Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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2.0043 The enzymes are induced when nitrogen limits

growth (e.g., the induction of proline oxidase –

the enzyme which catalyzes the first step of proline

degradation – by proline in E. coli, even in the

presence of ample carbon supply).

0044 The enzymes are induced independently of carbon

and energy or nitrogen supply (e.g., the induction

of threonine dehydrogenase – an enzyme involved

in threonine catabolism – in E. coli by growth in

leucine, even in the presence of other carbon and

nitrogen sources). In some microorganisms, cata-

bolite repression can be bypassed by a nitrogen

limitation signal that allows the induction of a

particular amino acid catabolic pathway. This

nitrogen limitation signal is probably related to

the complex regulation mechanisms of glutamine

synthetase (see Regulation of Amino Acid Biosyn-

thesis, above).

0045 In animal cells, amino acid catabolism is also sub-

jected to control mechanisms. Thus, for example,

removal of amino groups from amino acids is regu-

lated mainly by control of glutamate dehydrogenase.

This enzyme is allosterically inhibited by ATP and

guanosine triphosphate (GTP) and stimulated by

ADP and guanosine diphosphate (GDP). Hence,

when cellular energy charge is low the rate of amino

acid oxidation increases. On the other hand, the urea

cycle is controlled by N-acetylglutamate. This com-

pound is a positive allosteric effector of carbamoyl

phosphate synthetase, which catalyzes the first and

rate-limiting step in the pathway. N-acetylglutamate

is also a precursor of arginine and its synthesis is

inhibited by arginine. However, the amino acid cata-

bolic enzymes of animal cells are much more often

subjected to a hormonal control than are the micro-

bial enzymes. Thus, for example, the synthesis of

tryptophan oxygenase, regulated by adrenal activity,

is developmentally controlled so that the enzyme is

formed only in certain tissues and at certain times

during development. A high-protein diet is also a

factor that is known to stimulate the formation of

a number of amino acid degradative enzymes in

liver, namely urea cycle enzymes and tryptophan

oxygenase.

Synthesis of Biologically Important

Compounds

0046 In addition to their role in protein synthesis, energy

production, and gluconeogenesis, many amino acids

serve as precursors for the synthesis of other amino

acids and other biologically important compounds.

0047 Many oligopeptides containing up to 20 residues,

including hormones, antibiotics, and antitumor

agents, are synthesized in living organisms by mech-

anisms different from the usual ribosome-dependent

processes of protein synthesis. The dipeptides carno-

sine (b-alanyl-histidine) and anserine (b-alanyl-1-N-

methylhistidine) are synthesized enzymatically from

b-alanine and histidine, and from carnosine and

S-adenosylmethionine (SAM), respectively.

0048Glutathione (g-glutamylcysteinylglycine) plays a

variety of roles in living organisms. This tripeptide is

synthesized by a two-step enzymatic pathway:

0049The formation of a peptide linkage between the

g-carboxyl group of glutamate and the amino

group of cysteine, to produce g-glutamylcysteine;

0050The condensation of this dipeptide with glycine, to

form glutathione.

0051Thus the order of the amino acids in glutathione is

specifically determined by the enzymes catalyzing the

formation of each peptide bond.

0052At least90 different peptide antibiotics are produced

by strains of Bacillus subtilis and B. brevis. Gramicidin

S, for example, is a cyclic decapeptide composed

of two identical pentapeptides (d-phenylalanine-l-

proline-l-valine-l-ornithine-l-leucine). Gramicidin S

is synthesized by a multienzyme complex, gramicidin

synthetase, composed of two enzymes, one of which,

serving asa template, specifies the amino acid sequence

in the antibiotic.

0053SAM, the metabolically activated form of methio-

nine, functions as an important source of methyl

and propylamino groups for a wide variety of

compounds, including alkaloids, choline, creatine,

epinephrine (adrenaline), N-methylated amino acids,

nucleotides, and polyamines, as well as for phospho-

lipids, proteins, polysaccharides, and nucleic acids. It

is synthesized from methionine and ATP, in a reaction

catalyzed by SAM synthase.

0054A wide variety of amines occurring in bacteria,

plants and animals are derived directly or indirectly

from amino acids by decarboxylation; these include

ethylamine (from alanine), agmatine (from arginine),

g-aminobutyric acid (from glutamate), methylamine

(from glycine), histamine (from histidine), cadaverine

(from lysine), putrescine (from ornithine), phenyl-

ethylamine (from phenylalanine), ethanolamine

(from serine), tryptamine, and 5-hydroxytryptamine

(from tryptophan), and tyramine and dopamine (from

tyrosine). These amines and their derivatives often

play a variety of physiologically important roles. For

example, g-aminobutyric acid, phenylethylamine,

tryptamine, 5-hydroxytryptamine, or serotonin,

tyramine, dopamine, norepinephrine (noradren-

aline), and epinephrine are all neurologically active

compounds, whereas histamine, a powerful vaso-

dilator, is involved in allergic reactions. (See Amines.)

