OUR INDUSTRY TODAY
D e t e r m i n i n g P r o t e i n C o n t e n t o f Ice Cream and F r o z e n Desserts I
MANFRED KROGER, EMANUEL E. KATZ, and J. C. WEAVER
ABSTRACT
Dye binding of protein is a suitable,
rapid, and relatively inexpensive method
for the determination of protein in ice
cream and frozen desserts. The presence
of proteins with reduced amounts of
basic amino acids will depress protein
percentage slightly as measured by dye
binding. Proteins with higher percentages
of basic amino acids tend to increase
apparent protein content. At this time,
only noncolored ice cream is recom-
mended for protein testing by dye bind-
ing because of the influence of coloring
matter on test results. The effect of food
coloring was studied with model systems
of milk and 2% food color solutions. With
amido black, dye binding values (protein
percentages) were depressed increasingly
by yellow to red to green to blue dyes.
With orange G dye, the effect on protein
results was decreased from yellow to
green to blue to red. Also, protein per-
centages decreased with increased concen-
tration of food color. Overall, results with
amido black dye binding procedure were
less affected than protein readings with
orange G. Colored dairy products can be
tested accurately for protein by dye
binding if the nature and concentration
of the added food color are known. A
factor then must be determined to enable
conversion from dye binding value to
protein content.
New Frozen DessertsProtein Regulations
New federal frozen dessert standards were
published April 12, 1977 (2); however, the final
form has not been issued in all its details.
Received August 9, 1977.
~Authorized for publication as Paper No. 5344 in
the Journal Series of the Pennsylvania State Universi-
ty. The work was partially supported by a grant-in-aid
from Dairy Research Inc., Rosemont, IL.
1978 J Dairy Sci 61:274-277
Department of Food Science
The PennsylvaniaState University
University Park 16802
Accordingly, "ice cream contains not less than
10% milkfat, nor less than 2.7% p r o t e i n . . . ,
except that when it contains milkfat at 1%
increments above the 10% minimum, it may
contain the following milkfat-to-protein levels:
Minimum
Percent percent
milkfat protein
10 2.70
11 2.55
12 2.40
13 2.25
14 2.10
Except in the case of frozen custard, ice
cream contains less than 1.4% egg yolk solids
by weight of the food, exclusive of the weight
of any bulky flavoring ingredients used. In no
case shall the milkfat content of the finished
food be less than 8% or the protein content be
less than 2.2%, or, in the case of frozen custard,
shall the content of egg yolk solids be less than
1.12%."
In the mix calculations of m i n i m u m milkfat
and protein requirements, the solids of choco-
late or cocoa are bulky ingredients.
With ice milk, the reduction in fat and
protein content from the addition of bulky
flavors applies also. In no case must the milkfat
content be less than 2%, nor the protein
content less than 1.75%. When the milkfat
c o n t e n t increases in increments of 1%, ice milk
may contain the following protein concentra-
tion:
Minimum
Percent percent
milk protein
2 2.55
3 2.40
4 2.25
5 2.10
6 1.95
7 1.80
274
 OUR INDUSTRY TODAY
The minimum protein requirement for ice
cream replaces the previous minimum for total
milk solids. The reasoning given by the Food
and Drug Administration is that analytical
methods now exist to determine the protein
content of frozen desserts while the only
realistic means of enforcing the milk solids-
non-fat requirement was through factory and
records inspection.
The protein must be provided by milk
solids-not-fat and/or other milk-derived ingredi-
ents with a protein efficiency ratio (PER) not
less than that of whole milk protein (108% of
casein).
For the determination of protein content an
official method must be chosen (1). The origi-
nal official order (2) referred to the wrong
sections in the AOAC Handbook (1). It is
understood that changes in and additions to the
methods since the 1975 edition are valid and
can be utilized. More improvements will be
forthcoming in milk protein determinations. A
review of milk protein determinations has been
published elsewhere (4).
Dye binding methods are suited for protein
determinations in ice cream. Capital outlay for
apparatus and expenses for supplies and labor
are such that the cost of one protein determina-
tion could be kept below one dollar, depending
on circumstances. One Kjeldahl analysis costs
about $10 in 1977.
For most laboratories, a Kjeldahl analysis is
wasteful, cumbersome, and takes 2 to 3 h; a
dye binding result is obtained in about a
minute. As a result, more and more centralized
laboratories are set up to include protein
determinations by dye binding (3).
Advantages of Protein Dye Binding Over Kjeldah|
The question of whether dye binding pro-
vides accurate results was well answered by
Sherbon (5). Dye binding methods like the
Kjeldahl procedure give relatively good esti-
mates of protein content, but only indirectly.
For example, the principle of amido black
dye binding is a chemical reaction between the
dye and the basic amino acids lysine, histidine,
and arginine. Similar proteins have relatively
constant proportions of these three amino
acids. Since casein, milk protein, and whey
protein are dissimilar proteins they also will
have different concentrations of basic amino
2 75
acids, have slightly different dye binding capaci-
ties, and provide slightly different protein
results when analyzed with a dye binding
procedure that has been calibrated for only one
of the proteins. Usually milk protein (roughly
an 80:20 casein to whey protein mixture) is
used in standardizations. Consequently, a prod-
