1. PROTEINS
2. PROTEIN DIGESTION AND METABOLISM
3. GROSS PROTEIN REQUIREMENTS
4. AMINO ACIDS
5. QUANTITATIVE REQUIREMENTS OF AMINO ACID
6. SUPPLEMENTING DIETS WITH AMINO ACIDS
7. REFERENCES
J. E. Halver
University of Washington
Seattle, Washington
1.1 Classification
1.2 Structure
1.3 Properties
1.4 Chemical Determination
Proteins are complex, organic compounds composed of many amino acids linked together through peptide bonds and cross-linked between chains by sulfhydryl bonds, hydrogen bonds and van der Waals forces. There is a greater diversity of chemical composition in proteins than in any other group of biologically active compounds. The proteins in the various animal and plant cells confer on these tissues their biological specificity.
Proteins can be classified as:
(a) Simple proteins. On hydrolysis they yield only the amino acids and occasional small carbohydrate compounds. Examples are: albumins, globulins, glutelins, albuminoids, histones and protamines.(b) Conjugated proteins. These are simple proteins combined with some non-protein material in the body. Examples are: nucleoproteins, glycoproteins, phosphoproteins, haemoglobins and lecithoproteins.
(c) Derived proteins. These are proteins derived from simple or conjugated proteins by physical or chemical means. Examples are: denatured proteins and peptides.
The potential configuration of protein molecules is so complex that many types of protein molecules can be constructed and are found in biological materials with different physical characteristics. Globular proteins are found in blood and tissue fluids in amorphous globular form with very thin or non-existent membranes. Collagenous proteins are found in connective tissue such as skin or cell membranes. Fibrous proteins are found in hair, muscle and connective tissue. Crystalline proteins are exemplified by the lens of the eye and similar tissues. Enzymes are proteins with specific chemical functions and mediate most of the physiological processes of life. Several small polypeptides act as hormones in tissue systems controlling different chemical or physiological processes. Muscle protein is made of several forms of polypeptides that allow muscular contraction and relaxation for physical movement.
Proteins can also be characterized by their chemical reactions. Most proteins are soluble in water, in alcohol, in dilute base or in various concentrations of salt solutions. Proteins have the characteristic coiled structure which is determined by the sequence of amino acids in the primary polypeptide chain and the stereo configuration of the radical groups attached to the alpha carbon of each amino acid. Proteins are heat labile exhibiting various degrees of lability depending upon type of protein, solution and temperature profile. Proteins can be reversible or irreversible, denatured by heating, by salt concentration, by freezing, by ultrasonic stress or by aging. Proteins undergo characteristic bonding with other proteins in the so-called plastein reaction and will combine with free aldyhyde and hydroxy groups of carbohydrates to form Maillard type compounds.
The nitrogen content of most proteins found in animal, nut and grain tissue is about 16 percent; therefore, protein content is commonly expressed as nitrogen content × 6.25.
Ingested proteins are first split into smaller fragments by pepsin in the stomach or by trypsin or chymotrypsin from the pancreas. These peptides are then further reduced by the action of carboxypeptidase which hydrolyzes off one amino acid at a time beginning at the free carboxyl end of the molecule or by aminopeptidase which splits off one amino acid at a time beginning at the free amino end of the polypeptide chain. The free amino acids released into the digestive system are then absorbed through the walls of the gastro intestinal tract into the blood stream where they are then resynthesized into new tissue proteins or are catabolyzed for energy or for fragments for further tissue metabolism.
Gross protein requirements have been determined for a few species of fish (see Table 1). Simulated whole egg protein component of test diets contains an excess of indispensable amino acids. These diets were kept approximately isocaloric by adjusting total protein plus digestible carbohydrate components to a fixed amount as the protein diet treatments were varied over the ranges tested. Tests in feeding fry, fingerling, and yearling fish have shown that gross protein requirements are highest in initial feeding fry and that they decrease as fish size increases. To grow at the maximum rate, fry must have a diet in which nearly half of the digestible ingredients consist of balanced protein; at 6-8 weeks this requirement is decreased to about 40 percent of the diet for salmon and trout and to about 35 percent of the diet for yearling salmonids raised at standard environmental temperature (SET). See Figures 1 and 2. Gross protein requirements for young Catfish appear to be less than those for salmonids. Initially feeding fry require that about 50 percent of the digestible components of the ration be protein, and the requirement decreases with size. Some feeding trials with salmon have indicated direct relationships between changes in the protein requirements of young fish and changes in water temperature. Chinook salmon in 7 C water require about 40 percent whole egg protein for maximum growth; the same fish in 15 C water require about 50 percent protein. Salmon, trout and catfish can use more protein than required for maximum growth because of efficiency in eliminating nitrogenous wastes in the form of soluble ammonia compounds through the gill tissue directly into the water environment. This system for eliminating nitrogen is more efficient than that available to fowl and mammals. Fowl and mammals consume energy to synthesize urea, uric acid, or other nitrogen compounds which are excreted through the kidney tissue and expelled in urine. Digestible carbohydrate and fat will spare excess protein in the diet as long as the protein requirement for maximum growth is met (Figures 1 and 2).
