ANALYSIS OF FIBRE

ANALYSIS OF FIBERS

Importance of fibre determination in foods

1. Estimate the quantity of fiber in foods, especially celluloses

2. Estimate crude fiber in order to ensure that no deception in food and products due to
additives or other alternatives.

3. Estimated fiber in foods allows to know the quality and specifications for these foods.

4. Fiber estimate is considered a good indicator on the amount of food freshness like
vegetables, the increase in the age and maturity lead to the large increase in the percentage
of fiber in it.

5. Estimating the fiber and not the color and ash is a real indicator of purity of the flour.

Difference between crude fibre and dietary fibre

Crude fibre refers to the residue of a feed that is insoluble after successive, boiling with dilute acid
and alkali i.e. Crude fibre is the portion of the total carbohydrate of a food that is resistant to the acid
and alkali treatment.

Dietary fiber is defined as plant polysaccharides that are indigestible by humans, plus lignin. The
major components of dietary fiber are cellulose, hemicellulose, pectin, hydrocolloids and lignin.
Some types of starch, known as resistant starch, are also indigestible by human beings and may be
analyzed as dietary fiber.

Methods of analysis

Gravimetric Methods

Crude Fiber Method

The crude fiber method gives an estimate of indigestible fiber in foods. It is determined by sequential
extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH. The insoluble residue is collected
by filtration, dried, weighed and ashed to correct for mineral contamination of the fiber residue.
Crude fiber measures cellulose and lignin in the sample, but does not determine hemicelluloses,
pectins and hydrocolloids, because they are digested by the alkali and acid and are therefore not
collected. For this reason, many food scientists believe that its use should be discontinued.
Nevertheless, it is a fairly simple method to carry out and is the official AOAC method for a number
of different foodstuffs.

Total, insoluble and soluble fiber method

The basic principle of this method is to isolate the fraction of interest by selective precipitation and
then to determine its mass by weighing. A gelatinized sample of dry, defatted food is enzymatically
digested with a-amylase, amyloglucosidase and protease to break down the starch and protein
components. The total fiber content of the sample is determined by adding 95% ethanol to the
solution to precipitate all the fiber. The solution is then filtered and the fiber is collected, dried and
weighed. Alternatively, the water-soluble and water-insoluble fiber components can be determined
by filtering the enzymatically digested sample. This leaves the soluble fiber in the filtrate solution,
and the insoluble fiber trapped in the filter. The insoluble component is collected from the filter,
dried and weighed. The soluble component is precipitated from solution by adding 95% alcohol to
the filtrate, and is then collected by filtration, dried and weighed. The protein and ash content of the
various fractions are determined so as to correct for any of these substances which might remain in
the fiber: Fiber = residue weight - weight of (protein + ash).

This method has been officially sanctioned by the AOAC and is widely used in the food industry to
determine the fiber content of a variety of foods. Its main disadvantage is that it tends to
overestimate the fiber content of foods containing high concentrations of simple sugars, e.g., dried
fruits, possibly because they get trapped in the precipitates formed when the ethanol is added.

Chemical Methods

In chemical methods, the fiber content is equal to the sum of all non-starch monosaccharides plus
lignin remaining once all the digestible carbohydrates have been removed. Monosaccharides are
measured using the various methods described previously.

Englyst-Cummings Procedure

A defatted food sample is heated in water to gelatinize the starch. Enzymes are then added to digest
the starch and proteins. Pure ethanol is added to the solution to precipitate the fiber, which is
separated from the digest by centrifugation, and is then washed and dried. The fiber is then
hydrolyzed using a concentrated sulfuric acid solution to break it down into its constituent
monosaccharides, whose concentration is determined using the methods described previously, e.g.,
colorimetrically or chromatographically.

The mass of fiber in the original sample is assumed to be equal to the total mass of monosaccharides
present. The concentration of insoluble and soluble dietary fiber can also be determined by this
method, using similar separation steps as for the total, insoluble and soluble gravimetric method
mentioned above.

This method can be used to determine the total, soluble and insoluble fiber contents of foods, but
does not provide information about the lignin content. This is because lignin is not a polysaccharide,
and so it is not broken down to monosaccharides during the acid digestion. For most foods this is not
a problem because they have low lignin concentrations anyway. If a food does contain significant
amounts of lignin then another method should be used, e.g., the gravimetric method or more
sophisticated chemical methods (e.g., the Theander-Marlett method).

Analysis of Ash and Minerals

Determination of the ash and mineral content of foods is important for a number of reasons:

 Nutritional labeling. The concentration and type of minerals present must often be stipulated
on the label of a food.

 Quality. The quality of many foods depends on the concentration and type of minerals they
contain, including their taste, appearance, texture and stability.
  Microbiological stability. High mineral contents are sometimes used to retard the growth of
certain microorganisms.

 Nutrition. Some minerals are essential to a healthy diet (e.g., calcium, phosphorous,
potassium and sodium) whereas others can be toxic (e.g., lead, mercury, cadmium and
aluminum).

 Processing. It is often important to know the mineral content of foods during processing
because this affects the physicochemical properties of foods.

Determination of Ash Content

Ash is the inorganic residue remaining after the water and organic matter have been removed by
heating in the presence of oxidizing agents, which provides a measure of the total amount of
minerals within a food. Analytical techniques for providing information about the total mineral
content are based on the fact that the minerals (the analyte) can be distinguished from all the other
components (the matrix) within a food in some measurable way. The most widely used methods are
based on the fact that minerals are not destroyed by heating, and that they have a low volatility
compared to other food components.

The three main types of analytical procedure used to determine the ash content of foods are based
on this principle: dry ashing, wet ashing and low temperature plasma dry ashing.