uct such as ice cream without these two
proteins in the 80:20 ratio cannot be tested
accurately. The inaccuracy, however, is toler-
able. Two extreme cases may exist. When a dye
binding procedure calibrated for milk protein is
used to determine the protein of an ice cream
containing 3.0% pure whey protein (more
expensive and of higher nutritional quality),
then the instrument readout will be 3.6%. If the
ice cream contained 3.0% casein, then the
readout would be 2.9% (5).
Dye binding values could be used to reflect
protein quality because they are dependent on
lysine concentrations, and a high-lysine content
is a mark of a high-quality protein. In the
comparison between whey protein and casein,
whey protein gives a seemingly higher protein
content than casein (it also has a higher PER)
because it has greater dye binding capacity and
value due to its higher lysine concentration.
Lysine is an essential amino acid and the major
amino acid responsible for the dye binding
mechanism. From a practical standpoint, as-
suming that the dye binding apparatus is
calibrated for milk protein, an ice cream mix
with 10% fat formulated to 2.25% protein
solely with whey protein would appear to
satisfy the legal minimum. The higher cost of
pure whey proteins would rule against such
practice. In the true sense of the Food, Drug,
and Cosmetic Act, such practice might not even
be called food adulteration since a cheaper
ingredient is not substituted for a more ex-
pensive one. From a nutritional standpoint, the
hypothetical 2.25% whey protein ice cream
(reading out at 2.70% protein with the usual
dye binding apparatus) is of considerably higher
quality, because of its higher PER value, than
an equivalent ice cream containing casein or
even average milk protein. A protein with high
percentages of lysine will always show a higher
protein percentage. With milk-derived protein
the theoretical maximum difference would be
20%.
Regulatory officials may want to establish
certain guidelines and "administrative toler-
Journal of Dairy Science Vol. 61, No. 2, 1978
276 KROGER ET AL.
ances" for ice cream protein values which take
into account the variability of ice cream protein
and current cost of protein ingredients or
milk-solids-non-fat. Since most samples to be
tested are derived from "average" mixes with
conventional ingredients, including milk-solids-
not-fat, dye binding results will reflect the true
protein content.
Lower than expected dye binding values
could be due to "cheaper" proteins, such as
casein, as well as to an actual irregularly low
protein percentage.
Effect of Food Coloring
on Dye Binding Value and
Protein Percentage
Since protein dye binding is a colorimetric
procedure, there are interferences by colored
substances. The dye binding procedure requires
some dye to be precipitated out of solution by
the protein in the sample. The resulting change
in light transmittance is related to the protein
content of the sample. Coloring matter in the
sample would counteract the effect of dye-pro-
tein precipitation and cause a lower read-out of
protein percentage on the instrument's galva-
nometer.
Although the milk sample preservatives mer-
curic chloride and potassium dichromate lend
color to the sample they are preserving for
future analysis, no such depressing effect on
protein content was observed.
Tarassuk and Abe (6) reported that steriliza-
tion of milk, but not condensing and heating up
to 90 C for 15 min, would lower dye binding
values. However, it is not necessarily the result-
ing color of the Maillard browning reaction but
a reaction and removal of the amino acids
TABLE 1. Effect of various food colors on milk protein dye binding values with Duncan's separation of means,a
Amido Black procedure
Whole
Food color milk
Blank (H2 O) 3.16 b
Yellow 3.15 b
Red 3.13 b
Green 3.05 c
Blue 2.92 d
aWithin each column, means followed by the same letter are not different (a .05).
Journal of Dairy Science Vol. 61, No. 2, 1978
necessary for dye binding (thus lowering the
available n u m b e r of protein-dye binding sites),
that is responsible for the seemingly lowered
protein content.
The following 2% aqueous solutions of food
colors were prepared: FD & C Blue No. 1
(~92% pure), FD & C Yellow No. 5 (~92%
pure), FD & C Red No. 40 (89% pure) (all from
Allied Chemical Corp., Morristown, NY), and
Mint Green Shade (94% pure) which contains
FD & C Yellow No. 5 and FD & C Blue No. 1
(from H. Kohnstamm & Co., Inc., New York,
NY). These solutions, as well as distilled water
for the blank, were added in 1, 5, 10, 20, 30,
40, and 50-drop portions to 20-ml samples of
whole milk (pasteurized, homogenized) and
also to skim milk (pasteurized, fortified with
2% MSNF).
Milk protein percentages were determined in
triplicate with a Pro-Milk MK II instrument
with amido black 10 B and with a Pro-Meter
MK II with Orange G dye (both instruments
A/S N. Foss Electric Millerod, Denmark).
A statistical analysis was made on the
various sets of data. The experiments were
viewed as 2-factor factorials with color (five)
and concentration (seven) as factors. After an
analysis of variance, Duncan's modified (Bay-
esian) least significant difference test was used
to separate the means.
Results of Experiments with Food Colors
The protein values demonstrating the effect
of the various colors, along with Duncan's
separation of the means, are summarized in
Table 1. The means between the various colors
showed significant differences. Yellow was the
color which affected the results the least for
%Protein
Orange G procedure
Skim Whole Skim
milk milk milk
3.82 b 3.15b 3.70b
3.77c 3.07 c 3.68bc
3.75 c 2.78d 3.33 e
3.67d 3.00 c 3.6 led
3.53 e 2.99 c 3.57d
 OUR INDUSTRY TODAY 277

