Meat Science 11 (1984) 253-262

The Quantitative Analysis of Fat and Protein in Meat

by Transmission Infrared Analysis

Bernice L. Mills,* F. R. van de Voort? & Y. Kakuda

Department of Food Science, University of Guelph,
Guelph, Ontario N1G 2W1, Canada

(Received: 15 August, 1983)

SUMMARY

Transmission inJrared analysis, which has been successfully applied to

milk analysis, was assessedjbr the quantitative analysis ofJht andprotein
in meat products. Meats of varying fat andprotein content were converted
into milk-like emulsions, which were, in turn, analyzed by standard
chemical methods and by a Multispec M infrared analyzer. The
performance of the instrument for meat analysis using a standard milk
calibration was also assessed and compared with the instrument set with a
meat calibration. Both approaches provided a good estimate of thefat and
protein content .[or a range of meat products, the meat calibration being
more accurate than the milk calibration. The infrared method allowedfor
rapid and accurate analysis of meat and has future potential in the meat
industry for quality control purposes.

INTRODUCTION

T h e successful a p p l i c a t i o n o f m i d - i n f r a r e d transmission to the r a p i d a n d

q u a n t i t a t i v e analysis o f milk has h a d a t r e m e n d o u s i m p a c t o n the d a i r y

* Present address: Universit+ de Montr6al, D~partement de Nutrition, Facult~ de

M6dicine, Case Postale 6128, Succursale A, Montr6al, Qu6bec H3C 3J, Canada.
t Present address: School of Food Science, Macdonald Campus, McGill University,
21111 Lakeshore Road, St. Anne de Bellevue, Quebec H9X 1C0, Canada.
253
Meat Science 0309-1740/84/$03-00 © Elsevier Applied Science Publishers Ltd, England,
1984. Printed in Great Britain
254 Bernice L. Mills, F. R. van de Voort, Y. Kakuda

industry (Biggs, 1967; Grappin & Jeunet, 1976). The speed of

multicomponent (fat, protein and lactose) analysis, along with the sound
correlation of the results with standard chemical methods, has led to its
world-wide use for producer payment and dairy herd improvement
programs. The infrared (IR) method is based on the absorption of IR
energy at specific wavelengths in the mid-IR region of the spectrum by the
carbonyl group (C-~-O) in the ester linkages of fat molecules, by the
peptide linkages (CONH) between amino acids of protein molecules and
by the hydroxyl linkages (C--OH) in lactose. More recently, a newer CH
stretch absorption measurement for fat has been advocated to correct for
possible differences in molecular weight and discrepancies at the carbonyl
absorption band (Nexo & Anderson, 1981).
The resounding success of the IRMA (Infra-Red Milk Analyzer) has led
to the introduction of simpler, and more reliable, filter instrumentation,
capable of determining component pecentages in a matter of seconds
(Biggs, 1979). The expansion of IR analysis to other food products is an
inevitable consequence of the evolution of these relatively inexpensive
filter instruments. An area where rapid determination of fat and protein is
most needed is in the meat industry. In Canada, the federal government
has established new and explicit standards on both product ingredients
and composition (Government of Canada, 1982) and one of the major
problems facing the meat industry today is that of compliance with these
regulatory standards of quality. Profit margins have provided another
driving force to control the quality characteristics of meat products in
terms of fat and protein content. From an analytical standpoint, the meat
industry has relied basically on solvent extraction for fat determination,
i.e. the Soxhlet method, and on the classical Kjeldahl method for protein
determination. A variety of automated modifications of these basic
methods have been introduced over the years; however, truly rapid
methods have not yet been developed. Near infrared reflectance (NIR)
spectroscopy has been assessed for the estimation of fat, protein and
moisture contents in meats (Ben-Gera & Norris, 1968; Hauser & Heber,
1978; Kruggel et al., 1981) but reflectance spectra are sensitive to particle
size and drastic changes in reflectance can occur when changing the
grinding parameters for the sample.
The success of mid-IR transmission in analyzing milk has been due in
great part to the relatively consistent composition of cow's milk and to its
fluid and homogeneous nature. In applying this technique to the analysis
of meat, the main concerns must be directed towards sample preparation
 Analysis of fat and protein in meat by infrared analysis 255

