

Food texture has been used to: (1) explain food structuralcharacteristics; (2) explain changes of physical properties of afood during processing and cooking processes; (3) improvefood quality and unique characteristics; (4) explore the rela-tionship between sensory analysis and the instrumentalmeasurement(s).

Rheological Properties

Like texture, rheological properties are also one of the sig-nificant assessment criteria of food quality. The rheologicalproperties of food, such as consistency, viscosity, and elasticity,are very important factors and are used to evaluate the stabilityof a food during processing, and the shelf-life during storage(Herh et al., 2000). A proper design of food formulation cantherefore improve the shelf-life and sensory characteristics bymeasuring the rheological properties.

Foods can be classified into solid, liquid, emulsion, andgel according to their rheological state (Rao, 2007). However,due to the complex structural features of different food com-ponents, a food often appears to be a mixture of solidand fluid with varying properties. Rheological properties ofa complex food matrix are obtained through disciplinaryapproaches, using theoretical and experimental information togain a better understanding of various properties of foodmaterials.

Color

Whether or not people decide to buy a food depends, to alarge extent, on the appearance of the product, among whichthe most important factor is color, and seems to have an in-nate allure that gives people the first impression and thegreatest influence on our judgment (Potter and Hotchkiss,1998). For consumers, the most intuitive feeling for productsis the color.

Improving and maintaining color stability is one of themost important practices in food processing. Colorants areoften added into products in the food industry to enhance theappeal of foods. Natural pigments are normally not requiredto be certified before they are added to foods because they aregenerally considered to be safe (Ronzio, 2003). Naturally de-rived pigments are typically more expensive than certified

synthetic colorants and may add unintended flavors to foods.However, certified colorants are synthetically produced and areused widely because they are less expensive, as well as theyimpart an intense, uniform color, and blend more easily infood to create a variety of hues (Potter and Hotchkiss, 1998).Under these circumstances, the levels of colorants addedshould be monitored to ensure that appropriate regulatoryguidance is complied with.

Flavors

People enjoy the products with good flavor which can, in turn,affect the digestion and absorption of nutrients. Flavor is justthe sensory impression of a food mainly caused by thechemical senses. In general, the chemical senses are dividedinto two types: taste and smell. Sour, sweet, bitter, piquant,salty, umami, and metallic are the seven basic tastes, whereassmell is only limited to fragrance and stink (Jeleń, 2011; John,1995).

A flavor is determined by a mixture of several compoundsin most foods. If one or several compounds represent thecharacteristic flavor of a particular food, these are consideredas the characteristic or key compound(s). These substanceshave the properties of poor stability, small quantity, and vul-nerability (Philippe et al., 2003).

Flavoring can be practices that are used to prevent un-desirable flavors produced during treatment, storage, andprocessing of food, or to create desired flavors for specificproducts such as candies and other snacks. Natural or artificialflavorants alter the taste of the food itself. Most commercialflavorants used in the food industry are chemically synthesizedrather than extracted from native materials, as artificialflavorants are much less expensive and more available(Chandrashekar et al., 2006). As a result, in food industries,flavor analysis must be performed to ensure food qualityand safety.

Sampling and Sample Preparation

Sampling

Food analysis always starts with sampling which is the processof taking a representative fraction from a large collection of aparticular product or item, for example, a small quantity froma full production batch.

Before sampling, the objective and target analytes orproperties of a food must be well established and understood,so an appropriate sampling method is determined for reliableresults. There are two methods of sampling: random samplingand representative sampling. Opportunities for drawing anyindividual sample from the population in the process at ran-dom must be equal (Rana, 2008).

Sample Preparation

To eliminate the interference of other components, and in-crease the concentration of the target components, samplesare often pretreated before analysis. Sample preparation alsoincreases uniformity thus leading to better extraction


efficiency. Below are some commonly used sample prepar-ation methods:

Methods for removal of organic compoundsThese methods are mainly used for the determination ofinorganic elements. The inorganic elements of the food oftenbind tightly with organic compounds such as proteins.Therefore, organic compounds are removed to reduce/elim-inate potential interference. The details are discussed in theSection Ash Content Analysis.

Solvent extractionSolvent extraction is commonly used to remove unwantedcomponents by taking the advantage of significantly differentsolubilities between the analytes and interfering compounds.Solvent extraction methods include lixiviation, supercriticalfluid extraction (SFE), ultrasonic extraction, microwave-assisted extraction, and solid phase extraction.

ChromatographyAlthough modern chromatography has been largely instru-mentalized and automated for analytical purposes, somemanual are still widely used in sample cleanup and preparation,and more details are discussed in the Section Chromatography.

GrindingFor sample preparation before analysis, grinding is important.It reduces the particle size of solids and semisolids, and im-proves the uniformity and increases extraction efficiency of thesample. Various mills are available for reducing the particlesize to achieve sample homogenization.

Other methodsConcentration: Concentration reduces sample size and en-riches the target components to make the samples easier to beanalyzed. Concentration of liquid samples is done by evap-oration under vacuum or atmospheric pressure.

Distillation: Distillation, including air distillation, reducedpressure distillation, and steam distillation, separates analytesfrom the interfering components based on their different boil-ing points. Usually, no reagents are needed during this process.

Precipitation: The appropriate chemical agents, such asbasic copper sulfate, isolate the target components or inter-fering compounds by settling them out of solution. Filtrationor centrifugation is carried out to either recover the precipitateor the supernatant for analysis.

Masking: A masking agent is added to bind selectively toan interfering component; therefore, only the analytes areavailable for extraction or further analysis.

Sulfonation and saponification: These two methods areused to remove lipids from samples.

wt of wet sample

Sample Preservation

Ideally, all samples should be analyzed upon arrival at thelaboratory. However, more often than not, samples are storedbefore analysis. In the latter case, samples must be keptunder appropriate conditions. Light, oxygen, temperature, andmoisture are the major factors that cause deterioration of the

sample and the analytes. Exposure to light and oxygen (air) leadsto oxidation including photodegradation of compounds, forexample, riboflavin and carotenoids. Temperature and moistureaffect microbial growth and enzyme activity in the samples.Preservatives and antioxidants can be added into the sampleson the condition that they will not affect the analysis results.Freeze-drying and ultralow temperature are good preservationtechniques for samples that cannot be analyzed timely. Properpreservation methods must be chosen based on the probabilityof contamination, storage time, and the target analytes.

