Food Protein Analysis

FOOD PROTEIN ANALYSIS
Quantitative Effects on Processing
R.K.Owusu-Apenten The Pennsylvania State University

University Park, Pennsylvania
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FOOD SCIENCE AND TECHNOLOGY
A Series of Monographs, Textbooks, and Reference
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EDITORIAL BOARD
Senior Editors
Owen R.Fennema University of Wisconsin-

Madison
Y.H.Hui Science Technology System
Marcus Karel Rutgers University (emeritus)
Pieter Walstra Wageningen University
John R.Whitaker University of California-Davis
Additives P.Michael Davidson University of Tennessee-Knoxville
Dairy science James L.Steele University of Wisconsin-Madison
Flavor chemistry and sensory analysis John H.Thorngate III University of California-
Davis
Food engineering Daryl B.Lund University of Wisconsin-Madison
Food proteins/food chemistry Rickey Y.Yada University of Guelph
Health and disease Seppo Salminen University of Turku, Finland
Nutrition and nutraceuticals Mark Dreher Mead Johnson Nutritionals

Phase transition/food microstructure Richard W.Hartel University of
WisconsinMadison
Processing and preservation Gustavo V.Barbosa-Cánovas Washington State

University-Pullman
Safety and toxicology Sanford Miller University of Texas-Austin

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For Mum and Dad, Elizabeth, James, Richard, Candida, and Afia
Preface
There is no book dealing with food protein analysis exclusively, that is, with the analysis
of proteins in the food system. This books attempts to fill this niche. Protein analysis
comes in two forms: 1) Quantitative analysis, and 2) fractionation and characterization.
The first activity is described here. This publication provides a reference for planning,
performing and interpreting assays for food proteins. Many approved methods derive
from the late-19th century, but they have undergone rigorous testing and modernization.
This book does not focus on reviewing the latest research methods for protein analysis.
With the exceptions of Chapters 6 and 7, each of the 14 self-contained chapters describes
one protein assay—principles, practices, and expected results.
This book describes the effect of food processing on protein assay results with the
emphasis on how to analyze proteins in real foods. A number of “Methods” sections
provide instructions for specific tests. Sample pretreatment and clean-up procedures are
described. General pretreatment strategies help in the avoidance of interference. More
specific clean-up methods apply to particular protein assays and are described along with
these. Example results, performance characteristics, case reports, and practical problems
and solutions related to a wide range of foods are detailed in numerous figures, tables,
and references.
Food protein analysis is a hugely important activity performed by thousands
worldwide. The book should appeal to professionals interested in food proteins and
anyone working in the food system formerly called the food chain. This includes
researchers and workers in agricultural production, food processing, and wholesale and/or
retail marketing. It provides information for the grain or dairy farmer, extension worker,
agricultural scientist, food scientists and technologists, or college professor. Some
techniques described in this book were first used by clinicians, nutritionists, and
veterinary scientists. The book may also be of interest to those in small businesses,
private or government laboratories, research institutes, colleges, and universities. It will
be useful to undergraduate, postgraduate, or postdoctoral students. Sections dealing with
mechanisms assume graduate level chemistry and/or analytical biochemistry.
Any shortcomings of this project are wholly my responsibility. I thank all those
colleagues worldwide whose research is reported here. My thanks to Anna Dolezal, Mr.
DeSouza and Professor Arthur Finch for teaching me to think for myself. I am grateful to
my past students: Drs. Yetunde Folawiyo, Despina Galani, Michael Anaydiegwu,
Kiattisak Duangmal, Pitaya Adulyatham, Kwanele Mdluli, Halima Omar and Sripaarna
Banerjee for raising my awareness of protein assay issues and for reading parts of the
manuscript. Thanks to Dr. Bob Roberts (The Pennsylvania State University) for his
advice on combustion methods. I am grateful to Dr. S. Khokhar and Marcel Dekker, Inc.,
for their commitment. I am also grateful to my family for their support.
R.K.Owusu-Apenten
Contents
Preface xi
Part 1. Fundamental Techniques
Chapter 1. Kjeldahl Method, Quantitative Amino Acid Analysis and 1

Combustion Analysis
Part 2. Copper Binding Methods
Chapter 2. The Alkaline Copper Reagent: Biuret Assay 43

Chapter 3. The Lowry Method 62
Chapter 4. The Bicinchoninic Acid Protein Assay 88
Part 3. Dye Binding Methods
Chapter 5. The Udy Method 109

Chapter 6. The Bradford Method—Principles 150
Chapter 7. Bradford Assay—Applications 173
Part 4. Immunological Methods for Protein Speciation
Chapter 8. Immunological Assay: General Principles and the Agar Diffusion 196
Assay
Chapter 9. Speciation of Meat Proteins by Enzyme-Linked Immunosorbent 218
Assay
Chapter 10. Speciation of Soya Protein by Enzyme-Linked Immunoassay 248
Chapter 11. Determination of Trace Protein Allergens in Foods 262

Part 5. Protein Nutrient Value
Chapter 12.Biological and Chemical Tests for Protein Nutrient Value 299
Chapter 13.Effect of Processing on Protein Nutrient Value 333
Chapter14.Protein Digestibility-Corrected Amino Acid Scores 358
Index 389
1
Kjeldahl Method, Quantitative Amino Acid
Analysis and Combustion Analysis
1. INTRODUCTION TO FOOD PROTEIN ANALYSES
Protein analysis is a subject of enormous economic and social interest. The market value
of the major agricultural commodities (cereal grains, legumes, flour, oilseeds, milk,
livestock feeds) is determined partly by their protein content. Protein quantitative analysis
is necessary for quality control and is a prerequisite for accurate food labeling. Proteins
from different sources have varying aesthetic appeal to the consumer. Compliance with
religious dietary restrictions means excluding certain protein (sources) from the diet. The
variety of protein consumed is also extremely important in relation to food allergy.
Detecting undeclared protein additives and substitutions is a growing problem. Proteins
show differing nutritional quality or ability to support dietary needs. In summary, protein
analysis has legal, nutritional, health, safety, and economic implications for the food
industry (1).
The estimated global food production total for 1988 was 4 billion metric tons.
Allowing an average of 10% protein in foodstuffs yields 400 million metric tons of
protein annually (2). Nonetheless, sensitivity is a major consideration for protein analysts.
Some immunological methods can detect nanomole (10−9 mole) amounts of protein.
Other important considerations when choosing a method for food protein analysis include
TABLE 1 Approximate Chronology for Methods
for Food Protein Analysis
Date Technique
1831 Dumasa
1843 Nessler’s reagenta
1849 Biuret method
1859 Alkali-phenol reagent or Bethelot’s methoda
1883 Kjeldahla
1927 Folin-Ciocalteau
1944 Dye bindinga
1951 Lowry
1960 Direct alkaline distillation
1960 Near-infrared reflectance (NIR)a
Food protein analysis 2
1971 Modified Berthelot reaction

1975 Modified Lowry method (Peterson)
1976 Bradford method (Coomassie Blue binding method)
1985 Bicinchoninic acid (BCA) method
a
Techniques for which semiautomated or fully automated apparatus has been manufactured.
high sample throughput, simplicity, and low capital costs. Some of the most significant
methods (Dumas, Kjeldahl, and biuret assays) date from the late 1800s (Table 1).
Techniques for food protein analysis are described in this book. I will focus on the
techniques that feature most often in the food science literature. Infrared analysis of food
proteins is not discussed here.
1.1. Characteristics of Food Protein Assays

Techniques for food protein analysis need to be robust. This means one of several things.
Foremost is compatibility with fresh produce (cereals, fruits, vegetables, meat, milk) and
processed foods. Samples in various physical states (powders, slurries, dilute liquids,
emulsions, gels, pastes) should be analyzable. A robust assay will also deal effectively
with foods from either animal or plant sources. Such techniques are unaffected by the
presence of dyes or pigments that absorb infrared, visible, or ultraviolet light. A robust
protein assay needs mimimal sample pretreatment, which increases error and decrease
analytical precision. Sample cleanup also increases the time per analysis (reduces sample
throughput) and adds to costs. In the worst-case scenario, pretreatment can be too
invasive, thereby invalidating results. In summary, a robust protein assay is simple,
quick, sensitive, and reliable. It is also compatible with a diverse range of foods. The
economic imperative leads to a preference for techniques requiring low capital
expenditure and minimum training. Laboratories handling more than 8000 analyses per
year tend to select techniques on the basis of their speed and ease of operation. A high
sample throughput is usually achieved by automation or continuous flow analysis (CFA).
A rough “time line” for some food protein assays is given in Table 1. Common
descriptive terms for protein analysis are defined in Table 2.
Kjeldahl analysis gives accurate protein readings no matter what the physical state of
the sample. This technique has approved status and is the reference method adopted by
many national and international organizations. However, the use of hazardous and
potentially toxic chemicals in Kjeldahl analysis is creating concern. The Dumas
combustion method is comparatively quicker, cheaper, easier to perform, safer, and more
environment friendly; it is now considered on equal terms with Kjeldahl analysis in the
United States, Canada, and Western Europe. Dye binding is another robust test for
proteins (3,4). The biuret method is widely used,
Kjeldahl method, quantitative amino acid analysis and combustion analysis 3
TABLE 2 Some Important Calibration Indices and

a Brief Explanation of Their Meaning
Calibration feature Explanation
Linear dynamic range Range over which signal is proportional to analyte concentration
Sensitivity Slope of the calibration graph; analytical response per unit change in
protein concentration. cf. parameters a, a′ in Eqs. (1)–(4)
Accuracy Degree of agreement of results with a true value
Precision, repeatability, or Agreement between repeated measurements taken with a single
reproducibility sample or with different paired samples
Specificity Ability to discriminate between protein and interfering substance.
Ratio of sensitivity for the analyte and interference
Reliability A composite parameter combining specificity, accuracy, precision,
and sensitivity
Lower limit of detection Minimal protein concentration detectable above background noise
(LLD)
Sample throughput (time Numbers of samples analyzed per unit time, speed of analysis
per analysis)
especially for cereal proteins (5). Procedures involving copper-based reagents (Lowry
and bicinchoninic acid assays) continue to be important. Finally, a range of empirical
(viscosity, refractive index, specific gravity) measurements are used for protein
quantitation within industry.
1.2. Calibration and Statistical Principles

The two common forms of calibration are (a) method calibration and (b) sample
calibrations. With method calibration a set of food samples are analyzed using a new test
method and a reference method that has been validated by a committee of the Association
of Official Analytical Chemists (AOAC). A calibration graph is then drawn by plotting
results from the reference method (% Kjeldahl protein) on the Y-axis and the test results
on the X-axis. The Xi and Yi observations are usually related by an equation for a straight
line:
Yi=aXi+b
(1)
where a is the gradient and b is the intercept for the calibration graph. For each Xi result
we can determine the calculated % Kjeldahl protein value (Ycalc) via Eq. (2).
Ycalc=aXi+b
(2)
Values for Yi and Ycalc can be compared in order to evaluate the test
method(see later). Some investigators choose to plot the Kjeldahl results
on the X-axis. Therefore, rather than Eq. (1) we get
(3)
where Xi* is % Kjeldahl protein and Yi* is the test result. To compare Eq. (1) and Eq. (3),
notice that a′=1/a and b′=Yi−(Xi / a).
For sample calibration, the assay technique is assumed to be valid. We analyze a set of
(standard) samples containing known amounts of protein. In Eq. (1), Xi now represents a
range of known protein concentrations and Yi are the corresponding instrument responses.
Calibration factors (a, b, etc.) can be determined from simple algebra or statistical
analysis of paired (Xi, Yi) results. From the principles of least-squares analysis,
(4)
and
b=Ym−aXm
(5)
where Xm and Ym are the mean values for all Xi and Yi observations.
Agreement between the reference and test results is measured by the correlation
coefficient (R); R≈1 shows excellent agreement. When Yi and Ycalc observations are
poorly correlated, R≈0. The squared correlation coefficient (R2) can be calculated from
Eq. (6). Most handheld calculators can perform this operation automatically.
(6)
Precision is another measure of the (dis)agreement between Yi and Ycalc values. This can
be expressed as the standard deviation (SD) or coefficient of variation (CV). High-
precision methods produce low values for the SD and CV.
(7)
CV=(SD/Ym)×100
(8)
We can also measure precision (commonly called error) from n-replicate (Yi)
measurements on a single test sample. Thereafter, the numerator in Eq. (7) becomes
(Yi−Ym)2, which is the square of the differences between individual observations and the
mean for all observations. A low CV implies good agreement between successive test
results.
1.3. Assay Performance

Calibration parameters can provide a great deal of other information about assay
performance (Table 2). The linear dynamic range is the concentration range over which a
linear relationship exists between the instrumental response and protein concentration.
Sensitivity is the slope of the calibration graph, and the lower limit of detection (LLD) is
smallest quantity of sample that triggers an instrumental response above the background
noise. The LLD is dependent on the instrument baseline quality and assay sensitivity.
It is common to refer to “sensitivity” when we mean the LLD. We differentiate
between sensitivity and LLD via the following exercise. Measure the instrument baseline
noise by recording the output (Yo) and the standard deviation (SDo) using a sample blank.
The smallest instrumental response that can be distinguished from “random noise” in
95% of all cases is Yo±2.326SDo. Now substitute for Yi (=Yo+2.326 SDo) and Xi (=LLD)
in Eq. (1), leading to the following expression:
(9)
Usually Yo and b are both set to zero when the analyst sets the instrument baseline
response to zero. Consequently, Eq. (9) becomes
LLD=2.326SDo/a
(10)
This relation shows that LLD decreases with increasing assay sensitivity and with
increasing baseline quality (see decrease in the value for SDo). In order to ensure high
sensitivity, it is important to obtain a stable instrumental baseline.
1.4. Calibrating Protein Assays

The Kjeldahl method is used for calibrating other protein assays. Duda and Szot (6)
evaluated six methods for analyzing porcine plasma protein during its manufacture. The
techniques are simple and therefore of wider interest (Table 3). The protein content of
porcine plasma was 5.58% (w/v). All techniques showed a good correlation with Kjeldahl
results (R=0.905− 0.952). The precision for density and Kjeldahl assays was the same
(CV=10.8%). The sensitivity of the former method was better. With appropriate
calibration, density or viscosity measurements could be suitable for the routine analysis
during the manufacture of plasma proteins.
TABLE 3 Some Simple Methods for Evaluating
Porcine Plasma Protein
Method Instrument
Densitometry Standard picnometer
Refractometry Laboratory refractometer

Modified refractometry Laboratory refractometer
UV absorbance (215/225 nm) UV spectrophotometer
UV absorbance (241 nm) UV spectrophotometer
UV absorbance (280 nm) UV spectrophotometer
Williams et al. (7) calibrated beer protein analyses using quantitative sodium dodecyl
sulfate polyacrylamide gel electrophoresis (QSDS-PAGE). A range of test methods were
investigated including biuret, bicinchoninic acid (BCA), Bradford, Kjeldahl, Lowry, and
pyrogallol-red molybdate (PRM) assays. QSDS-PAGE revealed that beer has between
0.5 and 1mgmL−1 protein. Only the Bradford and PRM assays gave accurate results (Fig.
1). The main sources of error were low-molecular-weight interferences. Beer contains
plant pigments, starch, sugars, alcohol, and natural dyes of barley origin. Both Kjeldahl
and combustion analyses were subject to interferences by nonprotein nitrogenous (NPN)
compounds. Dialysis did not improve accuracy for BCA, Lowry, and biuret assays, which
were affected by high-molecular-weight Cu- reducing agents such as pectin and starch.
Calibration issues are discussed in two articles by Pomeranz and co-workers (8,9).
They considered the reliability of several test methods (biuret, dye binding, infrared
reflectance, alkaline distillation method) for analyzing proteins in hard red winter wheat
varieties from the American Great Plains. The test methods were highly correlated with
the Kjeldahl assay (R=0.976− 0.992). The order of precision was Kjeldahl > biuret > dye
binding > infrared analysis. Pomeranz and More (9) also considered the reliability of four
“rapid” methods for barley or malt protein analysis.* A summary of assay performance
statistics is given in Table 4. For barley samples, the precision and sensitivity of analysis
were highest for the Kjeldahl and infrared analyses. The use of Kjeldhal analysis to
calibrate protein assays for dairy products was discussed by Luithi-Pent and Puhan (10)
and also Lynch and Barbano (11).
2. KJELDAHL ANALYSIS
Johan Kjeldahl was born on August 16, 1849 in the town of Jaegerpris in Denmark. In
1876 he was employed by the Carlsberg brewery to develop an improved assay for grain
protein. The Kjeldahl method was published in 1883. The original technique has been
extensively modified. Key steps for the assay are (a) sample digestion, (b) neutralization,
(c) distillation and trapping of ammonia, and (d) titration with standard acid. An
exhaustive
*For the purposes of calibration, 44 samples of barley and 49 samples of malt were analyzed with
biuret, dye binding, infrared, alkaline distillation, and Kjeldahl tests. Such results were the basis for
deriving calibration relations between Kjeldahl and each test method. Then a further 76 samples of
barley and 72 samples of malt were analyzed using only the rapid test methods. Each Xi result gave
rise to a corresponding Kjeldahl protein (Ycalc) value.
FIGURE 1 Apparent protein

concentrations in stout beer as
determined by seven methods. (Data
from Ref. 7.)
TABLE 4 Barley Protein Analysis Using a Range
of Techniques
Test method Analysis time Regression line and correlation Standard error of
(min) coefficienta analysisb
Biuret 10 Y= 0.857 cP + 1.942 R=0.972 0.336 and 0.2336
Dye bindingc 15
Infrared 0.5–1.0 Y=1.060 cP −1.03 R=0.96 0.838–1.980
Alkaline 90 Y=1. 070 cP −0.670 2.383 and 1.540

distillation
a
cP, crude protein determined by Kjeldhal method (N×6.25); Y, response from the test method.
b
Assay standard error calculated from
c
No information given.
Source: Ref. 8.
account of the Kjeldahl method can be found in the monograph by Bradstreet (12). The
book is divided into five chapters: 1, introduction to the Kjeldahl method; 2, the Kjeldahl
digestion; 3, digestion procedure (for fertilizers, leather, cereals, foods and proteins, coal
and fuels); 4, the distillation and detection of ammonia. Chapter 5 is an extensive
bibliography. A standardized Kjeldahl procedure appears in the International Standard
ISO-1871 (13). Further descriptions are given by Gaspar (14) and Osborne (15).
Initially, only sulfuric acid was used for sample digestion. Then solid potassium
permanganate was added to facilitate sample oxidation. Mercuric oxide was introduced as
a catalyst in 1885. During the acid digestion phase, the food sample is heated with
concentrated sulfuric acid, which causes dehydration and charring. Above a sample
decomposition temperature, carbon, sulfur, hydrogen, and nitrogen are converted to
carbon dioxide, sulfur dioxide, water, and ammonium sulfate [Eq. (11)].
NH2(CH2)p COOH+(q+1) H2SO4→(p+1)
CO2+q(SO2)+4p(H2O)+NH4HSO4 (11)
Digestion is complete when the mixture turns clear (light green color), usually after 20–
30 minutes of heating. A further (after-boiling) period of heating is necessary to ensure
quantitative recovery of nitrogen. Data from McKenzie and Wallace (cited in Ref. 14)
show that adding X (mg) of potassium sulfate per mL of sulfuric acid increases its boiling
point according to the relation Y (°C)=55.8X+ 331.2. A maximum boiling point elevation
of 130°C is achivable by adding 2mg (potassium sulfate) per mL acid. A high boiling
point reduces the sample digestion time. Sample digestion can also be facilitated by using
a catalyst; the order of effectiveness for metal oxide catalysts is Hg > Se > Te > Ti > Mo
> Fe > Cu > V > W > Ag (16). A proprietary brand of Kjeldahl catalyst (Kjeltabs from
Foss Electric Ltd.) comes as tablets. Each tablet contains 0.25g of mercuric oxide and 5g
of potassium sulfate. A working selenium catalyst can be formulated with potassium
sulfate (32 g), mercuric sulfate (5g), and selenium powder (1 g). Chemical oxidants
(hydrogen peroxide, perchloric acid, or chromic acid) can be added to the sulfuric acid to
speed up sample digestion.
Ammonium sulfate is first neutralized with alkali to form ammonia. This is then
distilled and trapped using 4% boric acid. Ammonium borate is then titrated with
standard acid in the presence of a suitable indicator. Low-cost Quick-fit glassware is
readily available for distillation and titration. Sophisticated semiautomatic distillation
systems are also available. The processes of neutralization, distillation, and titrimetric
analyses are summarized as follows.
(12)
(13)
(14)
Suitable titration indicators include methyl orange, methyl red, Congo red,and Tashiro
indicator (a 1:1 mixture of 0.2% methyl red solution and 0.1%methylene blue).
2.1. Nitrogen-to-Protein Conversion Factors