TABLE 2. Effect of food color concentration on milk protein dye binding values with Duncan's separation of
means, a

%Protein
Food color
Amido Black procedure Orange G procedure
concentration
(drops/ Whole Skim Whole Skim
20 ml milk) milk milk milk milk

1 3.31 b 3.91 b 3.40 b 3.80 b

5 3.23 c 3.86 c 3.31 c 3.78 bc
10 3.16 d 3.83 c 3.16 d 3.72 c
20 3.08 e 3.73 d 3.04 e 3.61 d
30 2.99 f 3.64 e 2.82 f 3.50 d
40 2.93g 3.54 f 2.68g 3.38 e
50 2.86 h 3.45g 2.62g 3.24 f

aWithin each column, means followed by the same letter are not different (a .05).

both dyes. Blue f o o d color had the m o s t
depressing effect on the a m i d o black p r o c e d u r e
protein percentages (7.4%). Red f o o d color
affected the Orange G results the m o s t (10.8%
depression).
Since the colors affecting the results the
m o s t have wavelengths nearest to those o f the
respective dyes and w o u l d interfere with the
t r a n s m i t t a n c e the most, these results w o u l d be
expected. In protein d e t e r m i n a t i o n s with the
amido black procedure, the order of decreasing
protein m e a n values was yellow, red, green, and
blue while with the Orange G p r o c e d u r e the
order was yellow, green, blue, and red.
The e f f e c t o f f o o d color c o n c e n t r a t i o n on
milk protein percentages is s u m m a r i z e d in
Table 2. Each c o n c e n t r a t i o n includes data f r o m
all the f o o d colors in this study. As e x p e c t e d ,
increasing color c o n c e n t r a t i o n s caused greater
depressions of dye binding values (protein
percentage). This was true on all tests, as in
Table 2, where five and seven groupings of the
seven m e a n s occur. The higher c o n c e n t r a t i o n s
in these e x p e r i m e n t s never w o u l d be in actual
use.
These data d e m o n s t r a t e variable and signifi-
cant effects in protein dye binding results
caused by f o o d coloring matter. Standardiza-
tion of ice cream protein c o n t e n t will require
control by manufacturers and regulatory agen-
cies. F o r several reasons, dye binding probably
will b e c o m e the preferred testing method. No
p r o b l e m s will occur with u n c o l o r e d products,
but significant decreases in protein values will
seem to occur in testing colored products.
C o r r e c t i o n factors could be worked out for the
various colors and color c o n c e n t r a t i o n s used by
manufacturers, but a regulatory agency not
k n o w i n g the types and a m o u n t s of color added
w o u l d be at a disadvantage and could use dye
binding only as a screening test to check
c o m p l i a n c e with the m i n i m u m protein standard
of ice cream.
REFERENCES
1 Association of Official Analytical Chemists. 1975.
Official methods of analysis. Washington, DC.
2 Federal Register 43(70):19127-37.
3 Kroger, M. 1972. Techniques for milk protein
testing. Food Product Development 6 (7):68, 77.
4 Kroger, M. 1973. Milk protein determinations.
Amer. Dairy Rev. 35 (9): 18, 40-43.
5 Sherbon, J. W. 1977. Does protein testing give
accurate results? Dairy & Ice Cream Field 160 (1):
38, 68.
6 Tarassuk, N. P., and N. Abe. 1963. Factors
affecting dye-binding capacity of milk proteins. J.
Dairy Sci. 46:612.

Journal of Dairy Science Vol. 61, No. 2, 1978