Thermal Generation of Sulfur-Containing Flavor Compounds in Beef

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Chapter 42

Thermal Generation of Sulfur-Containing Flavor Compounds in Beef

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J. R. Vercellotti, J . W. Kuan, A. M . Spanier, and A. J . St. Angelo Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 19687, 1100 Robert E. Lee Boulevard, New Orleans, LA 70179 Positive cooked-beef flavor components as perceived by descriptive sensory panelists are lost during free radical catalyzed meat flavor deterioration (MFD) while negativeflavornotes with descriptor definitions of cardboard and painty intensify. Although the cardboard and painty off-flavors correlate well with lipid oxidation products and can be measured easily by gas chromatography (1), much less is known about the fate of the positive flavors in this MFD process. Previously a purge and trap gas chromatographic method was reported by the authors for cooked beef heterocyclics (2). A method of extracting and concentrating positive cooked beef flavors has been developed. A flame photometric detector specific for sulfur compounds revealed a mixture of sulfur compounds in the cooked meat extract which quantitatively changed with storage. Three major sulfur compounds were identified as markers for flavor changes, namely methional, methyl sulfone, and benzothiazole. Although considerable effort has been expended on the identification o f sulfur-conteining flavor components in beef (as reviewed by MacLeod et al., (3); Shahidi et al., (4)), little effort has been expended upon the dynamic chemistry of these compounds during the cooking and storage phases of the meat. Using a previously developed concentration method, MacLeod employed trapping methods (5-7) to determine the effect of water activity on the production of cooked-beef aroma compounds. These were significant studies because they demonstrated that different compounds develop at 58% or 17% moisture in rehydrated cooked ground beef, contrasting this work to the report of Hartman (8), wherein maximumflavorvolatiles production was reported at a water activity of 0.72 (about 64% moisture). Since several studies identified the aliphatic and heterocyclic sulfur compounds as being important to cooked-beef flavor (9-11). the present report focuses upon a few key sulfur compounds that contribute to flavor of cooked meat as they change with time and temperature. Previous work has been directed to obtaining reliable correlation of sensory panel data with objective instrumental data describing meat flavor deterioration on storage (1). The present study is targeted toward obtaining a reliable, objective assay This chapter not subject to U.S. copyright Published 1989 American Chemical Society Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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for desirable beef flavor in cooked meat products using sulfur specific detection techniques in gas chromatography. Beef Preparation. Beef was obtained from a local supermarket as USDA "choice" grade, top round steaks. Excess fat was trimmed and the lean muscle used for these experiments. Beef patties were prepared with minor variation to the methods for sensory analysis as described in Johnsen and Civille (12) and Love (13). Only the semimembranosus muscle of the top round steaks was used. The sample was ground twice and then made into 85 g patties containing about 3% fat. The patties were cooked on a Farberware grill for 8 min on each side to a mean internal temperature of 168° ± 3.5° F (75 °C). Weight loss was 37.1% on cooking. For storage studies the freshly cooked patties were cooled to below 50 C and reground to prepare a more homogeneous mixture of the cooked muscle. Ground meat samples (100 g) were placed in Petri dishes and stored with glass covers at 5°C for zero days, 2 days, 4 days, and 7 days, respectively. Samples were reheated at full power for 1 minute in a microwave oven prior to proceeding withflavorvolatile analysis (60 ° C internal temperature). A reproducibility study was carried out on 5 identical, 100 g samples that had been stored for 3 days after cooking, except that they were not reheated in the microwave prior to analysis. An ad hoc panel convened for these experiments consisted of two trained meat flavor panelists who scored the samples for characterization of MFD according to descriptive sensory methods described by Johnsen and Civille (12) and Love (13). The panelists were also active members of a twelve member descriptive sensory panel at the Center. Two duplicate repetitions were carried out for each experiment (4 samples studied).