The Kjeldahl technique measures sample nitrogen (SN) as ammonia. The value for SN is
later converted to crude protein (cP) by multiplying by a Kjeldahl factor, FK.
cP(%)=SNFK
(15)
The units for SN are g-N 100g−1 (g-nitrogen released per 100g of sample). The Fk (g-
proteing−1 N) is the amount of protein that produces a gram of nitrogen. Fk is also called
the nitrogen-to-protein conversion factor. AOAC-recommended values for FK for meat
and other food are summarized by Benedict (17). Frequently, FK is given a default value
of 6.25 or 5.7 for animal and plant proteins, which are assumed to have an average N
content of 16% and 17.5%, respectively. In fact, most proteins deviate significantly from
these averages (18). FK is also affected by the presence of NPN (e.g., adenine, ammonia,
choline, betaine, guanidine, nucleic acid, urea, free amino acids). Soya beans have 3–10%
NPN, which increases to about 30% for immature seeds. The amount of NPN also
changes with growth conditions as well as with geographic factors. There is generally no
correlation between NPN and protein content (19). No single FK value applies to all food
types. Ideally, FK should be determined for each individual food type (Table 5).
FK can be calculated from amino acid data (18,20–24). Table 5 lists the 20 naturally
occurring amino acids along with their formula weight, number of nitrogen atoms,
percent nitrogen, and the value for FK. For arginine, FK is 3.11 (=100/32.15). An
idealized protein having all 20 amino acids in equal numbers has a nitrogen content of
14.73%. The FK value is therefore 6.79 (100g/14.73). Evaluating FK from amino acid
data (for
TABLE 5 Determination of the Kjeldahl Factor for
Milk Protein Using Amino Acid Composition Dataa
1 Amino 2 N ato 3 4 5 Am 6 AA AA- 7 8 AA 9 Mole 10 biX
Acid Formula ms % N ino compo N (moles/g frac
weight Acid sition (mg/g N) tion (Xi)
(bi) FK (mg/g N)
N)
Arginine 174.2 4 32.15 3.11 234.0 75.2 0.001343 0.027082 4.71761
Histidine 155.2 3 27.06 3.70 188.0 50.9 0.001211 0.024422 3.79022
Asparagine 132.1 2 21.20 4.72 292 61.9 0.00221 0.044564 5.88694
Glutamine 146.1 2 19.16 5.22 783 150.1 0.005359 0.108048 15.7859
Lysine 146.2 2 19.15 5.22 487.0 93.3 0.003331 0.067157 9.81828
Glycine 75.1 18.64 5.36 128.0 23.9 0.001704 0.034362 2.58058
Alanine 89.1 15.71 6.36 219.0 34.4 0.002458 0.049553 4.4152
Tryptophan 204.2 2 13.71 7.29 90.0 12.3 0.000441 0.008886 1.81447
Serine 105.1 13.32 7.51 338.0 45.0 0.003216 0.064837 6.81433
Proline 115.1 12.16 8.22 571.0 69.5 0.004961 0.100016 11.5118
Valine 117.1 11.96 8.36 428.0 51.2 0.003655 0.073687 8.6288

Threonine 119.1 11.75 8.51 278.0 32.7 0.002334 0.047059 5.60469
Cystine 121.1 11.56 8.65 47.0 5.4 0.000388 0.007825 0.94756
Isoleucine 131.2 10.67 9.37 290.0 30.9 0.00221 0.044563 5.84662
Leucine 131.2 10.67 9.37 600.0 64.0 0.004573 0.092199 12.0964
Aspartic acid 133.1 10.52 9.51 214.0 22.5 0.001608 0.032415 4.3144
Glutamicacid 147.1 9.52 10.51 574.0 54.6 0.003902 0.078669 11.5723
Methionine 149.2 9.38 10.66 148.0 13.9 0.000992 0.019999 2.98379
Phenylalanine 165.2 8.47 11.80 341.0 28.9 0.002064 0.041615 6.87481
Tyrosine 181.2 1 7.73 12.94 297.0 22.9 0.001639 0.033045 5.98774
Sum= 5669.7 943.5 0.04960 1.0 132.0
Average 14.73
=
FK(1)= 6.79 FK= 6.01
a
FK(1) is determined from the average nitrogen content for all amino acids, 14.73%. FK(2)=value of
whole protein or sum of amino acid nitrogen divided by sum of weight of nitrogen.
Fk=5669.7/943.5. Columns 8−10 contain data for calculating total protein content from
quantitative amino acid analysis (Sec. 4).
skimmed milk) can be achieved in the following steps: (a) express each amino acid as mg
per gram of total nitrogen (see column 6 of Table 5) and (b) calculate the mass of
nitrogen derived from each amino acid (column 7 in Table 5). FK is the weight of amino
acids divided by the weight of amino acid nitrogen (AA-N).
(16)
Some typical values for FK are listed in Table 6 for a range of foods. The use of FK values
for quantitative amino acid analysis is discussed in Sec. 4.
TABLE 6 Nitrogen-Protein Conversion (Fk)
Factors for Selected Food Protein Sources
Food product Fk Food product Fk
Dairy products and egg Roots and tuber
Casein 6.15 Carrot 5.80
Milk 6.02 Beet 5.27
Cheese 6.13 Potato 5.18
Egg 5.73 Potato protein 5.94
Egg white solids 5.96

Meat and fish products Fruit
Beef 5.72 Tomato 6.26
Chicken 5.82 Banana 5.32
Fish 5.82 Apple 5.72
Leafy vegetables Microbial and fungal
Lettuce 5.14 Yeast 5.78
Cabbage 5.30 Mushrooms 5.61
Cereals and legumes
Wheat 5.71–5.75 Buckwheat 5.53
Rice 5.61–5.64 Oats 5.50
Corn 5.72 Millet 5.68
Sorghum 5.93 Mustard seed 5.40
Field pea 5.40 Rapeseed meal 5.53
Dry bean 5.44 Sunflower meal 5.36
Soya bean 5.69 Flax meal 5.41
Source: Data from Refs. 18 and 24.
2.2. Macro- and Micro-Kjeldahl Analysis

Kjeldahl digestion methods are discussed in this section. Illustrative examples are given
to establish a pattern of work. Individual results are described in later parts of the chapter.
A. Grain and Cereals

Kaul and Sharma (25) analyzed a range of legume and cereal grains by micro-Kjeldahl
analysis. About 200 mg of each sample was weighed into several 75-mL Kjeldahl
digestion tubes. Concentrated sulfuric acid (3 mL), hydrogen peroxide (1.5mL), and one
Kjeltab tablet were added. The tubes were heated using a Tectator digestion block at
374°C for exactly 25 minutes and then allowed to cool. The contents of each tube were
diluted to 75 mL and any ammonia produced quantified by colorimetric analysis (Sec. 3).
B. Potatoes
Mohyuddin and Mazza (26) analyzed proteins from 14 potato cultivars. Potato tubers
were peeled, sliced, diced, and dried in a vacuum oven at 70°C and 48.8 mm Hg pressure.
Each sample was milled and sieved through a 40-mesh sieve. Potato flour (100mg) was
added to each 100-mL Kjeldahl flask, followed by concentrated sulfuric acid (3mL),
hydrogen peroxide (30% solution; 1.5mL), and commercial catalyst (500 mg; 10:0.7 w/w
ratio of potassium sulfate and mercuric oxide). Heating for 45 minutes digested the
samples. After cooling to room temperature, the contents of Kjeldahl flasks were diluted
to 75 mL and then subjected to colorimetric analysis to determine ammonia.
C. Dried Milk Powder

Venter et al. (27) described a semimicro-Kjeldahl analysis for low-fat, medium-fat, and
high-fat dried milk. About 200–250 mg of sample was mixed with 2.1 g of selenium
catalyst and digested by heating with 10mL of concentrated sulfuric acid. The digest was
cooled and diluted to 100 mL with distilled water. Then 60 mL of 45% (w/v) NaOH
solution was added and the liberated ammonia was distilled into 20 mL of 4% (w/v) boric
acid solution. Titration was with 0.02 M HCl to an end point of pH 4.8. The results
agreed well with the macro-Kjeldahl method [International IDF Standard (1962) No. 20].
D. Beer Protein
Concentrated sulfuric acid (2mL) was added to 50 mL of beer (bitter, lager, or stout) and
the mixture was heated until nearly dry (7). Kjeldahl catalyst (10g) and more sulfuric acid
(20mL) were added, followed by further heating for 25 minutes. After cooling for 2
hours, water (250mL) was added and the Kjeldahl flask was connected to a condenser
with one end immersed in a 2% boric acid solution (200mL). Bromocresol green was
used as indicator. Sodium hydroxide (10M, 70mL) was added, followed by heating until
the distillate tested neutral. The borate solution was later titrated with 0.1N HCl. The
nitrogen content in beer was calculated from the relation
%N=14Va/Wd
(17)
where Va (mL) is the volume of HCl required to neutralize ammonia and Wd (g) is the dry
weight of beer. Table 7 summarizes characteristics of the Kjeldahl method used in the
brewing and allied industries (28).
TABLE 7 Macro-Kjeldahl Procedures Currently in
Use in the Brewing and Allied Industries
Parameter Commenta
Instrumentationb Kjel-Tec 1030 (Auto), Kjel-Tec 1007/ 1002*; Kjel-Foss 16120†
Sample weight 0.92 ± 0.23 g(barley or malt) range 0.5–1.5 g, 14 ± 8.4 mL (beer) range
5–25 mL
Catalyst (weight range)c K2SO4+CuSO4+TiO2; 3.5–15.8 g
Volume of conc. H2SO4 16 ± 4.3 mL, range 10–25 mL
d
Digestion temperature 411±14°C, range 380–430°C
Digestion timee 75 min or 9.6 min
End-point detection Mainly colorimetric
a
Average values unless otherwise stated.
b
Local suppliers * Tectator Ltd. and Perstop Ltd., both of Bristol, UK, † Foss Electric (UK) Ltd.,
The Chantry, Bishopthorpe, York, UK.
C
A large amount of a single catalyst or a smaller quantity of a combination catalysts was used. HgO
was used in 2 of the 25 laboratories.
d
Digestion temperatures were not reported for seven laboratories using a manual Kjeldahl
technique.
e
Digestion time plus after-boiling time. The digestion time is 9.6 min when H2O2 is used as a
prooxidant.
Source: Summarized from Ref. 28.
2.3. Automated Kjeldahl Analysis

Kjeldahl analysis has undergone three forms of automation. The Kjel-Foss® instrument
mechanizes the entire micro-Kjeldahl procedure (digestion, neutralization, distillation,
and titration). The Kjel-Tec® technique uses a digestion block in conjunction with
apparatus for automated distillation and titrimetric analysis. The final form of automation
is the Technicon AutoAnalyzer Instrument®, which uses continuous flow analysis (CFA).
A. The Kjel-Foss Instrument

The Kjel-Foss instrument (N.Foss Electric Ltd., Hillerød, Denmark) performs the entire
Kjeldahl procedure automatically (29–31). Automation reduces the analysis time from 3
hours to 6 minutes. The first analysis is completed in 12 minutes and succeeding analyses
every 3 minutes. The sample throughput is 120–160 analyses per day. The Kjel-Foss
instrument requires a reliable supply of electricity and tap water for installation and
adequate drains and ventilation. A fume cupboard is not essential. Accuracy, precision,
and economics of the Kjel-Foss method were compared with those of the manual
Kjeldahl method, neutron activation analysis, proton activation analysis, combustion
analysis, and the Kjel-Tech method (32). Results of the Kjel-Foss and manual Kjeldahl
methods were highly correlated.
Fish meal was analyzed using the Kjel-Foss instrument by Bjarno (33). Seven
collaborators compared the efficiency of antimony versus mercuric oxide as catalyst.
After modifying the Kjel-Foss procedure slightly with higher acid settings, the
differences in recovery and repeatability of the two procedures were <±1%. Using
mercuric oxide catalyst poses environmental concerns if the effluent from the Kjel-Foss
instrument is to be disposed of through the sewers. McGill (34) compared the Kjel-Foss
method with an improved AOAC Kjeldahl method for meat and meat products. Over 80
analyses were performed with low (25%) fat, high (40%) fat, and dry sausage (50% fat).
As shown in Fig. 2 and Table 8, the two techniques were highly correlated
(Y=0.9904X+0.1797; R2=0.9896). There was no systematic error in the Kjel-Foss
technique over the range of protein concentrations examined by McGill. This work
validated the Kjel-Foss instrument for meat product analysis.
Suhre et al. (35) also evaluated the Kjel-Foss instrument for meat analysis using the
AOAC Kjeldahl method as reference. Twenty-three different laboratories analyzed six
meat products having 10–30% crude protein. Eight laboratories used the automated Kjel-
Foss instrument, five used the official AOAC method, and eleven used a block digester
with steam
FIGURE 2 Calibration graph for the

Kjel-Foss automated method for
protein determinations. (Data from
Table 1 of Ref. 34.)
distillation. Recommendations from this work led to the block digester-steam distillation
method being awarded “first action” status.
B. Automated Kjeldahl Continuous Flow Analysis

The Technicon AutoAnalyzer has two reaction modules (36). The first module digests
water-dispersible samples. The digest is then pumped to a second module (AutoAnalyzer
Sampler II). Colorigenic reagents are added in quick succession before the flow stream
passes to a delay coil to allow color formation. Ammonia is detected using Berthelot’s
reaction or the ninhydrin assay (Sec. 3). The AutoAnalyzer was applied for protein
determinations in plant material (37), feedstuffs (38), grain flour (39), instant breakfasts,
meat analogues (40), meat products (41), and over 40 assorted canned and processed
foods (42,43). In general AutoAnalyzer results agreed with micro-Kjeldahl analysis.
The AutoAnalyzer digestion unit is heated in two stages at 380–400°C and 300–
320°C. To achieve efficient digestion, the ratio of acid to sample is
TABLE 8 A Comparison of the Automated Kjel-

Foss and Approved Kjeldahl Method for Protein
Analysis in Sausage Samples
Sausage type Kjel-Foss (% protein) Kjeldahl (% protein)
Low-fat sausages
Bologna 14.64 14.54
Polish 14.42 14.43
Krakow 16.76 16.60
Liver 16.64 16.74
Hungarian 16.01 16.02
Medium-fat sausages
Breakfast 14.40 14.52
Italian 14.77 14.80
Pizza 17.42 17.33
Pork 14.09 14.21
Dry sausages
Pepperoni 14.33 14.41
Summer 18.87 18.86
Source: Ref. 34.
higher than normal for batch digestion. A superheated layer of acid forms, which
facilitates sample digestion (44). Later tests showed that the recovery of nitrogen from
refractory materials (arginine, creatine, or nicotinic acid) was only 70%. Davidson, et al.
(45) concluded that the AutoAnalyzer digestion module was not reliable if an accuracy of
1% was desired for Kjeldahl analysis. Over 70 different animal feeds (corn grain, wheat,
barley, rice, alfalfa, mixed feeds, feed concentrates) were analyzed using the
AutoAnalyzer digestion module. The recovery of nitrogen was 88–90% (46). In contrast,
using a Technicon block digest or followed by AutoAnalyzer Sampler II led to 100%
recovery of nitrogen from cattle supplement, swine ration, pig starter, and poultry ration
(47). Suitable catalysts include copper sulfate and oxides of mercury, selenium, or
titanium. Ammonia was detected using alkaline phenol reagent (Sec. 3.1). Quantitative
recovery of nitrogen was also demonstrated by Kaul and Sharma (25), who used a
Tectator® heating block to digest 23 assorted strains of rice and 15 other cereal-legume
mixtures. The electrically heated block digests 40 samples per hour under controlled
temperature conditions. Samples were then transferred to the AutoAnalyzer Sampler II
for ammonia detection using the alkaline phenol reagent.
3. COLORIMETRIC ANALYSIS OF KJELDAHL NITROGEN
Colorimetric analysis simplifies Kjeldahl analysis and increases the sample throughput.
Other benefits include increased sensitivity and a greater potential for automation.
Reagents for colorimetric Kjeldahl-N analyses include (a) alkali-phenol reagent (APR),
also called indophenol reagent; (b) ninhydrin (indanetrione hydrate) reagent; (c) Nessler’s
reagent; and (d) acetylacetone formaldehyde reagent. These colorimetric techniques are
reviewed next.
3.1. Alkali-Phenol Reagent (Indophenol) Method

The alkali-phenol reagent is frequently used for the Technicon AutoAnalyzer. Under
alkaline conditions, ammonia, sodium hypochlorite, and phenol react to form a blue
product. Berthelot first reported this reaction in 1859. The principles of the APR assay
have been reviewed (48–51) although the underlying reactions remain uncertain.
Ammonia probably reacts with hypochlorite to form chloramine (NH2Cl). This reacts
with phenol to form N-chloro-p-hydroxybenzoquinone monoimine or quinochloroamine
(I).
Alternatively, ammonia may first react with phenol to form p-aminophenol, which is then
oxidized by hypochlorite to form (I). Irrespective of how (I) forms, it reacts with 1 mole
of phenol to form indophenol (II). The yellow compound ionizes under strongly alkaline
conditions (pKa 13.4) giving a blue anion
Substituted phenols (o-chlorophenol, m-cresol, and guaiacol) undergo the indophenol
reaction. However, para-substituted phenols and some meta-derivatives do not react.
Color intensity and the rate of color formation increase in the presence of manganese (+2)
ions or acetone. Sodium nitroprusside (10–40 mg L−1) is another catalyst.
A simple APR assay suitable for detecting ≤3 ppm ammonia is described in Ref. 48
(Fig. 3). Indophenol formation is pH and temperature dependent. The linear dynamic
range for ammonia was 0.3–3 ppm with a sensitivity of 0.3284 (absorbance units/1 ppm
NH3). The assay precision (for 1 ppm NH3) was ±3%. Although performed with boric
acid as the background medium, the simple APR assay is probably not suitable for
FIGURE 3 Calibration graph for

