HEXANAL AS AN INDICATOR OF MEAT FLAVOR DETERIORATION

FEREIDOON SHAHIDI and RONALD B. PEGG

Department of Biochemistry Memorial University of Newfoundland

St. John’s, NF, Canada, A13 3x9

Received for Publication September 6 , 1993 Accepted for Publication December 16, 1993

ABSTRACT

Pentanal and hexanal were the dominant volatile aldehydes generated from cooked pork during 3 weeks of refrigerated storage. Moreover, hexanal concen- trations may serve as an index of meat flavor deterioration (MFD) during the early stages of storage; its concentration increased more rapidly than any other aldehyde. During the first 6 days, contribution of pentanal and hexanal to the total volatile aldehydes increased linearly by 350 and 650%, respectively, ajer which, their concentrations declined quite markedly. Reactions of pentanal and hexanal with meat components and/or their further oxidation may be responsible for this observation. Therefore, caution should be exercised when using hexanal as an indicator of lipid oxidation and MFD since a given hexanal level may cor- respond with two different points during storage of cooked meats.

INTRODUCTION

The oxidation of unsaturated lipids has been extensively studied since it relates to deterioration of muscle foods, production of both desirable and undesirable breakdown products and numerous reactions associated with other food consti- tuents (Wong 1989). In 1958, Tims and Watts observed that lipid oxidation in refrigerated cooked meats was more pronounced than that in raw or frozen un- cooked meat. To describe this rapid development of lipid-derived oxidized flavor, they coined the term “warmed-over” flavor (WOF). In the late 1980s, various researchers showed that WOF was not solely a consequence of lipid oxidation (Vercellotti et al. 1987a,b; St. Angelo et al. 1988; Spanier ef al. 1988). These

Journal of Food Lipids 1 (1994) 177-186. All Rights Reserved. 0 Copyright 1994 by Food & Nutrition Press, Inc., Tnrmbull, Connecticut. 177

178 F. SHAHlDI and R.B. PEGG

researchers suggested that there was strong evidence that protein degradation reac- tions were also involved and that heteroatomic compounds formed from these reactions may be implicated with the phenomenon of WOF, particularly with the deterioration of desirable meaty flavor notes. Therefore, meat flavor deteriora- tion (MFD) was proposed as a more accurate term to use.

Although there are many assays available for assessing the oxidativc status of meat and meat products, the 2-thiobarbituric acid (TBA) test is widely used for this purpose (Tarladgis et al. 1960; Melton 1983; Shahidi 1992). Malonaldehyde is a relatively minor product of autoxidation of polyunsaturated fatty acids and it reacts with the TBA reagent to produce a pink-colored complex with an ab- sorption maximum at approximately 532 nm. The concentration of this complex is then used as an objective measure for evaluation of the oxidative state of cooked meats. Significant correlations have been found betwecn the TBA values and sen- sory scores of meat (Poste et al. 1986; Salih et d. 1987), but the TBA method has its own limitations (Melton 1983). The test has never been adequately stan- dardized as a measure of undesirable meat flavor but this is also true for other indices of lipid oxidation such as content of carbonyl compounds. Malonaldehyde mat interact with other food constituents such as amino acids, proteins, glycogen and food ingredients (Gray and Monahan 1992) rendering it unavailable to react with the TBA reagent, thereby, resulting in an underestimation of the TBA values. The TBA test has also been reported to be unreliable in assessing the oxidative status of frozen foods and of certain cured-meat products (Ihekoronye 1990). The data are not translatable from species to species (Pearson et al. 1977) and are quite variable depending on the method of analysis employed (Rhee 1978).

An alternative approach for assessing lipid oxidation in meat products is to measure the carbonyl compounds formed upon degradation of fatty acid hydroperoxides. Carbonyl compounds have been associated as significant con- tributors to the flavor of uncured meats (Shahidi et af. 1986; Shahidi 1989; Ramarathnam et af. 1991a,b). Moreover, some of these aldehydes have been shown to correlate with MFD. The concentration of hexanal, in particular, has been suggested as being a useful primary marker of MFD (Bailey et al. 1980; Dupuy et al. 1987; Shahidi et al. 1987; Shahidi 1992).