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0

Gas Chromatography (GO. GC was performed using a Hewlett-Packard 5880 gas chromatograph equipped with a capillary splitter on the injection port. A 50 meter, 0.31 mm fused silica capillary column coated with 0.52 micron film thickness of SE-54 (Hewlett-Packard Ultra 2, 5% phenylmethyl silicone) was used for the separations with detection via flame ionization (FID) and flame photometric (FPD; Hewlett-Packard Model #19256A). Injections were performed manually with the injection port at 200 °C, splitter ratio 8:1, and 1 μΐ sample injected with temperature programming from 50 ° C (5 minute hold) at a 5°C/minute ramp to 180 C (10 minute hold). Carrier gas flow was 2.5 ml/min at 40 ° C and gas regulation on the FPD was hydrogen, 70 ml/min, and air, 80 ml/min for operation (310 ml/min total for ignition). FPD temperature was set at 200 °C. Standards of typical meat flavor sulfur aliphatics and heterocyclics were made from 5 ng/μΐ to 500 ng/μΐ in hexane to determine response factors as well as reproducibility in the flame photometric detector. Background sulfur compounds were checked in concentrated reagent blanks. GC peaks were identified using the Finnigan Ion Trap Detector (ITD) and its programs for library comparison. To aid in the positive identification of peaks, a library of ITD mass spectra was generated using standards of compounds equivalent to those found in this work. Capillary GC (Perkin-Elmer Sigma-300) for the ITD was carried out similar to the conditions above, i.e., splitter ration, 20:1, oven temperature, 50 °C (5 minute hold) followed by a 5 C/min ramp to 180 C; the 10 minute hold was followed by a 5° C/min ramp to 200 °C (20 minute hold to remove impurities). e

0

0

Extraction. The sulfur compounds of interest and other meat flavor principles in the cooked meat patties were extracted and concentrated

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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according to the procedures seen in F i g u r e 1. Prior to passing the final extract through silica gel, the internal standard, benzothiophene (2500 ng in 100 μΐ of hexane), was added to the volume of organic solvent. Note that the meat flavor principles of most interest are not hexane soluble but are water soluble, and are finally back-extracted into methylene chloridemethanol (9:1). Sulfur Compounds of Beef Flavor. Methional, which results from the degradation of methionine, is an important contributor to flavor in meat. Thiolanes, formed during the cooking of beef, have peculiar oniony flavors that also augment the quality of the meaty flavor. Thiophenes and thiofurans are also important to meaty flavors. Sulfides, such as methyl sulfide, are oxidized to methyl sulfoxide and methyl sulfone. Condensation reactions of Maillard browning products also result in thiazoles such as benzothiazole, an important component of meat flavor. From the literature, 19 commonly reported beef flavor aroma compounds containing sulfur were chosen as representative of the capillary gas chromatographic spectrum of substances active in the FPD and are noted in Table I. The reproducibility of the instrument was demonstrated by repeated injections to identify the range of sulfur volatiles in beef samples. The internal standard chosen, benzothiophene is also listed in Table I. The FPD detector is quite specific for sulfur compounds and enhances the signal several fold over the FID. The total ion current chromatogram of these same sulfur standards were run with capillary GC on a Finnegan ITD. The mass spectral identification of each compound was quite efficient with the ITD system, even at 25 nanograms sample per peak. Table I: Sulfur Compounds Chosen for this Study dimethyl sulfide thiophene thiazole dimethyldisulfide 2-methylthiophene 3-methylthiophene 4- methylthiophene

2-ethylthiophene 2,5-dimethylthiophene 2-methyl-2-thiazole 4,5-dimethylthiazole 2-propylthiophene dimethyltrisulfide 2,4,5-trimethylthiazole

2-acetylthiazole 2-butylthiophene 3-acetylthiophene 2-acetylthiophene BENZOTHIOPHENE* benzothiazole

* BENZOTHIOPHENE = internal standard injected at 25 ng Volatile profiles of raw and cooked-beef flavor samples, prepared by the procedures of F i g u r e 1, were obtained after capillary GC and FPD. Although the identification of these sulfur containing compounds is as yet incomplete, the chromatograms demonstrated that there were a number of new sulfur compounds produced on cooking that were not present in the raw beef. Three prominent sulfur compounds were identified as markers in subsequent meat flavor deterioration experiments, namely, methional (13.2 min), methyl sulfone (13.8 min), and benzothiazole (25.3 min). Each compound produced an adequate mass spectrum for spectral library search and positive identification. A typical cooked-beef flavor deterioration sample was prepared as described in B e e f P r e p a r a t i o n section to observe changes in key sulfur marker compounds over a period of 0-, 2-, 4-, and 7-day storage. Sulfur markers, methional, methyl sulfone, and benzothiazole were compared with benzothiophene as internal standard to follow the course of free radical reactions taking place in the stored cooked-beef. Results are plotted in

