NS170

(v.2.0)

EVALUATION OF ENERGY VALUES OF FOOD USING BOMB

CALORIMETRY

1.PRINCIPLE

Classification of Energy Systems

The energy value of a food can be expressed in a variety of ways, ranging from
the most easily determined measure, gross energy, to the net energy value. In practice,
gross energy is not used in evaluating foods. The various systems can be divided into two
classes according to whether the criterion used is, on the one hand, digestible or
metabolizable energy or, on the other hand, net energy.

Although the metabolizable energy value of a food is reasonably constant for a

particular species of an animal, the efficiency with which that metabolizable energy is
utilized varies according to the purpose for which it is required by the animal, so that net
energy values vary accordingly. This has led to modifications in net energy systems, and
in some cases to their rejection. The present position is that systems based on digestible
or metabolizable energy are used almost universally for pigs and poultry, and also for
ruminants in the Western Hemisphere and the U.S.S.R., while net energy systems are
preferred for ruminants in Europe (including the United Kingdom).

A difficulty which applies in some measure to all systems, but particularly to

those based on net energy, is that the evaluation of a food is a laborious and complicated
procedure. For this reason an essential feature of most systems is a method for predicting
energy value from some more easily measured characteristic of the food, such as its gross
or digestible composition.

Net energy values of foods for poultry were determined in the United States by
Fraps (1), who used the method of comparative slaughter to measure energy retention in
young chickens. The figures obtained were called productive energy values, in order to
emphasize that they were net energy values for growth, not maintenance. Fraps also
devised methods for predicting the productive energy values of foods from their gross or
digestible nutrients.

In both the United Kingdom and the U.S.A. it has been decided that the energy
value of poultry foods should be expressed as metabolizable energy. Metabolizable
energy is very easily measured in poultry--it is in fact easier to determine than digestible
energy, because feces and urine are voided together. In addition, there are reliable
methods for predicting the metabolizable energy of the more common poultry foods from
their chemical composition.

1
Utilization of Metabolizable Energy

a) Maintenance

For maintenance purposes, the animal oxidizes the nutrients absorbed from its
food principally to provide energy for work. If it is given no food, it obtains this energy
mainly by the oxidation of body fat. When food is given, in quantities insufficient to
provide all the energy needed for maintenance, the task of providing ATP is partially
transferred from the reserves of body fat to the nutrients absorbed. If the energy
contained in these nutrients can be transferred to ATP as efficiently as can that in body
fat, no extra heat will be produced by the animal apart from that associated specifically
with the consumption, digestion and absorption of food.

b) Productive Purposes

Although energy may be stored by animals in a wide variety of products--in body

fat, muscle, milk, eggs and wool--the energy of these products is contained mainly in fat
and protein (only in milk is much energy stored as carbohydrate). The efficiency with
which metabolizable energy is used for productive purposes therefore depends largely on
the energetic efficiency of the metabolic pathways involved in the synthesis of fat and
protein from absorbed nutrients. In general, the synthesis of either fat or protein is a
more complicated process than its catabolism. Not only must the building materials be
present in the right proportions, but they must arrive on the scene at the right time, and
the absence of a particular material may prevent or seriously impair the whole process.
For example, fatty acid synthesis is dependent on a supply of NADPH. Because of the
greater complexity of synthetic processes, it is more difficult to estimate their theoretical
efficiency.

Measurement of Food Calories

The potential energy value of foods and the energy exchanges of the body are
expressed in terms of the calorie, which is a heat unit. By definition, the large Calorie, or
kilocalorie (kcal), is the amount of heat required to raise the temperature of 1 kg water
1oC (from 15o to 16o). This unit is always used in nutrition and is 1000 times as large as
the small calorie used in chemistry and physics. Some attempts have been made to use
the term kilocalorie in writings on nutrition, but most of the nutrition literature continues
to use the shortened form calorie.

The fuel value of foods is readily determined by means of an apparatus known as

a bomb calorimeter (see Figure 2). A weighed sample of food is placed in a heavy steel
container called a "bomb". After the bomb is charged with oxygen, the sample is ignited
and the heat is dissipated into a known volume of water surrounding the bomb. By
noting the change in the temperature of the water, one can calculate the energy value of
the food by applying the definition for a calorie.
 The caloric values of foods burned in the bomb calorimeter are somewhat higher
than those realized in the body. First, certain small losses occur in digestion for all three
nutrients. For the typical American diet, 98% of the carbohydrate, 95% of the fat and
92% of the protein are digested and absorbed. The carbohydrates and fats which are
absorbed are entirely oxidized in the body as they are in the bomb calorimeter. In the
case of proteins, however, the end products of metabolism are partly eliminated as urea
and other organic nitrogen compounds which are combustible. In other words, the body
loses part of the potential energy value of protein so that the net value is less than that
measured by the calorimeter.