Enzyme inactivationRaw food material often contains endogenous enzymes, whichmay decompose the primary elements, that is, proteins,thereby leading to interference upon analysis. Therefore, theseenzymes must be eliminated or controlled. In general, thermaltreatment is the most common method to inactivate enzymes.Sometimes, samples are frozen during storage to limit enzymeactivities. However, activities of some enzymes are moreeffectively regulated by changing the ionic strength or pH.

Lipid oxidation protectionUnsaturated lipids are protected through storage in vacuum orunder nitrogen, as they are sensitive to oxidative degradation.Lipids may be stabilized by antioxidants that could be usedupon analysis (Kadoya, 1991). Low-temperature storage issuitable for protecting most foods. Light-initiated photooxida-tion of unsaturated lipids are avoided by being stored in thedark. Containers for lipid samples should be free of any tran-sition metals as these ions are catalysts for lipid peroxidation.

Microbial growth inhibitionMicrobial contamination can occur during sampling and evenprocessing for analysis; therefore, efforts must be made toavoid exposure to microorganisms at each step between sam-pling and analysis. Freezing, drying, and chemical preserva-tives are effective controls and often a combination of these areused (Nielsen, 2010b).

General Analysis Methods

Moisture Analysis and Water Activity Measurement

Oven drying: The moisture content of the samples is calculatedby the loss of weight. The loss of moisture from a sample is afunction of time and temperature in the process of analysis.Sometimes, the components in the food may be decomposedat a higher temperature; while the volatile constituents, such asacids, alcohols, and other flavor compounds, may also be lost.Moisture content can be calculated using oven drying pro-cedures, such as the following (Nielsen, 2010b):

% Moisture content ðwt=wtÞ ¼ wt H2O in samplewt of wet sample

� 100 ½1�

%Moisture content ðwt=wtÞ

¼ wt of wet sample� wt of dry sample� 100 ½2�

Figure 1 AquaLab 4TEV water activity meter.


Oven-drying methods generally include air, vacuum,microwave, and infrared drying. Drying under reduced pres-sure leads to more efficient and complete removal of water andvolatiles, less decomposition, and reduced drying time.Microwave drying, in which the samples must be uniform,centrally located and evenly distributed in order to preventuneven heating of the sample, has the advantages of rapidityand accuracy. Infrared drying techniques are more suitedfor qualitative online processes, because of the rapidity ofanalysis: however it has not yet been approved by Associationof Official Analytical Chemists.

Distillation: The principles have been discussed in theSection Sample Preparation. Distillation causes less thermaldecomposition of some foods compared with oven drying athigh temperatures.

The Karl Fischer Method: The principle of the Karl Fischermethod is schematically based on the following oxidoreduc-tion reaction:

SO2 þ I2 þ 2H2O⇄2I− þ 4Hþ þ SO−4 ½3�

Others: Infrared (IR) spectroscopy (see Section Infraredspectroscopy) has been successfully used in the laboratory and inonline applications. For water, the –OH stretching at 1400–1450 nm and 1920–1950 nm (near-IR (NIR) bands) are char-acteristic (Ozaki et al., 2006; Workman and Weyer, 2007).Dielectric method is used to determine the moisture content bythe electrical properties of water. Hydrometry is still widely usedfor routine analysis of moisture (or solids) content of numerousproducts. Refractometry is used commonly to measure thecontent of moisture in liquid sugar products with the charac-teristics of convenience, rapidity, and surprising accuracy. It isalso used online to monitor the Brix of products in real time.

Water activity: Water activity (aw) is one of the most im-portant physicochemical properties for predicting the growthand the survival of microorganism in foods. In addition,it significantly affects lipid oxidation, endogenous enzymeactivity, and nonenzymatic browning reactions. aw is a ther-modynamic concept, defined as the vapor pressure of a liquiddivided by that of pure water at a given temperature. Moredetails are in the reference (Barbosa-Cánovas et al., 2008).

There are various techniques to measure aw, among whichare the portable water activity meters. As shown in Figure 1,the developed portable water activity meters that are widelyused in the food industry can yield accurate measurements inless than 5 min, where the reading is directly displayed on thescreen of the instrument.

Ash Content Analysis

Ash is the inorganic residue after all organic components of afood sample are burnt. Two major types of ash analysis areused: dry ashing, which is primarily used for specific mineralanalyses and wet ashing, a method based on completely oxi-dizing organic compounds using chemical agents. Beforeashing, drying and degreasing must be done, especially forhigh-fat products. The ash content of foods is expressed oneither a wet or a dry weight basis.

Dry ashing refers to the method of burning organic sub-stances completely in the presence of oxygen by using a mufflefurnace capable of maintaining temperatures of 500–600 °C,

at which water and volatiles are vaporized (Sharma, 2006).Most minerals are converted into oxides, sulfates, and otherinorganics. This method requires no reagents, as well as, littleattention during combustion, but the drawback is that it istime consuming and losses may occur for some volatileelements and mineral components that may interact withcrucibles. Strong acids or other oxidizing agents, or theircombinations are used in wet ashing. During the procedure ofwet ashing, minerals are solubilized in a solution, and becauseit is done at an ambient temperature, no loss occurs fromvolatilization (Aras et al., 2006). This is a faster method be-cause of the quick oxidation time.

Recently, a novel method called microwave ashing hasbeen developed (Nollet and Toldrá, 2012). Assisted by amicrowave instrument, both dry ashing and wet ashing canbe done. The microwave-assisted method is mainly used foraccelerating the digestion of samples.

Carbohydrate Analysis

Total carbohydratesCarbohydrates can be destroyed by the addition of a strongacid and/or high temperature, where various furan derivativesare produced. The furan derivatives react with phenols toproduce reaction products that can be conveniently measuredby a spectrophotometer. This method, namely, phenol-sulfuricacid method, is rapid, simple, sensitive, accurate, and specificfor carbohydrates, and widely applied (Chandrasekaran,2012). The reagents are also readily available, inexpensive, andstable.

