FOOD AND NUTRITIONAL ANALYSIS

Contents

Overview

Sample Preparation

Additives

Antioxidants and Preservatives

Contaminants

Water and Minerals

Pesticide Residues

Mycotoxins

Soft Drinks

Coffee, Cocoa, and Tea

Alcoholic Beverages

Wine

Meat and Meat Products

Dairy Products

Vegetables and Legumes

Oils and Fats

Fruits and Fruit Products

Packaging Materials

Overview

V Jain and K Gupta

, Haryana Agricultural University,

Hisar, India

& 2005, Elsevier Ltd. All Rights Reserved.

Introduction

The science of food and nutritional analysis has
developed rapidly in recent years. Food scientists
analyze foods to obtain information about their
composition, appearance, texture, flavor, shelf life,
etc., and also to guarantee the quality of the product.
Nevertheless, the term food and nutritional analysis
is often thought only to be concerned with the de-
termination of food composition and its nutritive
value/quality.

The analysis of food began in the nineteenth

century utilizing microscopy. It was the first
analytical technique used by analysts such as
Accum and Hassall to identify food components
and to detect adulteration. The development of
improved analytical methods to determine the
composition of foods (such as Kjeldahl method

for

nitrogen

estimation

in

1885),

together

with the concern over adulteration resulted in the
introduction

of

statutory

legislation

over

the

composition of important food products in many
countries. These forms of controls continued for
B100 years until the 1970s, when rapid advances
in food science and technology revolutionized the
manufacture of food. Media attentions have also
brought a public awareness of the links and causa-
tion between diet and health. As a result, today’s so-
phisticated consumers have become more concerned
over the quality and compositions of their food pur-
chases, the contained ingredients, and the presence of
additives and contaminants. Therefore, knowledge
of the chemical and biochemical composition of
foods is important to the health, well-being, and
safety of the consumers. Analytical characterization
is also important for compliance with legal stand-
ards, quality assurance, and determination of nutri-
tional value.

Various

types

of

modern

food

analytical

techniques have been developed, including elec-
trophoresis, chromatography, spectroscopy, rheol-
ogical

techniques,

and

sensory

evaluation,

to

meet the challenge of providing information on
the diverse components of these complex food
materials.

202

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

Information Sources

Standard Official Methods and Modern Information
Retrieval Systems

Standard analytical methods approved by profes-
sional

associations/scientific

agencies

such

as

Association of Official Analytical Chemists, The
American Association of Cereal Chemists, and Amer-
ican Oil Chemist Society are used in food industry.
(These methods are being improved and updated
from time to time.) The computerized systems for
storing and retrieving scientific information are now
well developed (Table 1).

Sampling

Food is a heterogeneous mixture of chemical sub-
stances; therefore, its analysis requires adequate
methods of sampling and preserving. The analytical

method selected must be specific, accurate, precise,
and sensitive for the substance to be analyzed. The
sampling includes sample selection, preparation, and
labeling. If it is not carried out properly/correctly it
leads to variability of results. Ideally, the sample
analyzed should have exactly the same properties as
the bulk of the sample (population) from which it
was taken. The major steps in sampling are identi-
fication of the population from which the sample is
to be taken, location from which the sample should
be collected, and method of obtaining the gross
samples.

Techniques and Methods

Spectroscopic Techniques

A variety of spectroscopic techniques has been
developed to analyze food materials (Table 2).

Table 1

Organizations that issue standard food analysis methods and modern information retrieval systems

Name of organizations/acronym

Area of analysis

Name of organizations
American Association of Cereal Chemists (AACC)

Cereals

American Oil Chemists Society (AOCS)

Oil seeds

Analytical Methods Committee of the Royal Society of Chemistry, UK (AMC)

Various

Association Francaise de Normalization (French Standard Organization) (AFNOR)

General

Association Internationale de I’Industrie des Bouillons et Potages (International Association of the Stock

and Soup Industry) (AIIBP)

Soups

Association of Official Analytical Chemists, USA (AOAC)

Food, agriculture

Association of Public Analysts, UK (APA)

Various

British Standards Institution (BSI)

General

Corn Industries Research Foundation Inc. (CIRF)

Starch products

Deutsche Gesellschaft fur Fettwissenschaft (DGF)

Oils and fats

European Economic Community(EEC)

Various

Food and Agriculture Organization, UN (FAO)

Agriculture

Federation of Oils, Seeds and Fats Association (FOSFA)

Oils and fats

International Association of Seed Crushers (IASC)

Vegetables oils and fats

International Association for Cereal Chemistry (ICC)

Cereals

International Commission on Microbiological Specification for Food (ICMSF)

Food (general)

International Commission for Uniform Methods of Sugar Analysis (ICUMSA)