Table 1 - Estimated Dietary Protein Requirement of Certain Fish 1/
|
Species |
Crude protein level in diet for optimal growth (g/kg) |
|
Rainbow trout (Salmo gairdneri) |
400-460 |
|
Carp (Cyprinus carpio) |
380 |
|
Chinook salmon (Oncorhynchus tshawytscha) |
400 |
|
Eel (Anguilla japonica) |
445 |
|
Plaice (Pleuronectes platessa) |
500 |
|
Gilthead bream (Chrysophrys aurata) |
400 |
|
Grass carp (Ctenopharyngodon idella) |
410-430 |
|
Brycon sp. |
356 |
|
Red sea bream (Chrysophrys major) |
550 |
|
Yellowtail (Seriola quinqueradiata) |
550 |
1/Adapted from C.B. Cowey, 1978
Fig. 1. Protein requirement of chinook salmon at 47°F. Top curve: initial individual average weight of fish, 1.5g. Bottom curve: initial individual average weight of fish, 5.6g.
Fig. 2. Protein requirement of chinook salmon at 58°F. Top curve: initial individual average weight of fish, 2.6g. Bottom curve: initial individual average weight of fish, 5.8g.
(Both figures adapted from: DeLong, D.C., J.E. Halver and E.T. Mertz, 1958, J.Nutr., 65:589-99)
Basically the fish must be given a diet containing graded levels of high quality protein and energy and adequate balances of essential fatty acids, vitamins and minerals over a prolonged period. From the resulting dose/response curve the protein requirement is usually obtained by an Almquist plot. These differences in apparent protein requirement are thought to be due to differences in culture techniques and diet composition.
The relatively high dietary protein levels required for maximal growth of certain fish such as grass carp, Ctenopharyngodon idella, and Brycon spp. are surprising as these fish are omnivorous. Brycon spp. are grown on unwanted fruit and other plant material of low protein content and under these conditions there is presumably a substantial contribution to their protein intake from a natural food chain.
Protein requirement of eurythaline fish such as the rainbow trout, Salmo gairdneri, and the coho salmon, Oncorhynchus kisutch, reared in water of salinity 20 ppt is about the same as the requirement in freshwater. No data are available for the protein requirement of these species in full strength sea water.(35 ppt).
4.1 Essential and Non-essential Amino Acids
4.2 Essential Amino Acids and Protein Quality
The amino acids are the building blocks of proteins; about 23 amino acids have been isolated from natural proteins. Ten of these are indispensable for fish. The animal is incapable of synthesizing indispensable amino acids and must therefore obtain these from the diet.
Salmon, trout and channel catfish fed diets devoid of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan or valine failed to grow (Fig. 3). These same fish fed diets devoid of other L-amino acids grew as well as fish receiving all 18 amino acids tested (Fig. 4). The nitrogen component in the test diets was made up of 18 L-amino acids in the pattern found in whole egg protein. All fish on test recovered rapidly when the missing amino acid was replaced in the diet. The slope of the growth curve of the recovery group was identical with that of fish receiving the complete amino acid test diet.
Dispensable amino acids tested were alanine, aspartic acid, cystine, glutamic acid, glycine, proline, serine, and tyrosine. These amino acids were found to be not essential for the growth of salmon, trout and channel catfish.
Quantitative studies on the requirements of the 10 indispensable amino acids used a casein-gelatin mixture supplemented with crystalline L-amino acids. The test diet had an amino acid pattern of 40 percent whole egg protein for the nitrogen component. Experiments conducted with carp and eel showed a similar lack of growth when an indispensable amino acid was absent from the diet.
Fig. 3. Growth of arginine deficient fish. The deficient group was divided after six weeks on the deficient diet and the missing amino acid was replaced in one of the two sub-lots.
Fig. 4. Growth of cystine deficient fish.
(Both figures adapted from: DeLong, D.C., J.E. Halver and E.T. Mertz, 1958, J.Nutr., 65:589-99)
If the essential amino acid requirements of fish are known, it should be possible to meet these needs in culture systems in a number of ways from different food proteins or combinations of food proteins.
Phenylalanine is spared by tyrosine. It is not known to be chemically modified nor rendered unavailable by the harsh conditions to which feedstuff proteins are normally subjected during processing. Measurement of phenylalanine in proteins is uncomplicated so that the provision and evaluation of phenylalanine in proteins in practical diets presents little difficulty.
