Production of Off-Odor Volatiles from Liposome-Containing Amino Acid Homopolymers by Irradiation D.U. AHN AND E.J. LEE

JFS: Production of Off-Odor Volatiles from Liposome-Containing Amino Acid Homopolymers by Irradiation D.U. AHN AND E.J. LEE ABSTRACT: Irradiation not only generated many new volatiles but also destroyed some volatiles already present in nonirradiated amino acid homopolymer-in-liposome systems. The amounts of some volatiles greatly increased, but others significantly decreased after irradiation. The majority of newly generated and increased volatiles by irradiation were sulfur compounds, indicating that sulfur amino acids are the most susceptible to changes by irradiation. More than one site in amino acid side chains was labile to free radical attack, and many volatiles were produced by the secondary chemical reactions after the primary radiolytic degradation of side chains. Although nonirradiated samples also produced some sulfury notes, irradiated samples produced much a stronger and astringent sulfury odor than nonirradiated samples. Keywords: amino acid homopolymer, secondary chemical reactions, irradiation, volatiles, sulfur compounds Introduction IRRADIATION OF AMINO ACIDS PRODUCED DISTINCT VOLATILE COMpounds via radiolytic degradation, but the resulting odor was much stronger and astringent than that from irradiated meat (Jo and Ahn 2000). Patterson and Stevenson (1995) found that dimethyl trisulfide is the most potent off-odor compound in irradiated chicken meat, followed by cis-3- and trans-6-nonenals, oct-1- en-3-one, and bis(methylthio-)methane. Ahn and others (1999, 2000) found many more sulfur compounds were produced from irradiated meat. Irradiation produced many new volatile compounds in oil emulsion prepared from fatty acids, but had little effect on the sensory characteristics of oil emulsion (Lee and Ahn 2002). The amounts of aldehydes, the indicators of lipid oxidation, in oil emulsions did not increase by irradiation (Lee and Ahn 2002), and volatiles from lipids accounted for only a small part of the off-odor in irradiated meat (Ahn and others 1997, 1998a, 1999). The odor of irradiated meat was characterized as bloody and sweet, barbecued corn-like, hot fat, burned oil, or burned feathers (Heath and others 1990; Hashim and others 1995; Ahn and others 2000). Merritt and others (1975, 1978) observed that the odor found in a lipid or a component of meat irradiated separately was different from that of the meat. Diehl (1995) indicated that there was a substantial difference between the radiation chemistry of pure substances and that of the same substances when they were components of complex food systems. Many new volatiles were generated, and the amount of volatiles produced from amino acid homopolymers were changed after irradiation (Ahn 2001). More than 1 site of amino acid side chains was susceptible to free radical attack, and many volatiles were produced by the secondary chemical reactions after the primary radiolytic degradation of side chains. Only sulfur-containing volatiles, however, produced strong odor that was similar to or close to irradiation odor, and methionine was the most important amino acid in producing irradiation odor (Ahn 2001). The perception of odor from samples containing sulfur volatiles, however, changed greatly depending on the composition and amounts 2002 Institute of Food Technologists present in the sample (Ahn 2001). This suggests that