Food Analysis

Investigations in food science and technology, whether by the food industry, governmental agencies, or universities, often require determination of food composition and characteristics. Trends and demands of consumers, the food industry, and national and international regulations challenge food scientists as they work to monitor food composition and to ensure the quality and safety of the food supply. All food products require analysis as part of a

quality management

program throughout the development process (including raw ingredients), through production, and after a product is in the market. In addition, analysis is done of problem samples and competitor products. The characteristics of foods (i.e., chemical composition, physical properties, sensory properties) are used to answer specific questions for regulatory purposes and typical quality control. The nature of the sample and the specific reason for the analysis commonly dictate the choice of analytical methods.

Speed, precision, accuracy

, and

ruggedness

often are key factors in this choice.

Validation

of the method for the specific

food matrix

being analyzed is necessary to ensure usefulness of the method. Making an appropriate choice of the analytical technique for a specific application requires a good knowledge of the various techniques (Fig. 1-1). For example, your choice of method to determine the salt content of potato chips would be different if it is for nutrition labeling than for quality control. The success of any analytical method relies on the proper selection and preparation of the food sample, carefully performing the analysis, and doing the appropriate calculations and interpretation of the data. Methods of analysis developed and endorsed by several nonprofit scientific organizations allow for standardized comparisons of results between different laboratories and for evaluation of less standard procedures. Such

official methods

are critical in the analysis of foods, to ensure that they meet the legal requirements established by governmental agencies.

Government regulations

and

international standards

most relevant to the analysis of foods are mentioned here but covered in more detail in Chap. 2, and nutrition labeling regulations in the USA are covered in Chap. 3. Internet addresses for many of the organizations and government agencies discussed are given at the end of this chapter.

Knowledge of government regulations relevant to the chemical analysis of foods is extremely important to persons working in the food industry. Federal laws and regulations reinforce the efforts of the food industry to provide wholesome foods, to inform consumers about the nutritional composition of foods, and to eliminate economic frauds. In some cases, they dictate what ingredients a food must contain, what must be tested, and the procedures used to analyze foods for safety factors and quality attributes. This chapter describes the US federal regulations related to the composition of foods. The reader is referred to references (1–4) for comprehensive coverage of US food laws and regulations. Many of the regulations referred to in this chapter are published in the various titles of the

Code of Federal Regulations

(CFR) (5). This chapter also includes information about food standards and safety practices established by international organizations. Internet addresses are given at the end of this chapter for many of the government agencies, organizations, and documents discussed.

Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the

Food and Drug Administration

(FDA) and the

Food Safety and Inspection Service

(FSIS) of the

United States Department of Agriculture

(USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

The field of food analysis, or any type of analysis, involves a considerable amount of time learning principles, methods, and instrument operations and perfecting various techniques. Although these areas are extremely important, much of our effort would be for naught if there were not some way for us to evaluate the data obtained from the various analytical assays. Several mathematical treatments are available that provide an idea of how well a particular assay was performed or how well we can reproduce an experiment. Fortunately, the statistics are not too involved and apply to most analytical determinations.

Quality attributes in food products, raw materials, or ingredients are measurable characteristics that need monitoring to ensure that specifications are met. Some quality attributes can be measured online by using specially designed sensors and results obtained in real time (e.g., color of vegetable oil in an oil extraction plant). However, in most cases quality attributes are measured on small portions of material that are taken periodically from continuous processes or on a certain number of small portions taken from a lot. The small portions taken for analysis are referred to as

samples

, and the entire lot or the entire production for a certain period of time, in the case of continuous processes, is called a

population

. The process of taking samples from a population is called

sampling

. If the procedure is done correctly, the measurable characteristics obtained for the samples become a very accurate estimation of the population.

Moisture assays can be one of the most important analyses performed on a food product and yet one of the most difficult from which to obtain accurate and precise data. This chapter describes various methods for moisture analysis – their principles, procedures, applications, cautions, advantages, and disadvantages. Water activity measurement also is described, since it parallels the measurement of total moisture as an important stability and quality factor. With an understanding of techniques described, one can apply appropriate moisture analyses to a wide variety of food products.