ammonia determination using the
alkali-phenol reagent assay. (Drawn
from results in Ref. 48.)
Kjeldahl-N determination. Copper, zinc, and iron salts were found to act as interferences.
Tetlow and Wilson (49) added ethylenediaminetetraacetic acid (EDTA) to APR to
reduce metal ion interference. Temperature control was also improved using a
thermostated water bath. An outline protocol is described below.
Method 1
Analysis of ammonia using the APR assay (48,49).
Reagents
1. Phenol (crystalline, >85% pure)
2. Sodium hydroxide solution (5 N)
3. Sodium hypochlorite solution (or commercial bleach)
4. Ammonium chloride solid
5. EDTA (6% w/v)
6. Acetone
Procedure
Preparation of alkali-phenol reagent. Place 62.5 g of solid phenol in a 500-mL beaker
and add 135mL of sodium hydroxide (5 N) slowly with stirring. Caution: Use an ice bath
to avoid excessive heat buildup. Add 12mL of acetone and make up the volume to 500
mL with deionized water.
Sodium hypochlorite (1% w/v available chloride). Prepare by diluting commercially
available bleach.
Ammonium chloride standards (1000 ppm NH3 and 100 ppm NH3). Dissolve 314.1 mg
of solid NH4Cl in 100 mL of water and then dilute 10-fold. Prepare a working standard
solution (0.5 ppm NH3) daily.
The APR assay sequence. Place 1 mL of sample (or standard) in a test tube. Add
EDTA solution (100 µL) with gentle shaking. Next, add APR (1 mL) and hypochlorite
(0.5mL) in quick succession, mixing after each addition. Finally, add 2.5mL of water and
incubate at 25°C for 60 minutes. Take A625 readings for samples. Prepare a reagent blank
as described next.
Reagent blank (reverse addition method). First, mix hypochlorite (0.5mL) and APR (1
mL) solutions and allow to react for 5–10 minutes. Next add EDTA (100 µL) followed
by 3.4mL of water (or the designated blank solution).
When reverse addition is used, hypochlorite reacts with phenol first. Traces of NH3
present in the blank are not detected (48,49). Reverse addition is useful where ammonia-
free water is not available for sample preparation. After optimization, the linear dynamic
range for ammonia analysis was 50–500 ppb. Assay sensitivity was 200% greater than
the results shown in Fig. 2. Color formation with 50–800 ppb NH3 was virtually complete
after 15 minutes at 14–30°C. Temperature variations had little effect on the reaction.
Thermostating at 25°C for 60 minutes improved the precision.
Addition of acetone to APR increased the response to ammonia by 10-fold. The color
yield with 500 ppb ammonia declined by 2.65%, 4.8%, and 6.8% for 4.5-hour-, 1-day- or
5-day-old APR. Addition of EDTA prevented interference from 100 ppb copper. The
intervals between addition of various reagents must not exceed 1 minute to ensure
optimum precision. Comparing results for normal and reverse addition provides a means
for detecting very small amounts of NH3 in samples—such as water. Otherwise,
ammonia-free water is needed for preparing reagents and blanks. The calibration curve
for the APR assay is described by the equation A625=0.7120X, where X is the
concentration of nitrogen (ppm). The analytical sensitivity was 0.7120 (absorbance units)
for 1 ppm ammonia. The SD for the reagent blank was ±0.0005 ppm. These values lead
to an expected LLD for ammonia of 1.6
TABLE 9 The Comparative Costs of Manual APR
Assay and Other Techniquesa
Technique Capital costb Running cost per Analysis per Cost per
yearc year analysisd
Manual APR 6000 (1) 5200 (1) 8000 0.72 (1)
method
Micro-Kjeldahl 12000 (2) 7000 (1.3) 2000 4.1 (5.4)
AutoAnalyzer 81,000 (13.5) 17,000 (3.3) 32,000 0.78 (1)
APR test
a
Costs are given in deutsche marks. At current exchange rate 2.8 DM=$1.4=£1. All methods
employ a digestion unit costing DM6000.
b
Capital costs for the micro-Kjeldahl method include the cost of a distillation unit and an
autotitrator.
c
The running cost includes DM5000 for miscellaneous chemicals.
d
Calculated for a 10-year period as capital cost/10+running cost)/no. of samples. Ratios of costs are
given in parentheses for each column.
ppb. Assuming a default FK of 6.25, the LLD for protein is 10 ppb. The APR assay is
widely used in conjunction with the AutoAnalyzer.The composition of the APR used in
CFA is pretty much the same as described earlier (45,52).
Kaul and Sharma (25) describe a rare attempt to deploy a manual Kjeldahl-APR assay
for protein analysis. They used a Tectator heating block for micro-Kjeldahl digestion of
grain. Sample nitrogen was then analyzed by the APR assay. The analytical performance
was similar to results obtained with the AutoAnalyzer-APR assay or the conventional
micro-Kjeldahl analysis. From Table 9, the capital cost for the manual Kjeldahl-APR
assay was 2 times lower than for the micro-Kjeldahl and 13.5 times lower than for the
AutoAnalyzer method. Running costs were also lowest for the manual APR assay.
For laboratories handling 40 or more analyses per day, it may be worth investing in an
automated technique. The manual Kjeldahl-APR analysis was advantageous for small
laboratories lacking the wherewithal to purchase an AutoAnalyzer. Mohyuddin and
Mazza (53) used the manual Kjeldahl-APR assay to analyze potatoes (see Sec. II.B.2).
The mean protein content for 14 potato cultivars was 10.65 (±1.23)% by the manual APR
assay and 10.53 (±1.13)% using the AutoAnalyzer.
3.2. Nessler’s Reagent

Ammonia reacts with alkaline potassium iodomercurate II (Nessler’s reagent) to form a
colloidal complex (λmax=430–460). The linear range for analysis extends to 75 mg
(ammonia) ml−1. A possible reaction scheme for Nesslerization is
2K2HgI4+3KOH+NH3→OHg2NH2I+2H2O+7KI
(18)
Hach et al. (54) developed a commercial Nesslerization reagent for use in Kjeldahl
analysis. A sulfuric acid-digested sample (0.4 mL) is diluted with 24.6mL of 0.01%
(w/w) polyvinyl alcohol (PVA) solution. One ml of Nessler’s reagent is added and the
sample is agitated mechanically before absorbance measurements are recorded at 430 nm.
As the product of Nesslerization is colloidal in nature, spectrophotometric analysis is
sensitive to the degree of sample agitation. PVA acts as a colloidal stabilizer and
improves the precision of the Nessler method.
3.3. Acetylacetone-Formaldehyde Reagent

The acetylacetone-formaldehyde assay is based on the Hantzsch reaction for the synthesis
of pyridine (55). Prediluted digest is reacted with a mixture of acetyltacetone and
formaldehyde in the presence of sodium acetate. The yellow product (3, 5-diacety1–1, 4-
dihydrolutidine) is measured at 410 nm (∆ε=1.4×103 Lmol−1cm−1). The color-forming
reaction is shown in Eq. (19).
(19)
Acetylacetone-formaldehyde reagent was used for the analysis of medicinal agents such
as paracetamol, sulfanilamide, and chloropramide. The potential for colorimetric Kjeldahl
analysis is obvious.
Method 2
Determination of ammonia using acetylacetone-formaldehyde reagent (55).
Reagents
Acetylacetone-formaldehyde reagent. Place 15mL of formaldehyde (37% w/v) and
acetylacetone (7.8 mL) into a 100-mL flask. Make up to 100mL with distilled water.
Sodium acetate (2M). Dissolve sodium acetate (82 g) in 1 L of distilled water.
Procedure
Add prediluted Kjeldahl digest (<2mL; 25–100µgN) to a 25-mL conical flask followed
by sodium acetate solution (3mL) and acetylacetone-formaldehyde reagent (3mL).
Incubate the mixture at 97.8°C for 15 minutes and cool to room temperature.
Bring the total volume to 25 mL and record A412 values using a 1-cm cuvette.
The linear dynamic range for the preceding assay was 0.5–6.0 µg N (per final reaction
mixture). The calibration graph was described by
where X is the amount of
nitrogen present in the final (25-mL) reaction mixture. The new method shows levels of
accuracy and precision equal to those of the micro-Kjeldahl method.
3.4. Ninhydrin (Indanetrione Hydrate) Assay

Ninhydrin* reacts with amino acids (Fig. 4) in two stages: (a) the amino acid is oxidized
to aldehyde and ammonia while ninhydrin is converted to hydrindantin and (b)
hydrindantin and 1 mole of ninhydrin react with ammonia to form Ruhemann’s purple
(56,57). For ammonia determination an added reducing agent is necessary to convert
ninhydrin to hydrindantin. Ninhydrin solution is available commercially. Results from the
ninhydrin-Kjeldahl assay agree closely with those from Kjeldahl analysis (56–58). The
linear dynamic range for colorimetric Kjeldahl assay depends on the extent of sample
dilution just before ninhydrin analysis. A 2-mL standard ammonium sulfate solution
containing 5.6 µg (or 2.8 ppm) reacted with 2mL of ninhydrin solution yields an A570
reading of 0.805. Interference was noted for concentrations of selenium above 86 µgmL−1
(prediluted digest). No interferences were observed with Fe, Zn, Pb, Cu, Ca, Ba, A1, Mg,
Co, or
* Ninhydrin is often encountered in detective novels as a reagent for fingerprint analysis.

FIGURE 4 Reaction scheme between

amino acids and ninhydrin reagent.
FIGURE 5 Colorimetric ninhydrin

analysis of Kjeldahl nitrogen calibrated
against the conventional macro-
Kjedahl analysis. (Drawn from data in
Ref. 58.)
Ni at concentrations of 50 µg mL−1 or from Hg at 30 µg mL−1. The overall impression is
that the ninhydrin assay is resistant to metal ions.
Quinn et al. (58) analyzed rapeseed flour, rapeseed concentrate, soybean concentrate,
and bovine serum albumin using the ninhydrin assay in conjunction with an
AutoAnalyzer (Fig. 5). The precision of analysis was 1.40–1.76%. A manual Kjeldahl-
ninhydrin assay has not been reported recently. This seems a pity. Compared with other
colorimetric methods, the ninhydrin assay is more resistant to interferences from metal
catalysts. The color is also formed at a more easily buffered pH between 4.9 and 5.4.
4. QUANTITATIVE AMINO ACID ANALYSIS
The following steps are involved in quantitative amino acid analysis: (a) hydrolyze a
sample of food using concentrated hydrochloric acid, (b) determine the amino acid
profile, (c) calculate the concentration of each amino acid in the sample, and (d) calculate
the weight of each amino acid.
Quantitative amino acid analysis is reportedly one of the most reliable methods for
protein quantitation (59–61).
4.1. Principles of Quantitative Amino Acid Analysis

Crude protein (cP) is expressed by Eq. (20), where Ci (mole) is the amount of each amino
acid in the sample and bi (g mole−1), is the formula weight for each amino acid.
(20)
However, amino acid profiles are reported in terms of mole fraction of each amino acid
(Xi):
Xi=Ci/Cnet
(21)
where Cnet is the net concentration of amino acids found in the

sample.Substituting Ci=CnetXi in Eq. (20),
(22)
The term ∑(biXi) is called the mean residue weight, W (g mole−1). This is the average
formula weight for all amino acids in the sample adjusted for their frequency.*
(23)
and
cP=WCnet or cP=FCnet
(24)
In Eq. (24), F is the mean residue weight. Usually, W (g mole−1) is adjusted to take into
account two routine errors in amino acid analysis: (a) many colorimetric reagents for
amino acids do not react with proline and (b) tryptophan is destroyed during acid
hydrolysis of proteins. Proline and tryptophan are usually determined by separate
experiments. After correcting for such errors, one gets the conversion factor, F (g
mole−1):
(25)
where XPro and XTrp are the mole fractions of proline and tryptophan. For a
* Horstmann called this parameter the weight equivalent (WE).

range of meat products, F (g mole−1) was 10–20% larger than W [Eq. (23)]. Calculations
of F (g mole−1) appear in last three columns of Table 5. Typical values for F (g mole−1)
are given in Table 10. Values for F range from 100 to 125 g mole−1 for most
proteins.Most protein sources are now routinely analyzed for amino acid scores during
nutritional evaluations (Chapter 14). This yields all the information necessary for total
protein estimation. Zarkadas and co-workers in Canada are strong exponents of
quantitative amino acid analysis (Table 11).
4.2. Quantitation of Specific Proteins

Meat collagen was determined by measuring 5-hydroxylysine (5OH-Lys). This amino
acid is found in collagen and no other meat protein. The concentration of collagen (Pj)
was calculated from a modified Eq. (24) (64,65);
(26)
TABLE 10 Corrected Mean Residue Weights (F)

for Selected Food Protein Sources
Protein source F (g mole−1)
Barley flour 129.84
Soya bean flour 119.82
Pea flour 118.85
Fish meal 112.70
Beef sausage 107.06–109.01
Pig skin (rind) 94.02
Bone meal 104.21
Soya bean protein
Flour 114.43
Isolate 114.48
Concentrate 115.69
Wheat
Flour 113.13–116.00
Gluten 108.4–108.52
Egg white solids 118.43
Potato protein 108.52
Milk (nonfat) powder 112.98
Source: Data calculated or taken from references in Table 11.
TABLE 11 Protein Determination by Quantitative

Amino Acid Analysis
Sample/comments Reference
NASA—Skylab meals Heidelbaugh et al. (59)
Mushroom total protein Weaver et al. (62),
Braaksma and Schaap (63)
Porcine muscle and connective tissue proteins (myosin, actin, elastin, Zarkadas et al. (64),
and collagen Zarkadas et al. (65)
Actin, myosin, and collagen in composite meat products; mixed meat Karatzas and Zarkadas (66)
sausages, bologna, frankfurters, sausages, hamburgers
Additives and ingredients for meat products including soybean, Zarkadas et al. (67)
wheat products, potato protein
Porcine skin (rind) Nguyen et al. (68)
Chicken meat and connective tissue Karatzas and Zarkadas (69)
Apple flower buds Khanizadeh et al. (70)
Bone isolates Zarkadas et al. (71)
New soybean cultivars Zarkadas et al. (72)
where Ci is the concentration of 5OH-Lys in the meat hydrolysate, Wj

ismean residue weight from the amino acid profile for collagen (averaged
forthe different collagen types), nj is the number of 5OH-Lys residues per
1000residues in collagen, and Mi is the formula weight for 5OH-Lys.
Typically,Wj=91.01 g mole−1, Ni=10 residues, Mj=145.18, and
consequently
Amount of collagen=62.75[50H-Lys]
(27)
Similarly, the concentration of Nτ-methylhistidine and 4-hydroxyproline was the basis

for assessing the amount of myofibrillar protein and connective tissue (collagen and
elastin) in meat. These methods are satisfactory for meat and meat products. They may
have questionable validity for composite foods. Plant foods may contain 4-
hydroxyprolinerich glycoproteins (extensins, lectins, salt-extractable glycoproteins). For
example, alfalfa protein and potato protein contain significant levels of 4OH-Pro.
4.3. Examples and Relation to Kjeldahl Method

Quantitative amino acid analysis is arguably one of the most accurate methods for food
protein quantitation. One source of error is the high concentration of free amino acids in
some foods. The protein content in nine strains of Agaricus was 28% (±3.4)% by
quantitative amino acid analysis (62). The results were poorly correlated (R=0.4) with
Kjeldahl results (22.4% protein per dry weight basis). A more recent analysis of freeze-
dried mushroom powder led to estimates of 7.0% protein by dry weight (63). In the later
study, mushroom powder was extracted with 0.5M NaOH and precipitating with
trichloroacetic acid (TCA) before analysis. This removed large amounts of TCA-soluble
NPN associated with mushrooms. Of the total NaOH-soluble nitrogen extracted from
mushrooms, 20% was protein, 60% was urea or ammonia, and 20% was free amino acids.
Food samples are now routinely extracted with organic solvents to remove NPN before
quantitative amino acid analysis (Table 11). Advocates for quantitative amino acid
analysis point to its compatibility with plant proteins. There is no interference from
phenols, tannins, and lignin. By contrast, the Kjeldahl method is unsuitable for plant
tissues regardless of the conversion factor used (73).
5. COMBUSTION NITROGEN ANALYZERS
The Dumas assay predates Kjeldahl analysis by 50 years (Table 1). The former technique
was invented by Jean Baptiste Dumas. Early applications include the analysis of plant
materials (74,75), meat (76), casein, whole powdered milk, soybeans, and maize flour
(77). The first-generation instruments for the Dumas method were not user friendly. The
volume of nitrogen gas produced by combustion was determined with a manometer. The
advent of easy-to-use and highly accurate combustion nitrogen analyzers (CNAs)
rekindled interest in the Dumas method.
CNAs from various manufacturers work on the same principle. The sample is dropped
into a 950–1050°C furnace, purged free of atmospheric gas, and filled with pure (99+ %)
oxygen. Complete sample combustion leads to CO2, water, SO2, NO2, and N2. The
product gases are cooled and a portion is passed through tubing packed with hot lead
chromate, copper, sodium hydroxide (solid), or phosphorus pentoxide to remove SO2, O2,
CO2, and water, respectively. The NO2 is then reduced to N2 and measured with a thermal
conductivity detector (TCD). Sample protein content is calculated by taking into account
the mass of sample injected, the proportion of the combustion gases analyzed, and the
nitrogen-protein conversion factor (FK). The calculations are now automated.
5.1. Collaborative Studies and Approved Status for CNAs

CNAs were calibrated with the Kjeldahl method. Interlaboratory studies appearing after
1987 are listed in Table 12. Such trials led to CNAs receiving approved status from the
AOAC (Association of Official Analytical Chemists), AOCS (American Oil Chemists’
Society), ASBC (American Society of Brewing Chemists), AFI (American Feed
Industry), BRF-International (Brewing Research Foundation-International), IOB
(Institute of Brewing), and EBC (European Brewing Convention).
The Canadian Grain Commission and U.S. Department of Agriculture (USDA)
Federal Grain Inspection Services (FGIS) approved CNAs in
TABLE 12 Food Protein Analysis Using the Dumas

Combustion Method
Samplea Reference
Animal feedstuffs, fertilizers Sweeney and Rexroad (78), Schmitter and Rhihs
(79), Sweeney (80), Sachen and Thiex (81), Tate
(82)
Beer ASBC (83), Johnson and Johansson (84,85)
Brewing grains—barley, malt, rice ASBC (86), Buckee (28,87), Krotz et al. (88),
Johansson (89), Angelino et al. (90)
Cereal grains—wheat, barley, corn, Bicsak (91), Bicsak (92), Williams et al. (93)
sorghum
Dairy products—skimmed, powdered milk Wiles and Gray (94), Wiles et al. (95), Simonne et al.
etc. chocolate milkshake, cheeses, etc. (96,97)
Fruit—guava, peaches, plum Simonne et al. (96,97), Huang et al. (98)
Infant food Bellemonte et al. (99)
Meat and meat products, fish (raw, fish in King-Brink and Sebranek (100), Simonne et al.
oil, tuna) (96,97), Buschmann and Westphal (101)
Oilseeds (soybean, canola, sunflower, corn) Bicsak (91), Duan and DeClercq (102), Berner and
Brown (103)
Potatoes Young et al. (104)
Vegetables—cabbages, broccoli, ketchup, Simonne et al. (96,97)
tomato
a
Approximate sample classification; classes contain the other foodstuffs.
TABLE 13 Advantages of the Dumas Method

1. Greater ease of operation
2. Higher operator safety owing to the nonrequirement for harzadous chemicals
3. The absence of wetchemistry
4. Reduced time of analysis
5. Higher performance characteristics (greatar accuracy, repeatablility)
6. Absence of waste disposal concerns (Table 14)
7. Simple instrument installation without a requirement for specialized ventilation
8. Low cost per analysis
1994 and 1996, respectively (91–93). Trials for the combustion method usually follow
guidelines described by Youden and Sleiner (105):
1. The number of laboratories ranges from 7 to 12. Studies involving as few as three
laboratories have been reported.
2. All studies compare CNAs with Kjeldahl analysis.
3. Interlaboratory studies focus on a single food group. Therefore, CNAs tend to receive
approval for one food group at a time (Table 12).
4. Trials usually test a “generic combustion method” and are independent of the choice of
instruments.
Minimum performance guidelines for CNA instruments include (a) a furnace temperature
of 950°C, (b) a separation system for trapping CO2 and water, (c) a thermal conductivity
detector for nitrogen, (d) sufficient accuracy to produce results within +0.15% of the
mean (% nitrogen) results for 10 successive measurements using a standard compound,
and (e) sufficient precision to produce a relative standard deviation of 0.01%. The LECO
FP-428 analyzer was used by about 80% of the laboratories involved in collaborative
trials. The Foss-Heraue Macro-N analyzer, Carlo Erba NA-5000, and Perkin Elmer
PE2410 also feature. The LECO FP-2000 combustion analyzer appears in the latest trials.
5.2. Advantages of the Combustion Method

The modern CNA has advantages over the Kjeldahl method (Table 13). There is greater
speed of analysis and greater operator safety stemming from the nonuse of aggressive
chemicals. The estimated cost for analysis is $0.37-$0.50 per sample with the LECO FP-
2000 protein analyzer (LECO Corporation, Saint Joseph, MI) compared with $1.0 per test
for the Kjeldahl method (106–109).
TABLE 14A Comparison of Materials Reqirement
for the Kjeldhal and Dumas Methods (74,84)
Requirement Kjeldahl Dumas
Chemical Conc. H2SO4, 40% NaOH, Air, oxygen, helium,
requirements K2SO4, TiO2/CuSO4 (or HgO), H3BO3 copper, turnings, EDTA, nitrogen
KH, phthalate, methyl red, catalyst, Mg perchlorate, sodium
phenolphthalein, pumice, water hydroxide, alumina oxide pellets
Other suppliesa Kjeldahl and Erlenmeyer flasks, burettes, Tinfoil squares, brushes, tin capsules,
acid, alkali and water dispensers, stirring combustion, reduction and absorption
equipment, large containers for acid, etc. tubes, cotton wool, steel wool, particle
filters, tubing
Ancillary Ductwork for corrosive fumes, acid- Ductwork for warm airb
equipment resistant fans, fume washer, fans, etc.
Disposal of Collected, professionally disposed Nontoxic, wastebin or sink disposal

chemical
Time per 120 min (24 samples) 3 min

analysis
Degree of 6 2
hazardc
Accuracy 70–98 100
Precision (CV 1.2 0.7
%)
a
Does not include main equipment (Kjeldahl digester and distillation apparatus, or CNA instrument
b
Optional, but advisable for large-scale testing.
c
Arbitrary scale of 1–10, with 10 being extremely hazardous and 1 completely safe. There may be a
risk of burns when maintaining the combustion instrument.
A more detailed discussion of the relative costs of protein analysis by Kjeldahl or

combustion analysis has to consider factors such as number of analyses per year, capital
costs for instrumentation, depreciation, maintenance costs, and savings of labor,
chemicals, and other consumables costs (106). It has been suggested that the combustion
method provides cost savings of about 30% with a payback period within 2 years. For
research institutes, universities, and small-scale laboratories, the safety of modern CNAs
probably outweighs cost considerations. Further comparisons of the CNAs and Kjeldahl
analysis are summarized in Table 14.
5.3. Combustion Analysis of Feeds, Cereal Grains, and Oilseeds