204 AMINO ACIDS/Metabolism

0055 Tyrosine plays several important roles in animal

metabolism as a precursor to melanins, thyroid

hormones (thyroxine and triiodothyronine), and

catecholamines (dopamine, norepinephrine, and

epinephrine). In the synthesis of melanins, tyrosinase

catalyzes first the hydroxylation of tyrosine to 3,

4-dihydroxyphenylalanine (dopa), followed by the

oxidation of dopa to phenylalanine-3,4-quinone

(dopaquinone). Dopaquinone undergoes a sequence

of reactions, including polymerization, to yield both

red and black melanins. Another enzyme forming

dopa is tyrosine hydroxylase, which catalyzes the

first reaction in the sequential enzymatic pathway

leading to the biosynthesis of catecholamines. Dopa

is then decarboxylated to yield 3,4-dihydroxyphenyl-

ethylamine (dopamine). Dopamine is hydroxylated to

norepinephrine, which in turn is methylated by SAM

to give epinephrine. (See Hormones: Adrenal Hor-

mones; Thyroid Hormones.)

0056 Putrescine, or 1,4-diaminebutane, is synthesized by

decarboxylation of ornithine, in a reaction catalyzed

by ornithine decarboxylase, a highly regulated

enzyme. Another route to putrescine formation

involves the conversion of arginine to agmatine by

arginine decarboxylase, followed by cleavage of

agmatine to putrescine and urea by agmatine ureohy-

drolase. Putrescine is an intermediate in the biosyn-

thesis of two important polyamines, spermidine and

spermine. Spermidine is synthesized enzymatically by

the SAM-mediated transfer of a propylamino group

to putrescine. The enzymatic transfer of an additional

propylamino group from SAM to spermidine pro-

duces spermine. These polycations play multiple

roles in stabilizing negatively charged intracellular

components such as nucleic acids and membranes.

0057 Creatine phosphate, which serves as a source of

high-energy phosphate in mammalian muscle and

brain, is synthesized in three steps from arginine,

glycine, and methionine.

0058 There are four classes of tetrapyrrole compounds –

hemes, chlorophylls, phicobilins and cobalamins – all

of which are synthesized from a common precursor,

d-aminolevulinic acid (ALA). In bacteria and animals,

ALA is synthesized by the condensation of glycine

and succinyl-CoA, with loss of carbon dioxide, in a

reaction catalyzed by ALA synthase. In plants, how-

ever, ALA is formed from glutamate by a three-step

pathway.

0059 An enormous amount of carbon in the bio-

sphere passes through the pathway leading to

lignin biosynthesis, the major constituent of woody

tissue. In the first reaction, phenylalanine ammonia

lyase catalyses the cleavage of phenylalanine to

trans-cinnamic acid and NH

. Cinnamic acid is a

precursor for the synthesis of a huge number of

plant substances, including lignin, tannins, flavo-

noids, pigments, many of the flavor components of

spices, and various alkaloids, such as morphine and

colchicine. (See Lignin.)

0060In addition, the synthesis of a variety of other im-

portant molecules utilizes various amino acids as pre-

cursors. Thus b-alanine is a component of CoA,

asparagine is a major form of transport of organic

nitrogenous compounds in plants, and aspartate is

involved in purine and pyrimidine biosynthesis.

Glutamate is a precursor of folic acid; glutamine

contributes to the synthesis of a variety of substances,

including purines, pyrimidines, ATP, cytidine triphos-

phate, NAD, amino sugars, and glycoproteins;

cysteine is a precursor of taurine, isethionic acid,

CoA, vasopressin, various types of pigments, includ-

ing phaeomalins and trichochromes, and other sulfur-

containing compounds. Glycine also plays multiple

roles, including contributions to the one-carbon pool

and as a precursor of purines, glyoxylate, and various

conjugates such as hippurate and glycocholate. Histi-

dine is involved in ergothionine and homocarnosine

biosynthesis, and methionine, via SAM, is the precur-

sor of the plant hormone ethylene, which influences

plant growth and development and induces the

ripening of fruits. Serine is involved in the biosynthesis

of phospholipids, and tryptophan is the precursor of

several important physiological substances, including

NAD, NADP, and the plant hormone indole 3-acetic

acid. (See Niacin: Physiology; Ripening of Fruit.)

See also: Amines; Energy: Measurement of Food Energy;

Enzymes: Functions and Characteristics; Glucose:

Function and Metabolism; Hormones: Adrenal

Hormones; Thyroid Hormones; Lignin; Niacin:

Physiology; Protein: Chemistry; Ripening of Fruit

Further Reading

Devlin TM (1997) Textbook of Biochemistry with Clinical

Correlations. New York: Wiley-Liss.

Dey PM and Harborne JB (1997) Plant Biochemistry. San

Diego: Academic Press.

Fowden L, Lea PJ and Bell EA (1979) The non-protein

amino acids of plants. Advances in Enzymology 50:

117–175.

Garrett RH and Grisham CM (1995) Biochemistry. Fort

Worth: Saunders College Publishing.

Kulka HE and Schwarz RS (1988) A flexible genetic code,

or why does selenocysteine have no unique codon?

Trends in Biochemical Sciences 13: 419–421.

Miflin BJ and Lea PJ (1977) Amino acid metabolism.

Annual Review of Plant Physiology 28: 299–329.

Moran LA, Scrimgeour KG, Horton HR, Ochs RS and

Rawn JD (1994) Biochemistry. Upper Saddle River, NJ:

Prentice Hall.

AMINO ACIDS/Metabolism 205

Murray RK, Granner DK, Mayes PA and Rodwell VW

(1988) Harper’s Biochemistry. East Norwalk, Connecti-

cut: Appleton and Lange.

