A S S E S S M E N T O F R E L A T I V E C O N T E N T O F M Y O G L O B I N , O X Y M Y O G L O B I N A N D M E T M Y O G L O B I N A T T H E

S U R F A C E O F B E E F

KAROL KRZYWICKI

Meat and Fat Research Institute, ul. Grochowe Laki 4, 61-752 Poznan, Poland

(Received: 21 January, 1978)

S U M M A R Y

A method o f calculating the relative content o f myoglobin, metmyoglobin and oxymyoglobin at the surface o f beef is described. It is based on measurements o f reflex attenuance o f incident light at the isobestic points 572, 525, 473 and 730 nm. The latter value corresponds to the achromatic attenuance o f light at the meat surface and can be used as an objective measure of its lightness.

It was shown that the colour image o f the meat surface, and the relative amounts of" these three myoglobin derivatives, are influenced by the opacity o f the surface layer. A variability in the light diffusingproperties o f the meat surface may be considered to be the primary factor in creating differences o f colour perception o f meat derived from animals of the same species and o f the same approximate age.

INTRODUCTION

The significance of colour in the evaluation of meat quality follows from indirect correlations which exist between meat palatability and the lightness of its surface colour. A subjective assessment of meat colour, sometimes assisted by appropriate standards, is satisfactory for a selection of abnormalities such as PSE or D F D meat. However, studies of more subtle changes require spectrophotometric methods. Such methods are essential in studies of the connection between meat colour and the chemical status of meat pigments.

Amongst the methods used to study the changes of meat pigments the most common are those based on the calculation of K/S ratios (Stewart et al., 1965) although the assumptions on which they are based seem not to be fully justified.

1 Meat Science (3) (1979)--O Applied Science Publishers Ltd, England, 1979 Printed in Great Britain

2 KAROL KRZYWICKI

When light falls on a meat surface it is not only reflected from the uppermost layer but penetrates into the tissue to a depth of 3 to 4 mm (Lawrie, 1964). It is absorbed by meat pigments and also, in a series of refractions and random reflections, by various colourless structural elements of the muscle. Thus, the value of the absorption coefficient K, in the ratio K/S, is usually higher than in uniformly pigmented materials and varies from sample to sample, regardless of pigment concentration.

Another source of error often lies in setting limiting values of K/S or reflex attenuance ratios (Krzywicki, 1976). They are based on the assumption that fresh meat does not contain any appreciable amount of metmyoglobin and that a treat- ment of meat with ferricyanide converts ,all myoglobin and oxymyoglobin to metmyoglobin. In the case of beef slices, these conditions certainly a r e n o t met and thus the assumPtion gives rise to erroneous results. T h e wide range of K/$572 :K/$525 values reported in the literature for 100 ~o oxymyoglobin and 100 metmyoglobin (Franke & Solberg, 1971) may, to some extent, be a result of these shortcomings.

The method presented in this paper allows the proport ions ofmyoglobin forms to be calculated without the need to determine the limiting values specifying the complete saturation of the surface layer by one of the three myoglobin derivatives. The method was used to follow changes of meat pigments during prolonged storage of vacuum-packed beef. Some of the results illustrating the influence of pH on proport ions of met-, oxy- and myoglobin fractions are presented and a possible interpretation of the observed facts is given.

METHODS

(a) Materials and general procedure Beef animals of approximately the same age were used as a source of material.

Samples for measurement were taken from (fresh or vacuum-packed) muscles and stored for up to six weeks at 0 °C. Slices of beef 1 to 1-5 cm thick were used. Reflex at tenuance of incident light was measured by a Pye-Unicam SP1800 spectrophotometer provided with a diffuse reflectance accessory SP 890. Samples were illuminated at 90 _+ 10 ° and the diffuse reflected light measured at 45 _+ 5 o. The reflex attenuance was recorded continuously in the range 400 to 760 nm or at the following specific wavelengths: 473,525, 572, 630 and 730 nm. Meat samples were not covered in the course of the measurements. Usually the attenuance was measured 30 min after cutting a fresh surface. When the rate of oxymyoglobin formaticm was studied the measurements were started immediately after cutting. The time required for the completion of a single scan from 473 to 730 nm was about 40 sec.

To distinguish between attenuation of light due to both absorption and diffusion and that caused by light absorption by transparent materials (absorbance) only, the

MYOGLOBIN, OXYMYOGLOBIN AND METMYOGLOBIN AT THE SURFACE OF BEEF 3

term reflex attenuance (the logarithm of the reciprocal of reflectance, suggested by Shibata, 1966) has been used.