The "physiologic full values" proposed by Atwater are based upon the corrections
for losses of unabsorbed nutrients in the feces and for the caloric equivalent of the
nitrogenous products in the urine. From a large number of determinations, Atwater
derived the well-known physiologic fuel values: Protein = 4; fat = 9 and carbohydrate =
4 Calories per gram.

These values were approximate only, but they served reasonably well when
applied to foods in the average American diet. This method of calculating the calorie
value of individual foods had been used since 1900, although the inaccuracies were
recognized. In the Agriculture Handbook No. 8 published by the U.S. Department of
Agriculture in 1963 (2), calorie values are calculated by a modification of the procedure
that had been in use for over 60 years. Instead of applying the general caloric factors 4, 9
and 4 to the percentage composition of food, as had been done previously, more specific
factors were used. These new factors take into consideration further research data on the
fuel values of foods.
University of California,
BerkeleyNutritional Sciences 170
(ver 2.2)
Rat Experiment: Bomb Calorimetry

Our purpose in using a bomb calorimeter in this experiment is to determine the

digestible energy (DE) value of each of the experimental diets so that we can relate the
'digestible energy' consumed by the rats to their performance.

The bomb calorimeter combusts test samples completely in an atmosphere of pure

oxygen. The end-products of the combustion are mainly CO2, H2O, NO2, SO2. The heat
liberated in this way gives us the 'gross energy' (G.E.) or 'heat of combustion' of a
sample. This differs from the heat released from the metabolism of foods in the body
when they are used as a source of energy in two ways.

1.Although fats and carbohydrates are metabolized to the same end products (CO2
and H2O) in the body as in the bomb and therefore yield the same energy, protein
has different end-products. Nitrogen in proteins metabolized for energy in the
body can only be converted to, and excreted as, urea (plus a little uric acid). This
means that there is a lower release of energy than in the bomb. We could make a
small correction for this, but our diets are adjusted to be of equivalent protein
content, and we will not do so on this occasion.

2.Some of the food eaten is never absorbed, and therefore not available for
metabolism. Obviously, the energy value of this fraction should not be 'counted'
as being available to the rats. We can estimate this by measuring the gross energy
of the rats' feces and calculating what weight of feces are produced 'per g of food
fed'.

Bomb calorimeters are of two types. In the classical instrument one obtained an
absolute measure of the heat produced from the rise in temperature of a known mass of
water in which the bomb is submerged. The bomb which we will use is called a 'ballistic'
instrument. It is quicker to use and has sufficient accuracy for our purpose. Its use
depends on an empirically determined relationship between the heat liberated on
combustion of a sample and the rise in temperature (following the combustion) of the
casing on the bomb, as measured by a thermocouple embedded in it.

From a standard obtained by burning pure compounds with a known gross energy
value, we can plot 'known calories released' against 'temperature rise'. 'Temperature rise'
is not read in degrees by in a galvanometer reading traced on moving paper, but the units
do not matter so long as 'standards' and 'test materials' are being compared on the same
scale.
Equipment:

The following items are necessary for the use of the ballistic bomb calorimeter:

1.Pellet press for preparation of granular or powdered samples into pellets.

2.Balance which will weigh to the nearest 1 mg.

3.Stainless steel crucibles (oven-dried) for holding samples in

calorimeter. 4.Forceps for handling crucibles and pellets.

5.Ballistic bomb calorimeter (made by Parr Instrument Company, Mcline, IL).

6.Fine cotton thread (00 gauge) to act as a fuse.

7.Recorder with moving chart paper, to which the thermocouple can be connected
so that the temperature change which occurs at the top of the bomb can be
permanently recorded.

8.Transformer with timer to provide current for ignition.

Preparation of Samples

Samples (approximately 1 g) are made into pellets to prevent spraying of the

sample throughout the calorimeter during the influx of oxygen. Such spreading of the
sample may lead to incomplete combustion. Pellet making is accomplished with the aid
of a pellet press, which consists of a mold into which the sample is placed, and a pestle
which is then pushed into the mold, crushing the sample material into a compact pellet.