Orten JM and Neuhaus OW (1982) Human Biochemistry.

St Louis, Missouri: CV Mosby.

Rawn JD (1989) Biochemistry. Burlington, North Carolina:

Neil Patterson.

Singh BK (1999) Plant Amino Acids. Biochemistry and

Biotechnology. New York: Marcel Dekker.

Smith EL, Hill RL, Lehman IR et al. (1983) Principles of

Biochemistry: General Aspects. Singapore: McGraw-

Hill.

Stenesh J (1998) Biochemistry. New York: Plenum Press.

Stryer L (1995) Biochemistry. New York: WH Freeman.

Umbarger HE (1978) Amino acid biosynthesis and its regu-

lation. Annual Review of Biochemistry 47: 533–606.

Voet D and Voet JG (1995) Biochemistry. New York: John

Wiley.

Zapsalis C and Beck RA (1985) Food Chemistry and Nutri-

tional Biochemistry. New York: John Wiley.

Zubay G (1993) Biochemistry. Dubuque, Iowa: Wm C

Brown.

ANALYSIS OF FOOD

K Gupta , V Jain, S Jain, K Dhawan and G Talwar,

CCS Haryana Agricultural University, Haryana, India

Background

0001 The science of food analysis has developed rapidly

in recent years. Analysis of food not only provides

information about composition, appearance, texture,

flavor, shelf-life, safety, processibility, and micro-

structure, but also guarantees product quality. Know-

ledge of the chemical and biochemical composition of

foods is important to the health, well-being, and

safety of the consumers. Also, it is important for

compliance with legal standards, quality assurance,

nutritional value determination, and adulteration

detection.

0002 Analysis of food involves sample preparation, an-

alysis, and detection of major food components

(amino acids, peptides, proteins, enzymes, lipids,

phospholipids, carbohydrates, alcohols, fat-soluble

vitamins, water-soluble vitamins, organic acids, or-

ganic bases, phenolic compounds, bittering sub-

stances, pigments, aroma compounds, and dietary

fiber) and miscellaneous components (preservatives,

antioxidants, colorants, and nonenzymic browning

substances). A large number of different analytical

techniques have been developed to analyze the prop-

erties of food because of the complexity and diversity

of food components. These techniques can be divided

in to three general segments focusing on physical

techniques including spectroscopy, chromatography

and electrophoresis, biochemical analysis, and

sensory analysis.

Sources of Information

0003The food analyst has to identify and choose the best

analytical method available for analyzing a food

sample depending upon its complexity. For this,

the food analyst must be familiar with all the recent

advances in food analysis methods and must

know where to look for the needed information/

literature. The main sources of information and gen-

eral approach to a literature search are detailed

below.

Books

0004The best way to obtain general information about

food analysis is to consult textbooks. A number of

textbooks are available on food analysis, and these

textbooks generally provide information on the

background and basic principles of food-analysis

methods, sophisticated instruments, and classical

procedures that have been in use for many years.

The textbooks usually concentrate on the fundamen-

tal principles rather than detailed methodology.

0005For a wide range of information, several encyclo-

pedias exclusively for food chemistry or food tech-

nology are good sources of literature, such as The

Encyclopedia of Science and Technology. Mono-

graphs are basically comprehensive surveys of current

knowledge on a specific subject. Recently, a number

of books (monographs) have become available

for specialized areas like the use of spectroscopy

and/or HPLC for food analysis. Handbooks are an-

other source of comprehensive information. For

example, the Handbook of Food Analysis, in its two

volumes, deals with the methods for physical charac-

terization as well as for biochemicals, nutrients,

206 ANALYSIS OF FOOD

and sensory analysis for a diverse number of food

components.

Reviews

0006 Once general information has been obtained, the next

step is to consult a critical review of the current

knowledge on the particular field. A review may be

written about the determination of a specific com-

ponent, about the specific technique that can be

used for different food sample analyses or about the

current aspects of a new technique/instrument. Crit-

ical reviews pertinent to food analysis are usually

published in scientific journals like Advances in

Food Research, Annual Review of Nutrition,

Methods of Biochemical Analysis, Recent Advances

in Food Science, etc.

Periodicals

0007 Scientific journals are one of the best sources of infor-

mation as they deal with the articles/information

about current and new analytical methods. Periodic-

als provide information about the research work or

analytical methods developed by other scientists.

They cover all scientific aspects of the chemical com-

position of foods, e.g., Journal of Food Composition

and Analysis, Analytical Chemistry, Food Testing and

Analysis, etc. Related journal articles can now be

searched for on the internet on specific sites, such as

www.academicpress.com/jfca for Journal of Food

Composition and Analysis.

Official Methods

0008 Several standard analytical methods have been ap-

proved by the respective governments/scientific agen-

cies (e.g. Association of Official Analytical Chemists

(AOAC), American Association of Cereal Chemists,

American Oil Chemists’ Society (AOCS), etc.) for use

in the food industry. These standard methods are

generally compiled in volumes, and they are being

improved and updated from time to time.

Symposia

0009 Programs of conferences and symposia are published

in several journals and are included on websites.

In symposia, prominent scientists present invited

review papers in the area of their competence. Ab-

stracts of papers presented at scientific meetings are

generally published in the form of a book by the

sponsoring scientific society. The Asia Pacific Food

Analysis Network and American Chemical Society

hold regular meeting/symposia on topics related to

food chemistry/analysis, and programs and abstract

books are available from the organizing society office

bearers.