Hexanal is a seemingly ubiquitous component of food, both fresh and stored. This stems from the fact that practically all foods have some linoleic acid (18:2w6), the fatty acid from which hexanal is derived (Frankel et af. 1984). Hexanal has been characterized as having a powerful and penetrating fatty-green and grassy odor (Arctander 1969). It has a very low odor threshold concentration of 4.5 p g l k g (ppb) in water (Belitz and Grosch 1987) and it may be detected analytically at levels in the ppb range. Hexanal generation has been successfully used for evaluation of the oxidative state of red meats from different species as well as fish. Dupuy et al. (1987) noted that in cooked ground roast beef, chicken and

.

HEXANAL INDICATING MEAT DETERIORATION 179

turkey, the level of pentanal, hexanal, 2,3-octanedione, nonanal and the total volatiles increased appreciably during storage over 5 days at 4C as did the sen- sory scores and TBA values. The hexanal content increased more rapidly than any other aldehyde. Shahidi et al. (1987) reported that a linear relationship ex- isted between the hexanal content and both the TBA values and sensory accept- ability scores of cooked ground pork systems. Similar increases in hexanal levels during storage were reported for cooked beef, pork and fish (Morrissey and Apte 1988), chicken (Ang and Lyon 1990), chevon (Lamikanra and Dupuy 1990), salted and dried beef (Torres et al. 1989) and cooked beef (Drumm and Spanier 1991). Consequently, hexanal generation appears to be a sensitive and reliable indicator for evaluation of the oxidative state and flavor acceptability of meat and meat products.

In recent years, gas chromatographic (GC) determination of the volatile com- pounds of stored cooked meats has been studied (St. Angelo et al. 1987). Unfor- tunately, volatile analyses by GC methods are time consuming since they generally involve pre-isolation, purification and concentration steps. A rapid technique to overcome these time-consuming operations is headspace-gas chromatography (HS- GC) (Ang and Young 1989). Thus, the objective of the present study was to deter- mine changes in oxidative state of cooked ground pork during a three-week storage period as measured by HS-GC analysis of the dominant aldehydes and total volatiles.

MATERIALS AND METHODS

Materials

Aldehyde standards, namely acetaldehyde, propanal, isobutanal, butanal, isopen- tanal, pentanal, hexanal, heptanal, and octanal as well as 2-heptanone were ob- tained from the Aldrich Chemical Company (St. Louis, MO).

Preparation of Meat Systems

Boneless pork loins were obtained from the Newfoundland Farm Products Cor- poration (St. John’s, NF) and their subcutaneous fat was trimmed. Loins were comminuted twice using a Hobart 4146 Meat Grinder (Hobart MFG Co. Ltd., Don Mills, ON) with a 0.79 cm and then with a 0.48 cm plate. Comminuted pork was mixed with 20% (w/w) distilled water. Meat systems were cooked in an 85C thermostated water bath for 45 min or until an internal temperature of 75 f 2C was reached. Systems were cooled to room temperature, homogenized in a Waring Blendor, transferred to Whirl Pak bags (Systems Plus, New Ham- burg, ON) and refrigerated at 4C until used.

180 F. SHAHIDI and R.B. P E W

Proximate Analysis

mined using AOAC standard methods of analysis (AOAC 1990). Moisture, crude protein, total lipid and ash content of pork samples was deter-

Gas Chromatographic Analysis

A Perkin-Elmer 8500 gas chromatograph and HS-6 headspace sampler (Perkin- Elmer Corp., Montrkal, PQ) were used for analysis of cooked pork samples. A high polarity Supelco SP-2330 fused-silica capillary column (30 m X 0.25 mm ID, 0.20pm film, Supelco Canada Ltd., Oakville, ON) was used. Helium was the carrier gas employed at an inlet column pressure of 17.5 psig with a split ratio of 7:l. The oven temperature was maintained at 50C for 5 min and then programmed to 115C at 10C/min, held there for 1 min, and then ramped to 200C at 30C/min. The injector and flame ionization detector (FID) temperatures were adjusted to 250C.

For HS analyses, 2.0 g portions of homogenized pork samples were trans- ferred to 5 ml glass HS vials. The vials were capped with teflon-lined septa, crimped and then frozen at -6OC until used. To avoid heat shock after removal from storage, frozen vials were tempered at room temperature for 30 min and then preheated in the HS-6 magazine assembly at 9OC for an equilibrium time of 45 min. Pressurization time of the HS vials was 5 s, and the volume of the vapor phase drawn was approximately 1.5 . ml. An optimization for time- temperature parameters for the determination of volatile compounds of chicken meat using the static HS technique was reported by Ang and Young (1989). Chromatogram peak areas were expressed as integrator count units. Individual volatile compounds were tentatively identified by comparing relative retention times of GC peaks with those of commercially available standards. Quantitative determination of dominant aldehydes was accomplished using 2-heptanone, as an internal standard.

