Proximate analysis of foods

This system of analysis divides the food into six fractions: moisture, ash, crude protein,
ether extract, crude fibre and nitrogen-free extractives.
The moisture content is determined as the loss in weight that results from drying a
known weight of food to constant weight at 100 °C.This method is satisfactory for most
foods, but with a few, such as silage, significant losses of volatile material (short-chain
fatty acids and alcohols) may take place.Therefore, for silages, the moisture content can
be determined directly by distilling the water from the sample under toluene.The distillate
is measured and corrected for the presence of fermentation acids and alcohols.
The ash content is determined by ignition of a known weight of the food at 550 °C
until all carbon has been removed. The residue is the ash and is taken to represent
the inorganic constituents of the food.The major component of ash is silica but ash
may, however, contain material of organic origin such as sulphur and phosphorus
from proteins, and some loss of volatile material in the form of sodium, chloride,
potassium, phosphorus and sulphur will take place during ignition.The ash content
is thus not truly representative of the inorganic material in the food either qualitatively
or quantitatively. Animals do not have a requirement for ash per se but require
the individual mineral elements that it contains and are determined by
methods such as atomic absorption spectrometry (see p. 12).
The crude protein (CP) content is calculated from the nitrogen content of the food,
determined by a modification of a technique originally devised by Kjeldahl over
100 years ago. In this method the food is digested with sulphuric acid, which converts
to ammonia all nitrogen present except that in the form of nitrate and nitrite. This
ammonia is liberated by adding sodium hydroxide to the digest, distilled off and
collected in standard acid, the quantity so collected being determined by titration or
by an automated colorimetric method. It is assumed that the nitrogen is derived from
protein containing 16 per cent nitrogen, and by multiplying the nitrogen figure by 6.25
(i.e. 100/16) an approximate protein value is obtained.This is not ‘true protein’ since
the method determines nitrogen from sources other than protein, such as free amino
acids, amines and nucleic acids, and the fraction is therefore designated crude protein.
The ether extract (EE) fraction is determined by subjecting the food to a continuous
extraction with petroleum ether for a defined period.The residue, after evaporation
of the solvent, is the ether extract. As well as lipids it contains organic acids,
alcohol and pigments.This procedure is referred to as method A. In the current official
method, the extraction with ether is preceded by hydrolysis of the sample with sulphuric
acid and the resultant residue is the acid ether extract (method B).
The carbohydrate of the food is contained in two fractions, the crude fibre (CF)
and the nitrogen-free extractives (NFE).The former is determined by subjecting the
residual food from ether extraction to successive treatments with boiling acid and
alkali of defined concentration; the organic residue is the crude fibre.
When the sum of the amounts of moisture, ash, crude protein, ether extract and
crude fibre (expressed in g/kg) is subtracted from 1000, the difference is designated the
nitrogen-free extractives.The nitrogen-free extractives fraction is a heterogeneous mixture
of all those components not determined in the other fractions. The crude fibre
fraction contains cellulose, lignin and hemicelluloses, but not necessarily the whole
Starch and sugars
Inadequacies in the nitrogen-free extractives fraction have been addressed by the
development of methods to quantify the non-structural carbohydrates, which are
mainly starches and sugars. Sugars can be determined colorimetrically after combination
with a reagent such as anthrone. Starch is determined by dilute acid hydrolysis
of the sample followed by polarimetric determination of the released sugars.
This gives a figure for total sugars (i.e. those originating from the hydrolysed starch
plus the simple sugars in the food). Sugars per se are determined by extracting the
sample with ethanol, acidifying the filtrate and taking a second polarimeter reading.
The starch content is calculated from the difference between the two readings
multiplied by a known factor for the starch source. Starch can also be determined
enzymically. For example, in cereals starch is converted to glucose using -amylase
followed by amyloglucosidase and then the glucose is measured using the glucose
oxidase-peroxidase reagent.
Fibre
Alternative procedures for fibre have been developed by Van Soest (Table 1.2). The
neutral-detergent fibre (NDF), which is the residue after extraction with boiling neutral
solutions of sodium lauryl sulphate and ethylenediamine tetraacetic acid (EDTA),
consists mainly of lignin, cellulose and hemicellulose and can be regarded as a measure
of the plant cell wall material.The analytical method for determining NDF was
originally devised for forages, but it can also be used for starch-containing foods provided
that an amylase treatment is included in the procedure. By analogy with the
nitrogen-free extractives fraction discussed above, the term non-structural carbohydrate
(NSC) is sometimes used for the fraction obtained by subtracting the sum of
the amounts (g/kg) of CP, EE, ash and NDF from 1000.
The acid-detergent fibre (ADF) is the residue after refluxing with 0.5 M sulphuric
acid and cetyltrimethyl-ammonium bromide, and represents the crude lignin and
cellulose fractions of plant material but also includes silica.