Total reducing sugarReducing sugars naturally existing in foods or released byenzymes are determined by the dinitrosalicylic acid method.In this reaction, 3,5-dinitrosalicylate is changed to the reddishmonoamine derivative, which can be measured by a spectro-photometer. This reaction is not stoichiometric and must bedetermined with a standard curve of D-glucose (Robyt, 1998).Another often used is the Somogyi–Nelson method. It is alsoused for the determination of oligo- and polysaccharides(Chandrasekaran, 2012).

MAC_ALT_TEXT Figure 1


Mono- and oligosaccharidesMono- and oligosaccharides such as D-psicose, difructoseanhydride III and oligofructose are commonly analyzed usinghigh-performance liquid chromatography (HPLC) (Zhanget al., 2009; Zhao et al., 2011, Hocine et al., 2000). HPLCidentifies individual sugars with specific retention times andgives quantitative results with peak integration. It is a rapid,precise, and accurate method for a wide range of samples.

Ionic-exchange, normal-phase and reversed-phase chro-matography are widely used for the analysis of sugars in dif-ferent foods such as honey, beverage, cakes, and ice creams. Inanion-exchange chromatography, the general elution sequenceis alditols, monosaccharides, disaccharides, and higher oligo-saccharides (Nielsen, 2010b). The elution order is reversedin cation-exchange chromatography (Rassi, 1994). In normal-phase chromatography, the stationary phase is the silicagel derivatized with amino groups, and acetonitrile water(50–85% acetonitrile) is a commonly used mobile phase.The elution order is monosaccharides and sugar alcohols, di-saccharides, and higher oligosaccharides. In the reversed-phasechromatography, the hydrophobic stationary phase is silica gelwith alkyl chains or phenyl group, such as a C18 column orphenyl column. It is used for the analysis of mono-, di-, andtrisaccharides (Nielsen, 2010b).

Thin-layer chromatography (TLC) has been used for theanalysis of mono- and oligosaccharides. Although it providesboth quantitative and qualitative information of the sugars,TLC is not as sensitive and accurate as HPLC. More infor-mation in detail is provided in the reference (Sherma andFried, 2003).

Starch, food gum, and dietary fiberThe starch content is determined by the amount of D-glucosereleased from the hydrolysis reaction by purified enzymesspecific for starch. The enzymes for starch hydrolysis must bepurified to prevent the release of D-glucose by other enzymes.For example, cellulose can be converted into D-glucose bycellulases. The presence of nonstarch specific enzymes cancause an overestimation of the total starch content.

The degree of gelatinization of starch is measured by acombination of pullulanase and β-amylase, which is particu-larly sensitive and only able to act on gelatinized or pastedstarch. The determination of the content of food gum, likepectin, is discussed in detail in the reference (Nielsen, 2010b).Dietary fiber is usually determined gravimetrically. The Proskymethod is the principal procedure, which measures bothsoluble and insoluble fiber. The Englyst-Cummings methodis used for the chemical measurement of dietary fiber(Shils, 2006).

Protein Analysis

Kjeldahl method: In the Kjeldahl procedure, after digestion inconcentrated sulfuric acid, the total organic nitrogen is con-verted to ammonium sulfate. Ammonia is formed and dis-tilled into boric acid solution under alkaline conditions.The borate anions formed are titrated with standardizedhydrochloric acid, by which is calculated the content ofnitrogen representing the amount of crude protein in the

sample. Most proteins contain 16% of nitrogen, thus theconversion factor is 6.25. However, the nitrogen from non-protein additives or contaminants in the food, such as mela-mine in milk, is also measured.

Biuret method: Under alkaline conditions, peptide bondsreact with cupric ions to produce a violet-purplish color, theintensity of which at 540 nm is correlated to the proteincontent of the sample (Owusu-Apenten, 2002). This method isquick and simple with little interference from nonpeptide ornonprotein sources.

Lowry method: The Lowry method is developed based onthe biuret method. Proteins react with Folin–Ciocalteu phenolreagent to produce a blue conjugate at 750 nm. The intensityof color is proportional to the protein content of the sample(Owusu-Apenten, 2002). Although this method is very sensi-tive, it is interfered by sucrose and lipids.

Ultraviolet 280 nm Absorption Method: Proteins, at280 nm, show strong absorption primarily for tryptophan andtyrosine residues. This technique is mainly applied in a puri-fied protein system, because absorbance will be falsely in-creased if particulates or other substance like nucleic acids arein the solution (Scopes, 1994). This method is noninvasive;thus samples can be used for other analyses after proteindetermination.

Other methods: In addition to the aforementionedmethods, the Dumas, IR spectroscopy, bicinchoninic acid,anionic dye-binding, Bradford dye-binding, Ninhydrin, andimmunological methods are some other techniques com-monly used for protein analysis (Nielsen, 2010b; Owusu-Apenten, 2002; Ozaki et al., 2006). Choosing an appropriatemethod depends on the properties of proteins present in thesamples as well as the characteristics of analytical approaches.

The nutritional value of a protein is determined by theamino acid composition and the digestibility of that protein.The most common parameters for protein quality are the AAS,BV, TD, and NPU. The procedure for calculation is furtherelaborated in the reference Owusu-Apenten (2002).

Lipid Analysis

Solvent extraction method: The total lipid content of a food iscommonly determined by its organic solvent extract. Thechoice of solvents is highly critical for total fat analysis. Themost commonly used solvents are ethyl ether and petroleumether, but pentane and hexane are used for extracting oil fromsoybeans. In addition, lipids in food may be conjugated withproteins and polysaccharide; thus, a successful extraction re-quires that the lipids be freed by alkaline or acid hydrolysisand then extracted into the organic solvents. The Goldfishmethod is commonly used as a continuous solvent extractionapproach. In this procedure, solvent from a boiling flaskcontinuously flows over the sample. Fat content is measuredby the weight of fat loss of the sample. Soxhlet method is asemicontinuous solvent extraction approach. In this pro-cedure, the sample is soaked completely for 5–10 min ina solvent and then siphoned back into the boiling flask.A commercial Soxhlet apparatus is shown in Figure 2. TheMojonnier test is an example of the discontinuous solventextraction method. SFE (Anklam et al., 1998) is a relatively

Figure 2 Soxhlet extraction apparatus.


new solvent-free technology developed to replace or reduce theuse of toxic organic solvents.