RESULTS AND DISCUSSION

To characterize the pork used, a proximate analysis of the fresh meat was car- ried out. The pork contained 73.3 f 0.6% moisture, 20.2 f 0.4% crude pro- tein, 5.7 f 0.4% total lipids and 1 .O f 0.3% ash. Since variations in moisture and fat levels of cooked samples may affect the stability of water- and fat-soluble flavor compounds, respectively, moisture and lipid contents were monitored during the storage period to see if any changes were evident (Ang and Lyon 1990). No significant changes in either level were observed during the course of this study.

HEXANAL INDICATING MEAT DETERIORATION

123

4

7

9

181

I . I . I . I . I . I . I

0 2 4 6 a 10 12

Time (min)

FIG. 1. A HS-GC CHROMATOGRAM OF THE FLAVOR VOLATILES OF COOKED GROUND PORK STORED FOR 5 DAYS AT 4C

A typical chromatogram of the headspace (HS) volatiles of cooked pork after 5 days of storage is presented in Fig. 1. The rapid GC-FID method used in this investigation did not allow for the analysis of all possible compounds related to MFD. However, most of the HS volatiles determined by the present method were

182 F. SHAHIDI and R.B. PEGG

low-molecular weight aldehydes. All volatiles were eluted within 20 min. The automated sampling features of the HS-6 analyzer and integration of the micro- processor controlled chromatographic and data management systems facilitated reproducibility between replicates. The dominant aldehydes detected were pen- tanal (peak #6) and hexanal (peak #7). Uncooked pork samples contained negligible amounts of these aldehydes, as determined in preliminary tests. Other aldehydes tentatively identified by retention time matching included acetaldehyde (peak #l), propanal (peak #2), isobutanal (peak #3), butanal (peak #4), isopentanal (peak #5), heptanal (peak #8) and octanal (peak #9).

The HS volatile profiles were qualitatively similar during the period of study, however, quantitatively they were quite different. The numbers assigned to the peaks in Fig. 1 were only used to mark the major components and do not reflect the total number of peaks observed. The peak areas of several volatile compounds increased substantially during the early stages of storage. Pentanal (peak 6) and hexanal (peak #7) levels increased by 350 and 650%. respectively, by day 6, reached a maximum and then declined. The increase in the content of pentanal, hexanal and total volatiles observed during the first 6 days of storage is presented in Fig. 2. Many studies have illustrated the increase in hexanal content during the first several days of storage (0 to 5) of cooked muscle foods and its correla- tion with TBA values or sensory scores (Morrissey and Apte 1988; Ang and Lyon

0 c FIG. 2. THE CONTENT OF PENTANAL, HEXANAL, AND TOTAL VOLATLES DETECTED BY HS-GC IN COOKED GROUND PORK DURING THE FIRST 6 DAYS OF STORAGE AT 4C

HEXANAL INDICATING MEAT DETERIORATION 183

1990; Spanier et al. 1992), but after this period, neither the content nor the fate of hexanal has been reported. Since aldehydes are quite reactive, they continu- ously oxidize. Palamand and Dieckmann (1974) subjected hexanal to autoxi- dation and reported that it underwent oxidation, polymerization and degradation resulting in the production of a large number of flavor-active compounds, most notably of which was hexanoic acid. However, a decrease in the concentration of hexanal may also be due to cross-linking reactions with various components in the meat matrix.

Based on the use of 2-heptanone, the concentration of pentanal and hexanal in the pork volatiles reached a maximum of 8.0 and 29 ppm, respectively, on day 6 (Fig. 3). The increase in pentanal and hexanal concentrations was linear over this period (i.e., days 0 to 6), after which, a linear decreasing trend was observed. A given hexanal level may correspond with two different points dur- ing storage of cooked meats. Therefore, caution should be exercised when using hexanal as an indicator of lipid oxidation and MFD. Nonetheless, hexanal levels do correspond well with a single point during the early stages of storage.