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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EXTRACTION OF SULFUR HETEROATOMIC COMPOUNDS FROM COOKED MEAT C o o k e d M e a t S a m p l e (100 g . g r o u n d o r 50 m l gravy)

Hexane solution of excess lipids

- Homogenize with hexane; decant solvent. Repeat 2X

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Slurry o f ground meat and hexane

Press out residual hexane from meat slurry with spatula Muscle fiber meat residue-

Suspend slurry in 500 ml of water and stir 0.5 hr Filter through cellulose paper (Whatman #4) on sintered glass Buchner funnel under vacuum.

Aqueous solution o f water soluble meat

AQUEOUS LAYER: salts, peptides, sugars, free fatty acids, vitamins & cofactors

flavor

principles

- Extract with methylene chloride/methanol (90:10%, vol/vol) - Separate aqueous/organic layer by centrifuging - Dry organic layer by passing through bed of sodium sulfate. Add internal std. (benzothiophene; 2500 ng/.lml) - Pass organic layer through lg of silica gel

C o n c e n t r a t e w i t h n i t r o g e n s t r e a m t o 2 5 - 5 0 ul a n d i n j e c t 1-2 ul f o r g a s c h r o m a t o g r a p h y w i t h a sulfur specific detector (flame photometric) detector) o r G C - M a s s spectrometry.

Figure 1. Extraction/flow diagram of sulfur heteroatomic compounds from cooked meat.

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Figure 2 and listed in Table II for the time course of 7 days of storage with standard deviation limits for FPD responses of the three sulfur markers. Table Π: Effect of storage on the content of sulfur heterocyclics in ground-beef Daily concentration (ppb) after storage (4 C) 4 day 2 day

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e

Compound

0 day

methional methyl sulfone benzothiazole

19.31 32.31 1.83

52.19 27.06 2.41

61.04 28.18 2.82

7 day 41.33 27.39 3.04

Figure 3 plots intensity changes perceived by ad hoc descriptive sensory panelists in intensity analysis for character notes involved in meat flavor deterioration during storage of the grilled beef samples reported in Figure 2 and Table II (12, 13). The loss of intensity for certain descriptors in Figure 3 is in accord with sensory panel experience that the positive notes such as cooked-beef brothy diminish with formation of new off-flavor compounds represented as cardboard and painty. Discussion and Interpretation. The authors have previously reported a general screening method for beef flavor compounds with high impact and low thresholds of human perception (2). These sulfur, nitrogen, and oxygen heteroatomic or heterocyclic compounds are present in meat in the low parts per billion range. Preliminary data on the fate of sulfur compounds during meat flavor deterioration reactions was reported by St. Angelo et al. (14). Of greater importance in the present paper is the question of the dynamics of the sulfur flavor compounds made by thermal generation in meat (10, 11, 15-17) and rapid degradation of these key substances through a variety of free radical mechanisms during storage and reheating of meat. Positive cooked-beef flavor components as perceived by descriptive sensory panelists are reduced during free radical catalyzed meat flavor deterioration (MFD) while negative flavor notes with descriptor definitions of cardboard and painty intensify, as reviewed recently by Love (12). Although the cardboard and painty off-flavors correlate well with lipid oxidation products and can be measured easily by gas chromatography (1, 14, 18), much less is known about the fate of the positive cooked-beef flavors in this MFD process (13). A large concentration differential exists between lipid oxidation products to beefy flavors (high ppm for lipid oxidation products vs low ppb for cookedbeef aromatics, respectively). Therefore, a method of extracting and concentrating positive cooked-beef flavors has been developed (Figure 1). The cleanup steps so described removed protein and a good deal of fat soluble materials, but the final residue upon drying and solvent removal remained quite identifiable as a representative roast beef aroma according to Johnsen and Civille (12) and Love (13). A mass spectral chromatogram for the extract from grilled beef is quite complex with over 100 peaks. In spite of the complexity of this chromatogram, several sulfur-containing compounds are consistently identifiable by ITD from these cooked meat extracts, which correspond to the FPD-active peaks. The three principal marker compounds identified