Nonsolvent wet extraction methods: The Babcock methodand Gerber method are commonly used for milk fat analysis.In these procedures, carbohydrate and protein are removed bycentrifugation after addition of H2SO4. Milk fat is separatedand measured volumetrically, and the result is expressed as apercentage of fat by weight.

Gas chromatography (GC): GC is the most sensitive andaccurate method for fat analysis. A triglyceride, triundecanoin(C11:0), is added as an internal standard. More information isin the Section Gas chromatography.

Other methods: Details of some other methods such as IR,specific gravity, nuclear magnetic resonance, and ultrasonicmethod can be found in the reference (McDonald andMossoba, 1997).

In addition to the total lipid content, physicochemicalproperties of the lipids in a foodstuff, such as stability andsafety of fats, are also important to be characterized. Refractiveindex, melting point, smoke point, flash point, fire point, andcloud point are commonly measured. Degree of unsaturationof fats or oils is indexed by the iodine value. Higher iodinevalue is indicative of a higher degree of unsaturation. Sa-ponification is an alkaline hydrolysis process by which neutralfats (triglycerides) break into glycerol and fatty acids. Saponi-fication value is an index of the mean molecular weight of the

triacylglycerides in the samples. The amount of fattyacids hydrolyzed from triacylglycerides is reflected by the acidvalue. Peroxide value tests the peroxide products after lipidoxidation. The thiobarbituric acid test measures the secondaryproducts of lipid oxidation, primarily malondialdehyde.Details on these and other indexes also can be found in thereference (Nielsen, 2010b).

Mineral Analysis

Atomic absorption spectroscopy is the most commonly usedmethod for mineral analysis, and it will be discussed in detailin the Section Atomic absorption and emission spectroscopy.Inductively coupled plasma MS (ICP-MS) is introduced in theSection Raman spectroscopy. Only traditional titration meth-ods are briefly introduced here.

Gravimetric analysis: This method is only for insolubleminerals. The mineral content of the sample is weighed afterproper precipitation, washing, and drying. It is not suitable forthe determination of trace elements.

Ethylenediamine tetraacetic acid complexometric titration:Ethylenediamine tetraacetic acid (EDTA) forms stable 1:1complexes with numerous mineral ions except alkali metals,and this principle has been the basis of the widely usedcomplexometric titration. The endpoint of a complexometricEDTA titration is the color change from pink to blue usingeither Calmagite or Eriochrome black T as the indicator(Nielsen, 2010a).

Precipitation titration: It is well suited for any foods thatmay be high in chlorides (Dieter and Multon, 1997). However,it ultimately has few applications in the analysis of food.

Colorimetric methods, redox reaction, and ion-selectiveelectrodes may also be used in mineral analysis (Nielsen,2010b). These procedures generally require only inexpensivechemicals and equipment readily available in an analyticallaboratory.

Vitamin Analysis

HPLC, owing to its simplicity, and good accuracy and pre-cision, is a preferred method for vitamin analyses (Leenheerand Lambert, 2002; Nollet, 2012). HPLC is suitable for mostlipid-soluble and water-soluble vitamins such as vitamins A,C, D, and E and the B vitamins, respectively. Liquid chro-matography (LC) in combination with MS is particularlyuseful for vitamin analysis. Detection by LC–MS leads toincreased sensitivity as well as unequivocal identification andcharacterization of vitamins. In addition to the UV/Vis ordiode array detector, fluorescence spectroscopy detector isalso used in HPLC, especially for B vitamins such as thiamin(vitamin B1).

Physicochemical Analysis

pH and Acidity

In food analysis, pH is different from titratable acidity, but thereis a close relationship between them. Titratable acidity indicatesthe total acidity contained within a food, whereas pH onlymeans the free H3O

+ concentration. Total acids, which give



more impact on flavor, are determined by exhaustive titration ofthe intrinsic acids with a standard base. The growth of micro-organisms is more dependent on pH than total acids. In nature,the value of pH can span from 1 to 14 orders of magnitude.

pH is usually determined using a pH meter or pH indicator.Titratable acidity is determined by neutralizing the bases pre-sent in the sample using a standard acid. When the colorof a pH-sensitive dye changes suddenly, the titration reachesthe endpoint. The endpoint of the target pH is identified bythe pH meter. The advantage of the potentiometric method isthat the precise equivalence point is identified. The titration ofacids or bases is also determined by the conductometricmethod, which is also applied for the determination of hal-ides, sulfides, and mercaptans (Paré and Bélanger, 1997).

Thermal Analysis

A set of physical properties of foodstuff and food products,such as texture and storage stability, can be analyzed by ther-mal analysis, which provides information on the structuresand qualities of the material. Thermogravimetric analysis(TGA) and differential scanning calorimetry (DSC) (Nielsen,2010b; Pomeranz and Meloan, 2002) are two typical methodsin thermal analysis.

Thermogravimetric analysis: TGA measures the thermal oroxidative stability of the components of the samples. Whensamples are heated from room temperature to 1000 °C orhigher, even small weight changes due to the decompositionof components are detected by TGA instruments (Figure 3).However, it does not identify the gaseous compounds evolvedfrom the sample.

Differential scanning calorimetry: DSC can measurestructures such as amorphous, crystalline, or semicrystallinecomponents of food, based on the fact that structural changesoccur as a result of heat absorption or release (Figure 4). It isperhaps one of the most useful of the thermal techniques foridentifying the structures and their changing characters.

Texture and Rheological Analysis

There are no better ways to quantify the sensory properties of aproduct in the mouth than by measuring the physical

Figure 3 TGA Spirit Drinks Trade Act (SDTA) 815.

properties related to the sensory texture of foods. Theseproperties are determined by rheological methods and textureanalysis. Instrumental and physiological methods are used todetermine a food’s quality and structural characteristics, thusproviding a more accurate description of a product, whichsubsequently can help control the quality and structure offood.

