Lipid and myoglobin oxidation in pork stored in oxygen- and carbon dioxide-enriched atmospheres

LIPID AND MYOGLOBIN OXIDATION IN PORK STORED IN OXYGEN- AND CARBON DIOXIDE-ENRICHED ATMOSPHERES J. A. ORDONEZ* & D. A. LEDWARD~

*Departmento de Higiene y Tecnologia de los Alimentos, Facultad de Veterinaria, Leon, Spain t Department of Applied Biochemistry and Nutrition, Food Science Laboratories, School of Agriculture, University of Nottinghara, Sutton Bonington, Loughborough LEI2 5RD, Great Britain

(Received: 21 October, 1975) SUMMA R Y

The formation of malonaldehyde and metmyoglobin in pork muscles stored in oxygenand carbon dioxide-enriched atmospheres at I°C was followed. The formation of metmyoglobin at the surface of the muscles was independent of carbon dioxide concentration. However, increased oxygen concentration caused a significant decrease in the rate of metmyoglobin formation; the surface concentration of metmyoglobin was below 30 ~o even after 15 days" storage in 80 % oxygen~20 % carbon dioxide. Lipid oxidation, as measured by malonaldehyde production (TBA Number), occurred at the same rate in air and mixtures containing 80, 90 and 100 ~o oxygen. In some muscles, the rate was such that rancidity was apparent within 6 days at I°C and, for pork, lipid oxidation, and not bacterial spoilage or metmyoglobin formation, may be the limiting factor in the use of oxygen-containing atmospheres for storage.

INTRODUCTION

Meat under refrigeration is normally spoiled by the growth of psychrotrophic bacteria. The meat is contaminated during butchering and there is a systematic relationship between the initial number of bacteria on the meat and its storage life (Haines, 1933). The result of the proliferation of micro-organisms is the production of slime and odour when the number is above about 10 v bacteria/cm 2 (Ayres, 1960). With an initial population of about 103 bacteria/cm 2 this occurs in 12 days at 0°C and in 6 days at 4°C when stored at a relative humidity o f 9 5 % (Schmid, 1931). Carbon dioxide inhibits the growth of psychrotrophic micro-organisms and the effect is more marked as the temperature is reduced, e.g. at 0°C the shelf life of beef slices is.more than doubled when stored in 10% CO2/air compared to air alone 41

Meat Science (1) (1977)--© Applied Science Publishers Ltd, England, 1977 Printed in Great Britain

42

J. A. O R D O N E Z , D. A. L E D W A R D

while at 5~C the increased shelf-life is only about 75°~ greater in 10 ~°,~CO2/air than in air (Shaw, 1972). Oxidative rancidity can be another cause of meat deterioration (e.g. Webb et al., 1972; Greene et al., 1971 ; Owen, 1973). However, it is rarely thought to be a serious problem in the storage of unfrozen meat. Although the growth of putrefactive micro-organisms and oxidative rancidity are the main causes of meat spoilage, the product is selected by the consumer on the basis of the colour and there is an inverse relationship between the degree of discoloration and the rate of sale (Hood & Riordan, 1973). The inhibition of micro-organisms and fat oxidation may be achieved by packaging the meat anaerobically, but the bright red colour of oxymyoglobin quickly changes to the undesirable purple of reduced myoglobin. On the other hand, oxidative rancidity can be inhibited and the red colour maintained by adding antioxidants and packaging in oxygen permeable bags (Greene et al., 1971). In this case, the meat will spoil by the growth of psychrotrophic bacteria. Elevated CO2 levels can be used to inhibit bacterial growth, although at the 0 2 concentrations present in air oxidation of myoglobin to the undesirable brown metmyoglobin (metMb) will occur. However, the external layer of oxymyoglobin in fresh beef stored in 80~o Oz and 2 0 ~ CO2 is oxidised only slowly so that the meat colour remains attractive for over 10 days at I-5°C (Taylor & MacDougall, 1973; MacDougall & Taylor, 1975) while the microbial population is maintained at an acceptable level ( < 107 bacteria/cmZ). Most of the work reported in the literature on the effect of different gaseous atmospheres on meat storage has been carried out on a variety of beef muscles and the present work was undertaken to extend these studies to pork. Longissimus dorsi and bicepsfemoris muscles were studied as, in beef, it is known that these muscles form metMb at vastly different rates (Ledward, 1971). Atthough the effect of 0 2 and CO2 levels on metMb formation and bacterial growth in meat has been extensively studied, little information is available on the rate of lipid oxidation under these conditions and so, as pork is susceptible to lipid oxidation, this parameter was also studied.