(b) Calculation of myoglobin (myo), oxymyoglobin (ox) and metmyoglobin (met) fractions of total pigment

The reflex attenuance of light falling on a meat surface is composed of two terms:

A ~ = A~ + A~ (1)

where A, represents achromatic absorption caused by refraction and internal reflections at the structural elements of the meat (Shibata, 1966; Clydesdale, 1975) and A~ represents the fraction of light absorbed by the pigments present in the tissue.

The achromatic absorption of light depends on the light diffusing properties of muscle proteins, cell membranes and fat particles. It increases in media of low diffusing power, with anincrease of light path length, as well as in well hydrated meat of high pH, and decreases with the growth of visible marbling or protein denaturation.

Myoglobin and its oxygenated and oxidised forms constitute the main pigment of

z

(:13

~,600

1,LO0

1,200

1,000

0.800

0,600

0/,130

A ~ , . . . . . . . . .

Fig, 1.

200 300 z.00 500 600 700 '~00 nm

WAVELENGTH

Spectra of light reflected from the surface layer of beef. A--fresh meat; B--meat after exposure to UV radiation (containing over 50 % of metmyoglobin).

4 K A R O L K R Z Y W I C K I

meat. Their maxima of absorption lie in the band 400 to 630 nm. Each reflectance spectrum of beef shows a minimum at approximately 730 nm (Fig. 1) where it is not dependent on the pigment concentrat ion and can be taken as the at tenuation of light by pigment-free meat. The presence of other colour compounds can be ignored because of their comparat ively low concentrations in meat. The term A, can be replaced, in eqn. (1), by a reflex at tenuance measured at 730nm:

A ~ = A 73° + A~ (2)

Following the Lambert-Beer law, absorpt ion of light by myoglobin pigments at the meat surface can be expressed as:

= e . c . d ( 3 )

where e ~ is the molar absorbance coefficient (MAC) at 2 nm; c is molar concentrat ion of pigments and dis the length of the light pa th in the surface layer. For a mixture of the three derivatives of myoglobin the MAC is:

C2 A A A : X . emy o -[- y . eme t + Z . eox (4)

where: emyo,~" emet,2 eoxZ denote the MAC of myoglobin, metmyoglobin and oxymyoglobin, respectively and x, y, z are the fractions of myoglobin, metmyo- globin and oxymyoglobin, respectively. Thus:

x + y + z = 1 (5)

At the 525 nm isobestic point all three M A C values are equal. The inherent pigment absorbance at 525 nm is equal to the difference in reflex at tenuance measured at 525 and 730nm:

A~ 25 : A 525 - - A 730 (6)

and depends only on the total concentrat ion of all myoglobin derivatives, while it is independent of the propor t ion in which the individual forms occur. When the average length of the light pa th is the same in two meat samples, the difference in their A~ 25 absorbances is directly propor t ional to the difference in pigment concentration in the samples. At the 572 nm isobestic point the MAC of myoglobin and oxymyoglobin are equal. The MAC of a mixture of the three derivatives at 572 nm is:

e 5 7 2 _572 (1 y) -t'- - 5 7 2 : e'my . . . . " - - e'rnet .y (7)

To calculate y, the fraction of metmyoglobin in total pigment, let us consider the ratio:

A 5 7 2 P (8)

a 1 - - A 5 2 5 - - p

MYOGLOBIN, OXYMYOGLOBIN AND METMYOGLOBIN AT THE SURFACE OF BEEF

TABLE 1 M1LLIMOLAR ABSORBANCE COEFFICIENTS OF MYOGLOB1N, OXYMYOGLOBIN AND

METMYOGLOBIN AT SOME 1SOBEST1C POINTS (AFTER BROIAMAND el al., 1958)

Pigment Jorm Wavelength (nm) 473 525 572

Myoglobin 4.4 7-6 10-6 Oxymyoglobin 7-6 7.6 10-6 Metmyogiobin 7.6 7-6 3-0

The r a t i o o f two a b s o r b a n c e s o f the same mea t s ample is equa l to the r a t io o f the M A C values :

-572 (1 -- y ) + _s72 a l = ~'myo " e'met "Y (9)

e525

Af te r subs t i tu t ing the M A C values f rom Tab le 1 the fo l lowing e q u a t i o n for c a l c u l a t i n g y is o b t a i n e d :

y = 1.395 - a 1 (10)

where :

A572 __ A 7 3 o

al = A525 _ _ A73O (11)

The la t te r can b e ca lcu la t ed f rom reflex a t t enuances m e a s u r e d at 5 2 5 n m and 730 nm.