Viscosity and other rheological properties of materials aredetermined by rheometers. Viscosity for fluids is the mostimportant parameter, providing rapid and fundamental in-formation. Some common foods exhibit ideal Newtonianflow: however, the majority of fluid foods exhibit pseudo-plasticity. If the viscosity of a material increases in responseto increased rate of shear strain, such as cornstarch slurry,the material is called a dilatant (Rao, 2007; Steffe, 1996). Therheological properties of solid foods are determined by ex-tending, compressing, or twisting the samples. The process, bywhich samples are significantly deformed, strained, damaged,or possibly fractured by mechanical forces, simulates theprocess of chewing resulting in the change of food texture inthe mouth (Bourne, 2002).

Texture profile analysis (TPA) is an important techniqueusing the texture analyzer. Numerous sensory parameters, in-cluding hardness, springiness, and cohesiveness, are deter-mined by the TPA test (Bourne, 2002). For example, samplehardness has been strongly related to the highest force frac-turing the sample. Figures 5 and 6 show rheometer and textureanalyzer, respectively.

Color Analysis

The concept of color is defined as the sensation that can beinterpreted by our brain. In chromatology, the Munsell systemis probably the best known and the most widely used one(John, 1995; Lawless and Heymann, 2010). A food colorcan be mathematically represented by the International

Figure 4 DSC Q2000.



Figure 5 ARES-G2 rheometer.

Figure 6 TA XT plus texture analyzer.


Commission on Illumination tristimulus values, although suchvalues are difficult to be directly related to the observed color.

The colorimeters are commercially available today. Theyare portable, online for process control, and specialized forspecific commodities. However, quantitative analysis of arti-ficial and natural colorants has been a great challenge in foodprocessing. The properties of colorants and their derivativesand the interaction between colorants and other componentsmake the qualitative and quantitative analysis very complex.Sometimes, especially in thermal processing, colorants aredifficult to be extracted. In addition, they are also sensitiveto oxygen, heat, light, and metal ions. The common methodsfor colorant analysis are stand-alone UV–Visible spectro-photometry and HPLC using a UV–vis detector. More detailsare discussed in MacDougall (2002).

Sensory Analysis

Sensory analysis combines experimental methods and statisticalanalysis for the purpose of evaluating food products usinghuman senses (sight, taste, smell, touch, and hearing). It greatlydepends on the sensation of the assessors, which is related tothe psychological factors at that time (Carpenter et al., 2000). Itis possible to make inferences about the quality of productsunder test by applying statistical methods to the results.

The sensory testing environment is vital to the perceptionof analysts, because they more or less cause specific physio-logical sensations/ characteristics. According to standardizedprocedures, sensory specialists need to be very careful inexperimental design, selection of protocols, and preparation ofsamples before testing. The visual appearance, such as size,shape, the quantity, and even the container of the samplesshould be decided carefully. There are many methods for de-signing experiments; the experimental design is divided intotwo basic structures, namely treatment structure and designstructure (Lawless and Heymann, 2010).

In the process of testing, people’s sensory opinions of foodis collected and described. Normally, the number of expertsshould not be less than five. Further details regarding sensoryanalysis can be found in these references (Carpenter et al.,2000; Chambers, 1996; Lawless and Heymann, 2010).

Biological Analysis

Enzyme Application

Enzymatic analysis involves the measurement of the activityof specific added commercial enzymes and/or endogenousenzymes, which can be used to indicate the quality offood. The enzyme reactions require less reaction time and costless in contrast to complicated chromatographic separationtechniques.

There are several uses of enzymes in food science andprocessing. For instance, peroxidase and lipoxygenase, twoendogenous enzymes found in vegetable products, are usedas a measure of adequacy of blanching (Fennema, 1996). Inaddition, glucose content in the presence of other reducingsugars can be determined by the combination of the glucoseoxidase (GOX) and catalase in a food system (Aehle, 2007). It




is a useful tool in determining the amount of glucose becauseof the GOX specificity for glucose.

Immunoassays

Immunoassays are widely used for food toxins such as pesti-cide residues, mycotoxins, process-induced toxins and drugs,identification of bacteria and viruses, and detection of aller-gens in food, which attribute to their high specificity, sensi-tivity and simplicity (Tothill, 2003).

Enzyme-linked immunosorbent assay (ELISA) is a commonimmune analytical technique based on the high and specificaffinity binding of particular target antigens with antibodies.Horseradish peroxidase and alkaline phosphatase are mostcommonly used in immunoassays, because these enzymes canconvert colorless substrates to colored soluble products in orderto generate the detectable signals for the assay. Western blot isanother common method that combines two techniques:polyacrylamide gel electrophoresis (PAGE) and immunoassay.

Food Contaminants and Residues Analysis

Microbiological Analysis

Food products may be exposed to potential risks of microbialcontamination during the process of production, transporta-tion, and at the point of sale.

Traditional methods for the detection and identification ofmicroorganisms in food largely depend on the culturemethod. A general strategy to detect specific microorganisms isto identify first the target organism and determine the nu-tritional requirements for the optimal growth of the targetmicroorganism. Optimum incubation conditions includingtemperature, time, and atmosphere are then established (Lundand Baird-Parker, 2000). Finally, assessment of the perform-ance against an established method using contaminatedfoodstuff is carried out.

In recent years, with the rapid advancement in molecularbiology and microelectronics technology, faster, more accurateand specific testing methods have emerged as novel alternativetools for the identification of microorganisms. The following isa few examples of the new technologies:

Electrical impedance method: During the growth of bac-teria, the inert macromolecules such as carbohydrates, pro-teins, and lipids in the culture medium will degrade intoelectrically active small molecules such as lactate and acetate,which can increase the conductivity of the medium, causingchanges in the impedance of the medium. Detection of suchchanges can be used as an indicator of the growth and char-acteristics of the corresponding bacteria in the medium(Harrigan, 1998). It has been used for the detection of the totalnumber of bacteria, fungi, yeast, Escherichia coli, Salmonella,and Staphylococcus aureus.

Nucleic acid probe technology: The nucleic acid probe,marked with specific deoxyribonucleic acid (DNA) fragments,is used to hybridize with target DNA, specifically. The probecan be labeled using either radioactive or nonradioactivelabeling. The nonradioactive labels can be biotin, digoxigenin,fluorescence labeling, and immunolabeling (Harrigan, 1998;

Jay, 1996). This method has been successfully used in thedetection of Salmonella and Listeria.