MATERIALS A N D M E T H O D S

Preparation and storage of samples The muscles were removed from the carcass 24 h after slaughter and trimmed of external fat. Cylinders of diameter 20 mm and thickness 8 mm were aseptically cut from them. All operations were performed at 4°C. All the muscles were of normal pH (5-3-5"5). The slices were stored in hermetically sealed plastic containers (volume 22 litres) in a cold room at I + 0.5°C. The atmosphere within the containers was maintained

43

LIPID AND MYOGLOBIN OXIDATION IN PORK

at the desired composition by periodic flushing with the appropriate gas mixtures. The relative humidity was maintained at 99.3~, by bubbling the gas mixtures through 0.02M NaCI (Ledward, 1970). [n some experiments, the slices were submerged for 30-60 sec in 0.15°~ chlortetracycline hydrochloride (CTC) prior to storage.

Analysis The surface concentration of metMb was measured using a Perkin Elmer 124 reflectance spectrophotometer (Stewart et al., 1965). Values for 0°/o metMb were determined on 24-h postmortem samples (Owen, 1973) and for 109~ metMb on ferricyanide oxidised samples (Ledward, 1970). Each determination was the mean of at least 10 samples. Oxidative rancidity was estimated by the reaction of malonaldehyde with thiobarbituric acid (TBA), as described by Tarladgis et al. (1960). Total bacterial numbers were estimated using plate count agar as the culture media. Quarter-strength Ringers solution was used for dilutions and two plates of each dilution were incubated at 22°C for 2 days.

RESULTS

Metmyoglobin formation Results for Iongissimus dorsi and biceps femoris muscles, from four different animals, stored in air, air + CTC, air/COz mixtures, O,/CO2 mixtures and Oz + CTC are shown in Figs 1, 2, 3 and 4. In all the muscles studied, provided the

,°°75 Tt

T

.~ 50

,I,

J'

25

~'

y

"~

t

5

10

15

20

Storage Time (Doys) Fig. 1. Effectof storage on metMb formation in pork 1. dorsi muscles stored in air (A), air + CTC (A), 10% COz + 90% Oz (O) and 20% CO2 + 80% 02 (C_.))at 1 5: 0"5°C. The arrows on the lines indicate the time for the bacterial population to reach 107-10s organismlcm2; these levels were not reached in the air + CTC and 209/. COz + 80% O., stored samples.

44

J. A. ORDONEZ, D. A. LEDWARD

I00 75

-o5O

25

5

I0

15

20

Storage Time (Days) Fig. 2. Effect of storage on metMb formation in pork b. femoris muscles stored in air (A), air + CTC (A), I 0 ~ CO2 + 9 0 ~ O., (Q) and 2 0 ~ CO., - 8 0 ~ 0 2 (.~) at I ± 0-5:C. The arrOws on the lines indicate the time for the bacterial population to reach 107-108 organism/cruZ; these levels were not reached in the air ~- CTC and 2 0 ~ CO., + 80% 0 2 stored samples.

100

50

25

1~0

'1'5

2O

Storage Time (Days) Fig. 3. Effect of storage on metMb formation in pork b. fvmoris muscles stored in air ~- CTC (A), CO2 2 0 ~ + air 8 0 ~ (A), Oz + CTC (O) and CO., 20% + O2 80% ( ~ ) at I --- 0.5"C. The bacterial population was less than 107/cm 2 during the whole of the storage period.