Ash

refers to the inorganic residue remaining after either ignition or complete oxidation of organic matter in a foodstuff. A basic knowledge of the characteristics of various ashing procedures and types of equipment is essential to ensure reliable results. Two major types of ashing are used: dry ashing, primarily for proximate composition and for some types of specific mineral analyses; wet ashing (oxidation), as a preparation for the analysis of certain minerals. Microwave systems now are available for both dry and wet ashing, to speed the processes. Most dry samples (i.e., whole grain, cereals, dried vegetables) need no preparation, while fresh vegetables need to be dried prior to ashing. High-fat products such as meats may need to be dried and fat extracted before ashing. The ash content of foods can be expressed on either a wet weight (as is) or on a dry weight basis. For general and food-specific information on measuring ash content, see references (1–11).

Lipids, proteins, and carbohydrates constitute the principal structural components of foods. Lipids are a group of substances that, in general, are soluble in ether, chloroform, or other organic solvents but are sparingly soluble in water. However, there exists no clear scientific definition of a lipid, primarily due to the water solubility of certain molecules that fall within one of the variable categories of food lipids (1). Some lipids, such as triacylglycerols, are very hydrophobic. Other lipids, such as di- and monoacylglycerols, have both hydrophobic and hydrophilic moieties in their molecules and are soluble in relatively polar solvents (2). Short-chain fatty acids such as C1–C4 are completely miscible in water and insoluble in nonpolar solvents (1). The most widely accepted definition is based on solubility as previously stated. While most macromolecules are characterized by common structural features, the designation of “lipid” being defined by solubility characteristics is unique to lipids (2). Lipids comprise a broad group of substances that have some common properties and compositional similarities (3). Triacylglycerols are fats and oils that represent the most prevalent category of the group of compounds known as lipids. The terms lipids, fats, and oils are often used interchangeably. The term “lipid” commonly refers to the broad, total collection of food molecules that meet the definition previously stated. Fats generally refer to those lipids that are solid at room temperature and oils generally refer to those lipids that are liquid at room temperature. While there may not be an exact scientific definition, the US Food and Drug Administration (FDA) has established a regulatory definition for nutrition labeling purposes. The FDA has defined total fat as the sum of fatty acids from C4 to C24, calculated as triglycerides. This definition provides a clear path for resolution of any nutrition labeling disputes.

Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure. Food proteins are very complex. Many have been purified and characterized. Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Daltons. They are composed of elements including hydrogen, carbon, nitrogen, oxygen, and sulfur. Twenty α-amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide bonds. Nitrogen is the most distinguishing element present in proteins. However, nitrogen content in various food proteins ranges from 13.4 to 19.1% (1) due to the variation in the specific amino acid composition of proteins. Generally, proteins rich in basic amino acids contain more nitrogen.

Carbohydrates are important in foods as a major source of energy, to impart crucial textural properties, and as dietary fiber which influences physiological processes. Digestible carbohydrates, which are converted into monosaccharides, which are absorbed, provide metabolic energy. Worldwide, carbohydrates account for more than 70% of the caloric value of the human diet. It is recommended that all persons should limit calories from fat (the other significant source) to not more than 30% and that most of the carbohydrate calories should come from starch. Nondigestible polysaccharides (all those other than starch) comprise the major portion of dietary fiber (Sect. 10.5). Carbohydrates also contribute other attributes, including bulk, body, viscosity, stability to emulsions and foams, water-holding capacity, freeze-thaw stability, browning, flavors, aromas, and a range of desirable textures (from crispness to smooth, soft gels). They also provide satiety. Basic carbohydrate structures, chemistry, and terminology can be found in references (1, 2).

Vitamins are defined as relatively low-molecular-weight compounds which humans, and for that matter, any living organism that depends on organic matter as a source of nutrients, require small quantities for normal metabolism. With few exceptions, humans cannot synthesize most vitamins and therefore need to obtain them from food and supplements. Insufficient levels of vitamins result in deficiency diseases [e.g., scurvy and pellagra, which are due to the lack of ascorbic acid (vitamin C) and niacin, respectively].