Sugar

International Dairy Federation (IDF)

Dairy products

International Office of Cocoa, Chocolate and Sugar Confectionery (IOCCC)

Cocoa, confectionery

International Organization of the Flavour Industry (IOFI)

Flavor

International Organization for Standardization (ISO)

General

International Union of Pure and Applied Chemistry (IUPAC)

General

Institute of Brewing, UK (IOB)

Beer

Nordisk Metodik-kommittee for Livsmedal (NMKL)

Food

Nederlands Normalisatie-Instituut (NNI)

General

Office Internationale de la Vigne et du Vin (OIV)

Wine

Modern information retrieval systems
Database
AGRICOLA, BIOSIS PREVIEWS, CAB abstracts, CRIS, Food science and technology abstracts,

Science citation index

Website
http/agrifor.ac.uk, www.altavista.com, www.biacore.com, www.bmn.com, www.rediff.com,

www.cabi.com, www.google.com, www.yahoo.com, http://www.worldmedicus.com

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

203

Rheological Techniques

Rheology is the study of flow of matter and defor-
mation and these techniques are based on their stress
and strain relationship and show behavior inter-
mediate between that of solids and liquids. The
rheological measurements of foodstuffs can be based
on either empirical or fundamental methods. In the
empirical test, the properties of a material are related
to a simple system such as Newtonian fluids or
Hookian solids. The Warner–Bratzler technique is an
empirical test for evaluating the texture of food ma-
terials. Empirical tests are easy to perform as any
convenient geometry of the sample can be used. The
relationship measures the way in which rheological
properties (viscosity, elastic modulus) vary under a

specific system of applied forces. These techniques
provide a measure of baking quality of flours, thick-
ening properties of biopolymers, texture, hardness,
flakiness, consistency in food, tenderness of meat,
and texture of fruits and vegetables. Rheological
techniques have now become essential tools in food
science and technology.

Biological Techniques

Biological

techniques

encompass

two

method-

ological approaches: live systems and biochemical
techniques. Live systems employ (1) whole animal,
(2) microorganisms, and (3) cell and tissue culture
methods as tools in food analysis. Biochemical tech-
niques include uses of enzymes and immunochemical
techniques (Table 3).

Table 2

Spectroscopic methods for food analysis

Type of spectroscopy

Information obtained

Nature of material required

Atomic absorption and emission

Analysis of elements

Solution

Atomic fluorimetry

Analysis of elements

Solution

Circular dichroism

Conformation of proteins

Solution of optically active molecules

Dielectric

Moisture content

Native

Electron spin resonance

Physical state

Native material

Nuclear magnetic resonance

Physical state and chemical analysis

Native material or solid or liquid extract

Near-IR and mid-IR

Physical state and chemical structure

Native material depending on type of IR

Mass spectroscopy

Analysis of elements and structure determination

Usually solid extracts

Raman

Physical state

Molecules

X-ray

Structure composition

Native material

UV–visible absorption

Chemical quantitative analysis

Native

Table 3

Techniques for biological evaluation of food

Analysis using live system

Analysis using biochemical techniques

Whole animal as

analytical tool

Microbiological analysis

Tissue and cell culture

analysis

Immunochemical

techniques

Enzymatic techniques

Assay on body
Body weight changes

Turbidimetric
Gravimetric method

Cell culture
Organ or tissue culture

Agglutination
Immunodiffusion

Substrate assay

methods

Digestibility assays

Serial dilution assays

Endpoint methods

Radioimmunoassay

Kinetic methods

Analysis of food protein

and energy (PER,
NPR), and net energy
assay

Acidimetric and

metabolic product
assay

Diffusion plate assay

Enzyme immunoassay

Immobilized enzymes

assay

Tubular enzyme

reactions

Enzyme electrodes
Rapid analysis strips

Changes in nutrient

content of body

Energy balance
Bv, NPU

Immunoenzymatic

methods

Enzyme-linked

immunosorbent
assay

Enzyme multiplied

immunoassay
technique

204

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

Live systems as analytical tools

Different life sys-

tems have been utilized in assessing composition and
nutritional quality of foods.

The whole animal techniques are used in the nu-

tritional and toxicological evaluation of foods by
providing a combined assessment of digestibility,
metabolism of food, and nutritional evaluation of
quality of foods. These approaches are aimed at
determining the changes in the nutrient content in
animal body in response to feeding a diet or meal
containing that nutrient. The animal used in con-
ducting food analysis assays include albino rats and
mice, and guinea pigs, chicken, ducks, hamsters,
gerbils, dogs, and monkeys.