45

LIPID AND MYOGLOBIN OXIDATION IN PORK

bacterial load was less than 107/cm z, high O , levels ( > 80 °,o) maintained the surface metMb concentration at less than 25S~, during 12-20 days' storage at I°C. During this storage period air stored samples formed from 45-80 ~ metMb. These observations agree with previous studies on beef(Taylor & MacDougall. 1973 ; MacDougall & Taylor, 1975). At bacterial levels above 107/cm z (Figs' 1 and 2) accelerated rnetMb formation was observed in agreement with other experiments on beef (Ledward, 1973). !00

T5

.o 50

25

5

10 Storage Time

15

211

[Days)

Fig. 4. Effect of storage on metMb formation in pork 1. dorsi muscle stored in air + CTC (A), COz 2 0 % + a i r 80% ( 4 ) , O z - ' CTC (Q) and COz 20%" -- Oz 80°,,(~.) at I ± 0 5 C. The

bacterial population was less than 107;'cmz during the whole of the storage period.

The results of Figs I, 2, 3 and 4 verify the observation of Hutchins et al. (1967) that CTC has no effect on levels of metMb accumulated in meat and Figs 3 and 4 show that elevated CO2 concentrations do not influence the rate of metMb formation in pork as the rate of formation in air and in mixtures of 20'~,] COz/80°,; air is the same. Ledward (1970) found that elevated CO2 levels did not affect the rate of metMb formation in beef slices. Compared to air storage, both storage in CO2-enriched atmospheres and treatment with CTC inhibited microbial growth. With COz levels of 1 0 ~ the storage life was extended by 2 to 3 days at I°C (Figs 1 and 2). This extension of storage life is less than that predicted from the results of Shaw (I 972), who found the storage life to more than double on addition of 1 0 ~ COz to air at 0"C. In 2 0 ~ COz and on treatment with CTC the meat ~vas not spoiled (less than 10 7 organisms/cm z) even after 20 days at I°C. As would be expected from the numerous studies on beef, metMb accumulated at different rates in the different muscles but, on the limited results reported in this

46

J.A. ORDONEZ, D. A. LEDWARD

study, it is not possible to say if the formation in pork bicepsfemoris is, in general, more or less rapid than in pork Iongissimus dorsi.

TBA Number The TBA Numbers were determined on the same samples as used for spectral analysis. Duplicate 10-g samples were Used and the values quoted are the means of two measurements on each of these samples. All measurements were within + I0 ~o of the mean. 14

12

"7 ~C m 6

,'~

5

10

15

19

Storage Time (Days) Fig. 5. Effect of storage on TBA Numbers in pork b. f e m o r i s ( .) and I. d o r s i ( . . . . . . . ) muscles in air (A), air -- CTC (A), lOgo CO., + 90~/o O2 (O) and 20'Y. COz + 8 0 ~ O l (©) at 1 ___0-5:C.

From Figs 5 and 6 it is seen that there is a steady increase in TBA value during storage. However, there is no significant difference between the rate of oxidation in air, 80?/o O2/207~ CO2 and 9 0 ~ O z / 1 0 ~ COz (Fig. 5). However, the rate is apparently decreased in air plus 20~o CO2, although the effect is more marked in the I. dorsi muscle than in bicepsfemoris (Fig. 6). In all the muscles studied a rancid odour was apparent when a TBA Number of about 5 was achieved. If this is taken as the spoilage level it is seen that, with storage in air or high O2 atmospheres, the shelf-life varies from 6 to 19 days at I°C (Figs 5 and 6). The times taken for the different air stored muscles to reach metMb levels of 4 0 ~ or a TBA value of 5 are summarised in Table I. The results in Table 1 suggest that, although both pigment

47

LIPID AND MYOGLOBIN OXIDATION 1N PORK

(b) 6 4

~,2 ¢-~

L

Z I.-.-

4 2

5

tO Storage

15

ig

Time (Days)

Fig. 6. Effect of storage on TBA Numbers in pork b. femoris (a) and I. dorsi (b) muscles in air ~ CTC (A), COz 20% + air 8 0 ~ (A), Oz + CTC (Q) and CO_, 20°,/0 + Oz 80% (O) at 1 -*- 0-5°C.

and lipid oxidation are occurring, the rates are not related. This is contrary to several reports in the literature (e.g. Greene et al., 1971) but agrees with the conclusions reached by Ledward & MacFarlane (1971)and Buckley & Kearney (1975) following studies on frozen beef slices. TABLE I TIMES TAKEN FOR PORK MUSCLES TO ATTAIN 40~/o metMb AND A TBA VALUE OF 5 DURING AIR STORAGE AT I'~C