L

0 7 14 21

Storage Period (Days)

FIG. 3. THE CONCENTRATION OF PENTANAL, 0 , AND HEXANAL, V, IN COOKED PORK VOLATILES

DURING STORAGE AT 4C

184 F. SHAHIDI and R.B. PEGG

Although hexanal was used as an index of lipid oxidation and MFD, it is not intended to imply that it is mainly responsible for the characteristic off-flavor of stored meat or that it should be used as an index of lipid oxidation at the ex- pense of the TBA test. The relationship between hexanal concentration and off- flavor notes, perceived by sensory means, is statistical and does not offer any physiological or psychophysical explanation of changes that happen in meat (Ihekoronye 1990). Nonetheless, HS-GC has potential for being used as an in- dicator for quality control during processing and storage of meat products. It may also be used for evaluating frozen and cured-meat products where oxidation pro- ceeds slowly or when the TBA methodology may lead to erroneous results.

ACKNOWLEDGMENTS

We are grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support.

REFERENCES

ANG, C.Y.W. and LYON, B.G. 1990. Evaluations of warmed-over flavor dur- ing chill storage of cooked broiler breast, thigh and skin by chemical, in- strumental and sensory methods. J. Food Sci. 55, 644-648, 673.

ANG, C.Y.W. and YOUNG, L.L. 1989. Rapid headspace gas chromatographic method for assessment of oxidative stability of cooked chicken meat. J. Assoc. Off. Anal. Chem. 72, 277-281.

AOAC. 1990. Official methods of analysis, 151h edition. (K. Helrich, ed.), Association of Official Analytical Chemists, Inc., Washington.

ARCTANDER, S. 1969. Perfume and Flavor Chemicals. pp. 1592, 1596, Published by S. Arctander, Elizabeth.

BAILEY, M.E., DUPUY, H.P. and LEGENDRE, M.G. 1980. Undesirable meat flavor and its control. In 7he Analysis and Control of Less Desirable Fluvors in Foodr and Beverages, (G. Charalambous, ed.) pp. 31-52, Academic Press, New York.

BELITZ, H.-D. and GROSCH, W. 1987. Lipids. In Food Chemistry, pp. 128-2O. Springer-Verlag, Berlin.

DRUMM, T.D. and SPANIER, A.M. 1991. Changes in the content of lipid autox- idation and sulfur-containing compounds in cooked beef during storage. J. Agric. Food Chem. 39, 336-343.

DUPUY, H.P., BAILEY, M.E., ST. ANGELO, A.J., VERCELLOTTI, J.R. and LEGENDRE, M.G. 1987. Instrumental analyses of volatiles related to

HEXANAL INDICATING MEAT DETERIORATION 185

warmed-over flavor of cooked meats. In Warmed-Over Flavor of Meat, (A.J. St. Angelo and M.E. Bailey, eds.) pp. 165-191, Academic Press, Orlando.

FRANKEL, E.N., NEFF, W.E. and SELKE, E. 1984. Analysis of autoxidized fats by gas chromatography-mass spectrometry. IX. Homolytic vs. heterolytic cleavage of primary and secondary oxidation products. Lipids 19, 790-800.

GRAY, J . I . and MONAHAN, F.J. 1992. Measurement of lipid oxidation in meat and meat products. Trends Food Sci. Technol. 3, 315-319.

IHEKORONYE, A.I. 1990. Direct sampling gas-liquid chromatographic-mass spectrometric analysis of hexanal from non-nitrite and nitrite-cured bacon dur- ing storage. Nahrung 34, 81-88.

LAMIKANRA, V.T. and DUPUY, H.P. 1990. Analysis of volatiles related to warmed over flavor of cooked chevon. J. Food Sci. 55, 861-862.

MELTON, S.L. 1983. Methodology for following lipid oxidation in muscle foods. Food Technol. 37(7), 105-1 11, 116.

MORRISSEY, P.A. and APTE, S. 1988. Influence of species, haem and non- haem iron fractions and nitrite on hexanal production in cooked muscle systems. Sci. Alimentes 8, 3-14.

PALAMAND, S.R. and DIECKMANN, R.H. 1974. Autoxidation of n-hexanal. Identification and flavor properties of some products of autoxidation. J. Agric. Food Chem. 22, 503-506.

PEARSON, A.M., LOVE, J.D. and SHORLAND, F.B. 1977. “Warmed-over” flavor in meat, poultry, and fish. Adv. Food Res. 23, 1-74.

POSTE, L.M., WILLEMOT, C., BUTLER, G. and PATTERSON, C. 1986. Sensory aroma scores and TBA values as indices of warmed-over flavor in pork. J. Food Sci. 51, 886-888.