This chapter describes traditional methods for analysis of minerals involving titrimetric and colorimetric procedures, and the use of ion selective electrodes. Other traditional methods of mineral analysis include gravimetric titration (i.e., insoluble forms of minerals are precipitated, rinse, dried, and weighed) and redox reactions (i.e., mineral is part of an oxidation–reduction reaction, and product is quantitated). However, these latter two methods will not be covered because they currently are used little in the food industry. The traditional methods that will be described have maintained widespread usage in the food industry despite the development of more modern instrumentation such as atomic absorption spectroscopy and inductively coupled plasma-atomic emission spectroscopy (Chap. 24). Traditional methods generally require chemicals and equipment that are routinely available in an analytical laboratory and are within the experience of most laboratory technicians. Additionally, traditional methods often form the basis for rapid analysis kits (e.g., Quantab

®

;

for salt determination) that are increasingly in demand. Procedures for analysis of minerals of major nutritional or food processing concern are used for illustrative purposes. For additional examples of traditional methods refer to references (1–6). Slight modifications of these traditional methods are often needed for specific foodstuffs to minimize interferences or to be in the range of analytical performance. For analytical requirements for specific foods see the

Official Methods of Analysis

of AOAC International (5) and related official methods (6).

There are two interrelated concepts in food analysis that deal with acidity:

pH

and

titratable acidity

. Each of these quantities is analytically determined in separate ways and each has its own particular impact on food quality.

Titratable acidity

deals with measurement of the

total acid concentration

contained within a food (also called

total acidity

). This quantity is determined by exhaustive titration of intrinsic acids with a standard base. Titratable acidity is a better predictor of acid’s impact on flavor than pH.

Methods for characterizing edible lipids, fats, and oils can be separated into two categories: those developed to analyze bulk oils and fats, and those focusing on analysis of foodstuffs and their lipid extracts. In evaluating foodstuffs, it is usually necessary to extract the lipids prior to analysis. In these cases, if sufficient quantities of lipids are available, methods developed for bulk fats and oils can be utilized.

Many protein separation techniques are available to food scientists. Several of the separation techniques described in this chapter are used commercially for the production of food or food ingredients, whereas others are used to purify a protein from a food for further study in the laboratory. In general, separation techniques exploit the biochemical differences in protein solubility, size, charge, adsorption characteristics, and biological affinities for other molecules. These physical characteristics then are used to purify individual proteins from complex mixtures.

Enzymes are protein catalysts that are capable of very great specificity and reactivity under physiological conditions. Enzymatic analysis is the measurement of compoundswith the aid of added enzymes or themeasurement of endogenous enzyme activity to give an indication of the state of a biological system including foods. The fact that enzyme catalysis can take place under relatively mild conditions allows for measurement of relatively unstable compounds not amenable to some other techniques. In addition, the specificity of enzyme reactions can allow for measurement of components of complex mixtures without the time and expense of complicated chromatographic separation techniques.

Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

The food chain that starts with farmers and ends with consumers can be complex, involving multiple stages of production and distribution (planting, harvesting, breeding, transporting, storing, importing, processing, packaging, distributing to retail markets, and shelf storing) (Fig. 18.1). Various practices can be employed at each stage in the food chain, which may include pesticide treatment, agricultural bioengineering, veterinary drug administration, environmental and storage conditions, processing applications, economic gain practices, use of food additives, choice of packaging material, etc. Each of these practices can play a major role in food quality and safety, due to the possibility of contamination with or introduction (intentionally and nonintentionally) of hazardous substances or constituents. Legislation and regulation to ensure food quality and safety are in place and continue to develop to protect the stakeholders, namely farmers, consumers, and industry. [Refer to reference (1) for information on regulations of food contaminants and residues.]

Analysis for extraneous matter is an important element both in the selection of raw materials for food manufacturing and for monitoring the quality of processed foods. The presence of extraneous material in a food product is unappealing and can pose a serious health hazard to the consumer. It also represents lack of good manufacturing practices and sanitary conditions in production, storage, or distribution. The presence of extraneous materials in the product ingredients may render the final product adulterated and not suitable for human food.