Muscle

Days to 40% metMb

Days to TBA calue of 5

L. dorsi A L. dorsi B B. femoris C B. femoris D

9 5 8 9

12 13 6 19

DISCUSSION

The results obtained in this study on pork are in general agreement with those observed in beef. Thus elevated C02 levels effectively inhibit bacterial growth

48

J . A . ORDONEZ, D. A. LEDWARD

without detriment to metMb formation whilst high O : concentration decreases the rate o f m e t M b formation at the surface of the muscle while not affecting the inhibitory effect o f C O , on bacterial growth. There is little information available on the level o f m e t M b at which consumers will reject pork meat but Greene et al. (1971) have claimed that consumers will reject beef at metMb concentrations greater than 40 °/ ,o. If this criterion is applicable to pork then it is apparent that the colour remains acceptable for five to nine days during air storage but in 80 or 90}0 O2 the colour remains attractive for at least 14 days. F r o m the TBA values measured it is seen that the lipids of the muscles studied in this work oxidise quite rapidly, although the rate varies from muscle to muscle. During air storage, rancidity was apparent in 6 to 19 days and may ~ell become the limiting factor governing the shelf-life o f pork stored in oxygen-containing atmospheres. Thus it would appear that the use o f 0 2 C O , mixtures to improve the colour stability o f pork and to minimise microbial proliferation may not be particularly useful because of the tendency o f some pork muscles to become rancid before or a r o u n d the same time as m e t M b formation becomes undesirable during air and air/COz storage. Ho~ever, it must be remembered that all these quality characteristics (i.e. bacterial population, pigment state and lipid oxidation) are surface effects, It may well be that in samples o f larger volume to surface area ratios (i.e. commercial cuts), the a m o u n t of lipid oxidation may be relatively unimportant so that surface discoloration and bacterial gro~th become, in all cases, the factors limiting the storage life. If this is so then increased storage life may be achieved by the use o f Oz- and CO:-enriched atmospheres. REFERENCES AYRE5, J. C. (1960). Fd Res., 25, I.

BUCKLEr, J. & KEAR,'qEY,L. (1975). 21st European .~feetint, Meat Research lfbrkers, p. 230. GREE~E, B. E., HSlN, 1. & ZIPSEk, M. W. (1971). J. FdSci., 36, 940. HAINES, R. B. (1933). J. Hyg. Camb., 33, 175. HOOD, D. I. & RIORDAN,E. B. (1973). J. Fd Technol.. 8. 333. HtJTO-IINS, B. K., LJu, T. H. P. & WArrs, B. M. (1967), J. FdSci., 32,214. LEDWARO,D. A. (1970). J. Fd Sci., 35, 33. LEDWARD,D. A. (1971). J. FdSci., 36, 138. LEDWARD, O. A. (1973). X I X eme Reunion Europeene des Chercheurs en Viamh,. Communication 13/4, p. 259. LEt)WARD, D. A. & MACFARLANE,J. J. (1971). J. FdSci., 36, 987. MAcDOUGAt-L,D. B. & TAYLOR,A. A. (1975). J. Fd Technol., 10, 339. OWEN, J. E. (1973). Oxidatit'e rancidity in porcine muscle. Doctoral Thesis. School of Agriculture, University of Nottingham. SCH.~It~, W. (1931). Beih. Z. ges. Kalteindustr., Reihe 3, Heft 6. SHAW, B. G. (1972). in Meat chilling--Why and who? bleat Research Institute, Langford, Bristol. MRI Symposium No. 2. STEWART,M. R., ZIPSER,hi. W. & WATTS, B. M. (1965). J. FdSci., 30, 464. TARLADGIS,B. G., WATTS,B. M., YOUN,',THAN,M. T. & DtZGAN,L. (1960). J. Am. Oil. Chemist's Soc., 37, 44. TAYt.OR, A. A. & MACDOVGALL,D. B. (1973). J. Fd Technol., 8, 453. WEBn, R. W., MARION,W. W. & HAYSE,P. L. (19721. J. FdSci., 37, 853.