The changes in nutrient content and energy value

of foods required for animal body involves the meas-
urements on animal body itself. These assays are
based on body weight changes for analysis of food
protein and energy and include protein efficiency ra-
tio (PER), net protein ratio (NPR), and multilevel
assays. The second approach to estimate change in
the nutrient content of the body involves measure-
ments of intake and output of nutrients from the
body and assay includes nitrogen or energy balance,
net protein utilization (NPU), biological value (BV),
and net energy.

The animal assay for evaluating toxic constituents

(natural or added) of food uses lethality as an index
and determines the dose of that toxic chemical to
kill/affect 50% of the test animal, referred to as
LD

50

dose. The response criteria can be toxicity,

carcinogenicity, mutagenicity, reproduction, and
metabolism.

Microbiological food assay

Microbiological food

assays involve the use of microorganisms as a substi-
tute for a higher animal. Since some of the nutritional
requirements of microorganisms and experimental
animals are similar, it is possible to use micro-
biological food assays to determine the substances
that are essential constituents of living cells. These are
based on the principle that in the presence of limiting
amount of specific nutrients, the amount of growth
is a function of concentration of this nutrient. These
are used for determination of amino acids, vitamins,
nucleic acids, heavy metals, growth factors, and the
nutritional value of proteins and antibodies. The mi-
croorganisms used for the assay are bacteria, fungi,
algae, yeast, and protozoa. The test organism selected
is based on nonpathogenicity, sensitivity, and specifi-
city to the nutrient to be assayed, its rapid and repro-
ducible

growth,

and

ease

in

making

growth

measurements. It can be assayed after adding the food
sample extract to a liquid medium or gel medium,
inoculated with the microbial culture followed by

growth stimulation, and then by turbidimetry meas-
urement, gravimetry measurement, diffusion plate
assay, and metabolic product assay.

Cell and tissue culture methodology

This method

uses cell, tissue, or organs for analytical purposes.
These have been used in food toxicology and safety.
Toxic chemicals like pesticides, food additives, and
chemicals produced during processing and cooking
of foods are tested for their toxicity, mutagenicity,
and oncogenic properties using tissue culture
techniques.

Biochemical techniques

These techniques include

immunochemical and enzymatic techniques.

Immunochemical techniques are based on specific

interactions of antibodies with antigens. Since macro-
molecules are present in microorganisms, food, and
agricultural products and are good antigens, the an-
tibodies against these molecules can be obtained in
response to the immunization with antigens, the sub-
stances foreign to animals. The antibodies produced
in response to a particular antigen are capable of
recognizing and binding due to the complementary
binding site on the antibody with that of antigen.
The methods employed in food analysis include
hemagglutination, precipitation, immunodiffusion,
immunofluorecence, immunoeletrophoresis, radio-
immunoassays, and enzyme immunoassays. Enzyma-
tic analysis means analysis with the aid of enzymes
using specific enzymatic reactions. The enzymatic
techniques are highly useful because of specificity and
sensitivity for measuring the concentration of com-
pounds. This reduces the time of analysis and avoids
lengthy separations of the components. The immobi-
lized enzyme systems with electrochemical detection
methods are used for analytical determinations
of food components that would be laborious or
otherwise impossible. These techniques require small
amounts of sample and do not require components to
be extracted from their matrix. Enzymatic methods
are used in the determination of structure of poly-
meric macromolecules, quality indices of foods, and
nutritional and physical induced changes in human
beings.

Sensory Evaluation

Sensory evaluation is a scientific discipline used to
evoke, measure, analyze, and interpret reaction to
those characteristics of food material as they are
perceived by the senses of sight, smell, taste, touch,
and hearing (sound). The sensory attributes of qual-
ity of food are measured to determine consumer
acceptance/preference in order to manufacture an

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

205

acceptable and affective product at maximum pro-
duction economy. The sensory attributes include ap-
pearance (color, size, shape, and consistency of liquid
and semisolid products), kinesthetic (texture, con-
sistency, and viscosity), and flavor (taste and odor).
The evaluation is done to determine quality criteria
by which raw materials and finished products may be
graded and classified. The sensory techniques help
food scientists to determine the conformity of a food
with established government or trade standards and
food grades and product development while main-
taining desirable sensory characteristics. The tech-
nique of sensory evaluation uses humans as data
generators; hence, it is influenced by cultural, psy-
chological, religious, social, and climatic factors,
physical and educational status of an individual,
availability, and nutritional knowledge. Highly
trained groups of experts are employed for evaluat-
ion to minimize the effects of such factors. For
complete sensory evaluation of food, two types of
tests – analytical sensory tests and affective tests
(like–dislike) are performed (Table 4).

The analytical sensory tests are confined to sen-

sitivity, discriminatory, quantitative (intensity), and
descriptive or qualitative analysis of the sensory at-
tributes of food.