Trade Publications

0010The purpose of the companies that trade in chemicals

and instruments is to sell products, but advertise-

ments are also sources of valuable information.

These publications are the best source on the proper-

ties and application of specialized equipment and

chemicals. Several manufacturers periodically issue

bibliographies and abstracts of technical-scientific

articles in a specific area, and some publish peri-

odicals that reproduce specialized articles from regu-

lar scientific journals. Even detailed handbooks

giving specification, properties, and details of analyt-

ical procedures in selected areas are provided by these

companies. The advertisements or lists of major com-

mercial companies for chemicals and instrumentation

appear in several scientific and trade journals (Sci-

ence, Nature, Prepared Foods, etc.). The instruction

manuals supplied with instruments are very useful for

installing, using, and servicing equipments.

Modern Information Retrieval Systems

0011Nowadays, computerized systems have a great cap-

acity for storing and retrieving scientific information.

A number of websites are available, on which one can

search information on a particular subject. These sites

can be accessed from any computer terminal linked

to the internet. The proper use of these systems re-

duces the time, cost, and effort involved in searching

the literature. Some of these sites/search engines in-

clude http://agrifor.ac.uk, www.altavista.com,

www.biacore.com, www.bmn.com, www.cabi.org,

www.google.com, www.ncbi.nlm.nih.gov, www.na-

tional academics.org, http://highwire.stanford.edu,

and www.yahoo.com.

0012Several more such sites/databases are available for

information on food analysis, such as Agricola

(USDA, National Agricultural Library, Beltsville,

MD), Dissertation abstracts online (University

Microfilms International, Ann Arbor, MI), Science

Citation Index (Institute for Scientific information,

Philadelphia, PA), and Food Science and Technology

Abstracts (International Food Information Service,

Reading, UK).

Preparation of Samples

0013Food is a complex inhomogeneous mixture of chem-

ical substances and microorganisms (active or not).

Therefore, improper sampling may result in variable

analysis results. Ideally, the sample analyzed in a

laboratory should have exactly the same properties

as the total population it is supposed to represent.

There are three steps involved in sample preparation

for chemical analysis of foods.

ANALYSIS OF FOOD 207

Sampling

0014 An analyst has to decide on the number of samples of

a given population to test, the location from which

the sample should be selected, and the method used to

collect them. The choice of a particular sampling plan

depends on the purpose of the analysis, the physical

property to be measured, the nature of the total

population and of the individual samples, and the

type of analytical techniques used to characterize

the samples.

Homogenization

0015 The complex structure and composition of food sub-

stances necessitate homogenization prior to most

chromatographic analysis. Each sample should be

homogenized using methods proven to be effective

with a particular matrix from experience, from the

literature, or by experiment to a degree that meets

all the requirements of the assay procedure. This must

be confirmed by replicate assays, blanks, and re-

coveries.

Sample Preparation

0016 Physical and chemical manipulation of the test por-

tion prior to analytical measurements is carried out.

This includes storage, weighing, dilution, clean-up,

extraction, digestion, purification, separation, deriva-

tization, etc. It is also important to report the results

of any analysis in a clear fashion, stating the proced-

ures used, the number of replications performed, and

the estimated reliability of the measured value. The

analytical method selected must be specific, accurate,

precise, and sensitive for the substance to be analyzed.

Selection of a Method for Food Analysis

0017 Once proper sampling and sample preparation have

been completed, the food product is ready for analy-

sis. There are numerous analytical procedures avail-

able to analyze a particular food product. Selection of

the most suitable procedure is often the key to the

success of an analysis. Some of the important factors

that should be considered are as follows.

0018 Specificity Specificity relates to the absence of inter-

fering substances that give a measurement of the same

kind as the substance being determined or the ability

to detect and quantify specific components within the

food material, even in the presence of similar food

components.

0019 Accuracy This is a measure of how close one

can approach the true value of parameter being

determined.

0020Precision This is the ability to reproduce a result

between the determinations of a substance by the

same scientist (or group of scientists) using the same

equipment and experimental approach.

0021Reproducibility This is the ability to reproduce a

result by scientists using the same experimental

approach in different laboratories using different

equipment.

0022Economy This is the total cost of analysis including

reagents, time, and instrumentation.

0023Simplicity of operation This is the ease with which

the analysis may be carried out by other workers.

0024Sensitivity This is the smallest detectable amount of

the substance/sample that can be quantified/detected

by the given technique.

0025Safety This concerns the potential hazards associ-

ated with reagents and procedures used in analysis.

0026Speed This relates to the time required to complete

the analysis.

0027Destructive/nondestructive This indicates whether

the sample is destroyed during analysis or remains

intact.

Techniques and Methods

0028The analytical techniques involved in food analysis

can be categorized as physical, biochemical, and sens-

ory evaluation techniques.

Physical Techniques

0029A variety of physical techniques have been developed

to analyze the properties of food materials. These

have been categorized according to the principles on

which they are based.

0030Spectroscopy Spectroscopic methods involve the

quantitative evaluation of the phenomenon resulting

from the interaction between a certain form of radi-

ation (electromagnetic, acoustic, electron or neutron

beam) and matter (atoms, molecules, ions) to provide

information about the properties of a food. Radiation

energy interacts with the matter in a variety of ways,

including absorption, emission, reflection, refraction,

dispersion, and polarization. Spectroscopic methods

based on each of these interactions are used in food

analysis, and those methods based on absorption and

emission are the most common (Table 1).