irradiation odor is either a composite of the volatiles generated in the protein and lipid portion of meat or a result of products formed by the interactions between fat and proteins during and after irradiation. The objective of this study was to determine the volatile compounds produced from a mixture of liposome containing amino acid homopolymers by irradiation and the interactions of the volatile compounds on odor characteristics of the irradiated liposome/amino acid system. Materials and Methods Sample preparation Phosphatidylcholine, phosphatidic acid, amino acid homopolymers, glutathione, and Met-Gly-Met-Met were purchased from Sigma (St. Louis, Mo., U.S.A.). A phospholipid liposome system prepared with phosphatidylcholine and phosphatidic acid was used because it represents cell membranes of meat. Phosphatidylcholine (100 mg dissolved in chloroform) was evaporated from chloroform to thin film on the wall of a 40-mL sample vial. The vial was placed under a nitrogen stream to remove any chloroform. All 7 amino acid polymer groups were used in this study: aliphatic (poly-l-alanine, poly-lglycine, poly-l-leucine), aliphatic hydroxyl (poly-l-threonine), basic (poly-l-histidine, poly-l-lysine), acidic (poly-l-aspartic acid, poly-l-glutamic acid), aromatic (poly-l-tyrosine), amide (poly-l-asparagine), and sulfur-containing (Met-Gly-Met-Met, glutathione) side chain groups. Each combination of selected amino acid polymer groups was weighed into a vial coated with phospholipids and was hydrated with 20 ml citrate-phosphate buffer (100 mm, ph 6.0) by gently shaking the solution for 15 min. The milky suspension was then vortexed to disperse the phospholipids before use. The concentration of each amino acid homopolymer was 2 mg/ml buffer, and each amino acid polymer group was treated like a single compound. A liposome containing all 7 amino acid polymer groups (used as a reference ) and 7 liposome solutions containing 6 amino acid homopolymer groups Vol. 67, Nr. 7, 2002 JOURNAL OF FOOD SCIENCE 2659

Table 1 Volatiles and odor characteristics of an amino acid homopolymer mixture containing all amino acid groups after irradiation* Sulfur dioxide 0 b 1210 a 90 1-Butene 386 a 211 b 11 1,1'-Oxybis ethane 875 a 299 b 24 2-Propanone 37459 a 357 b 340 Dimethyl sulfide 0 b 223 a 7 Carbon disulfide 454 b 2421 a 112 Methyl thiirane 210 a 0 b 5 2-Methyl-2-propanol 0 b 324 a 15 1,1-Dimethylethyl 517 a 0 b 8 2-Ethoxy butane 292 a 0 b 4 2-Methyl propanal 0 b 115 a 4 Hexane 757 a 76 a 326 Butanal 261 a 107 b 26 2-Pentene 259 a 0 b 5 Methylthio ethane 0 b 54 a 5 Ethyl acetate 67 a 0 b 3 Benzene 0 b 5083 a 166 3-Methyl-butanal 108 a 139 a 10 1,4-Dioxane 304 a 0 b 4 3,3-Dimethyl-2-butanone 107 a 0 b 5 Dimethyl disulfide 57 b 34490 a 692 Toluene 251 a 0 b 3 Ethyl-benzene 48 b 1228 a 34 1,3-Dimethyl-benzene 160 a 43 b 3 Methyl ethyl disulfide 0 b 66 a 1 Xylene 0 b 145 a 3 Dimethyl trisulfide 0 b 7010 a 400 Odor characteristics of irradiated samples Hard-boiled egg, boiled sweet corn, sweet and sulfury, steamed vegetable a,b Means with no common superscript differ significantly (p < 0.05), n = 4. *Contains acidic, aliphatic, aliphatic hydroxyl, amide, aromatic, basic, and sulfur amino acid groups. SEM = standard error of the mean. Table 2 Volatiles and odor characteristics of the liposome without acidic amino acid group after irradiation* Sulfur dioxide 0 b 834 a 176 1-Butene 41 a 36 b 2 Methanethiol 0 b 9601 a 482 1,1'-Oxybis-ethane 