Protein quality determination in monogastric animals, we can determine which protein is better in case of monogastric animals, Sri Venkateswara veterinary university, Animal nutrition, Vishnu Vardhan Reddy

1. Protein quality
determination in
monogastric animals
Vishnu Vardhan Reddy.P
TVM/2015-029
Department of Animal nutrition
College of Veterinary Science, Tirupati
Sri Venkateswara Veterinary University

2. 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.

3. • 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.

4. Protein efficiency ratio
• This is defined as follows:
PER =
gain in body weight (g)
protein consumed (g)
The rat is the usual experimental animal.

5. Net protein retention
• This is calculated as follows:
NPR =
weight gain of TPG − weight loss of NPG
weight of protein consumed
where TPG = group given the test protein and
NPG = group of protein-free diet.

6. 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.

7. 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.

8. 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.

9. 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 =
N intake − (faecal N − MFN) − (urinary N − EUN)
N intake − (faecal N − MFN)
where MFN = metabolic (endogenous) faecal nitrogen and EUN = endogenous
urinary nitrogen.

10. Calculation of biological value for maintenance and
growth of the rat

11. • 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.

12. • 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.

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

14. 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.

15. 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.

16. 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.

17. 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.

18. • 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.

19. 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.

20. • 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.

21. 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.

22. • 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.

23. 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.

24. 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.

25. • 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.

26. • 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.

27. THANK YOU
Vishnu Vardhan Reddy.P
TVM/2015-029