A sensitivity test is a measure of sensitivity to a

human stimulus and is expressed as threshold, which
is a fixed value at a given moment of time. The
threshold may be absolute or differential, which is
measured using various psychological methods. The
threshold is determined by approaching and receding
from standard stimulus by short steps and the thresh-
old is that step where the judge’s response shifts from
one category to another. In the method of average
error (or the method of adjustment), the judge ad-
justs the concentration of the comparison with the

standard stimulus. The frequency method records
each comparison stimulus against a standard stimu-
lus many times, thereby creating a difference
response.

Discrimination Tests

Food analysis is based on difference testing, the fun-
damental approach to sensory analysis of food. A
simple difference test permits one of the two re-
sponses ‘Yes, there is a difference’ or ‘No, there is a
no difference’. In directional difference testing, a
judge is asked which sample is more in a predefined
characteristic. The predesignated standard must be
similarly understood and used by all the judges. A
large number of difference tests, single stimulus,
paired comparison, paired difference, triangle, dual
standard, multiple standard, and multiple pairs, are
used to detect the sensory differences (such as sweet-
ness, softness, color, etc.) between the two or more
samples.

Quantitative Test (Intensity)

This approach of sensory evaluation is based on de-
scription of scaling, i.e., ranking category (or inter-
nal) and magnitude estimation (or ratio). Ranking is
used for grouping of the products based on their
quality or order of preference. The scoring on
category scale involves the use of a limited number
of categories, designated in terms of numbers, letters,
or points on a line. The magnitude estimation is a
type of ratio scaling that measure the relationship
between physical and sensory criteria. Any number is
assigned to the first stimulus. A proportional number
is assigned to reflect its strength in comparison to the
first.

Descriptive Analysis

In descriptive analysis small groups of highly trained
judges with considerable experience with the com-
modity under study develop adjectives to character-
ize the qualitative properties of the product
(attributes like appearance, aroma, texture, taste,
etc., are analyzed). The data of sensory analysis ex-
periments are subjected to statistical analysis to get
reliable results. Instrumental methods are also used
to correlate physicochemical measurements with sen-
sory judgments (Table 5).

Effective testing/acceptance testing

By acceptance

testing we mean measuring liking or preference for a
product. Preference is that expression of appeal of
one product versus another that can be measured
directly by comparing two or more products with
each other. The nine-point hedonic scale method

Table 4

Human response of sensory systems corresponding to

selected physical properties of foods

Physical property

(stimulus)

Sensory system

Human sensation

Density

Kinesthetic, haptic

Heavy, light

Moisture content

Haptic, thermal

Dry, wet, soggy

pH

Gustation, pain

Harsh, sharp, sour

Radiant energy

Visual

Appearance, color

Shear

Kinesthetic, haptic

Hard

Surface abrasion

Haptic, pain

Rough, prickly

Solubility

Gustation

Taste

Temperature

Thermal system

Cold, hot

Texture

Olfaction

Odor

Vapor pressure

Kinesthetic, haptic

Thin, thick

Vibration/pitch

Auditory

Sound, e.g., crisp,

crunchy, sizzle

206

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

occupies a niche for the sensory evaluation of prod-
uct acceptance/preference. The measure of liking
preference is a sensory evaluation model for me-
asuring product acceptance and it represents the final
phase of test resources with discrimination and de-
scriptive analysis test. The sensory acceptance test is
a cost-effective resource that has a major role in the
development of successful products.

The scale was developed to access the degree of

acceptability of food items, beverages, cosmetics,
paper products, etc., by measuring the degree/
magnitude of like/dislike.

Nutritional Evaluation of Food Processing

Although processed foods are not considered to be as
nourishing as fresh and unprocessed foods, there is an
increasing demand for processed foods, which are
available throughout the year. Food processing
involves a wide variety of industrial processes with a
correspondingly large variety of products. The opti-
mum quality maintenance of the product attributes
are compared in relation to the origin as ‘fresh like’
characteristics of preserved fresh products and ‘just
cooked like’ attributes of preserved cooked products.
New techniques such as modified atmosphere pack-
ing, edible and biodegradable packing materials, high-
pressure short-duration treatment, biopreservation,
high-voltage inputs, and a combination of different
mild treatments help in preserving the quality at-
tributes of the processed food.

Food Components

Knowledge regarding the major constituents of food
(e.g., carbohydrates, amino acids, proteins, lipids,
dietary fiber, and nutritional and antinutritional
components) is necessary so as to guage the extent
of any structural change that occurs during process-
ing and storage of foods, which may affect the qual-
ity and safety of the food product.