220 a 175 b 11 2-Propanone 35977 a 18682 b 2667 Dimethyl sulfide 0 b 457 a 18 Carbon disulfide 436 b 1184 a 79 1,1-Dimethylethyl 61 a 0 b 6 2-Methyl propanal 0 b 58 a 8 Hexane 0 b 108 a 33 Butanal 268 249 16 2-Pentene 195 a 0 b 4 Methylthio ethane 0 b 105 a 2 1-Methoxy-1-propene 0 b 109 a 26 Benzene 0 b 6300 a 205 3-Methyl butanal 290 b 454 a 18 1,4-Dioxane 215 123 56 3,3-Dimethyl-2-butanone 60 a 0 b 5 Dimethyl disulfide 78 b 34734 a 907 Toluene 346 a 0 b 4 Ethyl benzene 46 b 1199 a 50 1,3-Dimethyl benzene 141 a 48 b 7 Methyl ethyl disulfide 0 b 69 a 5 Xylene 0 b 170 a 9 Dimethyl trisulfide 0 b 7891 a 1230 Odor characteristics of irradiated samples Hard-boiled egg, boiled sweet corn, boiled vegetable, solvent/burned plastic a,b Means with no common superscript differ significantly (p < 0.05), n = 4 *Contains aliphatic, aliphatic hydroxyl, amide, aromatic, basic, sulfur amino acid groups SEM = standard error of the mean. were prepared. Four 5-mL portions of samples were transferred to scintillation vials and irradiated at 0 or 5 kgy using a Linear Accelerator (Circe IIIR; Thomson CSF Linac, Saint-Aubin, France). The energy and power level used were 10 MeV and 10 kw, respectively, and the average dose rate was 92.8 kgy/min. The max/min ratio was approximately 1.09. To confirm the target dose, 2 alanine dosimeters per cart were attached to the top and bottom surfaces of a sample vial. The alanine dosimeter was read using a 104 Electron Paramagnetic Resonance Instrument (Bruker Instruments Inc., Billerica, Mass., U.S.A.). Samples were used to determine volatile profiles and odor characteristics before and after irradiation. The volatiles and odor characteristics between the reference and other liposomes containing 6 amino acid groups were compared. Volatile analysis A purge-and-trap apparatus (Precept II and Purge & Trap Concentrator 3000; Tekmar-Dohrmann, Cincinnati, Ohio, U.S.A.) connected to a gas chromatograph/mass spectrometer (GC/MS; Hewlett-Packard Co., Wilmington, Del., U.S.A.) was used to analyze the volatiles produced (Ahn and others 2001). Sample solution (1 ml) was placed in a 40-mL sample vial, and the vials were flushed with helium (40 psi) for 5 s. Samples were held in a refrigerated (4 C) sample-holding tray before analysis. The maximum holding time was less than 6 h to minimize oxidative changes (Ahn and others 1999). The sample was purged with helium (40 ml/min) for 13 min at 40 C. Volatiles were trapped using a 2660 JOURNAL OF FOOD SCIENCE Vol. 67, Nr. 7, 2002 Tenax column (Tekmar-Dohrmann) and desorbed for 2 min at 225 C, focused in a cryofocusing module ( 90 C) and then thermally desorbed into a column for 30 s at 225 C. An HP-624 column (7.5 m x 0.25 mm internal dia., 1.4 çm nominal), an HP-1 column (52.5 m x 0.25 mm internal dia., 0.25 çm nominal; Hewlett-Packard Co.) and an HP-Wax column (7.5 m x 0.25 mm i.d., 0.25?m nominal) were connected using zero deadvolume column connectors (J &W Scientific, Folsom, Calif., U.S.A.). Ramped oven temperature was used to improve volatile separation. The initial oven temperature of 0 C was held for 2.5 min. After that, the oven temperature was increased to 15 C at 2.5 C/min, increased to 45 C at 5 C/min, increased to 110 C at 20 C/min, increased to 210 C at 10 C/min, and then was held for 4.5 min at that temperature. Constant column pressure at 20.5 psi was maintained. The ionization potential of mass-selective detector (Model 5973; Hewlett-Packard