208 ANALYSIS OF FOOD

0031 Atomic spectroscopy is based on the transition of

outer shell electrons between the different energy

levels upon interaction of atoms with electromagnetic

radiation in the ultraviolet, visible, and infrared

regions. When the electron returns to its normal

state, it releases its acquired energy in the form of

radiation that is specific to that element, and emitted

radiation can then be measured using suitable detect-

ors. Each element has a specific set of energy levels,

and so the wavelength of the absorbed or emitted

radiation can be used to identify the element, and

the extent of absorption or emission can be used to

quantify this element. Atomic spectroscopy is used

to determine the concentration of mineral elements

in food. In atomic absorption spectroscopy, the inter-

action between the source of energy in the correct

frequency region and a sample is measured. An

atomic absorption instrument contains a source of

radiation with a wavelength that matches those re-

quired to excite atoms in the flame, thereby providing

a more efficient means of atomic excitation. In atomic

emission spectroscopy, a sample is heated to atomize

it and to excite electrons to a higher energy state. The

emission of radiation by the excited atoms is meas-

ured using suitable detectors. The disadvantage of

atomic absorption spectroscopy is that it can measure

only one element at a time. In comparison, atomic

emission spectroscopy (also called flame photometry)

can determine several elements simultaneously.

0032 Atomic fluorimetry is based on the absorption of a

photon by an atom that causes the passage of a va-

lence electron of one orbital to another orbital with a

higher energy. The atom, being unstable, returns to its

ground state, emits a photon with a different fre-

quency (usually lower than the absorbed photon)

causing fluorescence. This can be used to detect a

few mineral elements (Ca, Cd, Fe, Pb, Ni, Cr, Cu, etc.)

0033UV-visible absorption spectroscopy is one of the

most common spectroscopic techniques used to re-

solve problems of compound identification, quantifi-

cation, and qualitative analysis of food. Like atomic

spectroscopy, it is also based on the transition of

outer-shell electrons. A beam of monochromatic

radiation is transversed through the sample, and

reduction resulting from absorption is measured. A

spectrum is obtained by carrying out measurements

over a wide range of wavelengths that contain peaks

corresponding to the absorbing groups. A compound

can be characterized by its spectrum, provided that

the absorption coefficients of bands are known, or by

comparing it with reference spectra. The quantitative

aspects of this technique are based on the Lambert–

Beer law, which states that the absorbance of a solu-

tion (the log of the ratio of light entering a sample to

light leaving the sample) is proportional to its concen-

tration at a particular wavelength under a set of

standard conditions. The quantity of a substance

can also be determined by measuring the height of

one of these absorption peaks (if there is no interfer-

ence from other molecules). Derivative spectroscopy

can be used to eliminate any interference caused by

the reaction environment. Photodiode array spectros-

copy represents a great advance on absorption spec-

troscopy allowing simultaneous detection at a wide

range of wavelengths in a liquid flow. It provides

immediate information of spectra for each eluted

compound.

0034Electron paramagnetic resonance spectroscopy

arises from the fact that electrons have a quantum

mechanical property, called spin, that results in the

generation of a magnetic moment. Only atoms or

molecules with unpaired electrons possess a magnetic

moment. The transition at this moment when a

sample is placed in magnetic field can be detected

tbl0001 Table 1 Spectroscopic methods for food analysis

Type of spectroscopy Information obtained Materialrequired

Nature Quantity

Atomic absorption and emission

spectroscopy

Analysis of elements Solution Small

Atomic fluorimetry Analysis of elements Solution Small

UV-visible absorption spectroscopy Chemical and quantitative analysis Liquid extracts Small

Electron paramagnetic Physical state Native material Moderate

X-ray spectroscopy Structure and composition Native material Very small

Nuclear magnetic resonance Physical state and chemical

analysis

Native material or solid or

liquid extract

Large

Infra-red spectroscopy Physical state and chemical

structure

Native material depending on

the type of IR

Small to large

Mass spectroscopy Analysis of elements and structure

determination

Usually solid extracts Very small

The quantity very small, sub-microgram level; small, sub-milligram level; moderate, tens of milligrams; large, hundreds of milligrams.

ANALYSIS OF FOOD 209

and characterized. This technique is employed to

detect lipid peroxidation, molecular damage to

proteins, and nucleic acids.

0035 Nuclear magnetic resonance (NMR) techniques

are concerned with the transition of nuclei between

different energy levels when these are placed in a

magnetic field. The frequency at which the moment

of spin ‘wobbles’ allows the elementary segments of a

molecule to be identified. The nuclei having a net spin

angular momentum can be detected by NMR, the

most common being

Hand

C. NMR can be used

to determine the moisture, concentration, molecular

structures, phase transitions, diffusion, and image,

and can be used to check the authenticity of food

and drink ingredients whose commercial value is

related to their natural origin.

0036 Infrared (IR) spectroscopy techniques are based on

the absorption/emission of electromagnetic radiation

by a sample resulting from vibration or rotation of

molecules. The interaction of IR radiation with

matter leads to an IR spectrum containing detailed

information on different types of chemical groups of

the substance in the form of peaks. The location and

magnitude of a peak provide information about the

type and concentration of the groups present. IR

spectroscopic techniques are available to determine

the proteins, lipids, carbohydrates, moisture contents,

flavor, and toxic constituents in food.