Lysine is a basic amino acid. In addition to the a -amino acid group normally bound in peptide linkage, it also contains a second, a -amino group. This a -amino group must be free and reactive, otherwise the lysine, although chemically measurable, will not be biologically available. During the processing of feedstuff proteins the a -amino group of lysine may react with non-protein molecules present in the feedstuff to form additional compounds that render the lysine biologically unavailable.
Methionine is spared by cystine. However, measurement of the methionine content of feed proteins is not easy as the amino acid is subject to oxidation during processing. After processing, methionine may be present as such or as the sulphoxide or as the sulphone. The sulphoxide may be formed from methionine during acid hydrolysis of the feed protein prior to measurement of its any-no acid composition. Acid hydrolysis of proteins before analysis disturbs the original equilibrium between the two compounds so that the composition of the hydrolysate no longer reflects that of the protein. In determining the methionine content of pure proteins, oxidation of the amino acid to methionine sulphone is normally quantitative. In the case of feed proteins, however, this will not reveal how much methionine or methionine sulphoxide was present in the protein prior to performate oxidation and hydrolysis.
Methionine sulphoxide may have some biological value for fish which may have some capability of reconverting it to methionine and thus partially make up for some of the methionine oxidized during processing.
Methods have recently been reported for measurement of methionine in proteins using an iodoplatinate reagent before and after reduction with titanium trichloride, to give values for both methionine and the sulphoxide in the original protein. A method for measuring methionine specifically by cyanogen bromide cleavage has also been described. Both methods remain to be independently assessed. Microbiological assay of methionine in feed proteins is a valuable tool although there is the danger that oxides of methionine may differ in their activity for micro-organisms and misrepresent values.
Quantitative requirements by salmonids for the ten indispensable amino acids were determined by feeding linear increments of one amino acid at a time in a test diet containing an amino acid profile identical with whole egg protein except for the amino acid tested. Replicate groups of fish were fed the diet treatments until gross differences appeared in the growth of test lots. An Almquist plot of growth response indicated the level of amino acids required for maximum growth under those specific test conditions. Diets were designed to contain protein at or slightly below the optimum protein requirement for that species and test condition to assure maximum utilization of the limiting amino acid. A comparison of the requirements for the ten indispensable amino acids between species is shown in Table 2.
A recent innovation has been the use in test diets of proteins relatively deficient in a given essential amino acid. Thus combinations of fishmeal and zein have been used in test diets to define the requirement of rainbow trout for arginine. Diets containing different relative amounts of casein and gelatin showed that an increase in the level of protein-bound arginine from 11 to 17 g/kg resulted in a significant increase in the growth of channel catfish.
Table 2 Amino Acid Requirements of Seven Animals 1/
|
Amino acid |
Eel fingerling |
Carp fry |
Channel catfish |
Chinook salmon fingerling |
Chick |
Young Pig |
Rat |
|
Arginine |
3.9 (1.7/42) |
4.3 (1.65/38.5) |
|
6.0 (2.4/40) |
6.1 (1.1/18) |
1.5 (0.2/13) |
1.0 (0.2/19) |
|
Histidine |
1.9 (0.8/42) |
|
|
1.8 (0.7/40) |
1.7 (0.3/18) |
1.5 (0.2/13) |
2.1 (0.4/19) |
|
Isoleucine |
3.6 (1.5/42) |
2.6 (1.0/38.5) |
|
2.2 (0.9/41) |
4.4 (0.8/18) |
4.6 (0.6/13) |
3.9 (0.5/13) |
|
Leucine |
4.1 (1.7/42) |
3.9 (1.5/38.5) |
|
3.9 (1.6/41) |
6.7 (1.2/18) |
4.6 (0.6/13) |
4.5 (0.9/19) |
|
Lysine |
4.8 (2.0/42) |
|
5.1 (1.23/24.0) |
5.0 (2.0/40) |
6.1 (1.1/18) |
4.7 (0.65/13) |
5.4 (1.0/19) |
|
Methionine 2/ |
4.5 (2.1/42) 3/ |
3.1 (1.2/38.5) |
2.3 (0.56/24.0) |
4.0 (1.6/40)3/ |
4.4 (0.8/18) |
3.0 (0.6/20) |
3.0 (0.6/20) |
|
Phenylalanine 4/ |
|
|
|
5.1 (2.1/41)5/ |
7.2 (1.3/18) |
3.6 (0.45/13) |
5.3 (0.9/17) |
|
Threonine |
3.6 (1.5/42) |
|
|
2.2 (0.9/40) |
3.3 (0.6/18) |
3.0 (0.4/13) |
3.1 (0.2/19) |
|
Tryptophan |
1.0 (0.4/42) |
|
|
0.5 (0.2/40) |
1.1 (0.2/18) |
0.8 (0.2/25) |
1.0 (0.2/19) |
|
Valine |
3.6 (1.5/42) |
|
|
3.2 (1.3/40) |
4.4 (0.8/18) |
3.1 (0.4/13) |
3.1 (0.4/13) |
1/ Expressed as percent of dietary protein. In parentheses, the numerators are requirements as percent of dry diet, and the denominators are percent total protein in the diet2/ In the absence of cystine
3/ Methionine plus cystine
4/ In the absence of tyro sine
5/ Phenylalanine plus tyrosine
(Adapted from: National Research Council, 1977)
Arginine requirement of rainbow trout has been determined from a conventional dose/response (growth) curve and also by measuring the tissue (blood and muscle) levels of free arginine in groups of trout given increasing amounts of dietary arginine. After the dietary requirement of the trout for arginine has been met, any further increase in arginine intake led to an increase in the concentration of free arginine in blood and muscle. Good agreement was obtained between the two methods.