Co.) was 70 ev, and the scan range was 18.1-300 m/z. Identification of volatiles was achieved by comparing mass spectral data of samples with those of the Wiley library (Hewlett-Packard Co.). Standards, when available, were used to confirm the identification by the mass selective detector. The area of each peak was integrated using the ChemStation (Hewlett-Packard Co.), and the total peak area (pa*s x 10 4 ) was reported as an indicator of volatiles generated from the sample. Odor characteristics Ten trained sensory panelists were used to characterize odor in samples. Panelists were selected based on interest, availability, and performance in screening tests conducted with samples

Table 3 Volatiles and odor characteristics of the liposome without aliphatic amino acid group after irradiation* Sulfur dioxide 0 b 1394 a 86 1-Butene 309 a 157 b 31 Methanethiol 0 b 614 a 37 1,1'-Oxybis ethane 306 a 77 b 15 2-Propanone 36575 a 2131 b 1328 Dimethyl sulfide 0 b 299 a 14 Carbon disulfide 482 b 2048 a 108 Methyl thiirane 229 a 0 b 7 2-Methyl-2-propanol 0 b 374 a 3 1,1-Dimethylethyl 437 a 0 b 10 2-Ethoxy butane 352 a 0 b 3 2-Methyl propanal 0 b 96 a 1 Hexane 0 b 216 a 50 Butanal 230 a 0 16 Methylthio ethane 0 b 91 a 4 Dimethyl tetrasulfide 0 b 63 a 18 1,4-Dioxane 264 a 0 b 41 3,3-Dimethyl-2-butanone 98 a 0 b 11 Dimethyl disulfide 81 b 43671 a 1459 Toluene 48 a 0 b 1 Ethyl-benzene 0 b 1400 a 23 1,3-Dimethyl benzene 0 13 12 Methyl ethyl disulfide 0 b 59 a 2 Dimethyl trisulfide 0 b 15330 a 367 Odor characteristics of irradiated samples Hard-boiled egg, fermented vegetable, rotten vegetable a,b Means with no common superscript differ significantly (p < 0.05), n = 4 *Contains acidic, aliphatic hydroxyl, amide, aromatic, basic, and sulfur amino acid groups. similar to those to be tested. During training, a lexicon of aroma terms to be used on the ballot was developed. Samples were placed in glass containers, and the sample temperature was brought to 25 C before samples were tested. All treatments were presented to each panelist, and the order of presentation was randomized. Panelists characterized overall odor of each sample, and rated the intensity of the selected attributes on 15 unit linear scales. Statistical analysis Four replicated analyses were done for the volatiles of samples. Data were analyzed using the generalized linear model procedure of SAS software (SAS 1989): Student s t-test was used to compare differences between irradiated and nonirradiated means. Mean values and standard error of the means (SEM) were reported. Significance was defined at p < 0.05. Table 4 Volatiles and odor characteristics of the liposome without aliphatic hydroxyl amino acid group by irradiation Sulfur dioxide 0 b 1814 a 130 1-Butene 280 a 199 b 21 1-Methyl pyrrole 124 a 0 b 5 2-Propanone 38767 a 422 b 452 Dimethyl sulfide 0 b 231 a 5 Carbon disulfide 490 b 2793 a 123 Methyl thiirane 239 a 0 b 5 2-Methyl-2-propanol 0 b 373 a 2 1,1-Dimethylethyl 504 a 0 b 4 2-Ethoxy butane 390 a 0 b 11 2-Methyl propanal 0 b 135 a 4 Hexane 0 b 106 a 29 Butanal 265 a 119 b 16 2-Pentene 244 a 0 b 7 4-Methyl-3-hexanol 71 a 0 b 3 Ethyl acetate 95 a 0 b 3 Benzene 0 b 5779 a 106 3-Methyl butanal 190 b 272 a 11 1,4-Dioxane 306 a 0 b 15 3,3-Dimethyl-2-butanone 102 a 0 b 4 Dimethyl disulfide 62 b 31460 a 1731 Toluene 327 a 0 b 16 Ethyl benzene 44 b 1405 a 20 1,3-Dimethyl benzene 124 a 48 b 9 Xylene 0 b 160 a 3 Dimethyl trisulfide 0 b 10287 a 124 