0037 Mass spectroscopy techniques rely on the produc-

tion of ions by bombarding organic compounds

(in their vapor state) with high-energy electrons. The

ions formed are accelerated and separated as a

function of their mass and charge under the effect of

variable electric and/or magnetic fields, and their

relative abundance can be detected. These techniques

can be used to identify and determine components in

mixtures of organic materials, e.g., flavor compon-

ents, lipids, amino acids, carbohydrates, vitamins,

minerals, toxicants, antinutritional factors, and addi-

tives in food, and to identify food microorganisms.

0038 X-ray spectroscopy/spectrometry techniques are

based on the emission of X-rays when accelerated

electrons/X-rays collide with matter and eject an elec-

tron from atom’s internal layer. The vacancy is filled

by another electron from a higher orbital, and this

equilibration leads to the emission of X-rays. The

excitation can be produced by accelerated electrons/

X-rays, leading to emission spectroscopy or X-ray

fluorescence spectroscopy. These techniques provide

information about the structure, composition, and

purity of the food material.

0039 Optical techniques Analytical techniques utilize the

interaction of monochromatic beam of light with

matter to provide information about the composition

and quality of a food. These techniques include

photometry and turbimetry, which concern the dis-

persion of incident light by suspended particles in

liquid and even in gases to measure colloidal particles

in water, and clarity of juices, syrups, and wines.

Polarimetry relies on the rotation of the plane of

polarized light by an optically active solution and is

used to determine lactose in condensed milk and

sugars in juices. Refractrometry is based on the

change in refractive index caused by the displacement

of a molecule/substance in solution or by refraction

caused by that substance in solution. These tech-

niques can be used in the analysis of lipids, carbohy-

drates, and protein concentration in liquid and dry

matter. In combination with density determination,

these techniques can be used to determine unknown

substances or structures of known substances. For

very precise measurements, differential refractrome-

try and interferometry can be used.

0040Chromatography Chromatography designates a

variety of techniques that can separate mixture of

molecules into individual components on the basis

of their differences in size, shape, mass, charge, solu-

bility, and adsorption properties. It is used for purifi-

cation and constituent preparation in the food

industry. A solution of a mixture of molecules (usu-

ally in a liquid or gas carrier) is passed through a

porous medium. The carrier is called the mobile

phase, because it moves through the porous medium,

whereas the porous medium is called the stationary

phase. In this process, different solute components are

separated as a result of the different affinities of the

components for a stationary (solid or liquid) phase;

the stronger the interaction, the slower they move.

The affinity or strength of an interaction depends

on the physico-chemical properties of the substance

and the stationary phase. Molecules can be separated

according to their size, shape, mass, polarity, electric

charge, solubility, and molecular interactions. The

chromatographic techniques can be divided into dif-

ferent categories depending on their physico-chemical

basis of separation (Table 2).

0041Chromatographic separation can be carried out in

one of the three modes, depending on the nature of

the support that holds the stationary phase. In paper

chromatography, the stationary phase (liquid) is

supported by cellulose fibers of a paper sheet. The

sample is spotted on to the bottom of a sheet of paper,

and the paper is kept in an atmosphere saturated with

water vapors to form a thin film of a stationary phase.

An appropriate solvent system acting as a mobile

phase is then allowed to flow over the sample spot.

The various components of the sample are then parti-

tioned between the stationary and mobile phases.

210 ANALYSIS OF FOOD

Components with a higher affinity for the stationary

phase move less rapidly than those with a higher

affinity for the mobile phase. A more sophisticated

separation can be achieved using two-dimensional

paper chromatography.

0042 Thin-layer chromatography (TLC) has largely re-

placed paper chromatography, because it is more

convenient, quicker, and more sensitive, and it has a

better resolution and reproducibility. This technique

is similar to that of paper chromatography, except

that paper is replaced by a plastic or glass sheet

coated with a thin film of porous material that acts

as the stationary phase. In column chromatography,

separation is achieved by passing the sample through

a vertically fixed tubular glass or polypropylene

column using either gravity or a slight vacuum to

move the mobile phase through. Steel columns and

high pressures are now used to speed up the process.

In gas chromatography, the sample is volatilized and

then carried along by a gaseous mobile phase through

a column coated with the stationary phase. The effi-

ciency of separation can be controlled by selecting the

appropriate combination of stationary and mobile

phase. In high-performance liquid chromatography

(HPLC), the sample dissolved in the liquid mobile

phase is passed through a column containing the

stationary phase (which may be either solid or liquid).

This gives a faster and superior resolution, as high

pressure is applied to move the mobile phase.

0043 Electrochemical techniques Analytical techniques

involve measuring the electrical properties of food,

e.g., potential, current intensity or resistance, to

provide information on composition. Potentiometric

methods rely on measurements of the potential dif-

ference between a specific (an indicator) electrode

and a reference electrode, and this is directly pro-

portional to the concentration of the species being

measured.

0044 Polarographic methods are based on the potential

difference between a mercury electrode and a refer-

ence electrode immersed in a solution containing

electroactive species. These are used to determine

heavy metals polluting water or foods, pesticides,

and preservatives. Coulometric methods involve

measuring the quantity of electric current required

to electrolyze the substance being analyzed; the

greater the electric charge, the higher the concentra-

tion of the substance. The electrical conductivity is

based on the displacement of different components of

a substance in the solution, which is equivalent to the

current passing between a pair of electrodes. The

amount of current is measured and then extrapolated

to the composition of a specific component within

the food.