Polymerase chain reaction technology: It can detectSalmonella, E. coli O157, and Listeria monocytogenes (Patel,1994): Although it requires special equipment, polymerasechain reaction is a novel technology that has the advantage ofbeing rapid, sensitive, accurate, and is the promising methodfor detecting microorganisms in the future.

Enzyme-linked immunosorbent assay: The principal ofELISA, in detail, is given in the Section Immunoassays. Theamount of aflatoxin is usually measured by this technology.

Pesticide and Veterinary Drug Residue Analysis

Pesticides in food can be herbicides, insecticides, fungicides,acaricides, molluscides, nematicides, pheromones, plant growthregulators, repellents, and rodenticides. Results of pesticideresidue analyses are influenced by several factors includingfood components, the physical and chemical properties of thepesticides, and the interaction between the food matrix andthe pesticide molecules. A wide range of analytical methodshas been used in pesticide analysis. The enzyme inhibitionassays and immunoassays are commonly used for the meas-urement of pesticides. Testing kits are also commerciallyavailable. Pesticides with enzyme-inhibitory activities will de-velop color; thus their residues can be quickly detected usingthese methods. Chromatographic techniques, including TLC,GC, HPLC, GC–MS, and LC–MS, are widely used in the an-alysis of pesticides. Details of these techniques are discussed inSections Mass Spectrometry and Chromatography.

Veterinary drugs are used to reduce the risk of diseaseoutbreak in the animal population. In addition, they are usedto promote animal growth and increase feed conversion inanimals (Picó, 2008). A wide variety of analytical methodshave been developed and optimized for the analysis of thedrugs including antibiotics. Microbial growth inhibition assayis usually used for quantitative measurement of the veterinarydrugs. If the drugs are present in samples, the microbial growthis inhibited. Enzyme substrate assays, immunoassays, HPLC,LC–MS, and LC–MS/MS have all been applied for the analysisof feed additives and veterinary drugs in foods.

Other Chemical or Biological Contaminants

Genetically modified organisms: The safety of geneticallymodified foods (GMFs) is a concern not only to the generalpublic but also to the food industry and government regulatoryagents. Currently, soybeans, corn, and canola have been im-plicated in genetically modified organisms (GMO) products. Inaddition to these common GMO field crops that are modifiedfor pesticide and herbicide-tolerance, other GMO foods fromboth flora and fauna have been developed for improved orenhanced quality and nutritional value (James, 2001). For ex-ample, genetically modified fruits have better taste and poten-tially a longer/ extended shelf-life. Another example is ‘goldenrice,’ which has been engineered to produce vitamin A precursor(Paine et al., 2005). In general, there are two methods for de-tecting GMO. Immunoassays, for detecting the proteins ofGMO, are highly specific methods because they are based onthe use of antibodies. However, detection of the transgenic


DNA itself, such as the promoter sequence is another effectivemethod for the identification of GMFs (Nielsen, 2010b).

Melamine: Melamine was added illegally to milk productsand animal feed to falsify the protein content, which causedillness and death of infants. LC–MS is commonly used formelamine analysis. However, several rapid and less expensivemethods such as near- and mid-IR technologies and im-munoassays have also been developed (Picó, 2012; Hui et al.,2006; Vail et al., 2007).

Residues of Packaging Materials: The health risks of novelplastic food packing materials or the plasticizers/additivesthereof have become an increasingly serious food safety issuein recent years (Robertson, 2012). Chemicals such as bisphe-nol A and 4-methylbenzophenone are used in plastics, buttheir residues can migrate into food. To ensure food safety,residue levels of packing material in food are highly regulatedand monitored using GC–MS, GC–MS/MS, LC–MS, and LC–MS/MS. Other analytical methods include uses of com-mercially available immune-affinity columns and ELISA kits.

Figure 7 FTIR spectroscopy Nicolet iS10.

Modern Instrumental Analysis

Spectroscopy

Ultraviolet and visible spectroscopyUltraviolet and visible spectroscopy (UV–vis) is one of themost commonly encountered techniques in food analysis. Thewavelength of UV light ranges from 200 to 350 nm, and thatof Vis light ranges from 350 to 700 nm in the spectrum (Frostand Russell, 1993). Many food components that have no orweak UV or visible absorption are analyzed with UV/Visspectrophotometric methods after color generation throughderivatization. Analyses of total phenolic, total flavonoid, totalanthocyanin, and total carotenoid contents in food are goodexamples (Tsao and Li, 2013).

The concentration of analyte in a given sample solution canbe determined by the quantitative absorption spectroscopybased on the observation that the concentration is pro-portional to the amount of light absorbed from a referencebeam as it passes through the sample solution. The relation-ship between the absorbance of a solution and the concen-tration of the analyte is known as Beer’s law (Nielsen, 2010b).

In spectroscopy or spectrophotometry, the solution of thesample must be uniform, clear, and transparent. The referencesolution for food samples is normally the sample solvent,namely, water or an aqueous buffer. For good quality results,the cuvette for the measurements within the UV range shouldbe quartz, whereas that for the Vis range can be silicate glass orinexpensive plastic cells. The best results are obtained whenthe wavelength is the maximum UV/Vis absorbance of theanalyte and the absorbance is stable at that wavelength.

FluorimetryDuring the past 20 years, the fluorescence spectroscopy isprimarily considered to be a research tool in biochemistry. Theuse of fluorescence now has been expanded. The fluorescencespectroscopy is similar to UV–vis, but more sensitive. The in-strumentation is also similar to that of the UV–vis absorptionspectroscopy, except for the optical system. In a fluorometer,

there is a need for two wavelength selectors, one for theemission beam and the other for the excitation beam. Simi-larly, the quantitation is also based on the direct linearrelationship between the fluorescence intensity and the con-centration of the targeted compound in the solution. For ex-ample, the content of certain vitamins such as thiamin isdetermined by this method (Ball, 2005). In addition, con-formation changes of a protein in a solution is studiedqualitatively or quantitatively using fluorimetric methods(Lakowicz, 2009).