Combustion analysis first received AOAC approval for feeds in 1968. The classical
instruments (Coleman model 29A nitrogen analyzer) used a manometer for the
volumetric assay of nitrogen (110). Comprehensive testing using modern TCD-based
CNAs appeared in 0 (78). A nine-laboratory collaborative trial to determine nitrogen in
feeds was successfully completed in 1989 (80). The AOAC approved CNAs for animal
feed testing in 1990.
The small sample sizes (150–500 mg) used with modern CNAs raised concerns about
sampling. Extensive grinding and mixing are essential to ensure sample homogeneity and
representative sampling. Sweeney and Rexroad (78) analyzed 14 different animal feeds
using the LECO FP-228 instrument with a prescribed sample size of <150mg. Estimates
of feed nitrogen agreed closely with results from Kjeldahl analysis. The precision of
analysis was significantly lower (0.013–0.052%) for the combustion method as compared
with Kjeldahl analysis (0.006–0.035%). Schmitter and Rihs (79) increased the sample
size for the LECO-F228 analyzer from 150 mg to 1g by palletizing before loading into
the instrument port. Adding a few drops of polyethylene (2% w/w in ethyl acetate
solvent) prevented flaking of the pellets. Table 15 shows nitrogen and protein data for
feeds determined using the CNA (78,79). Results have been averaged for samples of
sizes 0.15–1.0 g. Protein values are calculated as %N×6.25. The results agree favorably
with Kjeldahl analysis (Fig. 6). There appeared to be significant positive bias for
feedstuffs having <2% nitrogen (Fig. 7). The bias was ascribed to plant-derived materials
containing high levels of nitrate. The Kjeldahl method achieves low recoveries of
nitrogen from refractory compounds (75) with N—O or N—N bonds (nitrite; nitrate;
oximes; azo, nitro, nitroso-compounds).
Sachen and Thiex (81) found that CNAs showed a ≤1.38% (protein) bias for hay
samples. They attributed such results to “atmospheric error” arising from air being
trapped in the interstices of the (fluffy) hay samples. Compressing samples to remove
trapped air led to agreement between the Kjeldahl and CNA results (81). The LECO FP-
2000 nitrogen analyzer was not subject to an atmospheric blank because of improvements
in instrument design and efficient purging of atmospheric gases before sample
combustion. Sachen and Thiex examined a range of pelleting equipment and procedures
for eliminating the atmospheric blank for the LECO FP-428 instrument. They proposed
that powdered cellulose could be analyzed to check for an atmospheric error (81). Not all
investigators agree about the nature of the atmospheric error.
TABLE 15 Nitrogen and Protein in Feedstuffs
Determined by Combustion Method
Sample N (g kg−1) Protein (%)
Straw 0.620 3.88
Corn silage 1.175 7.34
Porc soup 1.185 7.41
Hay 1.280 8.00
Corn grain 1.375 8.59
Barley 1.515 9.47
Grass silage 1.835 11.47
Oats 1.760 11.00
Triticale 1.835 11.47
Wheat 1.985 12.41
Dried grass 2.240 14.00
Cow premix 2.755 17.22
Alfafa pellets 2.770 17.31
Hog feed 3.390 21.19
Broiler finisher 3.420 21.38
Milk powder 4.365 27.28

Bone meal 4.325 27.03
Rapeseed 5.710 35.69
Protein conc. 6.045 37.78
Soybean meal 6.485 40.53
Cattle conc. 6.690 20.91
Yeast 6.895 43.09
Peanut meal 8.450 52.81
Meat meal 9.300 58.13
Fish meal 9.960 62.25
Gluten 11.730 73.31
Feather meal 13.610 85.06
Soy protein conc. 14.020 87.63
Bicsak (91) described a collaborative study to extend AOAC-approved status to cereal

grains and oilseeds. Seven laboratories analyzed 15 matched pairs of samples (soybean,
canola, sunflower, wheat, barley, corn, sorghum) having protein levels of 8–13%, 17–
23%, or 35–40%. Six of the seven collaborators used the LECO FP-428 instrument. With
210 samples the average protein reading was 28.26% by combustion analysis and 28.01%
by Kjeldahl analysis. Repeatability and reproducibility statistics were comparable. A
recommendation to extend the AOAC combustion method to cereal grains and oilseeds
was approved. The following non-English publications
FIGURE 6 Comparison of protein

results for feeds determined using the
combustion method and the Kjeldahl
method. Micro-CNA and macro-CNA
refer to the use of 150-mg and 1-g
sample sizes with the combustion
nitrogen analyzer. List of feedstuffs is
given in Table 15. (Data derived from
Ref. 78.)
FIGURE 7 Residuals from Fig. 6

showing no systematic differences in
results.
describe collaborative tests leading to approved status for combustion analysis of cereal
and cereal products including wheat, wheat bran, pasta, sorghum, and maize (111,112).
5.4. Barley, Malt, and Beer

The combustion method was subjected to an 11-member interlaboratory trial for brewing
grain (rice, barley, malt, spent grain) analysis. Agreement was reached in 1992 to include
the Dumas method in the methods of analysis for brewing grains (86). The trial results
showed that most commercial CNAs had a linear range of 0–9.5% nitrogen and an LLD
of 0.0321%. The repeatability CV ranged from 0.013 to 0.055% compared with a
reproducibility of 0.042–0.067%.
Further collaborative studies to evaluate CNAs for barley, malt, and beer analysis
were reported by the UK Institute of Brewing in 1996 (28). Fifteen of the 25 participating
laboratories employed the LECO FP-428 instrument. Another five laboratories used the
Foss Heraeus Macro-N apparatus. The range of Kjeldahl techniques used is shown in
Table 7. All laboratories examined eight samples each of barley, malt, and beer. CNAs
gave slightly higher values for total nitrogen as compared with the Kjeldahl method.
Essentially identical results were obtained when results from the Kjel-Foss instrument
were omitted. The reproducibility was 0.03–0.07% for barley and 0.036–0.065% for malt
analysis. These values were independent of total nitrogen over the range 1.17–1.71%
(w/w) (barley), 1.45–2.03% (w/ w) (malt), and 268–1020 mg L−1 (beer). Based on such
results, the IOB Analysis Committee (UK) approved combustion analysis for use
alongside Kjeldahl analysis. However, the precision for beer analysis was low, perhaps
because the trial participants lacked expertise with liquid samples. CNAs were judged
unsuitable for beer protein determination.
CNAs were approved for beer analysis in Europe in 1999 (84,85). In the collaborative
trial organized by the IOB and EBC, five samples of beer and malt were analyzed in
duplicate by 18 laboratories from the brewing and allied industries. The collaborators
used the following CNAs: LECO FP-428 instrument (11 laboratories), LECO FP-2000 (3
laboratories), and Macro-N analyzer (3 laboratories). Glycine, Tris, or EDTA was used as
the calibrant. Samples of beer were found to contain 362–1159 mg (nitrogen) L−1 and
malt samples had 0.534–0.706% nitrogen. Repeatability and reproducibility statistics for
beer analysis were deemed satisfactory, leading to method approval.
5.5. Milk and Related Dairy Products

Despite the recent widespread use of CNAs for food protein analysis, few applications in
the dairy field have been published. Wiles et al. (95) described an 11-member
interlaboratory study from New Zealand. They compared milk protein analysis by CNAs
and the Kjeldahl method. Samples included ultra-heat-treated (UHT) whole milk, infant
formulas, whole milk powder, skimmed milk powder, whey protein concentrate, casein,
and sodium caseinate. Nine of the eleven laboratories employed the LECO FP-428
instrument. Results for CNAs agreed closely with Kjeldahl findings. There was no
systematic bias associated with the former results. Indeed, no evidence was found for a
systematic difference between CNA and Kjeldahl results reported between 1968 and
1997 (95).
5.6. Baby Foods and Infant Formulas

Bellemonte et al. (99) analyzed five categories of baby foods using the Carlo Erba model
1500 nitrogen analyzer. This instrument uses a high furnace temperature (1800°C)
combined with an oxygen-rich atmosphere to achieve complete sample combustion.
Nitrogen is quantified using a TCD. The analysis time for this instrument is reportedly 3
minutes. All sample types were analyzed successfully. Results obtained by the Kjeldahl
method were 1–4% lower than those obtained with CNAs (Table 16). Results from both
techniques compared favorably with protein values declared by food manufacturers.
Compared with the Kjeldahl method, CNAs are convenient for baby food analysis. The
sample throughput and safety considerations favor the Dumas method as described in
Sec. 5.2.
5.7. Meat
King-Brink and Sebranek (100) described a 12-laboratory trial to evaluate CNAs for meat
product analysis. Participants in the trial used the LECO FP-428 instrument (9
laboratories), the Foss Heraeus Macro-N analyzer (2 laboratories), or the Perkin Elmer
PE2410 analyzer. In all, 15 pairs of meat products, purchased from 30 different
manufacturers, were analyzed. All participants used CNAs satisfactorily judging from (a)
the low number of data outliers and (b) the high precision of results for standard
compounds. The CNA results were slightly higher than Kjeldahl figures: 15.75% versus
15.59% (w/w). Estimates for repeatability and reproducibility were comparable. A
recommendation that the Dumas method should be adopted as a reference test for meat
proteins was approved by the AOAC. A 14-laboratory trial for analysis of meat and meat
products was reported in
TABLE 16 Analysis of Protein in Five Categories
of Baby Foods (97)
Samplea Declared Kjeldahlb Dumasb
protein
1. Formula milk (7) 15.55 14.94 15.36
2. Cereal-based products (6) Cream of rice, semolina+honey, 10.28 10.12 10.35
wheat flour+milk+oats, milk soup+cereal+fruit, milk
soup+cereal+apples
3. Biscuits (2) 9.50 9.40 14.70
4. Lyophilized products (2) Veal, ham, and eggs 54.20 52.15 53.20
5. Homogenized products (6) Beef, beef+ham, chicken, 9.52 9.62 9.72
veal+brain, turkey, chicken+carrot+potatoes
a
Numbers in parentheses represent number of different foods in the category analyzed. Protein
values are averaged for each food category
b
Protein values were calculated with a conversion factor (Fk)=6.38 for milk formula or 6.25 for all
other categories of baby food.
Germany. This trial, too, concluded that the performance of the Dumas method was
comparable to that of the Kjeldahl assay but that the former method was quicker and
more environment friendly (101).
REFERENCES
1. WH Tallent. USA current developments in protein food regulations—labeling. J Oil Chem Soc
56(3):239, 1979.
2. DK Salunkhe, SS Deshpande. Foods of Plant Origin. Production, Technology and Human
Nutrition. New York: Van Nostrand Reinhold, 1991.
3. H Frankel-Conrat, M Cooper. The use of dyes for the determination of acid and basic groups in
proteins. J Biol Chem 154:239–340, 1944.
4. DC Udy. Estimation of protein in wheat and flour by ion-binding. Cereal Chem 33:190–197,
1956.
5. AJ Pinckney. Wheat protein and the biuret reaction. Cereal Chem 26:423–439, 1949.
6. Z Duda, M Szot. A comparison of several methods of protein determination in pig blood plasma.
Proceedings of the European Meeting of Meat Research Workers 32, Vol II 9, 1986, pp 447–
450.
7. KM Williams, P Fox, T Marshall. A comparison of protein assays for the determination of the
protein concentration of beer. J Inst Brew 101:365–369, 1995.
8. Y Pomeranz, RB More, FS Lai. Reliability of five methods for protein determination in barley
and malt. J Am Soc Brew Chem 35:86–93, 1975.
9. Y Pomeranz, RB More. Reliability of several methods for protein determination in wheat. Bakers
Digest February:44 -58, 1975.
10. QQ Luthi-Peng, Z Puhan. The 4th derivative UV spectroscopic method for the rapid
determination of protein and casein in milk. Milchwissenschaft 54:74–77, 1999.
11. JM Lynch, DM Barbano. Kjeldahl nitrogen analysis as a reference methood for protein
determination in dairy products. J Assoc Off Anal Chem Int 82:1389–1398, 1999.
12. RB Bradstreet. The Kjeldahl Method for Organic Nitrogen. London: Academic Press, 1965.
13. International Standard ISO-1871 (1975). Agricultural food products—general directions for the
determination of nitrogen by the Kjeldahl method.
14. L Gaspar. General laboratory methods. In I Kerese, ed. Methods of Protein Analysis.
Chichester, UK: Ellis Horwood, 1984, pp 30–86.
15. BG Osborne. The determination of protein in cereals. In BJF Hudson, ed. Developments in
Food Proteins—4. London: Elsevier Applied Science, 1986, pp 247–290.
16. RA Osborne, JB Wilkie. A study of the Kjeldahl method IV. Metallic catalysts and metallic
interferences. J Assoc Off Anal Chem 18:604–609, 1935.
17. RC Benedict. Determination of nitrogen and protein content of meat and meat products. J Assoc
Off Anal Chem 70:69–76, 1987.
18. R Tkachuk. Nitrogen-to-protein conversion factors for cereal and oilseed meals. Cereal Chem
46:419–423, 1969.
19. OA Krober, SJ Gibbons. Nonprotein nitrogen in soybeans. J Agric Food Chem 10:57–58, 1962.
20. R Tkachuk. Note on the nitrogen-to-protein conversion factor for wheat flour. Cereal Chem
43:223, 1966.
21. JAD Ewart. Amino acid analyses of cereal flour proteins. J Sci Food Agric 18:558, 1967.
22. S Boisen, S Bech-Andersen, B Eggum. A critical view on the conversion factor 6.25 from total
nitrogen to protein. Acta Agric Scand 37:299–304, 1987.
23. MAJS van Boekel, B Ribadeau-Dumas. Addendum to the evaluation of the Kjeldahl factor for
conversion of the nitrogen content of milk and milk products to protein content. Neth Milk
Dairy J 41:281–284, 1987.
24. FW Sosulski, GI Imafidon. Amino acid composition and nitrogen-to-protein conversion factors
for animal and plant foods. J Agric Food Chem 38:1351–1356, 1990.
25. AK Kaul, TR Sharma. Rapid determination of nitrogen content in grain-meal samples with
alkaline-phenol reaction, manually and with an AutoAnalyzer. Z Anal Chem 280:133–138,
1976.
26. M Mohyuddin, G Mazza. Determination of potato protein by alkali-phenol, dye-binding and
other methods. Am Potato J 55:621–626, 1978.
27. BG Venter, HP Sheepers, J Floor, JH Snyman. Semi-micro Kjeldhal procedures for protein
determination of dairy products. S Afr J Dairy Technol 17(3):107–111, 1985.
28. GK Buckee. Determination of total nitrogen in barley, malt and beer by Kjeldahl procedures
and the Dumas combustion method—collaborative trial. J. Inst Brew 100:54–64, 1994.
29. MJ Brennan. Automated Kjeldahl yields poetry. Food Eng 46(5): 102–103, 1974.
30. JE Trevis. Seven automated instruments. Cereal Sci Today 19(5): 182–189, 1974.
31. R Oberrieth, NH Mermelstein. Instrument automates, accelerates nitrogen determinations. Food
Technol 28(6):40–41, 43, 1974.
32. PC Williams, KH Norris, RL Johnsen, K Standing, R Fricioni, D MacAffrey, R Mercier.
Comparison of physicochemical methods for measuring total nitrogen in wheat. Cereal Foods
World 23(9):544–547, 1978.
33. OC Bjarno. Kjel-Foss automatic analysis using an antimony-based catalyst: collaborative study.
J Assoc Off Anal Chem 63:657–663, 1980.
34. DL McGill. Comparison of automated method and improved AOAC Kjeldahl method for
determination of protein in meat and meat products. J Assoc Off Anal Chem 64:29–31, 1980.
35. FB Suhre, PA Corrao, A Glover, AJ Melanoski. Comparison of three methods for
determination of crude protein in meat: collaborative study. J Assoc Off Anal Chem 65:1339–
1345, 1982.
36. JF Marten, G Catanzaro. Fundamental studies in automatic nitrogen digestion. Analyst 91:42–
47, 1966.
37. JA Varley. Automatic methods for the determination of nitrogen, phosphorus and potassium in
plant material. Analyst 91:119–126, 1966.
38. LL Wall, CW Gehrke. An automated total protein nitrogen method. J Assoc Off Anal Chem
58:1221–1226, 1975.
39. DE Uhl, EB Lancaster. Automation of nitrogen analysis of grain and grain products. Anal
Chem 43:990, 1971.
40. KR Vincent, WF Shipe. The effect of calibration procedures on accuracy and precision of
automated Kjeldahl nitrogen analysis in some formulated foods. J Food Sci 41:157–162, 1976.
41. WM Gantenbein. Collaborative study of the automated determination of nitrogen in meat
products. J Assoc Off Anal Chem 56:31–35, 1973.
42. HG Lento, CE Daugherty. Automated determination of protein nitrogen in foods. Food Prod
Dev 5:86, 1971.
43. HG Lento, CE Daugherty. The automated protein-nitrogen analysis of foods. Adv Auto Anal
Technicon Int Congr 2:75–80, 1970.
44. JF Marten, G Catanzaro. Fundamental studies in automatic nitrogen digestion. Analyst 91:42–
47, 1966.
45. J Davidson, J Mathieson, AW Boyne. The use of automation in determining nitrogen by the
Kjeldahl method, with final calculation by computer. Analyst 95:181–193, 1970.
46. CW Gehrke, LL Wall Sr, JS Absheer. Automated nitrogen method for feeds. J Assoc Off Anal
Chem 56:1096–1105, 1973.
47. LL Wall, CW Gehrke. Feeds: an automated total protein nitrogen method. J Assoc Off Anal
Chem 58:1221–1226, 1975.
48. WT Bolleter, CJ Bushman, PW Tidwell. Spectrophotometric determination of ammonia as
indophenol. Anal Chem 33:592–594, 1961.
49. JA Tettlow, AL Wilson. An absorptiometric method for determining ammonia in boiler feed-
water. Analyst 89:453–465, 1964.
50. CW Gherke, FE Kaiser, JP Ussary. Automated spectrophotometric method for nitrogen in
fertilizers. J Assoc Anal Chem 51:200–211, 1968.
51. PL Searle. The Berthelot or indophenol reaction and its use in the analytical chemistry of
nitrogen. Analyst 109:549–568, 1984.
52. JE McNeal, A Karasz, E Gorge Jr. Automation of methods for meat and meat products. 1.
Determination of protein. J Assoc Anal Chem 53:907–910, 1970.
53. G Mohyuddin, G Mazza. Determination of potato protein by alkali-phenol, dye-binding and
other methods. Am Potato J 55:621–626, 1978.
54. CC Hach, SV Brayton, AB Kopelove. A powerful Kjeldahl nitrogen method using
peroxymonosulfuric acid. J Agric Food Chem 33:1117–1123, 1985.
55. MB Devani, CJ Shishoo, SA Shah, BN Suhagia. Spectrophotometric method for micro-
determination of nitrogen in Kjeldahl digest. J Assoc Off Anal Chem 72:953–956, 1989.
56. S Jacobs. The determination of nitrogen in organic compounds by the indanetrione hydrate
method. Analyst 85:257–264, 1960.
57. S Jacobs. The effect of temperature on the Kjeldahl digestion process. Analyst 89:489–494,
1964.
58. JR Quinn, JGA Boisvert, I Wood. Semi-automated ninhydrin assay of Kjeldahl nitrogen. Anal
Biochem 58:609–614, 1974.
59. ND Heidelbaugh, CS Huber, JF Bednarczyk, MC Smith, PC Rambaut, HO Wheeler.
Comparison of three methods for calculating protein content of foods. J Agric Food Chem
23:611–612, 1975.
60. H-J Horstmann. A precise method for the quantitation of proteins taking into account their
amino acid composition. Anal Biochem 96:130–138, 1979.
61. GL Peterson. Determination of total protein. Methods Enzymol 91:95–119, 1983.
62. JC Weaver, M Kroger, LR Kneebone. Comparative protein studies (Kjeldahl, dye binding,
amino acid analysis) of nine strains of Agaricus bisporus (Lange) imback mushrooms. J Food
Sci 42:364–366, 1977.
63. A Braaksma, DJ Schaap. Protein analysis of the common mushroom Agaricus bisporus.
Postharvest Biol Technol 7:119–127, 1996.
64. CG Zarkadas, EA Meighen, GC Zarkadas, CN Karatzas, AD Khalili, JA Rochemont, M
Berthelet. Determination of the myofibrillar and connective tissue proteins in the bovine
diaphragm. J Agric Food Chem 36:1105–1108, 1988.
65. CG Zarkadas, N Karatzas, AD Khalili, S Khanizadeh, G Morin. Quantitative determination of
the myofibrillar proteins and connective tissue content in selected porcine skeletal muscles. J
Agric Food Chem 36:1131–1146, 1988.
66. CN Karatzas, CG Zakardas. Determination of the myofibrillar and connective tissue protein
contents and amino acid composition of selected composite meat products. J Agric Food Chem
36:1109–1121, 1988.
67. CG Zarkadas, NJ Drouliscos, CN Karatzas. Comparison of the total protein, nitrogen and amino
acid composition of selected additives and ingredients used in composite meat products. J Agric
Food Chem 36:1121–1131, 1988.
68. Q Nguyen, MA Fanous, LH Kamma, AD Khalili, PH Schuepp, CG Zarkadas. Comparison of
the amino acid composition of two commercial porcine skins (rind). J Agric Food Chem
34:565–572, 1989.
69. CN Karatzas, CG Zarkadas. Comparison of the amino acid composition of the intracellular and
extracellular matrix protein fractions isolated from avian skeletal muscles. Poult Sci 68:811–
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70. S Khanizadeh, D Buszar, CG Zarkadas. Comparison of three methods for calculating protein
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71. CG Zarkadas, Y Ziran, GC Zarkadas, A Minero-Amador. Assessment of the protein quality of
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72. CG Zarkadas, HD Voldeng, YI Ziran, Keijin-Shang, PL Pattsion, Comparison of the protein
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73. S Khanizadeh, D Buszard, CG Zarkadas. Misuse of the Kjeldahl method for estimating protein
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74. R Fiedler, G Proksch, A Koepf. The determination of total nitrogen in plant materials with an
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76. YS Lee, CE Damon, JP Crisler. A micro method for the determination of protein and screening
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References
1 1: Kjeldahl Method, Quantitative Amino