The method chosen for a particular analysis depends on the reason for carrying out the analysis, the
type of food analyzed and the equipment available. Ashing may also be used as the first step in
preparing samples for analysis of specific minerals, by atomic spectroscopy or the various traditional
methods described below. Ash contents of fresh foods rarely exceed 5%, although some processed
foods can have ash contents as high as 12%, e.g., dried beef.

Sample Preparation

As with all food analysis procedures it is crucial to carefully select a sample whose composition
represents that of the food being analyzed and to ensure that its composition does not change
significantly prior to analysis. Typically, samples of 1-10g are used in the analysis of ash content. Solid
foods are finely ground and then carefully mixed to facilitate the choice of a representative sample.
Before carrying out an ash analysis, samples that are high in moisture are often dried to prevent
spattering during ashing. High fat samples are usually defatted by solvent extraction, as this
facilitates the release of the moisture and prevents spattering. Other possible problems include
contamination of samples by minerals in grinders, glassware or crucibles which come into contact
with the sample during the analysis. For the same reason, it is recommended to use deionized water
when preparing samples.

i. Dry Ashing

Dry ashing procedures use a high temperature muffle furnace capable of maintaining temperatures
of between 500 and 600 oC. Water and other volatile materials are vaporized and organic substances
are burned in the presence of the oxygen in air to CO2, H2O and N2. Most minerals are converted to
oxides, sulfates, phosphates, chlorides or silicates. Although most minerals have fairly low volatility at
these high temperatures, some are volatile and may be partially lost, e.g., iron, lead and mercury. If
an analysis is being carried out to determine the concentration of one of these substances, then it is
advisable to use an alternative ashing method that uses lower temperatures.
The food sample is weighed before and after ashing to determine the concentration of ash present.
The ash content can be expressed on either a dry or wet basis:

% Ash (dry basis) = Mash x 100

Mdry

% Ash (wet basis) = Mash x 100

Mwet

where MASH refers to the mass of the ashed sample, and MDRY and MASH refer to the original masses of
the dried and wet samples.

There are a number of different types of crucible available for ashing food samples, including quartz,
Pyrex, porcelain, steel and platinum. Selection of an appropriate crucible depends on the sample
being analyzed and the furnace temperature used. The most widely used crucibles are made from
porcelain because it is relatively inexpensive to purchase, can be used up to high temperatures (<
1200oC) and are easy to clean. Porcelain crucibles are resistant to acids but can be corroded by
alkaline samples, and therefore different types of crucible should be used to analyze this type of
sample. In addition, porcelain crucibles are prone to cracking if they experience rapid temperature
changes. A number of dry ashing methods have been officially recognized for the determination of
the ash content of various foods (AOAC Official Methods of Analysis). Typically, a sample is held at
500-600 oC for 24 hours.

Advantages:

1. Safe,

2. Few reagents are required,

3. Many samples can be analyzed simultaneously,

4. It is not labor intensive, and

5. Ash can be analyzed for specific mineral content.

Disadvantages:

1. Long time required (12-24 hours),

2. Muffle furnaces are quite costly to run due to electrical costs,

3. Loss of volatile minerals at high temperatures, e.g., Cu, Fe, Pb, Hg, Ni, Zn.

Wet Ashing

Wet ashing is primarily used in the preparation of samples for subsequent analysis of specific
minerals (see later). It breaks down and removes the organic matrix surrounding the minerals so that
they are left in an aqueous solution. A dried ground food sample is usually weighed into a flask
containing strong acids and oxidizing agents (e.g., nitric, perchloric and/or sulfuric acids) and then
heated. Heating is continued until the organic matter is completely digested, leaving only the mineral
oxides in solution. The temperature and time used depends on the type of acids and oxidizing agents
used. Typically, a digestion takes from 10 minutes to a few hours at temperatures of about 350oC. The
resulting solution can then be analyzed for specific minerals.

Advantages:

1. Little loss of volatile minerals occurs because of the lower temperatures used,

2. It is more rapid than dry ashing.

Disadvantages

1. Labor intensive,

2. It requires a special fume-cupboard if perchloric acid is used because of its hazardous nature,

3. Low sample throughput.

Low Temperature Plasma Ashing

A sample is placed into a glass chamber which is evacuated using a vacuum pump. A small amount of
oxygen is pumped into the chamber and broken down to nascent oxygen (O2 � 2O.) by application of
an electromagnetic radio frequency field. The organic matter in the sample is rapidly oxidized by the
nascent oxygen and the moisture is evaporated because of the elevated temperatures. The relatively
cool temperatures (< 150oC) used in low-temperature plasma ashing cause less loss of volatile
minerals than other methods.

 Advantages: Less chance of losing trace elements by volatilization

Disadvantages: Relatively expensive equipment and small sample throughput

Significance of Ash fractions

Total ash: Determined by weighing the dry mineral residue of organic materials heated at elevated
temp (550°C). Total ash content is a useful parameter of the nutritional value of some foods and
feeds.

Water-soluble ash: An index of the fruit content of jelly and fruit preserve.

Acid-insoluble ash: An index of mineral matter (dirt or sand) and determined after digesting the total
ash in 10% HCl.

Comparison of Ashing Methods

The conventional dry ashing procedure is simple to carry out, is not labor intensive, requires no
expensive chemicals and can be used to analyze many samples simultaneously. Nevertheless, the
procedure is time-consuming and volatile minerals may be lost at the high temperatures used.
Microwave instruments are capable of speeding up the process of dry ashing. Wet ashing and low
temperature plasma ashing are more rapid and cause less loss of volatile minerals because samples
are heated to lower temperatures. Nevertheless, the wet ashing procedure requires the use of
hazardous chemicals and is labor intensive, while the plasma method requires expensive equipment
and has a low sample throughput.