and instrument calibration. The sample presented to the instrument must

be a fluid homogeneous solution with a particle size less than 2/~m to
avoid scattering of IR radiation and clogging of the narrow 40/~m path
length cell. Homogeneity is particularly difficult to obtain with meat since
it is a fibrous material containing a relatively high percentage of insoluble
matter. The sample preparation procedures must also be simple to reduce
the preparation time to a minimum so as not to detract from the rapid
analysis by IR instrumentation.
A second and important area of consideration is the calibration
methodology which creates particular difficulties because of the wide
compositional range of meats, as opposed to milk. It is crucial that the
best primary quantitative methodology be used for instrument cali-
bration and that these analyses be performed by skilled and experienced
technicians.
Although the requirements for meat analysis are not nearly as rigorous
as tho~,;efor milk analyzed for payment purposes, the aim in developing a
new method is to obtain the best possible accuracy without major
sacrifices in terms of time. Taking into account these general
considerations, the purpose of this study was (a) to develop a simple
method for preparing a stable fluid meat emulsion for mid-IR
quantitative analysis, (b) to assess the primary chemical methods for the
analysis of fat and protein in meat for IR instrument calibration and (c) to
assess the performance of the IR milk analyzer set with a milk calibration
versus the same instrument set with a meat calibration for general meat
analysis.

M E T H O D S A N D MATERIALS

Twelve commercially prepared meats and meat products, including

ground pork, bologna, ham, turkey roll and ground beef, were obtained
at locall retail outlets. This selection provided a range of fat content and
included both raw and processed meats. The individual meats were
ground three times through a 3-2 mm plate, formed into 30 g balls, frozen
using liquid nitrogen, placed in sealed polyethylene bags and stored at
- 2 0 °C for use as required for basic experimentation.
The development of a sample preparation method concentrated mainly
on the use of alkali to solubilize the meat proteins. The standardized
procedure adopted provided a meat emulsion of milk-like consistency
ready for presentation to the IR instrument and is described below.
256 Bernice L. Mills, F. R. van de Voort, Y. Kakuda

Twenty grams of the ground meat were accurately weighed into a

beaker on a top loading balance to which 100g of 0.IN N a O H were
added. The mixture was heated in a waterbath at 70 °C for 5 min and then
immediately homogenized using a Polytron mixer (Brinkmann
Instruments, Rexdale, Ontario) at high speed for 4min. The Polytron
stem was washed with ca. 50 ml of prewarmed distilled water and the
weight of the solution brought to 200 g with additional distilled water.
The milk-like emulsion was passed through a 150pm sieve set in a
Buchner funnel and the filtrate collected in a 250-ml glass sealable
container. The filtration procedure was carried out to verify the complete
solubilization of the sample and to prevent the inadvertant clogging of the
cell should any extraneous matter be present (i.e. bone chips).
The meat emulsions prepared in this manner were also used for analysis
by the standard reference chemical methods. The emulsion remained
stable at room temperature and, due to its high pH, did not deteriorate
microbiologically.

Primary chemical methods

The primary methods to which IR results are compared are of the utmost
importance in determining the calibration accuracy of the instrument. In
previous work (Mills et al., 1983). the Mojonnier method was found to be
a superior reference method to the standard AOAC Soxhlet procedure for
fat determinations. Tests carried out to determine whether the meat
emulsion results obtained by the Mojonnier method were comparable
with those for the fresh meat samples evaluated by the Soxhlet method
indicated that there was no loss of fat in the preparation of the meat
emulsions. The Mojonnier method is very accurate and precise for the
analysis of fat in milk and has been used for many years to calibrate IR
milk analyzers. The Mojonnier procedure has a distinct advantage over the
conventional Soxhlet method in that the chemical analysis is performed
on the milk-like emulsion to be analyzed by the instrument, rather than
on the fresh meat sample.
The Mojonnier fat extraction was carried out with the Mojonnier
apparatus using the procedure developed for milk ( M o j o n n i e r Bulletin,
1925). Ten grams of the homogeneous meat emulsion (representing 1 g of
meat) was taken for the extraction procedure. For the first extraction,
1.5 ml ammonia and 10 ml ethanol, 25 ml ethyl ether and 25 ml petroleum
ether were added to the sample, shaking after each addition. For the
 Analysis of fat and protein in meat by infrared analysis 257