Acid Analysis and Combustion Analysis
1. WH Tallent. USA current developments in protein food

regulations—labeling. J Oil Chem Soc 56(3):239, 1979.
2. DK Salunkhe, SS Deshpande. Foods of Plant Origin.

Production, Technology and Human Nutrition. New York: Van
Nostrand Reinhold, 1991.
3. H Frankel-Conrat, M Cooper. The use of dyes for the

determination of acid and basic groups in proteins. J Biol
Chem 154:239–340, 1944.
4. DC Udy. Estimation of protein in wheat and flour by

ion-binding. Cereal Chem 33:190–197, 1956.
5. AJ Pinckney. Wheat protein and the biuret reaction.

Cereal Chem 26:423–439, 1949.
6. Z Duda, M Szot. A comparison of several methods of

protein determination in pig blood plasma. Proceedings of
the European Meeting of Meat Research Workers 32, Vol II 9,
1986, pp 447– 450.
7. KM Williams, P Fox, T Marshall. A comparison of protein

assays for the determination of the protein concentration
of beer. J Inst Brew 101:365–369, 1995.
8. Y Pomeranz, RB More, FS Lai. Reliability of five methods

for protein determination in barley and malt. J Am Soc
Brew Chem 35:86–93, 1975.
9. Y Pomeranz, RB More. Reliability of several methods for

protein determination in wheat. Bakers Digest February:44
-58, 1975.
10. QQ Luthi-Peng, Z Puhan. The 4th derivative UV

spectroscopic method for the rapid determination of
protein and casein in milk. Milchwissenschaft 54:74–77,
1999.
11. JM Lynch, DM Barbano. Kjeldahl nitrogen analysis as a

reference methood for protein determination in dairy
products. J Assoc Off Anal Chem Int 82:1389–1398, 1999.
12. RB Bradstreet. The Kjeldahl Method for Organic

Nitrogen. London: Academic Press, 1965.
13. International Standard ISO-1871 (1975). Agricultural
food products—general directions for the determination of
nitrogen by the Kjeldahl method.
14. L Gaspar. General laboratory methods. In I Kerese, ed.

Methods of Protein Analysis. Chichester, UK: Ellis
Horwood, 1984, pp 30–86.
15. BG Osborne. The determination of protein in cereals. In

BJF Hudson, ed. Developments in Food Proteins—4. London:
Elsevier Applied Science, 1986, pp 247–290.
16. RA Osborne, JB Wilkie. A study of the Kjeldahl method

IV. Metallic catalysts and metallic interferences. J Assoc
Off Anal Chem 18:604–609, 1935.
17. RC Benedict. Determination of nitrogen and protein

content of meat and meat products. J Assoc Off Anal Chem
70:69–76, 1987.
18. R Tkachuk. Nitrogen-to-protein conversion factors for

cereal and oilseed meals. Cereal Chem 46:419–423, 1969.
19. OA Krober, SJ Gibbons. Nonprotein nitrogen in soybeans.

J Agric Food Chem 10:57–58, 1962.
20. R Tkachuk. Note on the nitrogen-to-protein conversion

factor for wheat flour. Cereal Chem 43:223, 1966.
21. JAD Ewart. Amino acid analyses of cereal flour

proteins. J Sci Food Agric 18:558, 1967.
22. S Boisen, S Bech-Andersen, B Eggum. A critical view on

the conversion factor 6.25 from total nitrogen to protein.
Acta Agric Scand 37:299–304, 1987.
23. MAJS van Boekel, B Ribadeau-Dumas. Addendum to the

evaluation of the Kjeldahl factor for conversion of the
nitrogen content of milk and milk products to protein
content. Neth Milk Dairy J 41:281–284, 1987.
24. FW Sosulski, GI Imafidon. Amino acid composition and

nitrogen-to-protein conversion factors for animal and
plant foods. J Agric Food Chem 38:1351–1356, 1990.
25. AK Kaul, TR Sharma. Rapid determination of nitrogen

content in grain-meal samples with alkaline-phenol
reaction, manually and with an AutoAnalyzer. Z Anal Chem
280:133–138, 1976.
26. M Mohyuddin, G Mazza. Determination of potato protein
by alkali-phenol, dye-binding and other methods. Am Potato
J 55:621–626, 1978.
27. BG Venter, HP Sheepers, J Floor, JH Snyman. Semi-micro

Kjeldhal procedures for protein determination of dairy
products. S Afr J Dairy Technol 17(3):107–111, 1985.
28. GK Buckee. Determination of total nitrogen in barley,

malt and beer by Kjeldahl procedures and the Dumas
combustion method—collaborative trial. J. Inst Brew
100:54–64, 1994.
29. MJ Brennan. Automated Kjeldahl yields poetry. Food Eng

46(5): 102–103, 1974.
30. JE Trevis. Seven automated instruments. Cereal Sci

Today 19(5): 182–189, 1974.
31. R Oberrieth, NH Mermelstein. Instrument automates,

accelerates nitrogen determinations. Food Technol
28(6):40–41, 43, 1974.
32. PC Williams, KH Norris, RL Johnsen, K Standing, R

Fricioni, D MacAffrey, R Mercier. Comparison of
physicochemical methods for measuring total nitrogen in
wheat. Cereal Foods World 23(9):544–547, 1978.
33. OC Bjarno. Kjel-Foss automatic analysis using an

antimony-based catalyst: collaborative study. J Assoc Off
Anal Chem 63:657–663, 1980.
34. DL McGill. Comparison of automated method and improved

AOAC Kjeldahl method for determination of protein in meat
and meat products. J Assoc Off Anal Chem 64:29–31, 1980.
35. FB Suhre, PA Corrao, A Glover, AJ Melanoski. Comparison

of three methods for determination of crude protein in
meat: collaborative study. J Assoc Off Anal Chem 65:1339–
1345, 1982.
36. JF Marten, G Catanzaro. Fundamental studies in

automatic nitrogen digestion. Analyst 91:42– 47, 1966.
37. JA Varley. Automatic methods for the determination of

nitrogen, phosphorus and potassium in plant material.
Analyst 91:119–126, 1966.
38. LL Wall, CW Gehrke. An automated total protein nitrogen

method. J Assoc Off Anal Chem 58:1221–1226, 1975.
39. DE Uhl, EB Lancaster. Automation of nitrogen analysis

of grain and grain products. Anal Chem 43:990, 1971.
40. KR Vincent, WF Shipe. The effect of calibration

procedures on accuracy and precision of automated Kjeldahl
nitrogen analysis in some formulated foods. J Food Sci
41:157–162, 1976.
41. WM Gantenbein. Collaborative study of the automated

determination of nitrogen in meat products. J Assoc Off
Anal Chem 56:31–35, 1973.
42. HG Lento, CE Daugherty. Automated determination of

protein nitrogen in foods. Food Prod Dev 5:86, 1971.
43. HG Lento, CE Daugherty. The automated protein-nitrogen

analysis of foods. Adv Auto Anal Technicon Int Congr
2:75–80, 1970.
44. JF Marten, G Catanzaro. Fundamental studies in

automatic nitrogen digestion. Analyst 91:42– 47, 1966.
45. J Davidson, J Mathieson, AW Boyne. The use of

automation in determining nitrogen by the Kjeldahl method,
with final calculation by computer. Analyst 95:181–193,
1970.
46. CW Gehrke, LL Wall Sr, JS Absheer. Automated nitrogen

method for feeds. J Assoc Off Anal Chem 56:1096–1105,
1973.
47. LL Wall, CW Gehrke. Feeds: an automated total protein

nitrogen method. J Assoc Off Anal Chem 58:1221–1226, 1975.
48. WT Bolleter, CJ Bushman, PW Tidwell. Spectrophotometric

determination of ammonia as indophenol. Anal Chem
33:592–594, 1961.
49. JA Tettlow, AL Wilson. An absorptiometric method for

determining ammonia in boiler feedwater. Analyst
89:453–465, 1964.
50. CW Gherke, FE Kaiser, JP Ussary. Automated

spectrophotometric method for nitrogen in fertilizers. J
Assoc Anal Chem 51:200–211, 1968.
51. PL Searle. The Berthelot or indophenol reaction and its

use in the analytical chemistry of nitrogen. Analyst
109:549–568, 1984.
52. JE McNeal, A Karasz, E Gorge Jr. Automation of methods

for meat and meat products. 1. Determination of protein. J
Assoc Anal Chem 53:907–910, 1970.
53. G Mohyuddin, G Mazza. Determination of potato protein

by alkali-phenol, dye-binding and other methods. Am Potato
J 55:621–626, 1978.
54. CC Hach, SV Brayton, AB Kopelove. A powerful Kjeldahl

nitrogen method using peroxymonosulfuric acid. J Agric
Food Chem 33:1117–1123, 1985.
55. MB Devani, CJ Shishoo, SA Shah, BN Suhagia.

Spectrophotometric method for microdetermination of
nitrogen in Kjeldahl digest. J Assoc Off Anal Chem
72:953–956, 1989.
56. S Jacobs. The determination of nitrogen in organic

compounds by the indanetrione hydrate method. Analyst
85:257–264, 1960.
57. S Jacobs. The effect of temperature on the Kjeldahl

digestion process. Analyst 89:489–494, 1964.
58. JR Quinn, JGA Boisvert, I Wood. Semi-automated

ninhydrin assay of Kjeldahl nitrogen. Anal Biochem
58:609–614, 1974.
59. ND Heidelbaugh, CS Huber, JF Bednarczyk, MC Smith, PC

Rambaut, HO Wheeler. Comparison of three methods for
calculating protein content of foods. J Agric Food Chem
23:611–612, 1975.
60. H-J Horstmann. A precise method for the quantitation of

proteins taking into account their amino acid composition.
Anal Biochem 96:130–138, 1979.
61. GL Peterson. Determination of total protein. Methods

Enzymol 91:95–119, 1983.
62. JC Weaver, M Kroger, LR Kneebone. Comparative protein

studies (Kjeldahl, dye binding, amino acid analysis) of
nine strains of Agaricus bisporus (Lange) imback mushrooms.
J Food Sci 42:364–366, 1977.
63. A Braaksma, DJ Schaap. Protein analysis of the common

mushroom Agaricus bisporus. Postharvest Biol Technol
7:119–127, 1996.
64. CG Zarkadas, EA Meighen, GC Zarkadas, CN Karatzas, AD
Khalili, JA Rochemont, M Berthelet. Determination of the
myofibrillar and connective tissue proteins in the bovine
diaphragm. J Agric Food Chem 36:1105–1108, 1988.
65. CG Zarkadas, N Karatzas, AD Khalili, S Khanizadeh, G

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Analysis of thermal stability of soya globulins using
monoclonal antibodies. Biochim Biophys Acta 1388:215–226,
1998.
11 11: Determination of Trace Protein
Allergens in Foods
Soybean Shibasaki et al. (21), Bucks et al. (22), Bush et

al. (23), Herian et al. (24), Ogawa et al. (25), E-F
Ebabiker et al. (26)
Peanut Sachs et al. (27), Barnett et al. (28), Bucks et al.

(29), Bucks et al. (30), Uhlemann et al. (31), Hefle et
al. (32), de Jong et al. (33)
Shrimp Hoffmann et al. (34), Nagpal et al. (35), Lehrer et

al. (36), Reese et al. (37)
Brazil nuts Hide (38), Morgan et al. (39), Nordlee et al.

(12), Borja et al. (40)
Almonds Bargman et al. (41), Hlywka et al. (42)
Egg white Hoffman (43), Langeland (44), Leduc et al. (45)
Milk proteins Ball et al. (46), Restani et al. (47),

Rosendal and Barkholt (48)
the absence of the allergenic individuals. Immunoblotting

is the default method for
detecting allergens in food materials. The main

disadvantages are that (a) care is needed
in the selection of human donors for pAb and (b) a

requirement for human pAb increases
costs. Once a major allergen is identified and isolated,

less expensive sources of pAb or
mAb may be developed. 2. SOYA BEAN PROTEIN ALLERGENS 2.1.

Structure and Characteristics of Soya Bean Allergens
Soybean allergy is common in children. The allergic

reaction is induced by soy protein,
with soybean oil being comparatively hypoallergenic (51).

The identities of soybean
protein allergens were established by SDS-PAGE and

immunoblotting with serum pAbs
from allergic patients. The same approach enabled the

identification of food allergens
associated with peanut, shrimp, almonds, Brazil nut, and
egg white (Table 3). Research
leading to the identification, characterization, and

quantitative assays for the major
soybean allergens is described in this section. Sixteen

soybean allergens were identified SDS-PAGE analysis,
Western transfer to a
nitrocellulose membrane, and immunoblotting using pAb

pooled from 361 patients
exhibiting atopic dermatitis (eczema). Protein bands were

visualized using iodine-125
labeled (rabbit) pAb for human IgE and autoradiography

(25). Approximately 20% of
patients with atopic dermatitis showed IgE production for

soy protein. The major soybean
allergens were associated with the 7S protein fraction

(7SF). Of the subset of 69 patients
showing sensitivity to soybeans, two thirds produced pAb

for a 30-kDa protein thereafter
named Gly m Bd 30K. About 23% of patients had circulating

pAb for a 70-kDa protein
designated Gly m Bd 70 (Table 4). Only 1.4% and 2.9% of

patients produced pAb for the
A subunit of glycinin and the Kunitz soybean trypsin

inhibitor, respectively. It has been
suggested that the pattern of IgE specificity can vary in

different populations depending
on (a) the age of the subjects, (b) history of

sensitization to the allergen, (c) route of
sensitization (airways vs. digestive tract), and (d) method

of sample pretreatment for
immunoblotting. It remains to determined whether Gly m Bd

30K is the major soy
allergen in other clinical populations. The foremost
soybean allergen (Gly m Bd 30K) seems identical to a 34-kDa
oil body
protein from soybean seeds. Ogawa et al. (52) found that

native Gly m Bd 30K forms a
350-kDa oligomer that elutes ahead of glycinin during gel

filtration column
chromatography. Treatment with SDS and 2-ME produced a

dissociated 30-kDa protein
with a pI of 4.5. The TABLE 4 Major Soybean Allergens

Detected by Immunoblotting with pAb from 69 Patients
(average age 6 years)
Protein size (kDa) Identity Patients with antibody (%)
30 7SF, Gly m Bd 30K 65.0
68–70 β-Conglycinin α-subunit 23.3
28 7SF, Gly m Bd 28K 23.3
63–67 7SF 18.8
52–55 7SF 14.5
47–50 7SF 10.1
43–45 7S globulin, /?-unit 10.1
33–35 7SF 15.9
35 11S globulin, α-unit 1.4
20 2S, KSTI 2.9
Source: Ref. 25.
N-terminal 15-amino-acid sequences for Gly m Bd 30K and

34-kDa oil body protein
were the same. The mAb for either protein cross-reacted

with the other. Following
established convention, Gly m Bd 30K, being the first

soybean allergen identified, was
designated Gly m 1. Kalinski et al. (53) independently
characterized the 34-kDa oil body protein. Within
intact cells, the protein was a vacuolar protein designated

P34. Like other storage
proteins, protein P34 undergoes post-translational

glycosylation and proteolysis. During
processing, P34 appears along the endoplasmic recticulum,

Golgi bodies, and eventually
within vacuoles or protein bodies. Protein P34 had partial

sequence homology with
cysteine proteases from the papain superfamily. It is not

certain that P34 has proteolytic
activity (54). Its association with soybean oil bodies was

an experimental artifact
produced by cell disruption. A survey of a large number of

soybean strains shows that
P34 is widespread. The possibility of eliminating P34 from

soybean lines by breeding
seems doubtful (55). The next important soybean allergen

(Gly m Bd 70K) is the 70-kDa β-conglycinin α
subunit. Antibody specific for the α-subunit did not

cross-react with the other two (a’ or
/?) subunits of β-conclycinin despite some sequence

homology between these
polypeptides (56). The third soybean allergen (Gly m Bd

28K) is a 26-kDa glycosylated
protein with a pI of 6.1 (57). It is apparently unstable

and present in very small quantities
in defatted soy flour (~ 15 ppm). Perhaps for these

reasons, Gly m Bd 28K was difficult
to detect in processed foods containing soy protein.

Finally, pAbs from some soybean
sensitive individuals recognized the A-chain residue from

glycinin. The epitope consisted
of a 114-amino-acid residue fragment (58). The epitope for
Gly m Bd 30K recognized by
mouse mAb was also identified by Hosoyama et al. (59).