The determination of ADF is particularly useful for forages as there is a good statistical
correlation between it and the extent to which the food is digested (digestibility).
In the UK the ADF method has been modified slightly, the duration of boiling
and acid strength being increased.The term modified acid-detergent fibre (MADF) is
used to describe this determination.
The acid-detergent lignin determination involves the preparation of aciddetergent
fibre as the preparatory step. The ADF is treated with 72 per cent sulphuric
acid, which dissolves cellulose. Ashing the residue determines crude lignin,
including cutin.
The Van Soest methods of fibre analysis are used in the system of food analysis
for ruminants developed at Cornell University (see Box 1.1).
In monogastric, and particularly human, nutrition the term dietary fibre is often
used and attention has been focused on its importance in relation to health. Dietary
fibre (DF) was defined as lignin plus those polysaccharides that cannot be digested
by monogastric endogenous enzymes. Initially epidemiological studies linked a lack
of DF to constipation, gut and bowel disorders, cardiovascular disease and type 2
diabetes; however, the causes of such diseases are multifactorial and in some cases
it is not just DF per se that has the beneficial effects but other aspects of the diet
also (e.g. antioxidants). Nevertheless, DF is a major component related to health in
humans and it has equally important effects in animals (see below).
The definition of DF has proved difficult, with definitions ranging through
physiological/botanical (derived from cell walls of plants, which are poorly digested);
chemical/botanical (non-starch polysaccharides (NSP) of plant cell walls); chemical
(NSP and lignin); and nutritional/physiological (NSP not digested in the small
intestine).The common features of DF definitions are carbohydrates (polysaccharides,
oligosaccharides and lignin) resistant to digestion in the small intestine but
that may be fermented in the large intestine and promote beneficial physiological
effects. By virtue of its definition, DF is difficult to determine in the laboratory.
The NSP in most foods, along with lignin, are considered to represent the
major components of cell walls. Methods for measurement of NSP fall into two
categories (with slight variations in the second category, depending on the research
laboratory):
■ Enzymic–gravimetric methods, which measure a variety of components and give
no details of polysaccharide type. In the method of the Association of Official Analytical
Chemists for total dietary fibre, samples are gelatinised by heating and
treated with enzymes to remove starch and proteins.The total dietary fibre is precipitated
with ethanol and the residue is dried and weighed.
■ Enzymic–chromatographic methods, which identify the individual carbohydrates
in the dietary NSP. The Englyst method can be used to determine total,
soluble and insoluble dietary fibre. Measurement of NSP by this method involves
removal of starch with the enzymes pullulanase and -amylase. After
precipitation with ethanol, the NSP residue is then hydrolysed with 12 M sulphuric
acid. The individual monomeric neutral sugar constituents are determined
by gas–liquid chromatography (see below) with separate determination
of uronic acids. Alternatively, the total sugars are determined colorimetrically
after reaction with dinitrosalicylate solution. Total NSP and insoluble NSP are
determined directly by analysis of separate subsamples and the soluble NSP are
calculated by difference. The major constituents of NSP are rhamnose, arabinose,
xylose, glucose, galactose, mannose and glucuronic and galacturonic acids.
Cellulose is the major source of glucose, and hemicellulose provides xylose,
mannans and galactose.The degradation of pectins releases arabinose, galactose
and uronic acids. Following the adoption of methods to determine NSP, it became
apparent that non-digestible oligosaccharides and resistant starch also
contributed to DF based on their physiological behaviour. In recognition of this,
enzymic procedures have been developed to determine these components. A
comparison of the dietary fibre contents for a range of food types .

In recent years attention has focused on the importance of both the soluble and
insoluble forms of fibrous material in the human diet.Water-soluble NSP is known to
lower serum cholesterol, and insoluble NSP increases faecal bulk and speeds up the
rate of colonic transit.This last effect is thought to be beneficial in preventing a number
of diseases, including cancer of the bowel.
The NSP of foods may be degraded in the gut of pigs by microbial fermentation,
yielding volatile fatty acids, which are absorbed and contribute to the energy supply.
A further benefit relates to the volatile fatty acid butyric acid, which is reported
to be an important source of energy for the growth of cells in the epithelium of the
colon; thus, the presence of this acid will promote development of the cells and enhance
absorption. The extent of degradation depends on the conformation of the
polymers and their structural association with non-carbohydrate components, such
as lignin. In addition, the physical properties of the NSP, such as water-holding capacity
and ion exchange properties, can influence the extent of fermentation. The
gel-forming NSPs, such as -glucan, reduce the absorption of other nutrients from
the small intestine and depress digestibility and adversely affect faecal consistency
in pigs and poultry. On a positive note, the water-holding properties lead to beneficial
effects on the behaviour of pregnant sows by increasing time spent eating and
resting owing to increased gut fill and by reducing inappropriate behaviour, such as
bar chewing.