The data shown in Table 2 suggest that real differences exist between fish species in their requirement for certain amino acids. This leads to difficulties in formulating the protein component of practical diets for those species whose amino acid requirements are not yet known. A possible solution is to use, for each amino acid, the highest level required by any of those species for which data is available. The need for further quantitative data on the amino acid requirements of fish, especially those actually or potentially useful as farm animals, is obvious.
One solution to the use of proteins that are relatively deficient in one or more amino acids is to supplement the protein with appropriate amounts of the amino acid needed in practical diets. Fish appear to utilize free amino acids at various degrees of efficiency.
Young carp, Cyprinus carpio, were shown to be unable to grow on diets in which the protein component (casein, gelatin) was replaced by a mixture of amino acids similar in overall composition. A trypsin hydrolyzate of casein was equally ineffective. However, if a diet containing free amino acids as the protein component is carefully neutralized with NaOH to pH 6.5-6.7 then some growth of young carp does occur. This growth was markedly inferior to that occurring on a comparable casein diet under the same conditions.
Channel catfish are also unable to utilize free amino acids given as supplements to deficient proteins. When soybean meal was substituted isonitrogenously for menhaden meal, growth and feed efficiency of channel catfish were substantially reduced. Addition of free methionine, cystine or lysine, the most limiting amino acids, to these soy-substituted diets did not enhance weight gain.
Raising the arginine level of catfish diets from 11 to 17 g/kg by isonitrogenous substitution of gelatin for casein enhanced weight gain significantly but the addition of free arginine, cystine, tryptophan or methionine to casein had little effect on growth or food conversion.
Salmonids are able to utilize free amino acids for growth. A zein-gelatin diet supplemented with lysine and trytophan was shown to be markedly superior to an unsupplemented zein-gelatin diet for rainbow trout when weight gain and protein utilization were used as criteria.
Several investigators have demonstrated the potential of supplementing amino acid deficient proteins with limiting amino acids in diets for salmonids. Casein supplemented with six amino acids produced feed conversion ratios with Atlantic salmon similar to those obtained when an isolated fish protein was used as the dietary protein source. Soybean meal supplemented with five or more amino acids (including methionine and lysine) was a superior protein source to soybean meal alone for rainbow trout. Single additions of methionine and lysine did not, however, improve the value of soybean meal. These results suggest that the amino acid spectrum of the isolated fish protein they used may possibly approximate the amino acid requirement of rainbow trout. The nutritional value of a soy protein isolate could be enhanced by supplementing it with the first limiting amino acid; i.e., methionine.
Diets containing, as protein component, fishmeal, meat and bone meal, and yeast and soybean meal could be improved by supplementing with cystine (10 g/kg) and tryptophan (5 g/kg) together. Fishmeal can be entirely replaced without a reduction in food conversion rate in diets for rainbow trout by a mixture of poultry by-product meal and feather meal together with 17 g lysine HCL/kg, 4.8 g DL-methionine/kg, and 1.44 g DL-tryptophan/kg.
Cowey, C.B. and J.R. Sargent, 1972 Fish nutrition. Adv.Mar.Biol., 10:383-492
Cowey, C.B., 1979 Protein and amino acid requirements of finfish. In Finfish nutrition and fishfeed technology, edited by J.E. Halver and K. Tiews. Proceedings of a World Symposium sponsored by EIFAC/FAO, ICES and IUNS, Hamburg, 20-23 June, 1978. Schr.Bundesforschungsanst.Fisch.,Hamb., (14/15)vol. 1:3-16
Mertz, E.T., 1972 The protein and amino acid needs. In Fish nutrition, edited by J.E. Halver. New York, Academic Press, pp. 106-43
National Research Council, 1977 Subcommittee on Warmwater Fishes, Nutrient requirements of warmwater fishes. Washington, D.C., National Academy of Sciences (Nutrient requirements of domestic animals) 78 p.