second extraction, 5 ml ethanol and 25 ml each of ethyl and petroleum

ethers were added. In the case of bologna, a precipitate formed using the
above procedure. The situation was corrected in this particular case by
decreasing the ethanol to 6 and 3 ml in the first and second extractions,
respec, tively, along with reversing the order of the addition of ethanol and
ethyl ,ether.
A standard semi-micro Kjeldahl procedure used for the analysis of
protein in milk (AOAC, 1970) was used for the protein determination of
the meat emulsions. The procedure was not modified for nitrite-
containing samples since the minute amounts present in the processed
samples were not considered to be significant enough to affect the results.
Preliminary tests had been carried out to determine whether the micro-
Kjeldahl results for the meat emulsions were comparable with those for
the fresh meat samples. These experiments indicated that there was some
loss of protein (0.3 %) in the.preparation of the meat emulsion but it was
not considered sufficient to warrant a change in methodology.

Instrumental methods

The commercial instrument used for this study was the Multispec M
(Biggs, 1979) (Shields Instruments, Osbaldwick, York, Great Britain)
equipped with four channel filter pairs (sample and reference wave-
lengths), i.e. a carbonyl pair (5.73 and 5.58 #m), a protein pair (6.46 and
6.68/~m), a lactose pair (9"61 and 7-68 #m) and a CH stretch pair (3.48 and
3.56/an). The instrument was linearized according to the manufacturer's
instruc, tions and optically zeroed with distilled water. Three emulsions for
each meat sample were prepared and preheated to instrument
temperature (43 °C) prior to measurement. Only the primary uncorrected
signals at each channel were recorded, obviating the effects of a set
calibration. The negative signals at the lactose channel were also recorded
and used in the calculations.
The overall precision of the instrumental method was obtained by
analyzing the three separate emulsions of the same meat and estimating
the variability or standard deviation (SD) for each instrument channel,
while the accuracy of the instrumental method was determined from the
mean difference (MD) and the standard deviation of the difference (SDD)
between the chemical and instrumental results (van de Voort, 1980).
To develop and assess calibrations, the Multispec M was used in
tandem with a Hewlett Packard HP 85 microcomputer capable of
258 Bernice L. Mills, F. R. van de Voort, Y. Kakuda

generating multiple regression calibration equations for estimating fat

and protein content. Meat-based and milk-based calibrations were
derived and tested on eight ground beef samples which had been
chemically pre-analyzed by the standard methods. SDDs and mean
differences (MD) were obtained to compare and determine the accuracy
of the calibrations.

RESULTS AND DISCUSSION

Table 1 presents the means for uncorrected instrument absorbance

signals obtained at the four channels for the selected meats.
Bologna was the only carbohydrate-containing product giving a
positive value at the lactose channel. For the other meat products, the
instrument variation at the lactose channel was most probably due to
water displacement effects by the fat and protein components.
An SD of +0.02~o for replicates indicated that the instrumental
method was very precise. It must be kept in mind that this value includes
the variability due to sample preparation, as well as the variability due to
the instrument itself. Since the range of values varies for each channel, a
better estimate of precision is expressed by the coefficient of variability

TABLE 1
M e a n * U n c o r r e c t e d I n s t r u m e n t A b s o r b a n c e Signals O b t a i n e d from the
Multispec M for Selected Meats