Odor characteristics of irradiated samples Hard-boiled egg, sulfury, boiled sweet corn, boiled cabbage a,b Means with no common superscript differ significantly (p < 0.05), n = 4 Contains acidic, aliphatic, amide, aromatic, basic, and sulfur amino acid groups. Results and Discussion IRRADIATION GREATLY INFLUENCED THE AMOUNTS AND PROFILES OF volatiles in amino acid homopolymer-in-liposome systems (Tables 1 through 8). Table 1 shows the volatiles produced from the reference sample (the amino acid homopolymer-in-liposome system that contains all 7 amino acid homopolymer groups) before and after irradiation. Many new volatiles including sulfur dioxide, dimethyl sulfide, 2-methyl-2-propanol, 2-methyl-propanal, methylthio ethane, benzene, methyl ethyl disulfide, and dimethyl trisulfide were generated, and the amounts of carbon sulfide, dimethyl disulfide, and ethyl benzene increased greatly after irradiation. On the other hand, methyl thiirane, 1,1-dimethylethyl, 2-ethoxy butane, 2-pentene, ethyl acetate, 1,4-dioxane, 3,3-dimethyl-2-butanone, and toluene disappeared, and the amounts of 1-butene, 1,1-oxybis ethane, 2-propanone, hexane, butanal, and 1,3-dimethyl benzene significantly decreased after irradiation. The majority of newly generated and increased volatiles by irradiation were sulfur compounds indicating that sulfur-containing amino acids are among the most susceptible amino acid groups to irradiation. Sensory panelists described the odor of irradiated amino acid homopolymers-in-liposome as hard-boiled egg, boiled sweet corn, sweet and sulfury, or steamed vegetable, typical odor characteristics of sulfur volatile-containing samples, indicating that sulfur volatiles played the major role in the odor of the irradiated meat sample. Although nonirradiated samples also produced some sulfury notes, irradiated samples produced much stronger and astringent sulfury odor than nonirradiated ones. Hashim and others (1995) described the characteristic of irradiation odor as a bloody and sweet aroma, and Ahn and others (2000) described it as a barbecued corn-like odor. All liposome groups containing the sulfur amino acids group produced similar odor characteristics, indicating that sulfur amino acids are mainly responsible for irradiation odor as suggested by Ahn (2001). The volatile profiles of all 6 liposome systems that contain 6 amino acid homopolymer groups (Table 2 to 7), except for the 1 without sulfur amino acid group (Table 8), were similar to that of the reference (Table 1). Most of the volatiles detected in the nonirradiated reference were also found in the liposome that contains the all amino acid homopolymers but acidic group ( without acidic group ), except that methyl thiirane, 2-ethoxy butane, hexane, and ethyl acetate found in the reference were not detected (Tables 1 and 2). After irradiation, methanethiol and 1- Vol. 67, Nr. 7, 2002 JOURNAL OF FOOD SCIENCE 2661

Table 5 Volatiles and odor characteristics of the liposome without amide amino acid group after irradiation* Sulfur dioxide 0 b 2365 a 52 1-Butene 307 a 236 b 14 1-Methyl pyrrole 56 a 0 b 1 1,1'-Oxybis ethane 1667 a 648 b 28 2-Propanone 39351 a 382 b 225 Dimethyl sulfide 0 b 239 a 1 Carbon disulfide 534 b 3649 a 61 Methyl thiirane 236 a 0 b 3 2-Methyl-2-propanol 0 b 394 a 3 1,1-Dimethylethyl 503 a 0 b 5 2-Ethoxy butane 345 a 0 b 5 2-Methyl propanal 0 b 165 a 2 Hexane 45 56 4 Butanal 0 b 115 a 3 2-Pentene 239 a 0 b 4 Methylthio ethane 0 