Infrared spectroscopyIR spectroscopy refers to the measurement of the absorption ofdifferent frequencies of IR radiation by any food component ina solid, liquid, or gaseous state (Settle, 1997). IR spectroscopycan be categorized into near-IR of which the wavelength is0.8–2.5 mm; mid-IR of which the wavelength is 2.5–15 mm;and far-IR of which the wavelength is 15–100 mm. The near-and mid-IR regions of the spectrum are both useful forqualitative and quantitative analysis of foods.

For food industry, the most important is attenuated totalreflectance in conjunction with Fourier transform IR (FTIR)technology. It is available for the analyses of fats and oils,meats, butter, milk, even sweetened condensed milk, andjuices (Downey, 1998; Van de Voort, 1992). FTIR (Figure 7) isconvenient, rapid and automatable, and has dramaticallysimplified sample handling.

NIR spectroscopy is more widely used for quantitative an-alysis of foods than mid-IR. This technique requires much lesstime and is widely used throughout the production of grainand cereal products, meat and fish products, and in oilseedprocessing industries. NIR spectroscopy has also been used toanalyze specific chemical constituents in a food, such as thetotal sugar content in fruit (Li-Chan et al., 2011; Ozaki et al.,2006), for monitoring changes that occur during processing,transportation, and storage.

Atomic absorption and emission spectroscopyAtomic spectroscopy is widely used for accurately measuringthe trace amounts of minerals in food. Although traditional



chemistry titration methods for mineral analysis, such as iron,chloride, calcium, and phosphorus, remain in use today, it hasbeen largely replaced by atomic spectroscopy. In theory, vir-tually all of the elements in food can be measured by atomicspectroscopy.

There are two types of atomic spectroscopy, namely atomicabsorption spectroscopy (AAS) and atomic emission spec-troscopy (AES). AAS is based on the absorption of ultravioletor visible radiation by free atoms in the gaseous state.Figure 8 is the photograph of AAS provided by the State KeyLaboratory of Food Science and Technology of Jiangnan Uni-versity. In contrast to AAS, the source of radiation in AES is theexcited atoms or ions in the sample rather than an externalsource (Lajunen and Perämäki, 2004; Wilson and Walker,2010).

AES has been revolutionized by adding an apparatus calledICP torch. The samples are made into an aerosol and carriedinto the plasma. They atomized and are excited into varioushigher energy levels (Clugston and Flemming, 2000; Lajunenand Perämäki, 2004). ICP–AES has a much larger analyticalworking range and are suitable for more elements.

Raman spectroscopyRaman spectroscopy, a vibrational spectroscopic technique, iscomplementary to IR spectroscopy. It is commonly used fororganic analysis. In general, the fingerprint region of organicmolecules is approximately in the wavenumber range of500–2000 cm−1. Another application of this technique is instudying the changes in chemical bonding, such as when asubstrate is added to an enzyme (Li-Chan et al., 2011). Incontrast to IR, low concentrations of organic molecules in anaqueous solution are allowed to be measured due to the weakRaman scattering of water. The instrument is shown inFigure 9.

Mass spectrometryMass spectrometry (MS), different from the above discussedspectroscopic techniques, is based on the detection of chargedmolecules or fragments of a molecule. The generated ions areseparated in the electrostatic field and then finally detected

Figure 8 Atomic absorption spectroscopy spectra 240FS AA.

according to their mass-to-charge ratio (m/z). The results fromion generation, separation, fragmentation, and detection aremanifested as a mass spectrum that can be interpreted to yieldmolecular weight or structural information of a compound.There are different ionization methods, but electrospray ion-ization and atmospheric-pressure chemical ionization are twoof the most frequently used methods, and both can be ineither positive or negative mode (Hoffmann and Stroobant,2013). MS is now interfaced with GC and HPLC, and thehyphenated analytical methods are powerful tools and nowwidely used for analyzing various food components in amixture. Structural information for the identification of un-known compounds separated and eluted from the HPLC col-umn can be obtained using an MS detector (McClatchey,2002). LC–MS has played important roles in the screening andidentification of bioactive compounds such as polyphenolsand carotenoids in fresh foods, and in functional foodsand nutraceuticals (Paré and Bélanger, 1997; Li et al., 2011; Liet al., 2012).

ICP-MS is a multielement analysis technology with excel-lent sensitivity. Atoms are first ionized and then driven into amass spectrometer, which accelerates the ions, and separatesthem according to their m/z. The resolution of modern massspectrometers is sufficient to separate ions with m/z differences(Nielsen, 2010b; Ötles- , 2011; Thomas, 2008). On the basis ofthe above principals, it can simultaneously measure mostelements in the periodic table. In addition, even ultratraceconcentrations of the samples can be detected and analyzed.

Nuclear magnetic resonanceNuclear magnetic resonance (NMR) spectroscopy providesimportant structural information for a wide variety of foodcomponents. NMR instruments (Figure 10) may be configuredto analyze samples in a solution or in a solid state. It may beused for the elucidation of the complete structure of complexmolecules (only NMR), the 3D-imaging of fresh tissues, andthe simple ingredient assays for quality assurance. The nucleiof protons (1H) and the 13C isotopes have a characteristiccharge and spin and are most commonly used in NMR offood components. The principles and the latest applications ofNMR in food analysis can be found in detail in the reference(Picó, 2012).

Figure 9 Nexion 300 ICP-MS. Photo by PerkinElmer Instruments(Shanghai) Co. Ltd. Copyright © 2013.



Figure 10 AVANCE III 400 MHz Digital NMR spectrometer.


X-ray methodsX-ray diffraction is mainly used to identify the structure oflipids, starch, as well as proteins and enzymes. The phases oflipids can also be determined by X-ray diffraction measure-ments. Trace amounts of metal ions in food are detected byX-ray fluorescence, whereas X-ray adsorption spectrometry isused to determine the structural environment around themetal ions in crystalline and amorphous structures (Pomeranzand Meloan, 2002).

Chromatography

Column chromatographyChromatography has found its use in nearly all areas of foodanalysis. Chromatography with a very high flow rate is knownas flash chromatography (FC) (Sensen, 1999). Several FCsystems have been commercially available. The FC technologymakes the separations easier and faster, and has been used forsemipreparation of food components.

