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Appendices

A. Specific procedures recommended for evaluation of early-generation and more advanced genetic material

General Considerations
Procedures for Chemical and Physical Evaluation - Tests Recommended for Field Laboratories
References

B. Selected books and other documents

1. Books and Reviews Related to the Evaluation of Protein Quality
2. Some relevant Documents from the Protein- Calorie Advisory Group of the U.N. System and from the World Hunger Programme of the U.N university

C. Joint UNU-IUNS working group

A. Specific procedures recommended for evaluation of early-generation and more advanced genetic material

General Considerations

The systematic approach recommended for evaluation of cereals in breeding programmes is predicated on the necessity to screen very large numbers of genotypes in a short period. The overall system is summarized in figure 6, which is a schematic diagram of the recommended screening and evaluation procedure.* Under such a system, the choice of tests, the order of tests in the screening procedure, and the arbitrary cut-off points recommended are in themselves important. For example, screening by means of the dye-binding capacity (DBC) for basic amino acids before assaying for total protein will eliminate about half the samples, thereby halving the number of protein assays needed. Similarly, a screening test that detects low tryptophan lines of maize will automatically eliminate the need for testing a large proportion of the material for Iysine, because the content of tryptophan, the second limiting amino acid in maize, has been shown to be closely related to Iysine, the first limiting amino acid.

It is recognized in plant breeding that rapid, large-scale screening techniques may result in the discarding of some lines with possibly useful characteristics. However, the need to save time and money more than justifies the relatively small amount of genetic material sacrificed in this manner.

Recomended Procedure for Screening Cereal Grains for Protein Content and Nutritive Quality

This appendix is taken from section IX of PAG Guideline No. 16, "Protein Methods for Cereal Breeders as Related to Human Nutritional Requirements," from PAG Bulletin, V (2): 22 - 48 (June 1975). The whole Guideiine is recommended reading for all engaged in protein evaluation on new cereal varieties.

Other scientists suggest that early generation material, F2 and F3, should be selected primarily on the basis of grain size and yield as well as other agronomically desirable characteristics. According to them, there is good correlation between grain weight and the total amount of protein per grain. They feel that only lines F4 or even Fs onwards need to be tested for protein quantity and quality.

Literature references are given to the specific analytical techniques recommended by the working group. Where necessary, the underlying principles of the tests are briefly described. Advice on equipment suitable for use in screening laboratories may be obtained on request from members of the working group.*

Certain crops, for example, maize, sorghum, and oats, require specialized analytical treatment due to oil and pigment contents higher than in most other cereals. In the case of maize, the oil interferes with certain extraction procedures and may also cause excessive frothing, e.g., during the digestion step in the micro Kjeldahl analysis. Any pigments present constitute a source of error in certain colorimetric tests. Most of these interferences can be minimized by extraction with a non-polar solvent.

In certain instances, it may be necessary to store samples while they await analysis. Inadequate storage, especially at high temperature and humidity, may lead to loss of valuable genetic material through chemical and biological changes. Deterioration by bacteria, fungi, and insects must also be prevented. Problems encountered in the storage of cereal grains are well covered in a recent monograph on the subject ( 1). Efficient aeration is necessary for the prolonged storage of any plant material; refrigeration, even if it is only partial, e.g., to 1 5°C 160° F) is recommended whenever Possible.

Procedures for Chemical and Physical Evaluation - Tests Recommended for Field Laboratories

Sampling and Sample Preparation

In the case of wheat, barley, triticale, and rice, a representative sample of not less than 5 9 is blended, airdried, and provided to the laboratory. This is the /east amount of sample required and is important in the case of early generation materials up to F., when only minimal quantitites of grain are available. For screening tests conducted on early generations, moisture content need not be determined unless precise comparisons are to be made with data from other laboratories, or when publication requires such values to be quoted.

The Udy or Weber cyclone mills are recommended for the grinding of these caryopsistype seeds. Due to their higher oil content, maize, sorghum, and oats tend to clog grinders of this type. These grains may be ground in any suitable impact mill, such as the Krups Model 75, the Moulinex mills, or even in a hand-grinder. After grinding before analysis, the ground material may be defatted with solvents such as hexane or petroleum ether, a procedure that also helps remove pigments that may interfere with some colorimetric tests (2). Further reduction in particle size may, in some cases, be advisable after defatting. The above impact mills are recommended for this purpose. For maize specifically, consult Villegas and Mertz (2).