1.Weigh approximately 1 g of sample.

2.Transfer it into the mold to make the pellet.
3.Very carefully remove the pellet from the press.
4.Weigh an oven-dried crucible. Note its number.
5.Immediately put the pellet into the crucible. Weigh.
6.Store in covered container.

Combustion

The crucible containing the sample is placed inside the calorimeter on a pedestal
and a platinum filament. A length of cotton thread is tied onto the filament, and its ends
placed into the crucible so that it will act as a fuse (the length of thread used at each
burning is the same so that the heat of combustion of the thread will be constant and will
thus drop out of calculation).
 The chamber is then closed, and the thermocouple put in place in the top of the
calorimeter. The chamber is filled with oxygen to a pressure of 400 lb/in3, and a current
run through the filament for 35 seconds, thus initiating the combustion of the sample.

Because of the obvious danger if the bomb is not correctly assembled, this
must always be the responsibility of a member of the staff in charge of this portion of the
practical work. (Do NOT attempt it yourself!)

The change in temperature of the top of the bomb is measured by the

thermocouple and recorded on chart paper. The chart paper is divided into 100 equal
units, and the number of units of rise (from baseline to peak temperature increase) is
recorded.

Final Actions

Once the sample has been burned and the maximum peak height determined, the
capsule is opened to the air through a small valve, allowing for equalization of the
internal and external pressures. Then, the chamber is opened and the crucible and interior
of the bomb inspected. You should expect to see condensed moisture on the walls and a
pellet of fused minerals in the crucible (if they were present in the sample). If unburned
organic material is seen, the determination must be repeated.

The top of the combustion chamber is then cooled with running water. Once
dried, it is then ready for the next burning.

Calculations

1.The common standard material is benzoic acid. It has a GE of 6.318 kcal/g and
is pure, cheap and non-hygroscopic. The calories given out by combustion of
unknown samples are calculated using the temperature rise of the standard.
Calculate the heat of combustion per gram of test material.

2.To calculate the digestible energy value of a diet, calculate the weight of dry
feces produced per g food eaten, and then work out how much indigestible gross
energy this corresponds to.

Example:

Imagine that your diet has a GE of 4.4 kcal/g and that during the period in which
you collect 6 g dry feces (which give a GE of 4.0 kcal/g), the rats in the cage have eaten
48 g food.

We can then say that if 48 g food gives rise to 6 g dry feces, then 1 g food gives
rise to 6 ÷ 48 = 0.125 g dry feces. This might contain 0.125 x 4.0 = 0.5 g GE. Therefore,
1 g food has 4.4 kcal GE of which 0.5 kcal is apparently indigestible (i.e. reappears in the
feces). By difference, the DE value of food is 3.9 kcal/g.
PELLET MAKING WITH A PARR PELLET PRESS

The steps required to produce pellets or tablets with a Parr Pellet Press have been
reduced and simplified by eliminating the die plug and receiving cup used in earlier
models. There are only two loose parts in this improved design: a polished stainless
steel die and a die holder with working surfaces on both ends. This new die
arrangement is an extension of a design previously used only for 1/8" and decimal size
pellets. All dies for the Parr press are now made to this design which will produce
pellets in four simple steps as described below.

Fill the die. Set the die and its holder on the base of the press with the beveled edge of
the die cavity facing upward and with the bottom of the die resting on
the flat surface in the reversible holder. Pour the charge into the die
cavity and tamp with a stirring rod, if necessary.

Compress the charge. Transfer the die and its holder to the press and push the lever
down to compress the charge. To obtain maximum compression, the
lever should require a firm push as it moves through its full stroke. If a
full stroke is not obtained, turn the anvil to lower the die until the full
mechanical advantage of the press can be utilized. Conversely, if the
lever moves through its full stroke without encountering sufficient
resistance, raise the die until firm compression can be applied.

Reverse the die holder. Raise the level and slide the die and its holder out of the press.
Reverse the holder to bring the deep cavity under the die and return the
parts to their original position. The clearance under the punch will be
limited when making thin pellets. In such cases it will be more
convenient to grasp the die with one hand and slide it upward on the
punch, holding it in that position while reversing the die holder with the
other hand.