Major Components

Moisture

Water content/moisture content is the

most ubiquitous substance in nature, the largest
single constituents of all living things and affects
quality, value, and freshness of food and is of major
concern in food, paper, and plastic industries. Mois-
ture determination is a widely used fundamental an-
alytical operation, which satisfies the technological,
analytical, commercial, and regulatory necessities in
the processing, testing, and storage of food products
and is an index of economic value, stability, and
nutritional quality of food products.

Removal of water for processing/storage purposes

either by conventional dehydration or freezing and
drying alters the native functional properties of
foods. Simple, rapid, and accurate methods for mois-
ture determination in raw, processed, and stored
food products are used to know the nutritive value of
food products. A homogenous food sample should
be prepared using a number of electrical/mechanical

Table 5

Analytical methds and consumer measurements of sensory attributes

Analytical tests

Stimulus concentration

Sensitivity

Quantitative

Qualitative

Threshold

Discrimination

Scaling

Duration

Descriptive analysis

Methods of limits

Paired comparison

Ordering or ranking

Time intensity

Texture profile

Methods of adjustments

or average error

Paired difference

Category (internal)

Flavor profile

Frequency method

Duo–trio

Magnitude estimation

(ratio)

Dilution profile

Dual standard

Quantitative descriptive

analysis

Multiple standard

Other methods

Triangle

Consumer tests

Effective

Acceptance

Preference

Hedonic test

Accept/reject what is

available

Select one over the

other

Degree of like and

dislike

FOOD AND NUTRITIONAL ANALYSIS

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207

devices like blenders, graters, grinders, homogeni-
zers, and mincers for the determination of moisture
by any of the analytical methods given in Table 6,
which are classified as direct and indirect procedures.
The weight of sample is taken before and after
it is dried, and the moisture content is calculated.
Instruments used for moisture determination are
simple to use and provide rapid and reliable measure-
ments and are suitable for routine quality control
applications.

Carbohydrates

Carbohydrates are a major source

of energy for humans and are present in all foods
(grains, vegetables, fruits, and milk), and vary
in form from simple monosaccharides (fructose,
glucose, galactose, sorbitol) to oligosaccharides
(maltose, sucrose, lactose, raffinose, stachyose, ver-
bascose), and more complex polysaccharides (starch,
cellulose, etc.).

The complete analysis of foodstuffs may require

the determination of simple sugars and reducing
sugars (fructose, glucose, galactose, sorbitol, mal-
tose, lactose), oligosaccharides (sucrose, raffinose,
stachyose,

verbascose),

polysaccharides

(starch,

cellulose, etc.), and fibers, which vary in amount/
form and all of which may play an important
role in the quality of the product. Methods of

carbohydrate determination in foods are summa-
rized in Table 7.

Neutral monosaccharides, uronic acids, hexosa-

mines, and sialic (neurominic acids) are identified
and determined by specific colorimetric reactions.
The principle behind the techniques rests on the con-
densation of the degraded products of the neutral
monosaccharides (hexose, pentose and methyl pen-
tose) by sulfuric acid with anthrone, cysteine hydro-
chloride, orcinol, and phenol reagents. Uronic acids
may be determined by colorimetric and manometric
procedures. While sialic acids are determined after
chemical/enzymatic hydrolysis, gravimetric and Van-
Soest detergent based methods are used to determine
cellulose, hemicellulose, and fiber.

Amino acids and proteins

A large variety of pro-

teins, either from animal origin (eggs, fish, meat, and
milk) or plant origin (cereal, pulses, fruits, and
vegetables) is available from food sources. There is a
practical need to determine amino acids, proteins,
and protein quality, and to monitor and regulate
protein quality in food production, processing,
storage, and marketing, which emphasizes the im-
portance of protein quality. The comprehensive nu-
tritional evaluation of protein quality of food and
food products begins with the determination of
nitrogen content, essential amino acid concentra-
tions, amino acids, protein types, and assessment of

Table 6

Classification of analytical methods for moisture

determination

Direct methods

Indirect methods

Direct methods usually yield

accurate and absolute value
and are manual and time
consuming

Indirect methods are rapid,

nondestructive and easily
automated

Drying method
Chemical desiccation
Freeze-drying
Oven drying
Vacuum drying

Physical and electrical

methods

AC and DC conductivity
Dielectric capacitance
Microwave absorption

Distillation method
Azotropic distillation

Spectroscopic methods
IR absorption
Near-IR reflectance
Nuclear magnetic resonance
Neutron and g-ray scattering
Refractometry
Sonic and ultrasonic

Chemical methods
Generation of acetylene
Heat on mixing with H

2

SO

4

Karl Fischer titration

Extraction method

Gravimetric method
Thermogravimetric analysis

Absolute methods
Dew point method
Gas chromatography
Manometric method
Psychrometry
Volumetric

