Protein quality determination in

monogastric animals

Vishnu Vardhan Reddy.PTVM/2015-029

Department of Animal nutritionCollege of Veterinary Science, TirupatiSri Venkateswara Veterinary University

Protein quality estimation in monogastric animals • Protein is mainly required in monogastric animals for building of body

tissue and for maintenance.

• Unlike ruminants all essential amino acids should come from food.

• To meet the protein requirements of the animals usually depend on the

digestible protein quantity of the feed but digestible protein figures are

not entirely satisfactory measures of the value of a protein

to an animal.

• Because the efficiency with which the absorbed protein is used differs

considerably from one source to another as the feed contain different sources

of protein.

• In order to allow for such differences, methods for evaluating proteins, such as

• The protein efficiency ratio (PER),

• The net protein retention (NPR) and

• The gross protein value (GPV),

• Which are based on the growth response of experimental animals to the

protein under consideration, have been devised.

Protein efficiency ratio

• This is defined as follows:

PER =

The rat is the usual experimental animal.

Net protein retention

• This is calculated as follows:

NPR =

where TPG = group given the test protein and

NPG = group of protein-free diet.

Gross protein value

• The live weight gains of chicks receiving a basal diet containing 80 g crude protein/kg

are compared with those of chicks receiving the basal diet plus 30 g/kg of a test

protein, and of yet others receiving the basal diet plus 30 g/kg of casein.

• The extra live weight gain per unit of supplementary test protein stated as a

proportion of the extra live weight gain per unit of supplementary casein is the GPV

of the test protein, i.e.:

GPV = A/A°

• where A = g increased weight gain/g test protein and A° = g increased weight gain/g

casein.

Nitrogen balance

• Live weight gains may not be related to protein stored, and a more

accurate evaluation of a protein may be obtained by using the results of

nitrogen balance experiments. In such experiments, the nitrogen

consumed in the food is measured, together with that voided in the

faeces, urine and any other nitrogen-containing products such as milk,

wool and eggs.

• When the nitrogen intake is equal to the output, the animal is said to be

in nitrogen equilibrium.

Balance trials are susceptible to several sources of error:

• Inadequate adaptation of experimental animals to the diet and the

environment;

• Collection and weighing of faeces and urine;

• Storage of faeces and urine;

• Preparation and sampling of faeces and urine for chemical analysis.

Biological value • This is a direct measure of the proportion of the food protein that can be utilised

by the animal for synthesising body tissues and compounds, and may be defined as

the proportion of the absorbed nitrogen that is retained by the body.

• A balance trial is conducted in which nitrogen intake and urinary and faecal

excretions of nitrogen are measured, along with the endogenous fractions in these

two materials. The biological value is then calculated as follows:

BV =

where MFN = metabolic (endogenous) faecal nitrogen and EUN = endogenous

urinary nitrogen.

Calculation of biological value for maintenance and growth of the rat

• The biological value of a food protein therefore depends upon the

number and kinds of amino acids present in the molecule: the closer

the amino acid composition of the food protein approaches that of

the body protein, the higher will be its biological value.

• The product of BV and digestibility is termed the net protein

utilisation (NPU) and is the proportion of the nitrogen intake

retained by the animal.

• Biological values are for the combined functions of maintenance, meaning the

replacement of existing proteins, and growth (i.e. the formation of new tissues).

Biological values for maintenance alone may be calculated from balance data.

• A linear relationship exists between nitrogen intake and balance below

equilibrium, which may be represented by the following equation:

y = bx – a

where y = N balance, x = N absorbed, a = N loss at zero intake and b = nitrogen

balance index.

• i.e. that fraction of the absorbed nitrogen retained by the body and is equal to the

BV for maintenance.

Biological values of the protein in various foods for maintenance and growth for the growing pig

Chemical score

• In this concept, it is considered that the quality of a protein is decided

by the amino acid that is in greatest deficit when compared with a

standard. The standard generally used has been egg protein, but many

workers now use a defined amino acid mixture, the FAO Recommended

Reference Amino Acid Pattern.

• The content of each of the essential amino acids of a protein is

expressed as a proportion of that in the standard (the standard pattern

ratio) and the lowest proportion taken as the score.

Calculation of chemical score

• In wheat protein, for example, the essential amino acid in greatest

deficit is lysine. The contents of lysine in egg and wheat proteins are 72

g/kg and 27 g/kg, respectively, and the chemical score for wheat

protein is therefore 27/72 = 0.37.