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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EFFECT OF REFRIGERATED STORAGE ON BEEF SULFUR HETEROCYCLICS

Ο — Ο Methional (/2) Ο — Ο Methylsulfone • — • Benzothiazole (xlO) 0

1

2

3

4

5

6

7

Refrigerated Storage Time [DAYS at 4°C]

Figure 2. Effect of storage on four FPD active sulfur compounds extracted from grilled beef stored up to 7 days at 4 ° C. SENSORY SCORE [Ground-beef patty] -OCBB - · PTY

Δ A

ACBD ABTR

-•BRC -•SWT

4.000 3.500 3.000 4-

CD

0

1

2

3

4

5

6

Days Refrigerated Storage Figure 3. Changes in panelists'(n = 2) perceived intensity of sensory descriptors from grilled ground beef patties during storage at 4 ° C. Aromatics: CBB: cooked beef/brothy; BRC: browned caramel; CBD: cardboard; PTY: painty. Tastes: BTR: bitter; SWT: sweet.

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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were methional, methyl sulfone, and benzothiazole, which were later quantitated from their FPD response curves using benzothiophene as an internal standard. The kinetics of marker sulfur compound turnover in grilled beef patties stored from time of cooking through 7 days as described herein (Table II and Figure 3) indicated that the free radical process is very dynamic for these sulfur compounds. Some sulfur flavor compounds in cooked-beef were destroyed during the storage and rewarming process while others were being produced by free radical reactions that may contribute to off-flavors. Although at low concentration methional can be a positive flavor contributor, when too high or out of balance it forms a boiled sweetcorn-like odor. Methional is formed from the Strecker degradation of methionine, and methional itself undergoes several free radical reactions to form further thiols or sulfides. Methional maximizes between days 2 and 4 of storage and then drops off in concentration. These concentrations reported for the storage study of grilled beef patties (Table II) are far above methionaPs threshold of human perceptibility (19), which is in the lower parts per billion range. The gradual formation of the stable end product, benzothiazole, during meat flavor deterioration is an interesting study in reactivity. Benzothiazole is a frequent roast beef positive flavor in low concentration. However, benzothiazole is a definite off-flavor (19) in the concentrations built up during the grilled beef patty storage process. Again, the blending and balance of these flavors is probably important to their overall impact. The nearly odorless and tasteless end-product of methionine oxidation, methyl sulfone, is practically unchanged throughout the storage period and, not surprisingly, is quite stable. This work would indicate that in the more severe thermal degradation conditions of roasting the beef methyl sulfone is formed whereas the kinds of free radical processes generated during storage further degrade methional and lead to the formation of the stable aromatic negativeflavoredend product, benzothiazole. The intensities of character notes in descriptive sensory analysis of these grilled beef patties are shown in Figure 3. Although these intensities were estimated by experienced meat flavor panelists, they should be viewed as preliminary characteristics of MFD. However, these results largely parallel the long experience of these panels with grilled beef patty flavor deterioration (12, 13). There are increases of the off-flavor character notes in Figure 4 that parallel changes in two of the dominant sulfur derivatives, methional and benzothiazole (Figure 2 and Table II). The desirable flavors, cooked beef brothy (CBB) and browned caramel (BRC), declined noticeably (cf., 12 and 13). Perhaps some of the cardboardy (CBD) off-flavor of MFD is attributable to changes in these methionine-related sulfur compounds. An interesting hypothesis from this work is that the descriptor cardboardy is perceived as wet, Kraft box material; paper mill odor; or sulfite liquors with a definite connotation of sulfury as conveyed by the sulfur-containing marker compounds found here. The painty (PTY) meat off-flavor, on the other hand, which is reminiscent of aged linseed oil, is more easily identifiable with the strong concentration of lipid oxidation products. Sweet (SWT) and bitter (BTR) tastes diverge during beef patty storage with SWT declining and BTR increasing. An obvious deficiency in the present marker study is that practically none of the other sulfur-containing molecules, known to be positive contributors to cooked-beef flavor, are detectable by this extraction and FPD gas chromatographic method (2-11, 20). The fate of more of these other contributors to the blend of cooked-beef flavor should also be studied to gain a better perspective. However, this extraction method is simple to use and is recommended for its reproducibility and high recovery of useful markers.