By ana logy , in view of the iden t i ty o f the M A C for o x y m y o g l o b i n and m e t m y o g l o b i n at 473 nm, the f rac t ion (x) o f m y o g l o b i n in the to ta l p i g m e n t can be ca l cu la t ed f rom the ra t io a 2 as fo l lows:

A473 _ A730

a2 - A52S _ A73O (12)

A473 _473 (1 X) -[- 473 p ~'.met:=ox . - - emv o . X (I 3)

a 2 = ~ = e525 " - -p

x = 2"375. (1 - a2) (14)

F ina l ly , the f r ac t ion o f o x y m y o g l o b i n in the to ta l p i g m e n t m a y be o b t a i n e d f rom:

z = 1 - (x + y) (15)

The accu racy o f the e s t ima t ion is d e t e r m i n e d by the a c c u r a c y a n d p rec i s ion o f the a p p a r a t u s used for the measuremen t s .

6 KAROL KRZYWICKI

RESULTS AND DISCUSSION

In the absence of metmyoglobin the validity of the above relationship can be checked using 1009/oo oxymyoglobin or myoglobin. However, metmyoglobin is seldom completely absent from beef. On the other hand, myoglobin is frequently absent at the surface of meat when, because the MAC of oxy- and metmyoglobin at 473 and 525 nm have the same values, the ratio a 2 should be very close to unity. Indeed, a z

values closely approaching unity are always found when the presence of the oxy- and met- forms only is expected (Table 2). One of the two absorption peaks of

TABLE 2 EXAMPLES OF REFLEX ATTENUANCE MEASUREMENT ON BEEF SAMPLES WITH EXPECTED ABSENCE OF MYOGLOBIN

AT THE SURFACE (30 mlN AFTER CUTTING; pH OF MEAT 5'5 TO 5"7)

Reflex attenuance at nm Fraction o f 730 572 525 473 a 1 a2 met myo ox"

0-690 1.225 1.215 1.205 1.019 0-981 0.38 0.04 0.58 0-360 1.045 0.915 0.915 1.234 1-000 0" 16 0.00 0.84 0-525 1.210 1-105 1.105 1.181 1.000 0.21 0-00 0-79 0-315 1.085 0-945 0.975 1.222 1.048 0.17 0-00 0-83 0-430 1.225 1.105 1-120 1-178 1-022 0-22 0-00 0.78 0.375 1-030 0.910 0-920 1.224 1.019 0-17 0-00 0.83 0-390 1.015 0.890 0-885 1-250 0.990 0-14 0.02 0.84 0.600 1.310 t.225 1.225 1.136 1.000 0.26 0.00 0.74 0-580 1-205 1.100 1.105 1.202 1-010 0.19 0.00 0.81 0.490 1.190 1.080 1.100 1.186 1.034 0.21 0.00 0.79

Values of a 2 > 1 result from a reading error when using the 0-2.0 extinction scale. a met, myo, ox = metmyoglobin, myoglobin and oxymyoglobin, respectively.

metmyoglobin is located at 630 nm and its intensity is known to be in direct relation to the concentration of this compound (Franke & Solberg, 1971). A comparison of results calculated by eqn. (10) with the inherent pigment absorbance measured at 630nm 63o (Ap ) is given in Fig. 2. To obtain samples with a broader range of metmyoglobin content, the surface of meat was exposed to UV radiation for 3 to 5 min. The observed agreement is completely satisfactory and the coegicient of correlation was high (r ---- + 0-96). The method was applied to over 200 samples of beef. The fractions of total pigment represented by myoglobin, oxymyoglobin and metmyoglobin at the meat surface were calculated. The collected results (Table 3) were divided into four groups according to the pH of the meat. During six weeks storage of vacuum-packed beef no significant colour changes were observed.

Almost all samples of the first two groups (with mean pH below 6.0) were of good colour. Of the third group only about 40 ~ was acceptable and of the fourth group (with pH over 6.4) all samples were rejected.

In earlier experiments (Krzywicki, unpublished) it was found that subjective evaluation of beef colour correlates well with the reflex attenuance at 730nm

MYOGLOBIN, OXYMYOGLOBIN AND METMYOGLOBIN AT THE SURFACE OF BEEF 7

0.7

0,6

0,5

0,~

0,3

02

0.1

J o a ° o oXY

Oooo~ o

o8~ o o~o~ 8o~ o

~70= O

/07 o ° / o =7/00

o~°oo

~ o O

i i

0.1 0.2 0.3 0/. 0.5 0.6 0.7 y[rnet]

Fig. 2. The fraction.of metmyoglobin plotted against the inherent metmyoglobin absorbance at 630 n m 6 3 0 525 (Ap ) related to the total p igment absorbance at 525nm (Ap ). Correlat ion coefficient r = +0 '96 .