0045Electrophoresis relies on differences in the move-

ment of charged macromolecules in an electrical field

applied across a substance. It is used to separate

molecules on the basis of a net charge, size, and/or

shape. The sample to be analyzed is added to a buf-

fered porous matrix (usually a strip of paper or gel),

and a current is applied. Charged molecules move

through the matrix in a direction that depends on

the sign of their electric charge. The rate of movement

depends on the magnitude of the charge and resist-

ance to movement through the matrix. The resistance

is determined by the relationship between the effect-

ive size of the substance and the size of the pores in

the matrix. The larger the size of the pores or the

smaller the size of molecules to be separated, the

lower the resistance, and therefore, the molecules

will move faster through the matrix. Isotachoelectro-

phoresis (separation of sample components based on

differences in net mobility) has a high resolving

power, because of its concentrating effect. Compon-

ents with different isoelectric points are separated

better by isoelectric focusing (IEF). Two-dimensional

electrophoresis can be carried out to improve the

resolution. Substances are separated in one direction,

according to their charge by IEF, and in a second

dimension (perpendicular to the first), on the basis

of their size.

0046Rheological techniques These techniques are based

on the relationship between the stress and strain in a

material, with a behavior between that of solids and

tbl0002 Table 2 Classification of separation techniques of chromatography on physico-chemical basis

Molecular

characteristics

Physical principles Example

Polarity Differences in partitioning coefficient between polar and

nonpolar solvent

Partition, i.e., liquid–liquid; liquid–solid

gas–liquid chromatography and HPLC

Ionic Differences in electrostatic interaction between charged groups

on molecules in the mobile phase and stationary phase

Ion-exchange chromatography

Size Differences in the size and shape of molecules Gel-permeation chromatography

Affinity/shape Specific interactions between molecules in the mobile phase and

stationary phase

Affinity chromatography

ANALYSIS OF FOOD 211

liquids. Measurements of these properties of foods

can be based on either an analytical or integral ap-

proach. In the analytical approach, the properties of a

material are related to a simple system such as New-

tonian fluids or Hookian solids. In the integral ap-

proach, a simple empirical relation between stress,

strain, and time is established. The relationship

measures the way in which rheological properties

vary under a specific system of applied forces. The

instruments used provide a measure of baking quality

of flours, thickening properties of biopolymers, tex-

ture, hardness, flakiness, consistency of food, tender-

ness of meat, and texture of fruits and vegetables.

Rheological techniques are now essential tools, espe-

cially as a quality-control tool in food science and

technology.

0047 Biochemical techniques There are a number of bio-

chemical techniques available that can be used to

determine the identity and/or concentration of spe-

cific components of food. Enzymatic analysis means

analysis with the aid of enzymes utilizing specific

enzyme reactions. The major advantage of enzymatic

techniques lie in their ability to react specifically with

individual components of a mixture. This avoids

lengthy separation of the components and reduces

the time required for analysis. These techniques re-

quire small amounts of sample and do not require

components to be extracted from their native

medium.

0048 Immunochemical techniques These rely on specific

interactions between an antigen and an antibody.

Since macromolecules found in microorganisms,

food, and agricultural products are good antigens,

antibodies against these molecules can be obtained.

The potential applications for immunological tech-

niques are numerous, as much for raw material and

final product quality control as for tracking produc-

tion and conservation effectiveness.

0049 Analytical microbiology involves the use of micro-

organisms as reagents for the quantitative determin-

ation of certain chemical compounds present in food.

Since some of the nutritional requirements of micro-

organisms and experimental animals are similar, it is

possible to use analytical microbiological techniques

to determine some substances that are essential con-

stituents of living cells. These are used for vitamins,

nucleic acids, amino acids, heavy metals, growth

factors, and nutritional values of proteins and anti-

bodies. The basic principle is that in the presence of

limiting amounts of certain compounds, the amount

of microbial growth is a function of the concentration

of those compounds. The microorganisms used for

assay are primarily bacteria, but yeast, fungi, algae,

and protozoa have also been used. These can be

assayed after diffusion of the substance through

solid inoculated culture media by turbidimetric, dilu-

tion, gravimetric, and metabolic response methods.

0050Sensory evaluation The sensory attributes of the

quality of a food are measured to determine consumer

preference in order to help in the manufacture of an

acceptable product at maximum production econ-

omy. Sensory attributes include appearance (color,

size, shape, and consistency of liquid and semisolid

products), kinesthetic (texture, consistency, and vis-

cosity) and flavor (taste and odor). The evaluation is

also done in order to determine the conformity of a

food with established government or trade standards

and food grades. The technique of sensory evaluation

involves using individuals as the data generator, and

so, it is influenced by factors such as social, cultural,

religious, psychological, and climatic factors, phys-

ical status of the individual, availability, and nutri-

tional education. Large, highly trained groups of

experts are employed for evaluation to minimize the

effects of such factors. Laboratory tests may be con-

ducted to study the human perception of food attri-

butes, to correlate sensory attributes with chemical

and physical measurements using principal compon-

ent analysis, to evaluate raw material selection; to

study the processing effects and means of maintaining

uniform quality, to establish shelf-life stability, and to

reduce costs.