RAMARATHNAM, N., RUBIN, L.J. and DIOSDAY, L.L. 1991a. Studies on meat flavor. 1. Qualitative and quantitative differences in uncured and cured pork. J . Agric. Food Chem. 39, 344-350.

RAMARATHNAM, N . , RUBIN, L.J. and DIOSDAY, L.L. 1991b. Studies on meat flavor. 2. A quantitative investigation of the volatile carbonyls and hydrocarbons in uncured and cured beef and chicken. J. Agric. Food Chem.

RHEE, K.S. 1978. Minimization of further lipid peroxidation in the distillation 2-thiobarbituric acid test of fish and meat. J. Food Sci. 43, 1776-1778, 1781.

ST. ANGELO, A.J., VERCELLOTTI, J.R., DUPUY, H.P. and SPANIER, A.M. 1988. Assessment of beef flavor quality: A multidisciplinary approach. Food Technol. 42(6), 133-138.

ST. ANGELO, A.J. etal. 1987. Chemical and instrumental analyses of warmed- over flavor in beef. J . Food Sci. 52, 1163-1 168.

SALIH, A.M., SMITH, D.M., PRICE, J.R. and DAWSON, L.E. 1987. Modified extraction 2-thiobarbituric acid method for measuring lipid oxidation in poultry. Poultry Sci. 66, 1483-1488.

39, 1839-1847.

186 F. SHAHIDI and R.B. PEGG

SHAHIDI, F. 1989. Flavor of cooked meats. In Flavor Chemistry. Trends and Developments, (R. Teranishi, R.G. Buttery and F. Shahidi, eds.) pp. 188-201, ACS Symposium Series 388. American Chemical Society, Washington.

SHAHIDI, F. 1992. Prevention of lipid oxidation in muscle foods by nitrite and nitrite-free compositions. In Lipid Oxidation in Food, (A.J. St. Angelo, ed.) pp. 161-182, ACS Symposium Series 500. American Chemical Society, Washington.

SHAHIDI, F., RUBIN, L.J. and D’SOUZA, L.A. 1986. Meat flavor volatiles: A review of the composition, techniques of analysis, and sensory evaluation. CRC Crit. Rev. Food Sci. Nutr. 24, 141-243.

SHAHIDI, F., YUN, 1. RUBIN, L.J. and WOOD, D.F. 1987. The hexanal con- tent as an indicator of oxidative stability and flavour acceptability in cooked ground pork. Can. Inst. Food Sci. Technol. J. 20, 104-106.

SPANIER, A.M., EDWARDS, J.V. and DUPUY, H.P. 1988. The warmed-over flavor process in beef A study of meat proteins and peptides. Food Technol.

SPANIER, A.M., VERCELLOTTI, J.R. and JAMES, C. , JR. 1992. Correla- tion of sensory, instrumental and chemical attributes of beef as influenced by meat structure and oxygen exclusion. J. Food Sci. 57, 10-15.

TARLADGIS, B.G., WATTS, B.M., YOUNATHAN, M.T. and DUGAN, JR., L.R. 1960. A distillation method for the quantitative determination of malonaldehyde in rancid foods. J. Am. Oil Chem. SOC. 37, 44-48.

TIMS, M.J. and WATTS, B.M. 1958. Protection of cooked meats with phosphates. Food Technol. 12, 240-243.

TORRES, E., PEARSON, A.M., GRAY, J.I., KU, P.K. and SHIMOKOMAKE, M. 1989. Lipid oxidation in charqui (salted and dried beef). Food Chem. 32, 257-268.

VERCELLOTTI, J.R., KUAN, J.W., LIU, R.H., LEGENDRE, M.G., ST. ANGELO, A.J. and DUPUY, H.P. 1987a. Analysis of volatile heteroatomic meat flavor principles by purge-and-trap/gas chromatography-mass spec- trometry. J. Agric. Food Chem. 35, 1030-1035.

VERCELLOTTI, J.R., KUAN, J.W., SPANIER, A.M. and ST. ANGELO, A.J. 1987b. Thermal generation of sulfur-containing flavor compounds in beef. In Thermal General ofAromas, (T.H. Parliment, R.J. McGorrin and C.-T. Ho, eds.) pp. 452459, ACS Symposium Series 409. American Chemical Soci- ety, Washington.

WONG, D.W.S. 1989. Lipids. In Mechanism a d Theory in Food Chemistry, pp. 147 , Van Nostrand Reinhold, New York.

42(6), 110, 112-1 18.