Table 7

Major methods of carbohydrate analysis

Traditional methods

Recent methods

Physical methods

Physicochemical methods

Determine the quantity of a

particular sugar present in
food

Provide more rapid analysis

with a greater precision and
specificity

Hydrometry
Polarimetry
Refractometry

Chromatography and

electrophoresis

Paper chromatography
Thin-layer chromatography
Gas chromatography
Liquid chromatography
Ion-exchange chromatography

Chemical methods
Classical methods

Ferricyanide method
Iodometry method

Colorimetric methods

Biochemical methods

Based on chemical reagents

used

Enzymatic methods: used for

starch and cell wall
carbohydrates

Anthrone
Clegg-anthrone
Copper reduction
Dinitrosalicylate
Phenol/sulfuric acid
Nelson-Somogyi
Neocuproine
Picric

Flow injection analysis

Automated method

208

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

nutritional value including digestibility (in vitro and
in vivo assays), and biological evaluation (PER,
NPU, BV, growth parameters, etc.) by taking albino
rats, guinea pigs, and human beings as experimental
models. Enzymatic and microbiological tests may al-
so evaluate nutritional quality of foods. Amino acids
can be determined by colorimetric and enzymatic
methods after hydrolysis of proteins in a specific
medium used to diminish destruction of essential
amino acids after separation by different techniques
(column chromatography, thin-layer chromatogra-
phy (TLC), gas chromatography (GC), high-perfor-
mance liquid chromatography, etc.). The availability
of all the essential amino acids in food proteins
can be measured with the enzymatic ultrafilteration
digest.

The determination of protein in food/food prod-

ucts depends on a measurement of a specific element
or chemical group in the proteins, which may be
carried out either directly by using chemical or phys-
ical properties. Based on nitrogen content, protein
content can be estimated by Kjeldahl’s method
(AOAC). Ammonical nitrogen produced after de-
struction of organic matter of foods is multiplied by a
coefficient of 6.25 for animal, vegetable, and pulse
proteins and by 5.7 for cereals to obtain protein
contents. Ammonia content can also be determined
by using an ammonia-specific electrode, based on the
difference in potential between a reference electrode
and a measurement electrode, and with the help of
Nessler’s reagent. Protein content in food/food prod-
ucts may be determined by various chemical and
physical methods as given in Table 8.

Specific protein components in food/food products

can be determined by chromatography (ion-exchange
chromatography, PC, TLC, GC, LC, etc.), elect-
rophoresis, and immunology or their combination. It
is also necessary to use high-resolution techniques
such as reversed phase LC, ion-exchange LC, size-
exclusion LC, slab electrophoresis, and capillary
electrophoresis (CE).

All protein determination methods described are

not absolute and demand some form of calibration.
The Kjeldahl’s method remains the only official
method currently available for calibration purposes
and maintains its position as the most frequently
used technique for the determination of organic nit-
rogen in food products. CE and immunochemical
(enzyme-linked immunosorbent assay) techniques
are most suitable for rapid separation and quantifi-
cation of individual food proteins and are promising
for widespread use in food protein analysis due to
their high sensitivity, specificity, and simplicity of
operation. There are numerous methods for the eval-
uation of the nutritional quality of food proteins,

which are already listed as determination of amino
acid composition and essential amino acids, and
enzymatic tests, microbiological methods, and bio-
logical measures.

Lipids

The analysis of lipids in food has three dis-

tinctive objectives: to determine (1) total lipid con-
tent, (2) the composition, and (3) the quality of
lipids.

1. Total lipid content. There are physical and

chemical procedures for oil/fat estimation. In phys-
ical procedures, lipids are not isolated and samples
are used directly, and lipids are estimated by nuclear
magnetic resonance (NMR) spectroscopy. In chem-
ical procedures, lipids are extracted by refluxing the
sample in suitable solvents (like petroleum ether)
by using standard methods (AOAC or AOCS).
Different classes of lipids such as neutral lipids
(by nonpolar solvents such as petroleum ether,
hexane), phospho- or glycolipids (by polar solvents
such as methanol) can be extracted and used for

Table 8

Methods of protein determination

Name of method

Principle used

Chemical methods
Biuret method

Based on binding of copper(II) to a

peptide bond in protein molecules
at alkaline pH values (measured
between 540 and 650 nm)

Lowry method

Based on reduction of Folin–

Ciocalteau reagent by oxidation
of tyrosine, tryptophan on
polypeptide side chain (measured
at 750 nm)

Dye binding

Based on the measurements of

excess dye remaining in solution
after removal of the precipitated
protein–dye complex

Physical methods
UV spectrophotometry

Based on absorption maximum at

280 nm, due to presence of a side
chain of aromatic amino acids
(Tyr and Try)

Fluorimetry

Based on emission of fluorescence

excited at 275 or 345 nm

IR reflectance

Based on characteristics

absorbance spectra at different
wavelengths

Refractometry

Based on change in refractive index

by the displacement of proteins

Turbidimetry

Based on change in intensity of light

due to protein diffusion

Immunological method

Based on interaction between an

antigen and its corresponding
antibody

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

209

quantitative determination. TLC, column chro-
matography, HPLC are specific techniques used for
separation of various classes of lipid.