• They are useful for grouping proteins but suffer a serious disadvantage

in that no account is taken of the deficiencies of acids other than that in

greatest deficit.

The essential amino acid index (EAAI)

• This is the geometric mean of the egg, or standard pattern, ratios of the

essential amino acids.

• It has the advantage of predicting the effect of supplementation in

combinations of proteins. On the other hand, it has the disadvantage that

proteins of very different amino acid composition may have the same or a

very similar index.

• Both the chemical score and the essential amino acid index are based upon

gross amino acid composition.

Biological assay of available amino acids

• The available amino acid content of a food protein may be assayed by

measuring the live weight gain, food conversion efficiency or

nitrogen retention of animals given the intact protein as a

supplement to a diet deficient in the particular amino acid under

investigation and the response to the test material is compared with

the response to supplements of pure amino acids.

• The method has been used successfully for lysine, methionine and

cystine.

• But, in addition to the usual disadvantages associated with biological

methods – time, technical expertise and supply of suitable animals –

there is the major problem of constructing diets deficient in specific

amino acids but adequate in other respects.

Microbiological assay of essential amino acids

• Certain microorganisms have amino acid requirements similar to those

of higher animals and have been used for the evaluation of food

proteins.

• The methods are based on measuring the growth of the microorganisms

in culture media that include the protein under test.

• Best results have been obtained with Streptococcus zymogenes

and Tetrahymena pyriformis.

• The former is used after an acid or enzymic predigestion of the food

protein; estimates of the availability of lysine and methionine have

agreed well with chick assays.

• T. pyriformis has intrinsic proteolytic activity and is used, for soluble

proteins, without predigestion.

• An improved method, using predigestion with the enzyme pronase and

measuring response in terms of the tetrahymanol content of the culture

medium, has given results for available lysine, methionine and

tryptophan that correlate well with those of biological assays.

Chemical methods

• The most widely used method is that for FDNB-reactive lysine, which

was originally proposed by K J Carpenter.

• Practically the only source of utilizable lysine in foods is that which has

the epsilon-amino group free to react with various chemical reagents.

• The protein under test is allowed to react under alkaline conditions with

fluoro-2,4-dinitrobenzene (FDNB) to give DNP-lysine, the concentration

of which can be measured colorometrically.

• In practice, the method has been found to agree well with biological

procedures for evaluating proteins as supplements to diets, such as those

containing high proportions of cereals, in which lysine is limiting.

• The correlation has also been good with diets based largely on animal

protein.

• With vegetable protein and diets containing high levels of carbohydrate,

the method is not so satisfactory, the results being too low owing to

destruction of the coloured lysine derivative during acid hydrolysis.

Dye-binding methods

• These have been used widely for estimating protein in such foods as cereals and

milk. The methods are rapid and give reproducible results, and attempts have

been made to use them for measuring total basic amino acids and reactive

lysine. The latter requires blocking of the epsilon-amino group to prevent

reaction with the dye.

• Orange G has been used, along with 2,4,6-trinitrobenzene sulphonic acid and

propionic anhydride as blocking agents, and has proved effective for estimating

the lysine content of cereals. It is less effective for fish and meat meals.

Interpretation of amino acid assays

Several factors may be responsible for a lack of agreement between

estimates of protein quality based on amino acid content and those made

in animal experiments:

• Even small changes in the concentration of one or more amino acids may

increase the amounts of others required to maintain growth rates.

• Certain acids, such as tryptophan and histidine, may be toxic, at

concentrations far greater than normally occur in food proteins.

• Antagonisms may exist between specific acids, which prejudice their utilisation.

Thus, the addition of as little as 20 g/kg of leucine to a diet deficient in isoleucine

may have deleterious effects on performance, and the arginine requirement of the

rat may be increased by giving higher levels of lysine.

• Antinutritional factors (ANF) are frequently present in foods used primarily as

protein sources. Chief among these are enzyme inhibitors, lectins, polyphenols

and certain non-protein amino acids. All are capable of lowering the absorption

and/or the utilisation of amino acids by the animal but are not taken into account

in evaluations of protein sources based on amino acid content.

• There is considerable evidence that growing animals, such as young

rats and chicks, do not fulfil their growth potential if the dietary

nitrogen is entirely in the form of essential amino acids. Additional

nitrogen is required and is best supplied as a mixture of non-essential

amino acids; glutamate, alanine and ammonium citrate are also

effective sources. Allowance must be made for these factors when a

protein source is being evaluated on the basis of its content of one or

more essential amino acids.

THANK YOU

Vishnu Vardhan Reddy.PTVM/2015-029