Eject the pellet. Bring the lever down gently to eject the pellet into the cavity in the
holder. If a thick pellet is not ejected by this stroke, turn the anvil to
raise the die. The pellet will then drop out freely. Remove the pellet with
tweezers or forceps; reverse the holder and repeat the cycle if additional
pellets are required.
CHARGING THE BOMB

Allowable Sample Size. Care must be taken to avoid overcharging the bomb for it must be realized that
the peak pressure developed during a combustion is proportional to the size of the sample and to the
initial oxygen pressure. To stay within safe limits, the bomb should never be charged with a sample
which will release more than 8000 calories when burned in oxygen, and the initial oxygen pressure
should never exceed 35 atmospheres (515 psig.). This generally limits the mass of the combustible
charge (sample plus benzoic acid, gelatin, firing oil or any combustion aid) to not more than 1.1 grams.
When starting tests with new or unfamiliar materials it is always best to use samples of less than one
gram, with the possibility of increasing the amount if preliminary tests indicate no abnormal behavior. To
avoid damage to the bomb and possible injury to the operator, it should be a standing rule in each
laboratory that the bomb must never be charged with more than 1.5 grams of combustible material.

Attaching the Fuse. Set the bomb on the A38A2 support stand and attach a 10 cm. length of fuse wire as
shown on the following page. Parr 45C10 nickel alloy wire will be used for most tests, but the same
procedure applies when using 36 ga. or finer platinum wire. In either case, a pair of forceps or tweezers
will be helpful for binding the wire to the electrodes. First, fasten one end of the wire to the loop
electrode (steps e through h). Pull the loop downward to tighten the connections; then bend the wire
upward as in detail i. Place the capsule in the loop holder and bend the wire down to touch the surface of
the charge as shown in detail j.

It is not necessary to submerge the wire in a powdered sample. In fact, better combustions will usually be
obtained if the loop of the fuse is set slightly above the surface. When using pelleted samples, bend the
wire so that the loop bears against the top of the pellet firmly enough to keep it from sliding against the
side of the capsule. It is also good practice to tilt the capsule slightly to one side so that the flame
emerging from it will not impinge directly on the tip of the straight electrode.

Water in the Bomb. Place 1.0 ml. of distilled water in the bomb from a pipet. Larger amounts of water
or alkaline solution will be required when using the bomb for sulfur or other determinations. The
required amounts are specified in each procedure.

Closing the Bomb. Care must be taken not to disturb the sample when moving the bomb head from the
support stand to the bomb cylinder. Be sure that the 104A3 contact ring is in place above the sealing ring
and that the sealing ring is in good condition; then slide the head into the cylinder and push it down as far
as it will go. It will be convenient (but not essential) to hold the bomb in an A124A2 bench clamp during
the closing operation and while filling the bomb with oxygen. Set the screw cap on the cylinder and turn
it down firmly by hand. Do not use a wrench or spanner on the cap. Hand tightening should be sufficient
to secure a tight seal.

Oxygen Filling Equipment. Commercial oxygen as supplied in a standard 1A cylinder with CGA No.
540 outlet is usually of sufficient purity for calorimetric work. Connections to the oxygen cylinder are
made with a Parr 1825 filling connection which is supplied by the calorimeter. This connection has a
needle valve which controls the flow into the bomb and a 0-55 atm. gage which shows the pressure to
which the bomb has been charged. It also has an automatic relief valve to prevent overcharging. To
attach the filling connection, unscrew the protecting cap from the oxygen tank and inspect the threads on
the valve outlet to be sure they are clean and in good condition. Place the ball end of the connection into
the cylinder outlet and draw up the union nut tightly with a wrench, keeping the 0-55 atm. gate in an
upright position.

Filling the Bomb. Press the fitting on the end of the oxygen hose into the inlet valve socket and turn the
knurled union nut finger tight. Close the control valve on the filling connection; then open or "crack" the
oxygen tank valve not more then one-quarter turn. Open the filling connection control valve slowly and
watch the gage as the bomb pressure rises to the desired filling pressure (usually not more than 30 atm.);
then close the control valve. The bomb inlet check valve will close automatically when the oxygen
supply is shut off, leaving the bomb filled to the highest pressure indicated on the 0-55 atm. gage.
Release the residual pressure in the connecting hose by pushing downward on the lever attached to the
relief valve. The gage should now return to zero. If the pressure drops slowly and a large amount of gas
escapes when the release valve is opened, the check valve in the bomb head is not operating properly.
This trouble will have to be corrected before the bomb can be used.