Oxygen demand is a commonly used parameter to evaluate the potential effect of organic pollutants on either a wastewater treatment process or a receiving water body. Because microorganisms utilize these organic materials, the concentration of dissolved oxygen is greatly depleted from the water. The oxygen depletion in the environment can have a detrimental effect on fish and plant life.

Spectroscopy

deals with the production, measurement, and interpretation of spectra arising from the

interaction of electromagnetic radiation with matter

. There are many different spectroscopic methods available for solving a wide range of analytical problems. The methods differ with respect to the species to be analyzed (such as molecular or atomic spectroscopy), the type of radiation–matter interaction to be monitored (such as absorption, emission, or diffraction), and the region of the electromagnetic spectrum used in the analysis. Spectroscopic methods are very informative and widely used for both quantitative and qualitative analyses. Spectroscopic methods based on the absorption or emission of radiation in the

ultraviolet

(UV),

visible

(Vis),

infrared

(IR), and radio (

nuclear magnetic resonance

,

NMR

) frequency ranges are most commonly encountered in traditional food analysis laboratories. Each of these methods is distinct in that it monitors different types of molecular or atomic transitions. The basis of these transitions is explained in the following sections.

Spectroscopy in the ultraviolet–visible (UV–Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV–Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

Infrared (IR) spectroscopy refers to measurement of the absorption of different frequencies of IR radiation by foods or other solids, liquids, or gases. IR spectroscopy began in 1800 with an experiment by Herschel. When he used a prism to create a spectrum from white light and placed a thermometer at a point just beyond the red region of the spectrum, he noted an increase in temperature. This was the first observation of the effects of IR radiation. By the 1940s, IR spectroscopy had become an important tool used by chemists to identify functional groups in organic compounds. In the 1970s, commercial near-IR reflectance instruments were introduced that provided rapid quantitative determinations of moisture, protein, and fat in cereal grains and other foods. Today, IR spectroscopy is used widely in the food industry for both qualitative and quantitative analysis of ingredients and finished foods.

Atomic spectroscopy has played a major role in the development of our current database for mineral nutrients and toxicants in foods. When atomic absorption spectrometers became widely available in the 1960s, the development of

atomic absorption spectroscopy

(AAS) methods for accurately measuring trace amounts of mineral elements in biological samples paved the way for unprecedented advances in fields as diverse as food analysis, nutrition, biochemistry, and toxicology (1). The application of plasmas as excitation sources for

atomic emission spectroscopy

(AES) led to the commercial availability of instruments for

inductively coupled plasma - atomic emission spectroscopy

(ICP-AES) beginning in the late 1970s. This instrument has further enhanced our ability to measure the mineral composition of foods and other materials rapidly, accurately, and precisely. More recently, plasmas have been joined with

mass spectrometers

(MS) to form inductively coupled plasma-mass spectrometer ICP-MS instruments that are capable of measuring mineral elements with extremely low detection limits. These three instrumental methods have largely replaced traditional wet chemistry methods for mineral analysis of foods, although traditional methods for calcium, chloride, iron, and phosphorus remain in use today (see Chap. 12).

Nuclear magnetic resonance

(NMR)

spectroscopy

is a powerful analytical technique with a wide variety of applications. It may be used for complex structural studies, for protocol or process development, or as a simple quality assay for which structural information is important. It is nondestructive, and high-quality data may be obtained from milligram, even microgram, quantities of sample. Whereas other spectroscopy techniques may be used to determine the nature of the functional groups present in a sample, only NMR spectroscopy can provide the data necessary to determine the complete structure of a molecule. The applicability of NMR to food analysis has increased over the last three decades. In addition to improved instrumentation and much lower costs, very complex and specialized NMR techniques can now be routinely performed by a student or technician. These experiments can be set up with the click of a button/icon, as all the basic parameters are embedded into default experiment files listed in the data/work station software, and the results are obtained in a short time.