Sample C--O CO N H C--O H CH

Bologna 2.26 0.95 0.21 3.12

Ham 0.19 1,70 - 0'06 0-77
Pork 2.02 1.42 - 0'22 2.84
Turkey 1.16 1.62 - 0.08 1-72
Beef 1 1.00 1.83 -0"23 1.69
Beef 2 1.12 1,83 -0.25 1.82
Beef 3 1.04 1.81 -0.20 1.71
Beef 4 1.69 1.59 - 0.22 2.47
Beef 5 0.92 1.91 -0.23 1.58
Beef 6 1.65 1.72 -0.25 2.42
Beef 7 1.79 1-62 -0"25 2.65
Beef 8 2.13 1.54 -0.25 3.13

* The values each represent the m e a n of three separate emulsions.

 Analysis of fat and protein in meat by infrared analysis 259

(CV) which was found to be 1.41 ~ , 0.93 ~ and 1-22 ~o for the C ~ O , CH
and C O N H channels, respectively. Although the CV is greater at the
C-m-O than at the CH channel, the difference was not significant.
Equations used to calculate the component percentage from the
instrument ,signals were derived by multiple regression analysis of the
chemical results versus the uncorrected instrumental signals. All possible
channel combinations were assessed in the analysis of data in order to
determine the best combination of instrument values to use for fat and
protein determinations. The best SDDs were obtained using all four
channel values, as shown in Table 2 for fat, although the CH channel
values could be excluded without seriously affecting the SDD.

TABLE 2
Standard Deviation of the Difference Obtained Using
Individual Channel and Combined Channel Absorbance
Signals to Calculate the Fat Contents of Meat

Channel SDD

C=O 0.301 6
CH 0.740 9
C=O + CH 0.288 4
CH + CONH + C--OH 0.684 1
C~O + CONH + C--OH 02792
C~O + C O N H + C - - O H + CH 0-260 1

A comparison of standard chemical results with IR instrumental

estimates for fat and protein in the meats using the four channel equation
is presented in Table 3. The CVs and SDDs were 1"46 ~o and 0.260, and
1.41 ~,i and 0"250, for fat and protein, respectively.
Since a large number of infrared instruments presently in use have been
adjusted by the manufacturer for the cross interference effects which
occur in milk, a milk calibration was obtained and tested for meat
analysis. Calibration milks were supplied by the Central Milk Testing
Laboratory (Guelph, Ontario, Canada) and they provided a range of fat,
protein and lactose levels required for establishing a sound calibration. A
comparison of fat and protein estimates for eight ground beef samples
using the meat or milk calibration is presented in Table 4. The component
percentages for meat results obtained with the milk calibration were
260 Bernice L. Mills, F. R. van de Voort, Y. Kakuda

TABLE 3
C o m p a r i s o n of Chemical a n d Infrared Results for F a t a n d Protein in Meats

Sample Chemical IR estimate Error

Fat:
Bologna 27.26* 27.24 0.02
Ham 4.22 4.28 - 0.06
Pork 25.24 24.81 0.43
Turkey 15.02 15.11 - 0.09
Beef 1 13.10 13.08 0.02
Beef 2 14.39 14.42 - 0.03
Beef 3 13-81 13.52 0.29
Beef 4 20.40 20.92 - 0.52
Beef 5 12.27 12.09 0.18
Beef 6 20.30 20.31 -0.01
Beef 7 21.52 21.89 -0.37
Beef 8 25.65 25.51 0.14

Protein:
Bologna 12.17 12.15 - 0.02
Ham 18.05 18.08 -0.03
Pork 16.42 16.42 0.00
Turkey 18.06 17.95 0.11
Beef 1 19.10 19.24 -0.14
Beef 2 19.89 19.30 0.59
Beef 3 19.07 19.11 -0.04
Beef 4 17.18 17.56 -0.38
Beef 5 19.58 19.84 -0.26
Beef 6 18.39 18.50 -0.11
Beef 7 17.88 17.60 0.28
Beef 8 16.68 16.72 -0.04

* Each value is the m e a n of three separate emulsions.