2.2. Quantitative Analysis of Soybean Allergens
Quantitative ELISA for Gly m Bd 30K was developed by Tsuji

et al. (60) using mAb
produced by conventional means. Eventually, two cell lines

producing mAb (mAb-F5
and mAb-H6) were isolated. Immunoblot analysis showed that

mAb-F5 and mAb-H6
were both specific for Gly m Bd 30K. Sandwich ELISA for Gly
m Bd 30K employed
mAb-H6 as the capture antibody and an HRP conjugate with

mAb-F5 as detector. The
linear range for analysis was 2–200, 5–500, or 10–500 ng

protein for reduced/
carboxymethylated-allergen, SDS/2-ME-treated sample, or

native soy protein,
respectively. Ten soybean products and five meat products

containing soy protein isolate
(SPI) were analyzed by a sandwich ELISA for Gly m Bd 30K

(61). Results from this
study are summarized in Table 5.* From such data it may be

surmised that (a) high concentrations of Gly m Bd 30K are
detectable within a range of processed soybean products,

(b) fermented soy products
(miso, soyu, and natto) are free of allergen, (c) Gly m Bd

30K can be accurately
determined in cooked meat products known to contain soy

protein isolate, (d) sandwich
ELISA results were corroborated by immunoblot analysis

using pAbs from soybean
sensitive patients, and (e) Gly m Bd 30K is heat resistant,

accounting for its detection in
cooked products. The thermal stability of soybean antigens

is discussed in Chapter 10.
The allergen was also resistant to digestion by

chymotrypsin and trypsin. Levels of Gly m
Bd 30K declined to negligible values in fermented soy

products, probably as a result of
digestion by microbial (acid) proteases. Treating soybean

samples with added microbial
proteases reduced the level of allergen, leading to

hypoallergenic soybean products (62).
Clearly, the preceding assay has potential uses for

monitoring soybean allergen in
processed foods. The ELISA test could also find use as a

quality control tool for
hypoallergenic soybean products.
* As pretreatment each (5-g) sample was homogenized with

Na- phosphate buffer (50 mM, pH 8.0
with 1% w/v SDS and 20mM 2-ME). The extract was centrifuged
and SDS was precipitated with
KCl (1 M final concentration). After centrifugation, the

SDS-depleted sample was analyzed by
sandwich ELISA. TABLE 5 Determination of the Major

Allergen (Gly m Bd 30K) in Soybean-Containing Products
using Sandwich ELISA
Product Sandwich ELISA results (mg allergen g− 1 N)

Immunoblot analysis a
Soybean 126 +ve
Soymilk 106 +ve
Tofu (kinugoshi) 89 +ve
Tofu (momen) 65 +ve
Kori-dofu 64 +ve
Kinako 29 +ve
Abura-age 59 +ve
Yuba 66 +ve
Miso np b
Shoyu np
Natto np
Meatballs 17 +ve
Beef croquettes 21 +ve
Fried chicken 9 +ve
Fish sausages np
Hamburger np
a SDS-PAGE/immonoblotting with IgE from soy-sensitive

patients.
+ve=positive, (−) negative results.
b np, no allergen present.
Source: Adapted from Ref. 61. 2.3. Indirect Monitoring of

Soybean Allergens by ELISA
ELISA for bulk soy protein analysis (Chapter 10) may be

useful for monitoring soybean
allergens indirectly. Yeung and Collins (63) described a

competitive ELISA using
(rabbit) pAb for whole soybean extract. The assay, which

was virtually identical to those
used by Hitchcock and co-workers (64–67), was intended to

detect bulk soy protein as
opposed to trace allergen. The antigen for immunizing

rabbits was prepared from
soybean flour defatted with cold acetone.* The competitive

ELISA for soy protein (63)
had the following characteristics:
1. Specificity. There was no cross-reactivity with 10 other

legumes, 8 varieties of nuts, 11 common food ingredients,
and 2 phytoestrogens.
2. Acuity. The concentration of soy proteins that produced

50% inhibition of pAb-antigen binding (IC 50 ) was 35ngmL
−1 and the linear range was 3–117ng mL−1. For various
real foods, the detection limit was 2 ppm.
3. Recovery. For processed tuna fish having 13.5–54ppm of

soy protein, the recovery was 77–95%. Similar recoveries
(63–96%) were observed with hamburger matrix.
4. Precision. For model solutions and hamburger matrix

containing soy protein, the interassay precision was
3.5–4.2% and 3.6–8%, respectively. The corresponding
intraassay precision 2.4–5.1% or 1.5–2.6%. Features such as
assay specificity and acuity were not routinely reported
for ELISA designed to quantify soy protein in meat samples
(Chapter 10).
* Soy flour was extracted with cold Tris-HCl buffer (10 mM,
pH 8, with 1% w/v SDS and 10mM
2-ME) at 4°C for 16 hours. 3. PEANUTS 3.1. Peanut Allergy
Peanut (groundnut) allergy occurs in about 1 in 200 of the

general population in the
United States. The incidence rate is probably similar in

Western Europe. Emmett et al.
(68) found a partial association between peanut allergy and

allergy to tree nut. Peanut
allergy also persists for life. Allergic subjects appear in

all age groups and with no gender
bias (69). About 25–28% of all cases of food allergy

involve peanuts. The figures for tree
nuts are Brazil nut (10.2%), almond (7.8%), and hazelnut

(7.1%). The incidence of
peanut allergy also appears related to the frequency of

exposure (70). In a study of 868
children in Singapore, 27% of subjects showed sensitivity
to bird’s nest soup. The
incidences of other food allergies were 24% (crustacean),

11% (eggs and cow milk), and
7% (traditional Chinese herbs). There was no recorded

adverse reaction to peanuts or tree
nuts. The low incidence of peanut allergy was attributed to

the low per-capita
consumption of peanuts and other nuts in Singapore (71).

Symptoms of anaphylactic shock arise in approximately 6% of
allergic reactions to
peanuts. The onset of fatal and near-fatal anaphylaxis can

be very rapid. The first reaction
may appear within 5–30 minutes of contact with the allergen

(72). More frequent clinical
effects include atopic dermatitis or eczema (40%),

angioedema (37%), and asthma
(14%); digestive symptoms occur more rarely (1.5%). The

quantity of peanut necessary
to produce an adverse reaction ranges from a few micrograms

to 1 g (73). The
anaphylactic response can arise from topical contact with

peanut residue on another
individual, by inhalation (74), or by exposure to peanuts

in vegetarian “beef” burgers
(75). Small amounts of peanut protein associated with

peanut oil can also induce an
allergic episode. Peanut protein was found in some infant

formula prepared from peanut
oil (76,77). Highly refined protein-free oil apparently

poses little threat (51). Reported fatalities due to
peanut allergy are of the order of 125 persons per year in
the
United States (84). Figures from Pumphrey et al. (78)

suggest that there were 21 deaths
from peanut and tree nut allergy in the United Kingdom
during the 5- to 6-year period
following 1992. Of 541 patients showing sensitivity to

nuts, 90% had serum IgE specific
for peanut. About 67% of patients showed serum IgE for

another nut besides peanut,
while 34% of patients had immunoglobins for all nuts

tested. The threshold quantity
necessary to induce allergy varies for different subjects.

Exposure to peanut allergens in
early infancy (<1 year old) or before birth may contribute

to sensitization (70). It is not
yet certain whether pregnant and lactating women should

avoid peanuts and tree nuts in
their diet (78–80). Avoidance of peanuts is the main

strategy for managing the diet of sensitive
individuals. Hidden allergens from peanut-derived

ingredients are a major issue. Eating
outside the home setting can also be problematic.

Restaurateurs cannot provide strict
assurances that their ingredients or recipes are free from

peanut allergens. People thought
to be highly sensitive to peanut protein are also advised

to have rapid access to
epinephrine (adrenaline) by injection. In summary, peanut

and nut allergies present
serious health threats. Reviews by Sampson (81), Hourihane

(82), Burks et al. (83,84),
and also Warner (85) deal with the clinical and public
health aspects of peanut allergy.
Some distinctive features of peanut allergy are (a) high

incidence in the population, (b)
speed and severity of the symptoms, (c) lack of an

effective treatment, (d) widespread use
of peanuts and peanut ingredients in the food chain, and

(e) persistence of peanut
sensitivity with age. The availability of tests for trace

amounts of peanut will reduce the
risk of exposure to peanut allergens. 3.2. Structure and

Characteristics of Peanut Allergens
Proteins from peanuts are predominately globulins (~ 90%

total). Approximately 10% of
the storage proteins are albumins. Other classes of storage

proteins (glutelins, prolamins,
etc.) are not present. Johns and Jones (86) divided the
peanut globulins into two groups,
arachin and conarachin. The foremost peanut allergen (Ara h

1) is thought to be the 60–
65 kDa subunit of conarachin. Allergin Ara h 2 corresponds

to a 21-kDa arachin subunit
(33). Therefore arachin and conarachin are legitimate

targets for ELISA tests designed to
monitor peanut allergens. SDS-PAGE analysis of peanut

protein treated with 2-ME
revealed 14 bands with molecular masses of 10–70kDa.

Western transfer to nitrocellulose
membranes and immuno-blotting with human pAb led to

visualization of 13 of the 14
polypeptides; a 26kDa protein was not allergenic, probably

due to denaturation by heat
and SDS treatment. Six major allergens (i.e., recognized by

antibody from ≥50% of
patients) had molecular masses ranging from 17 to 44 kDa

(Table 6). Fractionation of arachin and conarachin can be
achieved by three strategies (87,88):
1. Cryoprecipitation. Extract crude peanut protein by

stirring defatted flour with 0.2M phosphate buffer (pH
7.9) for up to 4hours. Dialyze against several changes of
tap water at 4°C. Arachin precipitates while conarachin
remains soluble. This can be recovered by a range of
methods, e.g., acid precipitation or freeze-drying.
2. Ammonium sulfate fractionation. Precipitate arachin and

conarachin from solution by adjusting to 0–40% and 60–80%
saturated ammonium sulfate, respectively. When this is
preceded by cryoprecipitation, the samples of arachin and
conarachin obtained are homogeneous. Chiou’s procedure
(89) for isolating conarachin is simple and effective. Add
peanut (acetone) powder directly to 60% saturated ammonium
sulfate solution. Centrifuge and then adjust the
supernatant to 85% saturation by adding more ammonium
sulfate. The precipitated protein is conarachin.
3. Ion-exchange chromatography. Fractionate crude peanut

protein using low-pressure chromatography on a DEAE
column. With FPLC, use a Mono-Q support. Three peaks (P1,
P2, and P3) are TABLE 6 Major Peanut Allergens
Molecular size (kDa) Patients with antibody (%)
26.0, 0
29.0, 28.0, 16.0 20
36 20–30
71.0, 60–64, 17.0 30.0–40.0
44.0, 40.0, 33.0 50–60
21.0, 20.0, 17–18 60–80
Source: Adapted from Ref. 33.
eluted with a 0–0.6 M gradient of NaCl. Protein peaks P2

(conarachin) and P3 (arachin)
normally elute at 0.23 M and 0.33M NaCl. Native arachin

(11–128 globulin) has a molecular mass of 350 kDa (90).
Treatment
with 2-ME followed by SDS-PAGE generates seven polypeptides

with molecular masses
of 72.4, 60.3, 39.8, 33.1, 29.0, and 21 kDa.* Native

conarachin is a 7S globulin with a
molecular mass of 180kDa. The quaternary structure is a
trimer of 60-kDa subunits. SDS
PAGE analysis of 2-ME-treated conarachin yields eight bands

(72.4, 39.8, 33.1, 26.9,
24.0, 21.9, 18.6, and 16kDa). These results from Monteiro

and Prakash (90) suggest that
peanut 7S protein has a different architecture from soybean

7S protein. The a, a’, and β
subunits of conglycinin appear to be devoid of S—S bonds.

In contrast, peanut 7S protein
is affected by 2-ME, indicating the presence of S—S bonded

subunits (91). 3.3. Thermal Denaturation of the 11S Peanut
Allergen (Arachin)
Ara h 2 is either the A or B subunit of arachin.

Investigations employing dry and moist
heat treatment show that arachin is very heat resistant.

Neucere (92,93) identified 14
peanut antigenic species by immunoelectrophoresis using

(rabbit) pAb. Moist heat
treatment (40% w/w moisture, 110°C) or dry heat treatment

(5% w/w moisture, 110–
150°C) for 60 minutes reduced the antigenicity of

nonarachin proteins monitored by
immunoelectrophoresis, rocket immunoelectrophoresis, or

AGID. To achieve the same
effect for arachin required moist heat treatment at 150°C

or dry heat treatment at 175°C.
Heating also reduced protein solubility. SDS-PAGE analysis

of peanut proteins heated at
100°C for 15–210 minutes showed little changes in the

patterns for arachin (94). Dry and
moist heating studies by Chiou (89) also confirm that

arachin is heat resistant. Shokraii
and Esen (91) found that treating peanut protein with SDS
and 2-ME had no effect on the
immunological characteristics. This raises the possibility

that some epitopes for peanut
allergens are continuous. That is, antibody binding occurs

with consecutive sequences of
amino acids. Discontinuous epitopes constructed from higher

order protein structure
would be destroyed by denaturants. 3.4. Thermal

Denaturation of the 7S Peanut Allergen (Conarachin)
The effect of thermal treatment on the conformation and

immunological characteristics of
Ara h 1 (β-subunit of conarachin) was examined by Koppelman

et al. (95). The Ara h 1
was isolated by cation-exchange chromatography of whole

peanut protein. Gel filtration
and sedimentation velocity measurements showed that native

Ara h 1 was a 180-kDa
trimer. An SDS-PAGE analysis confirmed the presence of

63-kDa subunits. The far-UV
spectrum for native Ara h 1 was consistent with there being

31% α-helix, 36% /β-sheets,
* Only six from seven bands were prominent.
and 33% aperiodic structure. The fluorescence spectrum for

Ara h 1 indicated that
tryptophan residues were solvent-exposed in the native

protein. Differential scanning
calorimetry thermograms showed a denaturation temperature

(T D ) equal to 87°C with a
small transition enthalpy (∆H m ) of about 120kJ mol −1

for an undefined irreversible
structure change. Upon heating samples of Ara h 1 for 15

minutes at 20, 50, 80, 90, 110, or 140°C,
protein solubility decreased to 75% and 32% at the last two

temperatures. SDS-PAGE
analysis showed the same molecular weights for heated and

unheated protein. Thermal
treatment did not produce significant changes in IgE

binding avidity for the soluble
protein fraction. Such results are consistent with one of

the following explanations: (a)
Ara h 1 is heat resistant, (b) the epitopes for IgE binding

are in parts of the three
dimensional structure not affected by denaturation, or (c)

the epitopes of Ara h 1 are of
the continuous type (confirmed in the following). The 3D

structure of Ara h 1 was elucidated from computer modeling
studies using the
crystal structure of phaseolin as template (96). The

locations of 23 linear/continuous
epitopes for human IgE binding to this allergen were also

established. For the native
protein, antigenic sites were clustered in the outer

exposed region of the 3D structure.
Most epitopes of IgE binding comprised nine amino acids.

There was no other common
structural feature for the epitopes. Single amino acid

substitution within epitopes
abolished antibody binding. 3.5. Digestibility of Peanut

Allergens
Protein stability and/or resistance to digestion appears to

be a feature of allergenic
proteins. Compared with a random group of plant proteins,

peanut allergen (Ara h 2) was
more resistant to digestion by simulated gut fluid (97).

The same was true for allergens
from soybean (β-conglycinin /β-subunit, KSTI, Gly m 1), egg

(ovalbumin), or milk (β
lactoglobulin, casein, and BSA). Analysis by enzyme
allergosorbent test (EAST) or
immunoblot analysis showed that simulated gut digestion

reduced the allergenicity of
peanuts and hazelnuts by 50 and <10% (98). The digestion of

soy proteins by rats
proceeded within 3 days to immunoreactive peptides (99).

Allergic reactions are liable to
be invoked by contact with raw, cooked, or roasted peanuts.

Peanut and other food
allergens will also have a tendency to survive common food

processing operations. 3.6. Determination of Peanut
Allergens
Peanut allergens were initially identified by

immunoblotting with human pAb (Table 3)
or using the RAST assay (17,18). Both techniques use

iodine-125 as label. These
approaches have limited general utility for several

reasons: (a) there is limited availability
of human antibody, (b) human antibody is difficult to

obtain as a standard reagent, (c)
there are possible biohazards associated with handling

human body fluid as a reagent, and
(d) RAST employs radionuclides for visualization (100).

More convenient ELISAs for
peanut allergen are now being developed by research groups

in several countries
including the United States (100), Netherlands (101),

Canada (102), United Kingdom
(103), and Germany (104). Keck-Gassenmeier et al. (105)

evaluated several ELISA kits
for peanut allergens before adapting one for

semiquantitative analysis of confectionery
products (see later). The first of the new generation of
tests for peanut allergens* was developed by Hefle
et al. (100). Results from the sandwich ELISA were

positively correlated with RAST
results for a range of samples including cheesecake mix,

chocolate peanut cookies,
chocolate-nut granolar bars, confection-peanut brittle, and

peanut butter. The high
correlation (R=0.8 5) is to be expected because the mAb

used for the ELISA test was
specific for peanut allergens recognized by human IgE. The

working range of the ELISA
was 3.2–3162µg mL −1 of peanut extract. With ice cream,

the LLD for peanut protein was
40 µg mL −1 . This level of acuity is higher than observed

with the traditional skin test for
peanut allergens. A competitive indirect ELISA for trace

amounts of peanut protein was developed by
Yeung and Collins (102). The IC 50 was adopted as an index

of specificity. The linear
range for analysis was 1–63 ngmL −1 extract (R 2 >0.98)

with an LLD equal to ~400ng g −1
(food sample). Negligible responses were obtained for

protein extracts from the following
legumes and nuts: soybean, green pea, chick pea, lupine,

hazelnuts, Brazil nuts, pine nut,
pecan, almonds, walnut, and cashew nut. Also, no

interferences occurred with general
foodstuffs such as corn, cocoa, chocolate, milk, sugar, soy

lecithin, peanut lectin, and
coconut. Peanut proteins were quantitatively determined in

over 40 processed food items
belonging to the following food groups: potato chips

(crisps), cooking oil, pasta sources,
plain cookies, ice cream, and milk chocolate bars. All
products labeled as containing
peanut protein were correctly identified with no

false-positive results. A sandwich ELISA using (rabbit)
pAb for purified Ara h 1 was described by
Koppleman and co-workers (101). The assay characteristics

can be summarized as
follows:
1. Range. The linear dynamic range for peanut protein was

0.005–1 µg mL−1. Fried peanut samples gave 45–80% of the
response obtained from raw samples.
2. Specificity. No cross-reactivity occurred with 40 food

products including, 9 legumes, 9 nuts, 7 cereals, 4 oil
seeds, and 9 animal proteins. In tests involving 31
processed goods (10 cookie brands, 6 chocolates, 3
cereals, 3 baby foods, and 8 sources and soups) all
products labeled as containing peanuts were accurately
identified.
3. Recovery. For samples spiked with 0.0001–1.0% peanut

protein, recoveries were typically 85–105%. Reproducible
recoveries were obtained from high-fat foods.
The competitive indirect ELISA described by Holzhauser and

Vieths (104) employed
commercial pAbs for unheated peanut protein. Before use,

pAbs were purified by
immunoadsorption with soy protein, white bean, and

marzipan. Precooking peanuts had
little effect on the levels of Ara h 1 and its binding by

both human and rabbit pAbs. The
final assay was successfully applied to over 30 commercial

food products. Three commercial ELISA kits for peanut
allergen were evaluated (105) at the Nestle
Research Center (Lausanne, Switzerland). The kits were

those from Cortecs Ltd. (UK),
Prolab Ltd. (Canada), and the TNO (Netherlands). The last

two kits appear to be
* Whole peanut protein was used as antigen. An mAb and
biotinylated (rabbit) pAb were used as
capture antibody and for detection, respectively.

Visualization was with an HRP-streptavidin
conjugate assayed with OPD-hydrogen peroxide substrate.
derivatives of the assays developed by Yeung and Collins

(102) and Koppleman and co
workers (101). Detailed results concerning the preliminary

evaluation were not reported.
The Cortecs kit was further evaluated for a

semiquantitative determination of trace
amounts of peanuts in a range of confectionery products.

The particular kit employs a
sandwich ELISA format with antibody for concanavalin A for

capture. Biotinylated
antibody for concanavalin is used for detection. Bound

antigen is visualized with
streptavidin-labeled HRP assayed using

tetramethylbenzidine. Recovery of peanut
allergen from dark chocolate was only 2–3% when using the
manufacturer’s extraction
buffer (TBST). Inclusion of 12.5% fish gelatin in the

extraction solvent increased
immunogen recovery to 65–97%. With the modified sample

extraction procedure, the
Cortecs ELISA kit was successfully applied to a wide range

of foods including milk
chocolate, chocolate confectionery, ice cream, sherbet,

ready-to-use refrigerated cookie
dough, lemon curd, apple sauce, piccalilli, honey, jelly,

and a frozen meal. The test kit
originally intended for a qualitative assay of peanut

protein could be adapted for a
semiquantitative analysis (105). In conclusion, several
ELISA assay formats are available for detecting trace
amounts
of peanuts and tree nut allergens (106). These highly

sensitivity assays can detect as little
as 2mg (allergens) per kg of food (2 ppm). Making sure that

allergen can be effectively
solubilized from highly processed foods is a limiting

factor. Present assays can readily
provide a yes/ no indication for hidden peanut allergens.