b 75 a 1 Ethyl acetate 327 a 0 b 4 Benzene 0 b 5875 a 31 Dimethyl tetrasulfide 0 b 86 a 13 3-Methyl butanal 271 244 10 1,4-Dioxane 216 b 277 a 10 3,3-Dimethyl-2-butanone 83 a 0 b 3 Dimethyl disulfide 72 b 31407 a 1141 Toluene 501 a 0 b 12 Ethyl benzene 69 b 508 a 4 1,3-Dimethyl benzene 224 a 47 b 7 Methyl ethyl disulfide 0 b 63 a 1 Xylene 0 b 244 a 52 Dimethyl trisulfide 0 b 10226 a 540 Odor characteristics of irradiated samples Hard-boiled egg, boiled sweet corn, sweet and sulfury, steamed vegetable a,b Means with no common superscript differ significantly (p < 0.05), n = 4 *Contains acidic, aliphatic, aliphatic hydroxyl, aromatic, basic, and sulfur amino acid groups. Table 6 Volatiles and odor characteristics of the liposome without aromatic amino acid group after irradiation* Sulfur dioxide 0 b 2610 a 133 1-Butene 299 a 219 b 14 1,1'-Oxybis ethane 51 a 207 b 4 2-Propanone 38847 a 416 b 513 Dimethyl sulfide 0 b 267 a 5 Carbon disulfide 406 b 3542 a 47 Methyl thiirane 191 a 0 b 0 2-Methyl-2-propanol 0 b 365 a 4 1,1-Dimethylethyl 456 a 0 b 37 2-Ethoxy butane 322 a 0 b 1 2-Methyl propanal 0 b 146 a 1 Hexane 0 a 149 a 57 Butanal 234 a 105 b 3 2-Pentene 240 a 0 b 3 Methylthio ethane 0 b 86 a 4 4-Methyl-3-hexanol 61 a 0 b 1 Ethyl acetate 85 a 0 b 1 Benzene 0 b 6353 a 109 Dimethyl tetrasulfide 0 b 91 a 7 3-Methyl butanal 161 b 195 a 5 1,4-Dioxane 446 a 0 b 24 3,3-Dimethyl-2-butanone 94 a 0 b 2 Dimethyl disulfide 67 b 41409 a 1264 Toluene 309 a 0 b 3 Ethyl benzene 50 b 1380 a 21 1,3-Dimethyl benzene 152 a 44 b 4 Methyl ethyl disulfide 0 b 67 a 2 Xylene 0 b 141 a 3 Dimethyl trisulfide 0 b 12058 a 175 Odor characteristics of irradiated samples Cooked/spoiled cabbage, sulfury, soy sauce, sewage a,b Means with no common superscript differ significantly (p < 0.05), n = 4. *Contains acidic, aliphatic, aliphatic hydroxyl, amide, basic and sulfur amino acid groups. methoxy-1-propene, not found in the reference, were produced from the without acidic group sample. The amount of 2-propanone in the without acidic group decreased by about 50% after irradiation, but was much higher than that in the irradiated reference. Methyl thiirane was not detected in the liposome without acidic group, suggesting that methyl thiirane was formed from the secondary reaction between sulfur compounds and acidic amino acid side chain products. 2-Methyl-2-propanal was not detected in the irradiated liposome without acidic group, indicating that a product of the acidic group side chain is needed to form this compound. Toluene was detected in all nonirradiated liposomes, but disappeared after irradiation. It is difficult to pinpoint the main reason for this change. The new production of benzene and the disappearance of toluene by irradiation, however, suggest that the radiolytic cleavage of the methyl group from toluene may be the mechanism behind the benzene formation. Du and others (2001a,b) found benzene in both irradiated and nonirradiated broiler meats, indicating that benzene and toluene could be produced from the components naturally present in meat even without irradiation. The odor characteristics of the liposome in the without acidic group were similar to those of the reference, but some sensory panels received a solvent/burned plastic odor note from the irradiated liposome without acidic group sample (Tables 1 and 2). 