Preliminary Quality Screening Tests 1. Dye-binding capacity (DBCJ)

A solution of the acid diazo dye Acrilane Orange G (also known as Orange 12 [CI 15970], chemically as 1phenylazo-2-naphthol-6-sulphonic acid, monosodium salt) is mixed with the ground sample. The dye is bound quantitatively by the basic imidazole, guanidino, and amino groups of the protein, which occur in the polypeptide chain on histidine, arginine, and Iysine or as free terminal groups. The unbound dye remaining in the solution is measured colorimetrically after filtration or centrifugation. Because the proportions of basic amino acids and terminal groups are reasonably constant in cereal proteins, the correlations between DBC and total protein content are high. However, the level of Iysine and arginine is increased substantially in highlysine cereals, and the differing ratios of DBC to protein can be utilized to select these genotypes. The chief sources of error in DBC testing have been discussed by Williams (4). In addition, the presence in the test material of excessive chlorophyll, fibre, and certain tannins can give rise to erroneously high DBC values.

Different lots of dye may differ slightly in colour intensity due to batch-to-batch variability; the dye should be standardized before use. The method of Mossberg is recommended for DBC (3). Samples of early generation material showing low DBC values may be eliminated, whereas those showing satisfactory values (i.e., the samples that show values higher than the arbitrarily set cut-off point) should be further analysed for total protein and Iysine by more specific methods, as outlined in figure 6.

2. Ninhydrin screening method

It has been shown that high-lysine mutants such as Opaque-2 maize (5), high-lysine sorghum (6), and mutant 1508 barley (7) contain unusually high levels of free amino acids. The presence of abnormally high levels of free amino acids may therefore be regarded as indicative of above average levels of Iysine. Ninhydrin reagent reacts with the alpha amino groups of free amino acids to form a coloured complex, the intensity of which is proportional to the concentration of total free amino acids (8). As an alternative to the DBC procedure, this test may be applied either to a sectioned surface of single kernels or to an extract of the pulverized grains. The sectioning technique is non-destructive, in that the portion of the kernel with the embryo intact may be planted and grown. The non-destructive feature of this test recommends its use for preliminary field testing. When screening for high-lysine mutants, comparisons should be made using controls with "normal" levels of Iysine.

3. Lysine and tryp tophan

These colorimetric procedures are significant primarily for maize {2). Lysine and tryptophan are present in a roughly constant ratio of 4:1. Consequently, when tryptophan is determined, an estimate of Iysine is furnished. The defatted, finely ground endosperm is digested with papain to release the amino acids. The tryptophan colour is developed by means of reagents that, when combined, produce glyoxylic acid (2-9). In the case of Iysine, the papain digest is treated with a solution of 2-chloro-3-,5-dinitropyridine (DNP) (2).

4. Total protein

The micro Kjeldahl procedure is described in the latest editions of the analytical testing methods manuals of the Association of Official Analytical Chemists (10) and the American Association of Cereal Chemists ( 11), as well as in the CIMM YT Bulletin, no. 20 (2).

5. Verification of high Iysine

Samples that indicate DBC values higher than the cut-off point should be tested for total protein. These samples should then be reanalysed for DBC on a constant protein basis. Samples are weighed to give a constant amount of protein, e.g., 60 mg, and tested again by the DBC technique. The results of the DBC re-tests are then plotted against the total protein content of the original samples, at which time any segregates having re-tested DBC values that are significantly above the slope of the regression line (the "line of best fit") will indicate samples with an above average level of Iysine.

References

1. C.M. Christensen, ed .,Storage of Cereal Grains and Their Products (American Association of Cereal Chemists, St. Paul, Minn., USA, 1974).

2 E. Villegas and E T. Mertz, "Chemical Screening Methods for Maize Protein Quality at CIMMYT," Research Bulletin, no. 20 (1971), (Centro Internacional de Mejoramiento de Maiz y Trigo [CiMMYT], Mexico, D.F., Mexico).

3. R. Mossberg, "Evaluation of Protein Quality and Quantity by Dye Binding Capacity: A Tool in Plant Breeding," in New Approaches to Breeding for Improved Protein, Panel Proceedings Series 212 (International Atomic Energy Agency, Vienna, Austria, 1969), pp. 151-160.

4. P.C. Williams, "Errors in Protein Testing and Their Consequences," Cereal Sci. Today, 19 (7): 280-282 (1974).

5. E.T. Mertz, L.S. Bates, and O.K. Nelson, "Mutant Gene That Changes Protein Composition and Increases Lysine of Maize Endosperm," Science, 145: 279-280 (1974).

6. R. Singh and J.D. Axtell, "High Lysine Mutant Gene That Improves Protein Quality and Biological Value of Grain Sorghum," Crop Sci., 13: 535-539 (1973).

7. J. Ingverson, B. Koie, and H. Doll, "Induced Seed Protein Mutant of Barley," Experientia, 29 1151-1152 (1973).

8. E T. Mertz, P.S. Misra, and R. Jambunathan, "Rapid Ninhydrin Color Test for Screening High-Lysine Mutants of Maize, Sorghum, Barley and Other Cereal Grains," Cereal Chem., 51: 304-307 (1974).

9. A. Dalby and C.Y. Tsai, "Acetic Anhydride Requirements in the Colorimetric Determination of Tryptophan,"Anatyt. Biochem., 63: 283-285(1975).