Self-operated diagnostic tests for
peanut allergens would be useful for domestic use. 4.

WHEAT AND RELATED CEREALS 4.1. Wheat Allergy
Adverse reactions to cereals and cereal products include

dermatitis herpetiformis, baker’s
asthma, and celiac disease.* The last condition is related

to the consumption of the
alcohol-soluble cereal proteins (prolamins) from wheat,

barley, rye, or triticale. It is
doubtful whether oat is a source of celiac-active protein

(107,108). Rice, millet, sorghum,
maize, and buckwheat are reportedly nontoxic to

gluten-sensitive individuals (109,110).
Baker’s asthma is ascribed to inhalation of airborne cereal

amylase inhibitor proteins
(111). This section focuses on allergy to wheat gluten and

related proteins. Various
aspects of celiac disease have been reviewed by Goodwin and

Rawcliffe (112), Baldo and
Wrigley (113), Ciclitira and Ellis (114), Cornell (115),

Brozzone and Asp (116), Silano
and Vincenzi (117), Feighery (118) and Hadjivassiliou et

al. (119). Marsh (120) wrote a
monograph on this subject. Celiac disease appears in 1 in
300 of the general population in Western Europe except
in Italy, where the frequency is nearer 1 in 4700. The

incidence of nontropical sprue in
the United States is about 1 in 250 (121). One of the

highest occurrences of celiac disease
was reported for the Seharawi tribe from western Algeria

and parts of Morocco. Their
staple diet
* Celiac disease is also known as gluten-sensitive

enteropahathy, gluten-induced enteropathy, or
nontropical sprue.
consists of couscous and bread. Catassi and co-workers

(122) suggested that celiac
disease has adaptive value in preventing intestinal

adhesion and infection by pathogenic
microorganisms such as Vibrio cholerae. The major lesion

accompanying celiac disease is atrophy of microvilli from
the small
intestine, duodenum, and jejunum. Microscopic observation

shows a flattening of the
mucosal lining and reduced surface area. There is

malabsorption of most nutrients. The
intestinal absorptive layer, being compromised, may allow

the uptake of a greater range
of psychoactive peptides. Theories explaining the etiology

of gluten-sensitive enteropathy
are summarized by Cornell (115), who also describes in

vitro methods for assaying
celiac-reactive polypeptides or allergens. Bruzzone and Asp

(116) provide an informative
introduction to gluten-sensitive enteropathy starting from

its discovery in 1950–1953 by
Dickie and covering possible links between diet and the
immune system. Progress in the
study of gluten-sensitive enteropathy has suffered owing to

the lack of reliable assays for
the condition. Current methods for diagnosis for this

condition involve
1. Use of organ culture. Intestinal biopsy spceimens when

grown in cell-tissue culture show normal microvilli within
24 hours. Addition of gluten (peptides) to culture media
reintroduces abnormalities in microvilli.
2. Detection of circulating antigliadin antibodies. Blood

serum from sufferers from gluten-sensitive enteropathy is
used as the basis of indirect ELISA.
The symptoms of celiac disease in infants and young

children include growth
impairment, abnormal stool, abdominal distention, and

psychological effects, which show
up as generally poor temperament. Adults sometimes exhibit

diarrhea, flatulence, bulky
stools, anemia, fatigue, weight loss, bone weakness, and

neurological disorders including
depression and schizophrenia. Treatment for celiac disease

involves the exclusion of
gluten from the diet. United Kingdom regulations state that

gluten-free foods should
contain no more than 300 mg (gliadin) per 100 g foodstuff

(i.e., 300 mg % or 0.3% w/w).
European Union and WHO limits for gluten-free foods are

between 10 and 1 mg %. Wheat gluten is an inexpensive
protein and therefore widely used as a “vegetable
protein” ingredient for meat products (123). Wheat flour is

used for thickening soups and
other processed foods. Gluten appears in confectionery

products and is also used as a
binder for pharmaceutical tablets. Beer contains barley

protein. Rye bread is widely
consumed in parts of Eastern Europe. Raw meat products such

as sausages may contain
wheat proteins derived from rusk. Hidden allergens need to

be considered in attempts to
reduce celiac-active proteins and peptides in the diet.

There is generally inadequate
labeling information with respect to products having wheat

or barley protein. Levels of
potentially active celiac proteins and peptides in exotic

foods, ethnic cuisine, and
restaurant or cafeteria foods are ill defined. A number of

manufacturers specialize in the
production of gluten-free products. Most developed

countries have celiac disease
societies that coordinate information useful for sufferers.

4.2. Structure and Characteristics of Dietary Wheat
Allergens A. Classification of Cereal Proteins
Thomas Burr Osborne divided plant seed proteins into five

classes according to their
solubility in water, 0.1M sodium chloride, alcohol, or

alkali. Sequentially extracting flour
using this range of solvents recovers albumins (in water),

globulins (with 0.1 M salt),
prolamins (in 40–70% ethanol, and glutelins (with 0.1M

sodium hydroxide). A fifth class
of protein (called residue protein) can be extracted only

with severe solvents. The
glutelins are extractable in alcohol and 2-ME or DTT. About

70–90% of the storage
proteins in cereals consist of nearly equal proportions of

prolamin and glutelins.* An
important exception is cereal oat, which contains 80%

globulins. Rice has 1–5% prolamin
and 80–90% glutelin. The allergens responsible for celiac
disease are thought to be amino
acid repeat sequences found in cereal storage proteins.

Tatham et al. (124) devised a new classification scheme for
cereal proteins (Table 7).
The new scheme acknowledges fundamental structural TABLE 7

Cereal Prolamins Thought to Be Involved in Celiac Disease
Prolamin Wheat Rye Barley
Sulfur rich
Monomeric γ-Gliadin γ-Secalin γ-Hordein
β-Gliadin
α-Gliadin
Polymeric LMW glutenin B hordein
Sulfur poor ω-Gliadin ω-Secalin C hordein
HMW HMW glutenin HMW secalin D hordein
relationships between the different protein fractions.

First, all cereal protein fractions
(besides albumins) are soluble in 70% alcohol and are

therefore classed as prolamins.
Next, the storage proteins are grouped according to

similarities in their amino acid and
cDNA sequences into three classes: (a) sulfur-rich

prolamins (α-, β-, γ-gliadin), (b)
sulfur-poor prolamins (ω-gliadin), and (c)

high-molecular-weight (HMW) prolamins. The
sulfur-poor prolamins are monomers possessing only

intramolecular disulfide bonds. This
group includes ω-gliadin (wheat), ω-secalin (rye), and C

hordein (barley). Some sulfur
rich prolamins form HMW polymers. By contrast, α-, β-, and

γ-gliadins represent
* By contrast, legume seeds (soya bean, pea, kidney bean,
and peanut) contain globulins as the
major storage proteins. There is relatively little (0–10%)

prolamin or glutelin present.
monomeric sulfur-rich prolamins. The three classes of

prolamins have related
architectures. B. Gluten
Gluten is prepared by washing wheat flour with water to

remove the starch. This leaves a
viscoelastic protein network (gluten) that is responsible

for the gas-retentive properties of
wheat dough. Gluten is a network formed by interacting

wheat prolamins (Table 7). Each
of the four gliadins can be prepared on a large scale by

ion-exchange chromatography
(125).* A single chromatographic run involving about 54 g

crude gliadin yields 1 g of ω
gliadin and approximately 10 g each of γ-, β-, and

α-gliadin. Electrophoresis at low pH
separates of gliadin into four subtypes. SDS-PAGE analysis

shows the following order of
increasing electrophoretic mobility: α-gliadin > β-gliadin

>, γ-gliadin > ω-gliadin.
Glutelin is highly polymerized by S—S bonds. The

sulfur-rich prolamins possess N-terminal repetitive amino
acid sequences
containing high numbers of three amino acids: glutamine

(Q), proline (P), and
phenylalanine (F) (Figure 1). The C-terminal areas have

variable sequences bearing a
number of half cysteine residues normally involved in

intramolecular S—S bonding. Also
noteworthy is the rare occurrence of SH groups in

ω-gliadin. Regularities are also evident
in the 2° structure of cereal proteins (126,127). Thus,

α-gliadin, β-gliadin, and γ-gliadin
have 36–37% α-helix, 11–12% /?-sheet, and 52–53% aperiodic

structure plus β-turns.
The precise quantity of β-turn or hairpin loop structure is

uncertain because this does not
produce a distinct circular
* Stir 1 kg of flour with water-saturated n-butanol to

remove lipids. Next, extract with four volumes
of 70% (v/v) aqueous ethanol. Dry the extract by rotary

evaporation and redissolve the protein in
0.1 M acetic acid solvent. Dialyze against the same solvent

and fractionate with a CMC-cellulose
column (10cm×22cm, containing 1.7 kg support) equilibrated

with sodium acetate buffer (5 mM+1
M dimethylformamide and adjusted to pH 3.5 with acetic

acid). Elute the bound protein using
stepwise changes in NaCl concentration gradients. FIGURE 1

The primary structure of cereal prolamins. (Adapted from
Refs. 124 and 126.)
dichroism absorbance peak. However, numerical calculations

using amino acid sequence
data suggest that the repetitive sequences are β-turns.

Celiac toxic peptides have been identified at the
N-terminal regions of α-gliadin and β
gliadin (117,128,129). The epitopes recognized by

intestinal T cells appear to be two
tetrapeptides, PSQQ and QQQP. The first occurs in α-gliadin

at amino acid residues 13–
16, 50–53, and 213–216. QQQP occurs at residues 16–19,

33–36, and 188–191. Four out
of six potentially toxic sequences appear within the first

56 amino acids from the N
terminal. The epitope bound by mAb-WC2 is probably QQQP,
which is apparently
common to all celiac-active proteins (129). Tanabe et al.

(130), using antibody from
celiac sufferers, identified the sequence QQQPP as being

celiac active. Bromelain digests
proline-rich peptides leading to wheat flour that may be

useful for producing
hypoallergenic bread (131). Ginger protease has high

specificity for proline residues
(132). 4.3. Thermal Denaturation of Wheat Allergens
The thermal denaturation of gliadin was examined using 70%

(v/v) ethanol as solvent.
Heating γ-gliadin led to a conformational change at 20–60°C

as monitored by far-UV
circular dichroism. The spectral changes showed an

isobestic point implying that
denaturation was a two-state (helix-coil type) transition.

At elevated temperatures the
proportion of α-helix declined by 7% and the structure of

gliadin loosened to expose
phenylalanyl residues to the solvent. Purified α-, β-, and

ω-gliadin behaved similarly.
Conformational changes produced by the relatively short

(5-minute) thermal treatments
were reversible (126,127). Heating wheat flour matrix

revealed that ω-gliadin is more
heat resistant than the sulfur-rich gliadins.* The levels

of α/β-gliadin decreased
substantially after 20 minutes of heating. The γ-gliadin

was resistant for up to 50 minutes
of heating at 100°C. There was no change in the quantity of

ω-gliadin recovered after 0–
100 minutes of heating. This result is due to the virtual
absence of half cystine residues in
ω-gliadin. This protein can not undergo

sulfhydryl-disulfide exchange during heating
(133). 4.4. Determination of Wheat Allergens
Immunological assays for cereal proteins are generally

concerned with the detection of
celiac-active peptides. Some attempts to assess grain

varietal differences by EIA were
reported (134). Also of interest is the identification of

(bread making) quality-related
polypeptides by immunoassay (135,136); literature in this

area was reviewed by Skerritt
et al. (137). Sections 4.4 through to 4.10 consider ELISA

tests for trace (nanogram to
microgram) quantities of cereal proteins. ELISAs for wheat

allergens and bulk protein
adulterants are not fundamentally different in their

design. However, the former
techniques possess greater acuity. SDS-PAGE-immunoblot

analysis is another ELISA
format. 4.5. Sandwich ELISA Tests for Gluten Using pAb
McKillop et al. (141) provide a useful introduction to

ELISA of wheat gliadin. Sandwich
ELISA and competitive indirect ELISA formats were

implemented using (rabbit) pAb for
unfractionated gliadin. The two-antibody format was

selected for full evaluation. The
assay characteristics were as follows:
1. Linear range. 0.05–6µg (gliadin)mL −1 .
2. Detection limit. A concentration of 23 ng (gliadin) mL

−1 produced absorbances of 2 SD above the reagent blank.
3. Specificity. Gliadin and gliadin-containing foods. Wheat
flour had 5.7% (w/w) gliadin equivalent protein. The
values for oats, rice, and corn flour were 240, 46, and 47
mg %, respectively.
* A suspension of wheat flour in water was heated at 100°C

for 0, 10, 20, 30, 50, and 100 minutes.
Flour proteins were then extracted with 1 M urea solution

and analyzed by gradient gel
electrophoresis.
4. Precision. The within-assay reproducibility for analysis

ranged from 5 to 40%. Samples with >330ng (gluten) mL − 1
were determined with below+10% error.
The preceding assay enabled the identification of

“gluten-free” ingredients. No tests were
performed on heated samples. Table 8 summarizes other

ELISAs for wheat proteins. Fritschy et al. (140) used
(rabbit) pAb for total gliadin as the basis for a gluten
sensitive ELISA. Wheat flours (20 different types) were

found to contain 2.9–6.7% (w/w)
gliadin equivalent proteins. The gliadin contents of many

commercial gluten-free foods
were insignificant. Rice contained 57.5mg % (w/w) and

sorghum 12.5mg % (w/w)
gliadin on a dry weight basis. Food samples were extracted

with 70% (v/v) ethanol with a
recovery of 77–107%. The LLD was 10ng (gliadin) mL −l

sample extract. The assay was
specific for cereal proteins. It was possible to assay

rice, corn, oats, and barley protein
when 1–2% BSA was included in samples to avoid nonspecific

protein binding to
microwell plates. Troncone et al. (145) purified (rabbit)

pAb by affinity chromatography using
Sepharose 4B-immobilized gliadin. They then implemented a

conventional sandwich
ELISA using (rabbit) pAb for capture. The linear dynamic

range for gliadin analysis was
5–400 ngmL −1 with an LLD of 0.5 ng (3 ng for the

competitive assay). There was cross
reactivity with maize and oat prolamins, although these

were detected with 1000-fold
lower sensitivity. The response for gliadin was 10 4 -fold

higher than obtained for rice
prolamin. Rye and barley were not assayed. Interference

from maize, rice, or oat
prolamins could be avoided by using high protein dilution.*
* Equal amounts of prolamin from different cereals yield

1000- to 10,000-fold differences in assay
sensitivity. The ELISA response for wheat protein (cf.

maize, oats, and rice proteins) measures
“gluten”-like structure and functionality. ELISA results do

not necessarily show the absolute
amounts of prolamins present in the different cereals.

TABLE 8 Analysis of Gliadin and Other Prolamins by ELISA
Sandwich, pAb α-Gliadin, gliadin Windemann et al. (138),

Meier et al. (139), Fritschy et al. (140)
Sandwich, Competitive, pAb Gliadin McKillop et al. (141)
dot blotting, mAb ω-Gliadin, gliadin Skerritt and Smith

(133), Skerritt (142), Skerritt et al. (143), Freedman et
al. (144)
Sandwich, Competitive, pAb Gliadin Troncone et al. (145)
Sandwich, mAb Gliadin Freedman et al. (146)
Immunoblotting Gliadin Janssen et al. (147)
Sandwich, pAb Gliadin Ayob et al. (148)
Competitive, pAb Gliadin Friis (149), Chirdo et al. (150)

Sandwich, mAb α-Gliadin, γMills et al. (151) gliadin
Sandwich, mAb AOAC
approved laboratory test ω-Gliadin, glutenin subunits

Hill and Skerritt (152), Skerritt et al. (153), Skerritt
and Hill (154, 155)
Sandwich, mAb Home kit ω-Gliadin, glutenin subunits

Skerritt and Hill (156)
Sandwich-ELISA Gliadin Hekkens and Twist de Graaf (157)
Sandwich, mAb Competitive Gliadin peptides Ellis et al.

(158), Denery-Papini (159), Ellis et al. (160), Nicolas et
al. (161)
Gliadin was accurately determined in human milk. Eleven of

18 lactating mothers who
had consumed gluten meals 2 hours prior to testing showed

gliadin-reactive material in
their breast milk. Gliadin was detectable in human plasma

after heat treatment to denature
circulating human antibodies. Thermal treatment (121 °C, 5

minutes) had no adverse
effect on assay sensitivity. There was apparently no

immunoreactive material in blood
plasma from celiac patients (145). By contrast, Lane et al.

(162) found significantly high
levels of gluten in the serum of patients suffering from

dermatitis herpetiformis and
celiac disease (compared with normal controls). Different

assay conditions used in these
studies may explain the lack of agreement. It is perhaps

significant that Lane and co
workers calibrated their assay by adding gliadin to human

plasma. The performance of a low-cost sandwich ELISA test
for gluten was compared with
two commercial gluten tests produced by Cortecs Ltd. (UK)

and R-Biopharm (Germany)
(163). Results for the in-house test were highly correlated
(R=0.0.9967) with both
commercial tests. The former was more sensitive than the

Cortecs gluten test (see the
following). The in-house test had the advantage of a

10-fold lower cost. The linear range
for the in-house assay was 50–150ngmL −1 compared to 20–80

ngmL −1 for the R
Biopharm test. The LLD values of the two tests were 0.04
and 0.1% mg %, respectively. 4.6. Competitive ELISA for
Gluten Using pAb
A competitive ELISA test for gliadin (149) had a working

range of 10–250 ngmL −1 with
an LLD of 1 ngmL −1 . The highly accurate test had an

interassay precision of 33%. There
was specificity for α-, β-, γ-, and ω-gliadin as well as

barley, oats, and rye prolamins. No
responses were obtained for maize, soya, or millet protein.

Buckwheat gave a low
response. The competitive ELISA test for gluten was not

tested for heat-processed
samples. Chirdo et al. (150) used (rabbit) pAb for

competitive ELISA for gluten in a
range of processed foods including cake flour, breakfast

cereal, processed meat, soup,
and chocolate. The working range for gliadin standards was

0.6 ng mL −1 to 10µgmL −1 .
The LLD of 1ng mL −1 (0.002mg %) was the same as reported

by Friss (149). The average
within-assay precision was about 9%. It was claimed that

this test was useful for
thermally processed foods. 4.7. The AOAC-Approved ELISA

Test for Gluten
The AOAC-approved test for gluten employs mAbs for capture
and detection (154).
Skerritt and Smith (133) and Skerritt (142) found two mAbs
(mAb-401/21 and mAb
304/13) with specificity for ω-gliadin, HMW and LMW

glutenin subunits. Because ω
gliadin is heat resistant (Section 4.3), the preceding

tests may be suitable for the analysis
of heat-processed foods (see later). Either mAb-401/21 or

mAb-304/13 could be used for
capture or detection, leading to similar results.