multiplied by a factor of ten to account for the dilution in the preparation

of the meat emulsions.
The M D s and S D D s in Table 4 indicate that a meat calibration is more
suitable than a milk calibration for estimating c o m p o n e n t percentage in
meat, especially in the case of fat. The specifications for IR accuracy in the
case of milk require an M D of < 0.05 ~o and an S D D of < 0.06 ~o for fat
and protein. Considering that the fat content in meat is five to six times
greater than in milk, even an M D of 0"84 with an S D D of 0.33 obtained
with the milk calibration is acceptable for quality control purposes. The
 Analysis of fat and protein in meat by infrared analysis 261

TABLE 4
C o m p a r i s o n of Results O b t a i n e d U s i n g a M e a t versus
a Milk C a l i b r a t i o n (CaD for Estimating the F a t and
Protein C o n t e n t in G r o u n d Beef Samples

Chemical Instrumental %fat*

Meat Cal Milk Cal

% Fat
14.39 14.32 13.95
13.81 13.42 13.01
20' 30 20.19 19.43
25.65 25.27 24.94
17.78 17.53 16.86
24-99 24.79 23.43
24.11 23.88 23.17
15.35 15.28 14.77
M D = 0.21 M D = 0.84
S D D = 0.13 S D D = 0.33

YoProtein
19-89 19.19 19.60
19"07 19.00 19.35
18'39 18.41 19'06
16'68 16'65 17.77
18'76 18.61 19.13
17'15 17"58 18-05
16'41 17.17 17-89
18'48 18"98 19.44
MD= -0.10 MD= -0.68
S D D = 0-45 S D D = 0.55

* M D - - m e a n difference; S D D - - s t a n d a r d deviation of
the difference

greater SDDs for protein can be attributed to the greater difficulties in

acquiring accurate data and the technical expertise required in the case of
the K.jeldahl. The use of the 6-25 factor for conversion to protein based on
the average nitrogen content of meats in general may not be appropriate
in all cases. The fact that the chemical analysis is based upon the amount
of reduced nitrogen, rather than the number ofpeptide linkages, may also
account for some of the discrepancy. For quality control purposes, the
SDDs for protein, using either the meat or milk calibration, are
acceptable.
262 Bernice L. Mills, F. R. van de Voort, Y. Kakuda

CONCLUSION

F r o m this work, it is apparent that it is possible to obtain satisfactory

quantitative results by transmission IR for the analysis of meat products.
It has been demonstrated that the IR instrumental method can produce
analytical results for fat in meat with precision and accuracy comparable
with that for milk. The results suggest that a meat calibration is more
suitable than a milk calibration for IR instruments used for meat analysis,
although a milk calibration does provide a reasonable reflection of the
standard chemical results. When only one type of meat is to be analysed,
better accuracy would probably be achieved by calibrating with the type
of meat to be analysed. Further developments in this area should be
concentrated on improving the accuracy for protein, on testing other
meat products and in the evolution of inexpensive IR meat analyzers for
commercial use.

ACKNOWLEDGEMENTS

The financial support of the Natural Sciences and Engineering Research

Council of Canada and the McGill University Grants Office is gratefully
acknowledged.

REFERENCES

AOAC (1970). Method 16.035.

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Biggs, D. A. (1979). J. Assoc. Off. Anal. Chem., 62, 1202.
Government of Canada (1982). Health and Welfare Canada Bulletin
(November).
Grappin, R. & Jeunet, R. (1976). Le Lair, 56, 498.
Hauser, E. & Heber, U. (1978). Fleischwirs., 58, 397.
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(1981). J. Assoc. Off. Anal. Chem., 64, 692.
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Anal. Chem., 66, 1048.
Mojonnier Bulletin 101 (1925). Mojonnier Bros. Co.,iChicago, IL.
Nexo, S. A. & Anderson, H. R. (1981). US Patent 4,247,773.
van de Voort, F. R. (1980). J. Assoc. Off. Anal. Chem., 63, 973.