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Methods of Protein Analysis
and Variation in Protein Results
C. E. McDonald
Premiums on high-protein hard red spring wheat has created much interest in the protein test. The Kjeldahl method, a chemical procedure for nitrogen, is still the basic method used for protein analysis. The Kjeldahl method, the Udy dye binding method and the new infrared reflectance method for determining protein are described in this paper. In the analysis of wheat protein by the Kjeldahl method, the moximum amount of variance in results expected between different laboratories and within the same laboratory is larger than the increments of pro tein on which price premiums have been paid. The causes of protein variance and ways of reducing the variance are discussed.
In the past year, the price of hard red spring
wheat has been substantially influenced by the
protein content. Price premiums have been paid
on increased increments of 0.1 percentage points
of protein. The price expected for wheat on the
basis of protein as determined by a North Dakota
laboratory sometimes is changed because the pro
tein found later at Minneapolis or Duluth differs
more than the protein increment required for
premium. With barley, an unexpected discount
may be applied against the seller because the pro
tein is found to be over the discount protein level
at the marketing point. This paper discusses the
protein test, how much variance should be expect
ed between laboratories, and the causes of protein
variance.
Protein Methods
1. Kjeldahl Nitrogen Method. Amino acids are
the building blocks of protein, and they contain
carbon, hydrogen, oxygen, sulfur and nitrogen
(Table 1). In the basic method for protein, nitro
gen is determined by a method of chemical anal
ysis known as the Kjeldahl procedure. The nitro-
Dr. McDonald is professor, Department of Cereal Chemistry and Technology.
Table I. Basis of Kjeldahl Protein Procedure.
1. Protein made of amino acid building blocks.
2. Amino acids made of carbon, hydrogen, oxygen,
sulfur, nitrogen.
3. Protein calculated from measurement of nitro
gen.
- Per cent nitrogen x 5.7 = per cent crude pro
tein of wheat.
Per cent nitrogen X 6.25 = per cent protein of
other grains.
gen found, times a factor of 5.7 for wheat and 6.
for other grains, gives the content of crude pro
tein. Other protein methods like the Udy or the
new infrared reflectance method are calibrated
against the basic Kjeldahl method. The steps for
the Kjeldahl protein determination are given in
Table 2. This procedure requires the use of boiling
concentrated sulfuric acid and strong caustic.
2. Udy Dye Binding Method. In this procedure,
ground grain is shaken with an orange dye solu
tion. This acid dye forms an insoluble dye-protein
complex with the basic amino acid building blocks
of the protein. The dye-protein complex reduces
May-June, 1977
Table 2. Major Steps in Kjeldahl Protein Analysis.
- Grind and weigh 1 gram (rv 1/28 oz.) grain.
- Digest in boiling concentrated sulfuric acid to convert protein nitrogen to ammonia (NH3).
- Add 50 per cent caustic to make ammonia vol atile.
- Boil off ammonia and collect by distillation.
- Measure amount of ammonia collected by titra tion.
the amount of dye left in solution, the complex is removed by filtering, and the concentration of dye in solution is determined with a color measur ing instrument. The amount of dye left in solution is inversely related to the protein content of the sample.
- Infrared Reflectance Method. In 1971, in struments using infrared reflectance (Neotec and Infraalyzer) were introduced into the grain trade to measure protein, moisture and oil. In this meth od, infrared light of different wave lengths (ob tained by filters) is directed onto a sample of ground grain in a cell (Figure 1). The light reflect ed from the grain depends upon the chemical composition of the grain, and the reflected light at different wave lengths is measured by a photo cell detector. The response of the detector is fed into a small computer which calculates complex equations to obtain the protein, moisture or oil content of the sample. The instruments should gain rapid use in the grain trade, provided the accuracy is near that of the Kjeldahl method now used in protein laboratories.
Variance Expected In Protein Result
Discussion of protein variance will be con fined mostly to the basic Kjeldahl procedure, be cause almost all protein laboratories are still using this method. Protein for samples in the grain trade are obtained from a single analysis on each sam ple. With a single analysis there is probability that occasionally there will be a wide variation from the actual value. More than one analysis on each sample would reduce this number of varia tions.
Data shown in Table 3 indicate how much
variance in protein results should be expected between laboratories. Wheat flour samples were analyzed on a monthly basis by some 50 different laboratories in the United States. Wheat grain samples were also analyzed on a monthly basis by 13 different laboratories in the spring wheat area of the United States. The standard deviation, as a measure of deviation from the average value, was
I. R. LIGHT
DETECTOR
Figure 1. Illustration of infrarec:l reflectance method fOl protein analysis.
lower for flour than wheat grain. The step of grinding the wheat sample by each laboratory should have caused at least a part of the higher variation for wheat. Another factor is the uneven distribution of protein found in ground wheat, which will be discussed later.