2. Composition of oil/fat. To study the composi-

tion of oil/fat it is essential to test the purity of an oil/
fat for adulteration, accidentally or voluntarily. The
specific fatty acid in fat can be determined by GC by
preparing methyl esters with sodium methoxide.
Mass spectrometry coupled to GC (GC–MS) is the
most powerful tool for identification of fatty acids
separated by GC. Free fatty acids in oil (index of
rancidity) can be determined by titration against
standard alkali. Infrared (IR) spectroscopy, Raman
spectroscopy, and ultraviolet (UV) spectroscopy
(200–400 nm) are used to detect isomers (trans and
cis) of unsaturated fatty acids and conjugated double
bonds. It is important to study saponification value
(depict fatty acid chain length), iodine value (give the
degree of unsaturation), and hydroxyl value (free
fatty acids present in fats).

Ash and Mineral Components

The determination of ash/minerals in food contrib-
utes to assessment of a food’s nutritional value and
refers to elements and verifying if the food contains
some minerals in quantities dangerous to the health
of the consumer, whether their presence is natural or
adulteration of certain food stuffs/processed or
stored food products.

Ash content is a measure of total minerals and is a

reliable index of nutritional value for many foods
(tea, flour, edible gelatin, etc.) and feed (for poultry
and cattle) and is recognized as a useful tool in de-
termining the nature and distribution of mineral
constituents of food. Ash is the inorganic material
left after complete oxidation of organic material at
high temperatures (500–600

1C).

Ash is prepared by two direct procedures (dry and

wet ashing) and an indirect technique (conduct-
ometeric method that can determine the total
electrolyte content of foods), governed by the
following purpose: the particular constituents to be
determined and the method of analysis to be used.
Dry ashing is the standard method to determine ash
content of a sample (AOAC). For wet ashing the
sample is digested in a mixture of HNO

3

and

HCLO

4

(ratio 4:1).

Gravimetry (for sulfur), titrimetric (chloride), fluo-

rimetry (Se, Al, F), colorimetric methods, or atomic
spectroscopy are the traditional classical chemical
methods for the determination of individual ele-
ments. Atomic spectroscopy techniques have a much
higher sensitivity and specificity and provide a com-
plete profile of elements in a food/feed. Emission

spectrometry, polarography, X-ray spectrometry,
mass spectrometry, and activation analysis are im-
portant physical methods used for the determination
of minerals, and are suitable for determination of
alkaline metals, provide accurate and reproducible
results, and offers simultaneous determination of
numerous elements.

Other food components

It is also important to

analyze the toxic constituents/antinutritional factors,
which are present naturally in foods, produce a del-
eterious effect when ingested by humans or animals.
The toxic constituents/antinutritional factors, which
are present naturally in foods are flatous producing
factors, protease inhibitors, hemagglutinins (lectin),
and saponins present in legumes, and glucosinolates
(in oil seed crops), cyanogens (in bitter almond, lima
beans), gossypol (in cotton), and lathyrogen (seeds of
Lathyrus species). A large number of standard
(AOAC) analytical techniques are adopted to deter-
mine the food components such as bitter compounds
like anthocyanins, carotenoids (pigments), flavono-
ids, phenols, terpenoids, vitamins, aroma com-
pounds, alcohols, organic acids, antioxidants and
preservatives etc.

See also: Amino Acids. Carbohydrates: Overview.
Food and Nutritional Analysis: Sample Preparation;
Packaging Materials. Liquid Chromatography: Over-
view. Thin-Layer Chromatography: Overview.

Further Reading

AACC (1983) Approved Methods of Analysis, 8th edn. St.

Paul, MN: American Association of Cereal Chemists.

AOAC (1995) Official Methods of Analysis, 16th edn. As-

sociation of Official Analytical Chemists, Arlington,
Virgini, USA.

AOCS (1990) Official Methods of Recommended Practic-

es. Chicago, IL: American Oil Chemists Society.

Fennema OR (1996) Food Chemistry. New York: Dekker.
Gruenwedel DW and Whitakar JR (eds.) (1984) Food

Analysis: Principles and Techniques, vols 1–4. New
York: Dekker.