10.Official Methods of Analysis of the Association of Official Analytical Chemists, 11 th ed. (Association of Official Analytical Chemists, Washington, D.C., 1970).

11. American Association of Cereal Chemists Approved Methods, 7th ed. (American Association of Cereal Chemists, St. Paul, Minn., USA, 1962)

B. Selected books and other documents

1. Books and Reviews Related to the Evaluation of Protein Quality

Bodwell, C.E., ed. Evaluation of Proteins for Humans. Avi Publishing Co., Westport, Conn., USA 1977.

Bodwell, C.E., J.S. Adkins, and D.T. Hopkins, eds. Protein Quality in Humans: Assessment and In Vitro Estimation. Proceedinqs of a conference held at Airlie House, Warrenton, Va., USA, March 1980. Avi Publishing Co., Westport, Conn., USA, 1980 (in press).

Committee on Amino Acids, Food and Nutrition Board, National Research Council. Improvement of Protein Nutriture. Edited by A.E. Harper and D.M. Hegsted. National Academy of Sciences, Washington, D.C., 1974.

Dreyer, J.J. Contributions to the Biological Assessment of Dietary Protein Quality. National Food Research Institute, Scientia, Pretoria, South Africa,1979.483 pp.

Eggum, B.O. A Study of Certain Factors Influencing Protein Utilization in Rats and Pigs. Publ. no. 406. National Institute of Animal Science, Copenhagen, 1973. 173 pp.

Evans, E., and R. Witty. "An Assessment of Methods Used to Determine Protein Quality." In G.H. Bourne, ea., World Review of Nutrition and Dietetics: Human and Animal Nutrition, pp.1-26. S. Karger AG, New York, 1978.

Friedman, M., ed. Protein Nutritional Quality of Foods and Feeds. Part R Assay Methods: Biological, Biochemical and Chemical. Marcel Dekker, Inc., New York, 1975.626 pp.

Hulse, J.H., K.O. Rachie, and L.W. Billingsley, eds. Nutritional Standards and Methods of Evaluation for food Legume Breeders. IDRC-TS7e. International Development Research Centre, Ottawa, Canada, 1977.

McLaughlan, J.M., and J.A. Campbell, "Methodology of Protein Evaluation." In H.N. Munro, ea., Mammalian Protein Metabolism, vol. l 11, pp.391 -422. Academic Press, New York and London, 1969.

Mertz, E.T., R. Jambunathan, and P.S. Misra. "Simple Chemical and Biological Methods Used at Purdue University to Evaluate Cereals for Protein Quality." Interpretation Programs in Agriculture. Station Bulletin no. 70. Agricultural Experiment Station, Purdue University, West Lafayette, Ind., USA, 1975. 25 pp.

"The Midlands Conference: New Concepts for the Rapid Determination of Protein Quality." University of Nebraska, Lincoln, Neb., USA, 20-22 Feb.1977. Published in Nutr. Rep. Internat, 16 (2): 157-226 (1977); Food Technol., 31 (6): 69-96 (1977).

Munro, H.N., and A. Fleck. "Analysis of Tissues and Body Fluids for Nitrogenous Constituents." In H.N. Munro, ea., Mammalian Protein Metabolism, vol. 111, pp. 423-525. Academic Press, New York and London, 1969.

Nutritional Evaluation of Cereal Mutants. Proceedings of an Advisory Group meeting, Vienna, July 1976. ST1/Pub. 444. International Atomic Energy Agency, Vienna, Austria, 1977.

Pellett, P.L. "Protein Quality Evaluation Revisited." Food Technol., 30 (5): 60-76 (1978).

Porter, M.W.G., and B.A. Rolls, eds. Proteins in Human Nutrition. Proceedings of a NATO Advanced Study, Institute of the Chemistry, Biology and Physics of Protein Evaluation. Academic Press, New York and London, 1973. 560 pp.

Wilcke, H.L., D.T. Hopkins, and D.M. Waggle, eds. Soy Protein and Human Nutrition. Academic Press, New York and London, 1979.406 pp.

Young, V.R., and N.S. Scrimshaw. "Nutritional Evaluation of Proteins and Protein Requirements." In M. Milner, N.S. Scrimshaw, and D.l.C. Wang, eds., Protein Resources and Technology: Status and Research Needs, pp.136-173. Avi Publishing Co., Westport, Conn., USA, 1978.

Some relevant Documents from the Protein- Calorie Advisory Group of the U.N. System and from the World Hunger Programme of the U.N university

Table: PAG Statements

Table:Pag Guidelines

Table: Others PAG Documents

Table

From the UNU World Hunger Programme Food and Nutrition Bulletin

Joint UNU-IUNS working group

Workshop on the Evaluation of Protein Foods (Revision of NAS-NRC Publication 1100), sponsored by the United Nations University World Hunger Programme, 5-7 September 1979, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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