Mass spectrometry

(MS) is unique among the various spectroscopy techniques in both theory and instrumentation. As you may recall, spectroscopy involves the interaction of electromagnetic radiation or some form of energy with molecules. The molecules absorb the radiation and produce a spectrum either during the absorption process or as the excited molecules return to the ground state. MS works by placing a charge on a molecule, thereby converting it to an ion in a process called

ionization

. The generated ions are then resolved according to their

mass-to-charge ratio

$$(m/z)$$

by subjecting them to electrostatic fields (

mass analyzer

) and finally detected. An additional stage of ion fragmentation may be included before detection to elicit structural information in a technique known as

tandem MS

. The result of ion generation, separation, fragmentation, and detection is manifested as a mass spectrum that can be interpreted to yield molecular weight or structural information. The uniqueness of this process allows the method to be used for both detection and identification of an unknown compound.

Chromatography has a great impact on all areas of analysis and, therefore, on the progress of science in general. Chromatography differs from other methods of separation in that a wide variety of materials, equipment, and techniques can be used. [Readers are referred to references (1–19) for general and specific information on chromatography.]. This chapter will focus on the principles of chromatography, mainly

liquid chromatography

(LC). Detailed principles and applications of

gas chromatography

(GC) will be discussed in Chap. 29. In view of its widespread use and applications, high-performance liquid chromatography (HPLC) will be discussed in a separate chapter (Chap. 28). The general principles of extraction are first described as a basis for understanding chromatography.

High-performance liquid chromatography

(HPLC) developed during the 1960s as a direct offshoot of classic column liquid chromatography through improvements in the technology of columns and instrumental components (pumps, injection valves, and detectors). Originally, HPLC was the acronym for

high-pressure liquid chromatography

, reflecting the high operating pressures generated by early columns. By the late 1970s, however,

high-performance liquid chromatography

had become the preferred term, emphasizing the effective separations achieved. In fact, newer columns and packing materials offer high performance at moderate pressure (although still high pressure relative to gravity-flow liquid chromatography). HPLC can be applied to the analysis of any compound with solubility in a liquid that can be used as the mobile phase. Although most frequently employed as an

analytical

technique, HPLC also may be used in the

preparative

mode.

The first publication on gas chromatography (GC) was in 1952 (1), while the first commercial instruments were manufactured in 1956. James and Martin (1) separated fatty acids by GC, collected the column effluent, and titrated the individual fatty acids for quantitation. GC has advanced greatly since that early work and is now considered to be a mature field that is approaching theoretical limitations.

Food scientists are routinely confronted with the need to measure physical properties related to sensory texture and processing needs. These properties are determined by

rheological methods

, where

rheology is a science devoted to the deformation and flow of all materials

. Rheological properties should be considered a subset of the textural properties of foods, because the sensory detection of texture encompasses factors beyond rheological properties. Specifically, rheological methods accurately measure “force,” “deformation,” and “flow,” and food scientists and engineers must determine how best to apply this information. For example, the flow of salad dressing from a bottle, the snapping of a candy bar, or the pumping of cream through a homogenizer are each related to the rheological properties of these materials. In this chapter, we describe fundamental concepts pertinent to the understanding of the subject and discuss typical examples of rheological tests for common foods. A glossary is included as Sect. 30.6 to clarify and summarize rheological definitions throughout the chapter.

Thermal analysis is a term used to describe a broad range of analytical techniques that measure physical and chemical properties as a function of temperature, time, and atmosphere (inert or oxidizing gas, pressure, and relative humidity). Depending on the technique, test temperatures can range from − 180 to 1000°C or more, allowing investigation into a range of applications, including low temperature stability and processing (e.g., freezing and freeze-drying) to high temperature processing and cooking (e.g., extrusion, spray drying, and frying).

Color, flavor, and texture are the three principal quality attributes that determine food acceptance, and color has a far greater influence on our judgment than most of us appreciate. We use color to determine if a banana is at our preferred ripeness level, and a discolored meat product can warn us that the product may be spoiled. The marketing departments of our food corporations know that, for their customers, the color must be “right.” The University of California Davis scorecard for wine quality designates four points out of 20, or 20% of the total score, for color and appearance (1). Food scientists who establish quality control specifications for their product are very aware of the importance of color and appearance. While subjective visual assessment and use of visual color standards are still used in the food industry, instrumental color measurements are extensively employed. Objective measurement of color is desirable for both research and industrial applications, and the ruggedness, stability, and ease of use of today’s color measurement instruments have resulted in their widespread adoption.