James CS (1994) Analytical Chemistry of Foods. London:

Blackie Academic and Professional.

Kulp K and Ponte JG Jr (2000) Handbook of Cereal Sci-

ence and Technology, 2nd edn. New York: Dekker.

Linden G (ed.) (1996) Analytical Techniques for Foods and

Agricultural Products. New York: VCH.

Multon JL (ed.) (1997) Analysis of Food Constituents.

New York: Wiley-VCH.

Nollet LML (ed.) (1996) Handbook of Food Analysis, vols

1–2. New York: Dekker.

Nollet LML (ed.) (2000) Food Analysis by HPLC, 2nd

edn. New York: Dekker.

210

FOOD AND NUTRITIONAL ANALYSIS

/ Overview

Oliveira FAR, Oliveira JC, Hendrickx ME, Korr D, and

Gorris LGM (eds.) (1999) Processing Food: Quality Op-
timization and Process Assessment. New York: CRC Press.

Poumeranz Y and Meloan CE (1994) Food Analysis: Theory

and Practice, 3rd edn. New York: Chapman and Hall.

Sorensen

(2000)

Chromatography

and

Capillary

Electrophoresis

in

Food

Analysis.

New

York:

Springer.

Wilson RH (ed.) (1994) Spectroscopic Techniques for Food

Analysis. New York: Wiley-VCH.

Sample Preparation

Kiyoshi Matsumoto

, Kyushu University, Fukuoka,

Japan
Hiroyuki Ukeda

, Kochi University, Nankoku, Japan

& 2005, Elsevier Ltd. All Rights Reserved.

Sample Preparation

In many respects, the sampling and preparation of a
sample are critical steps in any technique of food
analysis. The objective of sampling is to ensure that
the sample taken for analysis is representative of a
defined whole, and the method of sampling depends
on the size and nature of the defined whole.

Obtaining a sample for analysis that is represen-

tative of the whole is referred to as sampling, and
the total quantity from which a sample is obtained is
called the population. An accurate and precise esti-
mate of the quality of a population will only be ac-
hieved by using an adequate sampling technique
because only a portion of the population is used for
analysis. With a proper sampling technique, the es-
timated value will reflect the characteristics of the
population.

Special care, however, is required in the sampling

and sample preparation for food analysis because of
the peculiarities of food. Foods are derived from
biological substances and contain various organic
compounds. Many of these components are influ-
enced by various external and internal influences
such as temperature, light, moisture, microorgan-
isms, metabolism, and ripening. Therefore, rapid
preparation of food samples enough to prevent com-
position change is very important in food analysis.
Preparation at low temperatures during processing or
storage in the frozen state is also effective for preven-
ting the composition change of food samples.

In food analysis, analyses are generally performed

on the edible portion of the defined food, discarding
the nonedible portion, unless the compositions with
respect to the total sample weight are required. Var-
iations in moisture of food are relatively large, even
among the same kinds of food. Therefore, values

corrected on the basis of dry matter (values of an-
hydrous material) or values of moisture contents are
used in evaluating the analyzed value.

The general steps for sample preparation and sam-

ple processing are (1) sampling and size reduction, (2)
comminution and homogenization, (3) pretreatments
of the sample, such as predrying or defatting, (4) pre-
servation of the prepared sample, and (5) cleanup of
the analytical sample for instrumental analysis. This
article covers the practical considerations of sample
preparation necessary for food analysis.

Sampling and Size Reduction

It is important to define clearly the population that is
to be sampled. Populations are generally finite, such
as the size of a defined lot, except for evaluating a
process. The sampling methods selected depend on
the purpose of the inspection, the nature of the
product, the nature of the test method, and the na-
ture of the population being investigated. The basic
principle for sampling is probability sampling. This
sampling plan provides a statistically sound basis for
obtaining representative samples while eliminating
human bias and, therefore, is the most desirable. The
most useful sampling method is random sampling in
which samples are simply taken at random from the
whole population. The random sampling process al-
lows all parts of the population to have the same
chance of being sampled. The goal of providing the
same chance can be achieved by means of random
number tables or computer-generated random num-
bers. Sample size is determined by lot size, the degree
of accuracy required, and the expense of the test
method. However, a general standard for the number
to be sampled is proposed. For instance, to select the
sample flour from sacks, the number of sacks to be
sampled is determined by the square root of the
number of sacks in the lot.

If the mass of the collected samples is too large for

analysis, it is subjected to size reduction to obtain a
smaller quantity for analysis. For granular or pow-
dered material, such as cereals, pulses, nuts, and

FOOD AND NUTRITIONAL ANALYSIS

/ Sample Preparation

211