Food Components

0051Structural changes occur during the processing and

storage of foods. Therefore, knowledge of the com-

position of a food is necessary in order to determine

the extent of any change in the concentration of

the nutrients. The use of agrochemicals and additives

also may affect the quality of the food product. In

order to obtain quality assurance, in accordance with

legal requirements, nutrition, and food safety, the

concentration of major and minor food components

must be known. The methods used for the analysis

of food components are discussed below, and these

methods form part of the techniques already de-

scribed.

Major Components

0052Moisture Water is the major component of most

foods and is an index of the economic value, stability,

and quality of food products. It is important to note

that the removal of water either by conventional de-

hydration or by separation locally in the form of fine

ice crystals (freezing) greatly alters the native proper-

ties of foods. Therefore, it is very important to use

212 ANALYSIS OF FOOD

simple, rapid, and accurate methods for moisture

determination in food products. The food sample

should be homogenized using a number of electrical/

mechanical devices like blenders, mincers, graters,

homogenizers, and grinders. Analytical methods of

moisture determination can be classified as direct

and indirect procedures. Direct procedures include

the determination of moisture by drying (oven,

vacuum, freezing, chemical desiccation), distillation,

chemicals (Karl Fischer titration), and extraction (gas

chromatography and refractrometry). The drying

method relies on the evaporation of water from the

sample and is one of the most commonly used pro-

cedures for determining moisture content (conven-

tional ovens, forced air ovens, vacuum oven,

microwave oven, and IR oven). The weight of the

sample is taken before and after it is dried, and the

moisture content is then calculated. In the distillation

method, the food sample is mixed with an organic

solvent, and water is evaporated and collected in a

graduated collection arm, in which its volume can be

determined. The most commonly used chemical

methods for moisture determination are Karl Fischer

titration and the gas-production method. These

methods rely on specific chemical reactions between

water and other substances that lead to some quanti-

fiable parameters. Indirect methods include rapid

and nondestructive methods, and these involve cali-

bration against standard moisture values that have

been precisely determined by using one or more

of the direct methods. These methods are based on

differences in the physical properties of water com-

pared with other components and are widely used

to determine moisture content including IR gas

chromatography, NMR, and several electrical

methods. These instruments are simple to use, pro-

vide rapid and reliable measurements, and are, there-

fore, particularly suitable for routine quality-control

applications.

0053 Mineral components Ash is a measure of total min-

eral components in a food and is regarded as a general

measure of quality in certain foods such as tea, flour,

and edible gelatin, etc. There are two major proced-

ures of ashing that are used to determine the mineral

content of the sample. These are dry ashing and wet

ashing. The nature of the ashing procedure is

governed by the purpose for which ash is prepared,

the particular constituents to be determined, and the

method of analysis to be used. In dry ashing, the

sample is ignited at 550–600



C to oxidize all organic

material without a flame, whereas in wet ashing, the

sample is digested in acid to determine mineral elem-

ents. The individual elements within ash can be deter-

mined by titrimetric methods, colorimetric methods,

or atomic spectroscopy techniques. Atomic spectros-

copy techniques can provide a complete profile of

different types of elements in a food material,

whereas the titrimetric and colorimetric methods are

usually designed to determine a particular element. In

addition, atomic spectroscopy techniques have a

much higher sensitivity and specificity.

0054Carbohydrates Carbohydrates are present in all

grains, vegetables, fruits, and other plant parts and

vary in form from simple monosaccharides to oligo-

saccharides and more complex polysaccharides.

These are a major source of energy for the human

body, and their varied functional properties are util-

ized by the food industry to enhance the palatability,

acceptability, and shelf-life of foodstuffs. There is

thus a continuous need to monitor levels of carbohy-

drates in foods. The complete analysis of foodstuffs

may require the determination of several groups of

compounds, e.g., simple sugars, reducing sugars,

polysaccharides, and fibers, all of which may play

an important role in the quality of the product. For

this purpose, a number of physical and chemical

methods have become well established and com-

monly used in many laboratories. Physical methods

include refractrometry, polarimetry, and hydrometry,

whereas chemical methods include titrimetric and

colorimetric, which determine the quantity of a par-

ticular sugar present in food. Chromatographic tech-

niques such as paper chromatography, TLC, gas

liquid chromatography (GLC), and HPLC provide

more rapid analyses with a greater precision and

specificity. Nowadays biochemical methods of analy-

sis are often used because these are specific, sensitive,

rapid, and reproducible. Flow injection analysis (FIA)

gives an excellent performance and has been the basis

of a large number of methods for the separation,

processing, and analysis of carbohydrates. The future

of carbohydrate analysis undoubtedly belongs to

three analytical techniques – HPLC, enzymatic

assays, and FIA – all having a close relationship to

one another.

0055Proteins The determination of proteins in foods and

food products has an important nutritional, func-

tional, and technological significance. The methods

for protein estimation depend on the measurement of

a specific element or chemical group in the proteins.

The elements or groups most commonly used are

nitrogen, aromatic amino acids, or peptide linkage.

Based on nitrogen content (approx. 16% of protein

weight), either protein can be estimated by Kjeldahl’s

method (AOAC) or ammonia can be directly deter-

mined in the digest by color-inducing compounds

such as ninhydrin, indophenol, or Nessler’s reagent.

ANALYSIS OF FOOD 213