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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As described in MacLeod and Coppock (20) subtleties in flavor do exist even between samples of the same meat cooked in different conventional ways, e.g., boiled versus roasted. The present method is a good route to screening many compounds in these samples also by FID or GC/Mass Spec after various treatments such as the use of preservative antioxidants. Acknowledgments The authors wish to thank Dr. Harold P. Dupuy, Visiting Professor, Dept. of Food Science, Virginia Polytechnic Institute and State University, Blacksburg, VA for volatile profiles analyses of grilled beef patties used in storage studies. Technical assistance by Mr. Charles James, Jr., is also recognized.

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Literature Cited 1. St. Angelo, A.J.; Vercellotti, J.R.; Legendre, M.G.; Vinnett, C.H.; Kuan, J.W.; James, C., Jr.; Dupuy, H.P. J. Food Sci. 1987, 52, 1163. 2. Vercellotti, J.R.; Kuan, J.W.; Liu, R.H.; Legendre, M.G.; St. Angelo, A.J.; Dupuy, H.P. J. Agric. Food Chem. 1987, 35, 1030. 3. MacLeod, G.; Seyyedain-Ardebili, M. CRC Crit. Rev. Food Sci. Nutr. 1981, 14, 309. 4. Shahidi, F.; Rubin, L. J.; D'Souza, L.A. ibid. 1986, 24, 141. 5. Galt, A. M.; MacLeod, G. J. Agric. Food Chem. 1984, 32, 59. 6. MacLeod, G.; Ames, J. M. J. Food Sci. 1986, 51, 1427. 7. MacLeod, G.; Ames, J. M. ibid. 1987, 52, 42. 8. Hartman, G.J.; Scheide, J.D.; Ho, C. T. ibid. 1984, 49, 607. 9. Brinkman, H. W.; Copier, H.; de Leuw, J. J. M.; Tjan, S. B. J. Agric. Food Chem. 1972, 177, 181. 10. Gasser, U.; Grosch, W. Z. Lebensm. Unter Forsch. 1988, 186, 489. 11. Katz, I. In Flavor Research: Recent Advances; Teranishi, R.; Flath, R. Α.; Sugisawa, H., Eds.; Marcel Dekker, Inc. New York, 1981; p 217. 12. Johnsen, P.B.; Civille, G. V. J. Sensory Studies 1986, 1, 99. 13. Love, J. Food Technol. 1988, 42, 140. 14. St. Angelo, A.J.; Vercellotti, J.R.; Dupuy, H.P.; Spanier, A.M. Food Technol. 1988, 42, 133. 15. Liebich, H.M.; Douglas, D.R.; Zlatkis, Α.; Muggler-Chavan, F.; and Donzel, A. J. Agric. Food Chem. 1972, 20, 96. 16. Brinkman, H.W.; Copier, H.; de Leuw, J.J.M.; and Tjan, S.B. J. Agric. Food Chem. 1972, 20, 177. 17. Mussinan, C.J.; Wilson, R.A.; Katz, I.; Hruza, Α.; and Vock, M.H. . In Phenolic, Sulphur, and Nitrogen Compounds in Food Flavors; Charalambous, G.; Katz, I., Eds.; ACS Symposium Series No. 26; American Chemical Society: Washington, DC, 1976; p 133. 18. Bailey, M. E.; Shin-Lee, S. Y.; Dupuy, H. P., St. Angelo, A.J.; Vercellotti, J. R. In Warmed-Over Flavor of Meat; St. Angelo, A. J.; Bailey, M. E., Eds.; Academic Press: Orlando, FL, 1987; p 237. 19. Fors, S. In The Maillard Reaction in Foods and Nutrition; Waller, G.R.; Feather, M.S., Eds.; ACS Symposium Series No. 215; American Chemical Society: Washington, D.C., 1983; p 268. 20. MacLeod, G.; Coppock, B.M. J. Agric. Food Chem. 1977, 25, 113. RECEIVED July 21, 1989

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.