(r = +0-84). A similar correlation was observed by Ockerman & Cahill (1969). According to Judd & Wyszecki (1963) lightness is an attribute related to the light- diffusing and light-transmitting properties of an achromatic equivalent of a sample. The observed correlation confirms the assumption that the reflex attenuance at 730 nm corresponds to the light absorption of pigment-free meat tissue. A dark meat

T AB L E 3 RELATIVE CONCENTRATIONS OF MYOGLOI~IN, METMYOGLOB1N AND OXYMYOGLOBIN AT THE SURFACE OF BEEF

(MEANS AND STANDARD DEVIATIONS)

Number of Mean value Reflex attenuance Fraction of total pigment samples and range of pH at 730 nm Myoglobin Metmyoglobin Oxymyoglobin

75 5.64 0-454 4- 0.058 0-05 ___ 0-06 0"20 __. 0-06 0.75 (5-55-5-80)

66 5-91 0-495___0-057 0-13+0.05 0 .22+0.02 0.65 (5-81-6-10)

63 6.24 0-585___0.058 0-23___0-01 0-23___0-03 0.54 (6.11-6-40)

39 6.60 0-616___ 0~045 0-27___0-02 0-24___0-02 0-49 (6.41~-80)

Correlation coefficient, between A 730 and myoglobin fraction, r = + 0.90.

8 KAROL K R Z Y W I C K I

colour (e.g. DFD) is brought about by a strong achromatic absorption of light. A darkening of meat with an increase of its pH is the result of deeper penetration of light into more hydrated and less opaque tissue. In addition, a strong influence o fp H on the equilibrium between myoglobin and oxymyoglobin content at the surface was noted. A high pH in meat was usually accompanied by an increased myoglobin fraction at the surface. The content of the metmyoglobin fraction was practically constant and not related to pH or time of storage.

The equilibrium between myoglobin and oxymyoglobin at the surface of meat with a pH value of around 5.6 is shifted entirely on the side of the oxygenated form but, at pH values near 6.6, the surface layer contains one third of the pigment in the reduced form. These changes of myogtobin content with meat pH cannot be explained on the grounds of chemical kinetics. Format ion of oxymyoglobin from myoglobin in the presence of oxygen is a rapid reaction and its equilibrium constant does not depend on pH (Haurowitz & Hardin, 1954). The half-time of myoglobin oxygenation was found to be 4 .10 -4 sec, and at partial oxygen pressures higher than 60 mm Hg only oxymyoglobin is present (Haurowitz & Hardin, 1954). Under these conditions the formation ofoxymyoglobin starts immediately after the surface is exposed to the atmospheric oxygen and is controlled by the rate of oxygen diffusion into meat. It is suggested that when oxygen reaches the maximum depth and the final equilibrium state is attained, the colour perception of a meat surface is essentially influenced by the more advanced of the two penetrating factors--l ight and oxygen. When oxygen penetration prevails, the surface of meat appears to be completely covered by bright oxymyoglobin. Otherwise, if the light penetrates the surface layer of meat beyond the oxymyoglobin and (thin) metmyoglobin (Hood, 1976) layers, the meat appears darker due to increased achromatic absorption and to the appearance of purplish myoglobin in the light path. The fraction of oxymyoglobin in the total visible pigment appears to fall according to the equation:

Z a

x + z b (16)

where: a is the thickness of the oxymyoglobin layer, and b is the depth of light penetration.

The observed high correlation (r = + 0-90) between the reflex attenuance at 730 nm and the fraction ofmyoglobin in the total pigment (Table 3) results from the fact that both these values are related to the depth of light penetration. The course of myoglobin oxygenation was followed with the aid of the method described during the first 30 min after the fresh surface was cut. It was found that in a cross-section of meat freshly exposed to atmospheric oxygen the maximum oxymyoglobin fraction was reached at room temperature in less than 20 rain. In the majority of the samples with pH lower than 6-0 full saturation of the surface layer with oxymyoglobin (no myoglobin present) was achieved in about 16min. In samples with pH over 6.0, where saturation was not achieved even in 30 or 40min, the maximum

MYOGLOBIN, OXYMYOGLOBIN AND METMYOGLOBIN AT THE SURFACE OF BEEF 9

oxymyoglobin fraction was similarly observed after about 16min. In only two samples was saturation observed after 10 min. These samples were pale and bright red.