Table 3. Variance in Protein Results Between Laboratories on Samples Analyzed Monthly.
Average Number (^) Range in standard Sample (^) samples Protein deviationS % % Wheat flour (1975)1 (^12) 11-12 0. Wheat grain (1975)2 10 12-18 (^) 0. '1\1 onthly check sample from the American Association of Cereal Chemists analyzed by some 50 laboratories. 2Monthly check sample of wheat grain from Ingman Laboratories, Inc., analyzed by 13 laboratories. 'Standard Deviation - a measure of the deviation from the average value expressed as percentage protein.
Normal variation expected in protein results where there is a standard deviation of 0.16 per cent is given in Table 4. There would be a 67 per cent probability that a laboratory doing a single protein analysis would obtain a value deviating from the actual value by one ± standard deviation (SD) unit. For example, for a wheat of 14.0 per
(^4) Farm Research
2. Cleanliness of Grain. The second factor
that might cause protein variance is the cleanli
ness of the grain (Table 6). The protein content of
some dockage material is given in Table 7. Small
amounts of high-protein soybeans and wild oats
in wheat or barley samples could increase protein
results, while low-protein straw and chaff could
Table 7. Protein Content of Dockage and Extrane
ous Material Found in Grain.
Material Protein content %
Buckwheat 13
Pigeon grass 14
Soybeans 40
Straw and chaff 3
Wild oats 19
decrease them. The protein of buckwheat and
pigeon grass is near that of wheat, so small
amounts probably would not have a significant
effect on protein. Routinely, grain is cleaned be
fore a sample is taken for grading or a protein
analysis.
3. Moisture Content of Grain. The third factor
that can affect protein results is the moisture con
tent of the grain. The extent of protein change in
grain due to differences in moisture is illustrated
in Table 8. First, a dry sample of 14.0 per cent pro
tein contains 86 per cent nonprotein dry matter.
This same sample with 10.0 per cent moisture now
contains only 12.6 per cent protein because the
sample has been diluted with water, and for the
same reason the sample of 14.0 per cent moisture
will contain only 12.1 per cent protein. When
large enough moisture changes occur in grain
there is also a change in the protein content. For
this reason, grain samples to be tested should al
ways be stored in air-tight containers.
Table 8. Protein Content at Different Moisture
Levels.
Moisture Per^ cent^ of^ dry^ sample^ Protein content water (^) protein nonprotein total wt. content' % % % % (^) % %
'Protein content = Protein as^ %^ of^ dry samEZe^ X 100 Total wt. as % of dry sample
Table 9. Moisture Loss on Grinding Wheat Grain
for Protein Analysis.
12.2% 16.2% Moisture grain i Moisture grain i Moisture Change of Moisture Change of after protein after protein grinding' content grinding' content Mill used (^) % % % %
Labconco Burr 12.4 0.0 16.1 0.
Hobart Burr 12.3 0.0 16.2 0.
U dy Cyclone 3 10.9 +0.2 13.4 +0.
I Determined with the Matomco moisture meter. 'Determined by the 1300 C. air-oven method. 3 A 0.5 mm. sieve was used in the mill.
are used in most protein laboratories and the Udy
cyclone mill for grinding samples for analysis by
the U dy method. The two burr mills caused very
little moisture loss on grinding wheat, even when
the moisture content of the wheat was 16.2 per
cent. The Udy cyclone mill caused considerable
moisture loss and a change in protein. For ex
ample, a sample having an initial 12.2 per cent
moisture in the wheat showed a 0.2 percentage
increase in the protein content. With the 16.2 per
cent moisture wheat, an increase of 0.5 percentage
of protein occurred due to moisture loss. Protein
changes due to moisture loss should be considered
when using the Udy mill or similar type mill.
In the grain market, wheat protein content is
expressed on an "as is" moisture basis. As already
shown, the protein content on this basis changes
with moisture content. The protein can also be
expressed on a specific moisture basis so that there
is no protein variance with moisture. Barley pro
tein in the grain market is expressed on a dry
weight basis (0 per cent moisture in sample).
Wheat and flour moisture is expressed in scientific
laboratories in the United States on a 14 per cent
moisture basis.
4. Protein Differences in Particles of Ground
Grain. The fourth factor that can affect protein
results is protein differences in particles of ground
grain (Table 6). The endosperm of grain is not
uniform in protein. In hard wheat, Kent (3) has
reported a protein content of 8 per cent in the
center portion of the endosperm to 43 per cent
near the bran layers. He also found that the flour
contained material of high and low protein con
tent. One would also expect ground grain to have
a mixture of particles that would differ in protein
content.