Preliminary tests (153) showed specificity
for wheat (bread or durum) >rye~barley maize oats. No

responses were observed for
oat, maize, or rice protein at levels comparable to those

for wheat gluten. Sorghum was
not tested. Assay performance depended on these factors

(152,153,154):
1. Extraction solvent. The optimal solvent for gluten

extraction from a range of food samples was 40 % (v/v)
ethanol. Using 70% (v/v) ethanol, 1 M urea, or 1 mM HCl as
solvent led to underestimation or overestimation of the
gluten content.
2. Form of extraction. Homogenization of flour samples

using an omnimixer or Ultraturrax mixer for about 30
seconds led to accurate analysis. Vortexing for 30 or 60
seconds duration (four times per hour) led to inaccuracy,
probably due to shearinduced precipitation of gluten.
3. Choice of gluten standard. Normal gluten is a suitable

standard. This is relatively soluble, stable in the
freeze-dried form, and usable over a wide concentration
range (dilutions of up to 10,000−1 were used for
analysis).
4. Choice of solid phase. Polystyrene microwell plates were

preferable to PVC plates, which produced high nonspecific
binding. Background adsorption of gluten was not
ameliorated by a range of blocking solutions including PBST
with 0.05–5% BSA, 0.65M NaCl, or 2% Tween.
5. mAb characteristics. The nature and concentration of the
capture and detector antibody and coating conditions (time
and temperature of coating) affected the assay.
Following optimization, the linear dynamic range of the

AOAC-approved ELISA test
was 0.0075–5 mgmL −1 (15mg % to 10% w/w gluten in actual

foods). The LLD was 0.1–
0.2µg mL −1 , which is equivalent to 0.2–0.38-mg gliadin

per 100 g of flour (0.2 -0.38 mg
%). A partial list of food types successfully analyzed

includes starches (wheat, maize),
cake flours (plain, self-raisin, bread crumb, cookie),

gluten-free bread mixes, processed
meat (cooked beef, ham sausages, salami), baby foods (beef

or chicken based with and
without flour thickener), breakfast cereals, baked goods

(bread crumb mix, sweet
cookies, crisp bread), soups, confectionery (caramel,

chocolate), and others (lentils, eggs,
milk powder). 4.8. Collaborative Testing of AOAC-Approved

ELISA Test for Gluten
Prior to AOAC approval, the previous ELISA was subjected to

a collaborative trial by 15
laboratories* (155). Eighteen samples including five

prestudy samples (three wheat
starches and two meat-gluten blends) with known amounts of

added gluten were tested.
Participating laboratories familiar with ELISA methodology

were supplied with
commercial versions of the Skerritt-Hill test. The linear

dynamic range for gluten was
16mg % to 11% (w/w) basis. Assay reproducibility was 24–33%

with a repeatability of
19–22%. Gluten was accurately determined in a range of
foods (164). The accuracy of
the Skerritt-Hill ELISA test is indicated by its high

precision. Gluten levels reported by
collaborators were less than ± 12% different from expected

values. The collaborative
study led to AOAC approval for the Skerritt-Hill ELISA test

for gluten.
Potential limitations of the AOAC assay for gluten have

been suggested (165). A
minicollaborative trial using a limited number of gluten

test kits was organized by the
Celiac Society of Great Britain. Participating groups were

major UK clinical laboratories
with longstanding interest in celiac disease.† Results from

five laboratories agreed with
respect to four gluten-free foods and nine

gluten-containing foods (spaghetti bolognese,
egg and bacon breakfast, crispbread, malted drink,

porridge, barley, plain flour, and
wheat starches). By contrast, large disparities (five- to

sixfold differences) were reported
for gluten levels in 11 foods including egg and bacon,

beer, low-alcohol lager, stout,
gluten-free flour, gravy powder, cornflour, and rice

pudding. An mAb-based gluten test
developed by Mills et al. (151) showed a linear range

comparable to that of the AOAC
test. They used pAbs from chicken‡ for capture while

mAb-IFRN 033 functioned as the
detector antibody. The mAb-IFRN 033 was specific for

α-gliadin and γ-gliadin, which
together constitute ~85% of gliadin. The linear range for

gluten was 0.25–2.5 µgmL −1
with the LLD being 0.1 µgmL −1 . The preceding tests may
be compared with the 1987 sandwich ELISA test developed
by Freedman et al. (146) from Guys and St Thomas’s

Hospital, London. They used
(rabbit) pAb for capture and mAb for unfractionated gliadin

for detection. The bound
mAb was visualized with alkaline phosphatase-labeled (goat)

pAb for murine IgG/IgM.
The linear dynamic range for this assay was 10ngmL −1 to 1

µgmL −1 . Strong wheat flour
contained 3.7% (w/w) gliadin-equivalent proteins. Several

brands of “gluten-free” flour
produced from wheat starch had 1.9–3.3 mg % of gliadin. The

day-to-day precision of the
assay was better than 5%. 4.9. A Cocktail mAb-Based

Sandwich ELISA Test for Gluten Gluten tests should be
equally sensitive to all celiac-active proteins from wheat,
*The list of participants includes M.Billington (City of

Birmighman Public Analysts, Birmingham,
UK), A.Crimes, (Unilever, Bedford, UK), C.Cuncliffer,

(Somerset County Council, Taunton, UK),
M.Cutrufelli, (USDA, Betsville, MD), T.McKenny (Cerestar,

Manchester, UK), M.Murlry, (Kraft
Research Center, Glenview, IL), N.Paterl, (Campden, Food

and Drink Research Association,
Chipping Campden, UK), J.Rhodes, and D.Lord, (Lancashire

County Lab., Preston, UK), B.Ritter,
(ABC Research, Gainesville, FL), M. Scooter, (Food Science

Division, MAFF, London, UK),
M.Smith, (Avon County Scientific Services, Bristol, UK),

C.Stanley, (Laboratory of Government
Chemists, Teddington, UK), P. Sutton, and S.Cooper,

(British Food Manufacturing Industries
Research Association, Letherhead, JK), and B.Taylor and
D.Ansell, (Greater Manchester Public
Analyst, Manchester, UK).
† Participating institutions were St James University

Hospital (Leeds), St Bartholomew’s Hospital
(London), Western General Hospital (Edinburgh), Radcliff

Infirmary (Oxford), and Bristol Royal
Infirmary (Bristol).
‡ The pAb is recovered from the eggs of immunized chicken.
barley, or rye. However, proteins from celiac-negative

cereals (maize, rice, millet, etc.)
shouldnot interfere. Single mAbs had different

sensitivities for different celiac toxic
prolamins (166). Combining different mAbs is one way to

ensure cross-reactivity for a
range of celiac-active prolamins. A cocktail antibody test

for gluten was developed by
mixing two capture mAbs (mAb-13B4 and mAb-Rye5) with

specificity for barley and
rye (167). A third mAb for rye protein (mAb-Rye3) was

chosen for detection. Results
compared favorably with the commercial AOAC-ELISA test for

gluten. The cocktail
sandwich ELISA produced comparable calibration graphs for

gliadin, hordein, and
secalin with a linear range of 3–100 ngmL −1 and an LLD of

1.5ng (gliadin)mL −1 , 0.05ng
(hordein)mL −1 , 0.15 ng (secalin)mL −1 , or 12ng (avenin)

mL −1 . The acuity toward barley
and rye prolamin was higher than obtained for wheat. The
response toward avenin was
10–100 times lower, which is as expected from the lower

toxicity of oats. Compared with
the AOAC test, the cocktail mAb test had an LLD for hordein
or gliadin that was ~25
fold and 4- to 10-fold lower. The mAbRye3 used for

detection in the cocktail ELISA had
low specificity for promlamins from wheat, barley, and rye.

This feature probably
explains the relatively high response for barley and rye.

4.10. A Home Test for Gluten
The Skerritt and Hill (156) home test kit for gluten
performed well with ordinary citizens,
who successfully identified gluten-free foods with 82–100%

accuracy. The home test
agreed closely with results from the AOAC-approved

laboratory test kit. The home test
kit can be usefully compared with the home pregnancy test

kits, which are now
commonplace. Similar self-use kits are possible for other

food allergens. The home
gluten test consists of the following components:
1. Polystyrene test tubes (Nunc, Denmark) precoated with

mAb-401/ 21 and blocked with 1% BSA. The antibody-coated
test tubes are stable at 4°C for 12 months or at 20°C for
6 months.
2. Graduated tubes for sample extraction.
3. Enzyme-labeled mAb-402/21 for detection.
4. Substrate solution (TMB/hydrogen peroxide).
The kit was field tested in eastern Australia by 5

dieticians or food technologists and 47
ordinary citizens registered with the Celiac Society.

Participants were 10- to 77-year-old
urban and rural dwellers of varied educational background.

The testers were given six
food samples to grade as having “low,” “borderline,” or
“high” gluten. Samples classed
in the borderline (40 mg %) or high (>150mg %) gluten

categories were not acceptable as
“gluten-free” foods, whereas those with <10mg % were placed

in the gluten-free
category. For sample pretreatment, 0.5 g of ground or

finely chopped foodstuff was
shaken with 5 mL of dilute hydrochloric acid (2mM) for 1

minute. There was 100%
protein recovery from wheat starch, 62% recovery from wheat

starch having 100mg %
gluten, and 18% recovery from wheat flour with 11% gluten.
Home testing for gluten involved five simple steps: (a) add
0.8mL of reaction buffer
(1% BSA solution in PBST) to the antibody-coated test tubes

provided, (b) add one drop
(30 µL) of food extract and mix gently for 30 seconds, (c)
add three drops of HRP-mAb
conjugate, (d) wait about 1½ minutes and rinse the tubes

with running tap water, and (e)
add enzyme substrate solution. A positive test gives a blue

coloration within 2 minutes.
Classify tests results as high, low, or borderline by

comparing results with color produced
by standard samples. 4.11. Gluten ELISA Tests Using

Peptide Antigens
As the benchmark technique, the AOAC-approved ELISA test

has been subject to a range
of criticisms, not all of which are justified:
1. Low accuracy. The approved method measures ω-gliadin.

However, the amount of ωgliadin fraction may be only weakly
correlated with the total gliadin content (168).
Determining total gluten by extrapolation from the
ω-gliadin may lead to error.
2. Low selectivity. There is some cross-reactivity with
cereal proteins not proved to be celiac toxic (e.g.,
starch granule proteins).
3. Poor limit of detection. The LLD achieved by the

AOAC-approved gluten test may be inadequate to deal with
future, more stringent legislation regarding gluten
content.
4. Unproven range of applicability. Although maize, rice,

and oats were tested by the approved AOAC method, it may
be desirable to test a wider range of materials.
5. Low reliability for certain foods. A commercial ELISA

kit based on the approved method gave discordant results
for 11 out of 24 foods measured in five laboratories
(Section 4.8).
Peptides are useful antigens for developing gluten ELISA

tests. A sandwich ELISA for
gluten was developed using mAb-WB8 with specificity for

peptide B3144 as the detector
(158). B3144 consists of the celiac-toxic N-terminal

residues 3–56 from α-gliadin (129).
The capture antibody was (rabbit) pAb for unfractionated

gliadin. The mAb-WB8 bound
α-gliadin, β-gliadin, γ-gliadin, ω-gliadin, glutenin

subunits, and unfractionated gliadin.
There was also high reactivity toward rye and barley

prolamins. The response for celiac
active proteins was 125–4000 times greater than that with

celiac-negative cereals such as
oats, maize, millet, or sorghum. Very low binding occurred

with ovalbumin or BSA (Fig.
2). FIGURE 2 Specificity of two mAbs for celiac-active

peptide B3144. Specificity was tested by noncompetitive
indirect ELISA using cereal proteins extracted with 50%
(v/v) ethanol. The Y-axis shows mAb titer from ascite
fluid. (Drawn from results in Ref. 129.) The working
range for gliadin detection was 2 ng mL −1 to 1 µg mL −1 .
The LLD (+1.5
SD above background absorbance) was 15ng mL −1 (extract)
or 0.03 mg % of gliadin in
wheat flour. The corresponding LLD values for other cereals

were 0.03 mg % (rye), 0.25
mg % (barley), and 0.5 mg % (oats). The LLD for gliadin is

four orders of magnitude
lower than the current UK upper limit (300 mg %) for

gluten-free foods. The preceding
assay was suitable for uncooked foods only. Prolamins were

extracted with 20 volumes
of 40–50% (w/w) ethanol before analysis. The peptide-based

ELISA test for gluten
underwent further refinements. A 19-residue peptide

fragment from α-gliadin N-terminal
(residues 31–49) was used as antigen. By comparison with

mAb-WB8, the new detector
antibody (mAb-PN3) (160) was more selective for

unfractionated gliadin and α-gliadin at
the expense of ω-gliadin, rye, and barley proteins (Fig.

3). The LLD for gliadin was 4 ng
mL −1 of extract or 0.008 mg % (flour). The assay

repeatability was 8.7–9.3% with a
reproducibility of 18.8–22.3%. Cooking reduced the

apparent gluten levels by 70% when measured by the mAb-PN3
based ELISA. An attempt to increase sample solubility by

FIGURE 3 Sandwich ELISA tests for gluten developed with
mAbs for peptide antigens. The capture antibody is
(rabbit) pAb. The dectector antibody is either mAb-WB8 or
mAb-PN3 with specificity for α-gliadin N-terminal
residues 3–56 or 31–49, respectively. (Drawn using results
from Refs. 158 and 160.)
extracting with a reducing buffer was unsuccessful.

Residual mercaptoethanol affected
the stability of antibody during ELISA. The inability to

assay gliadin in heat-processed
samples is a serious shortcoming. The test using mAb-PN3 is
also likely to underestimate
celiac-toxic potential when a product contains ingredients

from barley or rye. This is
because the limit of detection for wheat protein was

250-fold lower than values for
hordein or secalin. The latter proteins will remain

“hidden” in foods exhibiting relatively
low amounts of celiac-active proteins. Denery-Papini and

co-workers (159) produced pAbs for a number of synthetic
peptides. Each peptide matched a unique amino acid sequence

found in one of the four
classes of gliadins. Each 8- to 12-residue peptide was

covalently coupled to a protein
carrier for immunization. The (rabbit) pAbs for these

antigens were evaluated by ELISA
as well as SDS-PAGE and immunoblot analysis. A pAb specific

for the N-terminal
sequence from γ-gliadin or ω-gliadin bound only to the

corresponding protein. A
particular pAb specific for the N-terminal sequence of

α/β-gliadin was used as the basis
for a competitive ELISA test for gliadin (161). Gliadin

levels were found to be 2333–
5040 mg % using flour from 20 wheat cultivars. The results

were highly correlated with
gliadin determinations by reverse-phase HPLC (R=0.82) and

Kjeldahl analysis (0.86).
The linear range for analysis was 45–250 µgmL −1 (extract)

with an LLD of ~9 µgmL−1.
Stirring wheat flours with 70% (w/w) ethanol for 14 hours

led to the solubilization of
48% to the total protein. The solubilized protein was

gliadin with little contamination
from glutenin. Results from ELISA tests for gluten are

summarized in Table 9. This information was
compiled from the limited number of studies involving real

foods
(140,141,144,146,149,150,154,160). The agreement between

gluten values is reasonable.
Results are routinely reported in milligrams of gliadin

(equivalent protein) per 100 g dry
food (mg %). Measuring gluten in this fashion is convenient

because ELISA tests are
usually calibrated using whole gliadin extract. The ideal

assay for gluten should show
equal responsivity toward prolamins from all celiac-active

cereals. It is necessary to test
samples of alcohol-soluble proteins from each cereal. The

results in Table 9 do not
include contributions from glutelin subunits, which

(although celiac active) dissolve
poorly in 40–70% (v/v) aqueous alcohol. Glutenin could be

more effectively solubilized
for immunoassay using solvents containing SDS or guanidine

hydrochloride. Conroy and
Esen (169) found that zein was efficiently adsorbed by

polystyrene microwell plates from
acetic acid (60% v/v) or 6 M urea (dissolved in carbonate

coating buffer). Sample
extraction with these solvents could improve the recovery

of glutenin. TABLE 9 Apparent Gluten Levels in Range of
Foods Determined by ELISA for Gliadin
Foodstuff or commodity Gliadin (mg %)
Gluten-negative foods
Tropical cereals
Millet, sorghum, rice, jawar Negative
Legumes
Soya flour, chickpea flour, buckwheat, quinoa
Miscellaneous
Potato flour, molasses, skimmed milk powder, sugar
beet
Gluten-free (wheat starches)
Kinderm, Glutafin, Wheatex, Nutregen 0.5–7.75
Wheat starch-based products
Cooked foods a
Glutafin GF fiber loaf, Glutafin GF white bread with soya,

Loprofin white bread, low-protein chocolate chip cookies
0.12–0.24
Uncooked products
Glutafin GF white mix, fiber mix, Rite Diet low-protein

flour mix 0.9–1.4
Wheat-free products
Cooked products
Chocolate digestive biscuits, custard creams, tea biscuits,

orange flavor cream wafers, mince pies 0.144–1.18
Uncooked products
Glutafin pasta spirals, vermicelli, spaghetti, macaroni

0.07–6.7
Flours
Buckwheat b 4
Sorghum b 13
Maize b 46
Rice b 47–57
Oats b 100–240
Barley 400
Rye 580
Wheat (self-raising, plain) 4000–8100
a Levels can be underestimated by 70% after heating.
b Sometimes nil gliadin is found in these cereals.
A meaningful comparison of ELISA tests results for gluten

is difficult because successive
investigators aimed for innovation rather than

standardization (170). Slight changes in
assay conditions can produce substantial changes in assay

performance. The following
general observations seem relevant. The most sensitive

tests are ELISA using pAb. The
LLD for such assays is about 0.5–1 ngmL −1 . To achieve

high acuity it is necessary to
avoid nonspecific binding of gliadin to microwell plates.

Exposing microwell plates to
3% skimmed milk powder or BSA for 60–120 minutes reduces

nonspecific protein
binding. Precoating with extraneous protein is better than

adding the coating protein
together with the sample. Coating of microwell plates at

4°C overnight leads to better
assay performance in contrast to coating at 37°C for 90

minutes. Using mAb in place of
pAb increases the cost of ELISA tests by an estimated

10-fold. On the other hand, mAbs
exhibit increased specificity and decreased likelihood of

detecting celiac-negative cereals.
Immunoaffintity-purified pAbs show high selectivity for
celiac-active cereals and are
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FOOD PROTEIN ANALYSIS

Quantitative Effects on Processing

R.K.Owusu-Apenten The Pennsylvania State University

MARCEL DEKKER INC.

This edition published in the Taylor & Francis e-Library, 2005.

ISBN 0-203-91058-3 Master e-book ISBN

ISBN: 0-8247-0684-6 (Print Edition)

Owen R.Fennema University of Wisconsin-

Dairy science James L.Steele University of Wisconsin-Madison

Food engineering Daryl B.Lund University of Wisconsin-Madison

Food proteins/food chemistry Rickey Y.Yada University of Guelph

Health and disease Seppo Salminen University of Turku, Finland

Nutrition and nutraceuticals Mark Dreher Mead Johnson Nutritionals

Processing and preservation Gustavo V.Barbosa-Cánovas Washington State

Safety and toxicology Sanford Miller University of Texas-Austin

Additional Volumes in Preparation

Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz

Extraction Optimization in Food Engineering, edited by Constantina Tzia and George

Physical Principles of Food Preservation: Second Edition, Revised and Expanded,

Handbook of Vegetable Preservation and Processing, edited by Y.H.Hui, Sue Ghazala,

Food Process Design, Zacharias B.Maroulis and George D.Saravacos

Part 1. Fundamental Techniques

Chapter 1. Kjeldahl Method, Quantitative Amino Acid Analysis and 1

Chapter 2. The Alkaline Copper Reagent: Biuret Assay 43

Part 3. Dye Binding Methods

Chapter 5. The Udy Method 109

Part 4. Immunological Methods for Protein Speciation

Chapter 11. Determination of Trace Protein Allergens in Foods 262

Chapter 13.Effect of Processing on Protein Nutrient Value 333

Chapter14.Protein Digestibility-Corrected Amino Acid Scores 358

1. INTRODUCTION TO FOOD PROTEIN ANALYSES

1971 Modified Berthelot reaction

1.1. Characteristics of Food Protein Assays

TABLE 2 Some Important Calibration Indices and

1.2. Calibration and Statistical Principles

1.3. Assay Performance

1.4. Calibrating Protein Assays

Refractometry Laboratory refractometer

FIGURE 1 Apparent protein

Alkaline 90 Y=1. 070 cP −0.670 2.383 and 1.540

2.1. Nitrogen-to-Protein Conversion Factors

Valine 117.1 11.96 8.36 428.0 51.2 0.003655 0.073687 8.6288

Egg white solids 5.96

2.2. Macro- and Micro-Kjeldahl Analysis

A. Grain and Cereals

C. Dried Milk Powder

2.3. Automated Kjeldahl Analysis

A. The Kjel-Foss Instrument

FIGURE 2 Calibration graph for the

B. Automated Kjeldahl Continuous Flow Analysis

TABLE 8 A Comparison of the Automated Kjel-

3. COLORIMETRIC ANALYSIS OF KJELDAHL NITROGEN

3.1. Alkali-Phenol Reagent (Indophenol) Method

FIGURE 3 Calibration graph for

3.2. Nessler’s Reagent

3.3. Acetylacetone-Formaldehyde Reagent

3.4. Ninhydrin (Indanetrione Hydrate) Assay

* Ninhydrin is often encountered in detective novels as a reagent for fingerprint analysis.

FIGURE 4 Reaction scheme between

FIGURE 5 Colorimetric ninhydrin