High- or ultrahigh performance liquid chromatographyCompared with conventional column chromatography, HPLCis much faster. It is a convenient and widely used technologyfor sugar content, pesticide residues, amino acids, toxins, or-ganic acids, lipids, vitamins, and various phytochemicals infoods. The types of separation modes and its application in thefield of carbohydrate analysis have been described in theSection Mono- and oligosaccharides. Many hydrophilic foodcomponents such as vitamin C, amino acids, phenolic com-pounds, and many bioactive foods are analyzed by reversed-phase HPLC, whereas both normal and reversed HPLC areused for lipophilic compounds such as vitamin E and car-otenoids (Nollet, 2012; Li et al., 2012). The average molecularweight of proteins and molecular weight range of poly-saccharides are rapidly determined by size-exclusion chroma-tography. Many glycoproteins are purified using affinitychromatography (Nollet, 2012).

It is worth noting that in the past several years, two revo-lutionary technology advancements, the development ofultrahigh pressure pumps and sub-2-micron packing particles,have led to the state-of-the-art ultrahigh performance liquid

chromatography (UPLC or UHPLC) (Waksmundzka-Hajnosand Sherma, 2011; Xu, 2013; Li et al., 2012). UHPLC not onlysignificantly reduces the sample size, analytical run time, andthe solvent usage, but also produces exceptionally high sen-sitivity and better resolution. UHPLC is now also interfacedwith MS.

Gas chromatographyGC is a separation method used to analyze thermallystable volatile substances. For example, GC has been used forthe determination of fatty acids, triglycerides, flavor com-pounds, and many other food components, as well as pesti-cides, aroma compounds, polychlorinated biphenyls, andother volatile contaminants. Sample preparation is a criticalstep in GC analysis. It generally involves grinding, homogen-ization, and isolation of analytes from food samples, whichmay be achieved by headspace analysis, distillation, prepara-tive chromatography, or solvent extraction. More details arementioned in the reference (Poole, 2012).

Paper and thin-layer chromatographyAlthough paper chromatography is no longer widely used,TLC, because of its ease of use, relatively low cost, and greatersensitivity and reproducibility, is still used to analyze a varietyof compounds in food including lipids, carbohydrates, vita-mins, amino acids, and natural pigments. TLC plates withfluorescent indicators are commercially available. Compoundswith UV absorption are directly detected under a UV lamp.

Other Methods

BiosensorsA biosensor is a device composed of a biological sensingelement coupled to a suitable transducer. Enzyme–substrate,antibody–antigen, DNA–DNA, and aptamer–target are themost well-known interactions used in biological biosensordesign. The transducers include electrochemical, optical, or acombination of the two. A wide range of biosensors has beendeveloped for the detection of many food components suchas glucose and starch, or contaminants such as carbamate,pesticides, and herbicides. The references (Joshi and Joshi,2006; Mutlu, 2010) provide more details.

Electronic nose and tongueElectronic nose (e-nose) and electronic tongue (e-tongue) arewidely used for flavor, aroma, or odor analysis. Both techni-ques use different sensors for detection, and the componentsare grouped through pattern recognition using data analysistools. e-Nose may use either a metal oxide semiconductor orconducting polymer as sensors that can be selective and sen-sitive, whereas taste sensors for e-tongue are often chemicalsensors with cross-sensitivity to different components, thuslacking the sensitivity and selectivity. It should be pointed outthat none of these techniques provide compositional infor-mation of a food sample, but rather profiles of different typesof compounds. Using a solid-phase microextraction probe, theheadspace gas can be absorbed onto the fiber and injected to aGC or GC–MS for compositional analysis. Some applications



of e-nose and e-tongue in food analysis are discussed in thereferences (Berger, 2007; Deisingh et al., 2004).

UltrasoundUltrasound is a nondestructive testing method for character-izing food composition, monitoring some unit operationsduring production, and elucidating molecular processes infoods. Ultrasound is highly flexible and can be applied in anonline environment allowing real-time observation of thechanges in the product structure under processing conditions.More examples are described in the reference (Irudayaraj andReh, 2008).

ElectrophoresisElectrophoresis is a technology that separates proteins ac-cording to the migration of charged molecules in a solutionthrough an electric field. Each protein separated in a non-denaturing system has a characteristic mobility determined bya combination of the intrinsic charges located on the proteinsurface combined with physical characteristics such as shapeand molecular weight (Walker and Rapley, 2008). Unlikenondenaturing electrophoresis, sodium dodecyl sulfate-PAGEis used to separate protein subunits only by size.

Modern instrument such as capillary electrophoresis hasbeen developed and used for protein separation and analysis,as well as for some artificial sweeteners and preservatives insoft drinks. Niacin in food is determined by capillary zoneelectrophoresis (Perrett, 2002). More examples of instrumentalanalysis are described in the reference (Pomeranz and Meloan,2002).

Conclusion

Advances in food science and technology have brought in-novations and improvements in the analytical methods ortechniques used to ensure food safety, quality, and nutrition-related research and actual applications along the food pro-duction chain. Fundamental discussions such as those onthe comprehensive reviews on well-established conventionalchemical, physical, and microbiological methods were in-cluded in this article; furthermore, recent advances in sen-sorial, bioanalytical, and instrumental analysis techniques andtheir applications have also been addressed. Applications andtheir pros and cons of these novel technologies in the analysisof the food components and additives including carbo-hydrates, proteins, lipids, trace elements, vitamins, toxins,contaminants, and residues have been discussed. Technologiesrelated to the physical and chemical properties of food suchas texture, rheology, and color are also important aspects forfood-related research and development. Introduction to noveltechnologies and approaches, such as the noninvasive andnondestructive methods such as NMR, ultrasound, biosensorand e-nose and e-tongue, is believed to provide insights intofuture trends in food analysis.

See also: Advances in Pesticide Risk Reduction. Analyses of TotalPhenolics, Total Flavonoids, and Total Antioxidant Activities in Foods

and Dietary Supplements. Food Microbiology. Food Packaging. FoodSafety: Emerging Pathogens. Food Safety: Shelf Life ExtensionTechnologies. Food Security, Market Processes, and the Role ofGovernment Policy. Food Toxicology. Regulatory Challenges toCommercializing the Products of Ag Biotech

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