It is suggested that in the case of the first group of samples (with pH lower than 6-0) the depths of oxygen diffusion and light penetration are approximately the same. The second group represents a population of samples where the light penetrates the surface layer of meat deeper than oxygen and the full saturation of the surface with oxymyoglobin c a n n o t be achieved. In the case of those samples where saturation was attained in only 10min, the opposite might be true, i.e. due to the high diffusing power of the surface the penetration of the light stops above the point at which oxymyoglobin can still form. Taking as the starting point the equation given by Roughton (1959) for haemoglobin solutions, an empirical equation was derived which accurately describes the observed decrease of the myoglobin content (or increase of the oxymyoglobin content) at the surface of meat exposed to oxygen:

log (myo) = 0-24t 3/4 (17)

where:

x (myo) = (18)

X - k - z

t = time (in minutes) after exposing the fresh surface. The depths of light and oxygen penetration are assumed to be equal.

The maximum depth of oxygen diffusion was assessed at a fresh cross-section of meat. The borderline between oxy- and myoglobin is then sharply outlined. In some twenty samples the thickness of the bright oxymyoglobin layer was found to be very regular and close to 0-3 cm.

To illustrate the course of myoglobin disappearance from the surface of meat samples of normal or high pH, appropriate curves were calculated and are compared with experimental ]-esults in Fig. 3. Curve A was calculated according to eqn. (17) and illustrates changes in the group of samples with a pH of 5-5 to 5.7. The overall agreement with experimental results is satisfactory. In calculating curve B (samples with pH > 6.0) it was assumed that the rate and depth of oxygen diffusion are similar in both group A and group B.

The difference in appearance of the samples is caused by the increased thickness of the oxymyoglobin surface layer. When the state of final equilibrium is reached, the fraction of myoglobin, x, can be used to assess the depth of the light penetration, b, according to eqn. (16).

It can be concluded that the colour image of beef (in the absence of drastic variations of pigment concentration and with a metmyoglobin fraction not exceeding approximately 30 ~ of the total pigment) is controlled by the optical properties of the meat surface. All factors affecting the distance of the light path in meat, such as pH, amount of marbling and connective tissue, size of muscle fibres

I0 KAROL KRZYWICKI

N

×~

Z

c3

o >-

x

0.6

0.5

0.~

0.3

0.2

0.I

0

×

B

x

2 L 8 16 20 minutes

Fig. 3. The course of myoglobin oxygenation at the surface of freshly cut beef. Crosses denote mean values of experimental results and bars (curve A) give their confidence limits at P = 0-05. Full lines represent calculated course of change in normal beef (A) and in beef with a pH value of over 6.0 (B).

and denaturation o f sarcoplasmic proteins, may have a significant influence on the lightness of beef colour and, to some extent, on the ratio o f myoglobin to oxymyoglobin , i.e. on the hue o f meat colour.

REFERENCES

BROUMAND, H., BALL, C. O. & STIER, E. F. (1958). Food Technol., 12, 65. CLYDESDALE, F. M. (1975). In Theory, determination and control o f physical properties o f food materials

(ChoKyun Rha (Ed.)), p. 275, Reidel Publishing Co., Dordrecht, Holland. FRANKE, W. C. ~ SOLBERG, M. (1971). J. Fd. Sci., 36, 515. HAUROWITZ, F. & HARDIN, R. L. (1954). In Theproteins (Neurath, H. & Bailey, K. (Eds.)), Vol. 2, part A,

p: 279, Academic Press, New York. HOOD, D. E. (1976). Proc. 22nd Meet. European Meat Res. Workers, Malmoe, 2, K 01. JUDD, D. B. ~¢. WYSECKI, G- (1963). Colour in business, science and industry (2nd edition) Wiley & Sons,

New York. KRZYWICKI, K. (1976). Proc. 22nd Meet. European Meat Res. Workers, Malmoe, 2, K 5. LAWRIE, R. A. (1964). Meat science (First edition) p. 272, Pergamon, Oxford• OCKERMAN, H. W. & CAHILL, V. R. (1969). J. Anita. Sci., 28, 750. ROUGHTON, F. J. W. (1959). In Progress in biophysics and biophysical chemistry (Butler, J. A. V. & Katz,

B. (Eds.)), Vol. 9, p. 55, Pergamon, Oxford. SHIBATA, K. (1966). In Methods o f biochemical analysis (Glick, D. (Ed.)), Vol. 9, p. 217, Interscience,

New York. STEWART, M. R., ZIPSER, M. W. & WATTS, B. M. (1965). J. Fd. Sci., 30, 464.