Moisture can also be lost during the grinding
of the wheat with resulting changes in protein.
Table 9 illustrates the moisture changes with three
mills commonly used for grinding grain for pro
tein analysis. The Labconco and Hobart burr mills
The data of Table 10 shows 3 to 4 percentage
points difference in protein content between parti
cles of hard red spring wheat when the Labconco
and Hobart burr mills are used. The U dy cyclone
mill produced particles that were found to differ
only 1 percentage point in protein content. Thus
(^6) Farm Research
Table 10. Protein Content of Particles in Ground Hard Red Spring Wheat (Waldron vari ety).
Particle size, micrometers 1 Mill Used >1,000 920 670 398 358 223 < 149
Labconco Burr Mill % Protein 16.6 15.9 14.1 13.7 14.0 14.6 17. % of grain 3.6 9.4 44.4 7.4 11.3 10.1 13. Hobart Burr Mill % Protein 16.2 15.7 14.5 13.7 13.8 14.2 16. % of grain 7.5 11.0 36.3 7.3 13.5 11.4 12. Particle size, micrometers 1 )420 358 237 ( Udy Cyclone Mill % Protein 14.5 14.8 14.6 15. % of grain 13.1 60.8 13.7 12. 1 Particles of different sizes were separated on sieves.
a sampling error can occur when weighing 1 gram of ground sample for the protein test if the right amounts of different size particles are not select ed. The ground sample should be thoroughly mixed before weighing to avoid this type of error.
- Laboratory Techniques. Poor laboratory techniques can cause protein variance in the Kjel dahl method. These variances can be reduced to a minimum by properly trained and experienced technicians and by a continuous check on the ac curacy of the determination. An analysis should be made on check samples each day. Chemical stan dardization of each new solution or reagent, and frequent checks on the accuracy of instruments and glassware should also be made.
(Jarnagin ... from Page 2) is true of most scientific research reporting, pro vided that it is accurate and more or less complete. This is not something that has been "granted" to experiment stations, but has been earned over the years through the witness of experience and satis fied customers-the farmers and ranchers who ultimately use the information.
Information from the Experiment Station is believable and believed. It has withstood the ex treme tests of time, experimentation and practical application. It has successfully resisted the efforts of unscientific and non-scientific disbelievers to dent its armor of credibility.
But, we must remember that Experiment Sta tions are composed of people, too. And they suffer through all of the interpersonal communications problems that the rest of us do, too. The principal difference in their messages as reliable sources of information lies in their use of the methods of science in their search for truth. Once their methods uncover new evidence ann new "facts" that indicate what they believe is so, then the
Conclusions
Protein analysis of grain is a complex proce dure with many steps where errors can occur. A sampling error can be introduced prior to analysis when sampling the original grain, when an aliquot of the original sample is obtained for grinding, and when weighing the ground grain for the pro tein test. A change in moisture content of the grain before grinding or during grinding can also introduce an error prior to analysis. High or low protein dockage material in grain can cause a pro tein variance. Therefore, this material should be removed prior to grinding. Many factors can cause errors during the chemical analysis by the Kjel dahl method. To keep these to a minimum, techni cians should be properly trained, and there should be frequent checks made on instruments and glass ware for accuracy. Checks should also be made on all new solutions and chemical reagents used. Check samples should be analyzed daily to test the overall accuracy of the determination. Even with the best technician and equipment, a few results can still vary from the true or correct value.
Literature Cited
- Williams, P. C. 1974. Errors in protein testing and their consequences. Cereal Sci. Today 19:280-282, 286.
- Williams, P. C. 1975. Application of near infrared reo flectance spectroscopy to analysis of cereal grains and oilseeds. Cereal Chern. 52:561-576.
- Kent, N. L. 1966. Subaleurone endosperm cells of high protein content. Cereal Chern. 43:585-601.
communication problem becomes one of penetrat ing the perceptive shields of their intended audi ences with believeable and understandable inter pretations of that evidence.
In this task, they have the support of a group of skilled interpersonal communication craftsmen whose business it is to help create messages that can attract attention and have meaning for those who can make use of that information. This is a fascinating business. The challenges we constantly meet to use all of the skills and techniques at our command to compose effective messages keeps our work filled with interest and variability.
It is the relative success of our working to gether to overcome these challenges that has brought about the large measure of credibility and believability that comes along with information based on Experiment Station research. Our future success lies in our ability to keep up with the new human communication technologies and use them to their fullest as our society continuously expands its interests in and uses of interpersonal communi cation.
May-June, (^1977 )