2-Pentene, ethyl acetate and 3-methyl butanal detected in the nonirradiated reference were not found in the nonirradiated liposome that contained the all amino acid homopolymers but 2662 JOURNAL OF FOOD SCIENCE Vol. 67, Nr. 7, 2002 aliphatic group ( without aliphatic group, Tables 1 and 3). After irradiation, methanethiol and dimethyl tetrasulfide, not found in the reference, were detected in the without aliphatic group sample. Butanal, benzene, and 3-methyl butanal detected in the reference, however, were not found. This indicates that 2-pentene, benzene, 3-methyl butanal, and xylene are derived from the aliphatic amino acid side chain. Methanethiol could be formed in the presence of the acidic and aliphatic side chain groups. Benzene was not detected in liposomes when the aliphatic amino acid group was not present, indicating that benzene was derived from aliphatic amino acid side chains. The odor intensity and characteristics of the liposome without aliphatic group sample were similar to those of the reference (Tables 1 and 3). From the nonirradiated liposome that contained the all amino acid homopolymer but aliphatic hydroxyl group ( without aliphatic hydroxyl group ), 2 volatiles, namely, 1-methyl pyrrole, and 4-methyl-3-hexanol, not found in the nonirradiated reference sample, were detected (Tables 1 and 4). After irradiation, methylthio ethane and methyl ethyl disulfide that were found in the reference were not found in the liposome without aliphatic hydroxyl group. 1,1-Oxybis ethane found in the reference was not present in both the irradiated and nonirradiated liposome without aliphatic hydroxyl groups. An aliphatic hydroxyl group side chain is needed for 1,1-oxybis ethane, methylthio ethane, and methyl ethyl disulfide formation. The odor characteristics of the liposome without aliphatic hydroxyl group sample were

Table 7 Volatiles and odor characteristics of the liposome without basic amino acid group after irradiation* sulfur dioxide 0 b 2512 a 183 1-Butene 370 a 179 b 16 1,1'-Oxybis ethane 1248 a 242 b 30 2-Propanone 37517 a 358 b 607 Dimethyl sulfide 0 b 219 a 1 Carbon disulfide 472 b 3502 a 209 Methyl thiirane 232 a 0 b 7 2-Methyl-2-propanol 0 b 363 a 4 1,1-Dimethylethyl 519 a 0 b 9 2-Ethoxy butane 373 a 0 b 9 2-Methyl propanal 0 b 119 a 2 Hexane 60 b 314 a 56 Butanal 244 a 191 b 56 2-Pentene 274 a 0 b 10 Methylthio ethane 0 b 79 a 1 4-Methyl-3-hexanol 68 a 0 b 2 Ethyl acetate 69 a 0 b 2 Benzene 0 b 5295 a 21 3-Methyl butanal 151 b 217 a 8 3,3-Dimethyl-2-butanone 100 a 0 b 6 Dimethyl disulfide 69 b 52027 a 4210 Toluene 316 a 0 b 5 Ethyl-benzene 49 b 1232 a 8 1,3-Dimethyl benzene 147 a 42 b 6 Methyl ethyl disulfide 0 b 85 a 5 Xylene 0 b 161 a 3 Dimethyl trisulfide 0 b 7845 a 782 Odor characteristics of irradiated samples Strong hard-boiled egg, fermentation odor, sulfury, hospital odor a,b Means with no common superscript differ significantly (p 0.05), n = 4 *Contains acidic, aliphatic, aliphatic hydroxyl, amide, aromatic, and sulfur amino acid groups. Table 8 Volatiles and odor characteristics of the liposome without sulfur amino acid group by irradiation 1-Butene 295 a 132 b 19 1,1-Dimethyl cyclopropane 0 b 65 a 1 Pentane 0 b 183 a 1 2-Propanone 39551 a 377 b 785 2-Methyl-2-propanol 0 b 297 a 5 1,1-Dimethylethyl 455 a 0 b 4 2-Ethoxy butane 342 a 0 b 9 2-Methyl propanal 0 b 146 a 3 Hexane 48 b 122 a 13 3-Methylfuran 0 b 79 a 2 Butanal 0 b 156 a 6 2-Pentene 432 a 0 b 11 4-Methyl-3-hexanol 244 a 0 b 8 Benzene 0 b 9948 a 271 3-Methyl butanal 377 a 268 b 14 1,4-Dioxane 181 a 0 b 10 3,3-Dimethyl-2-butanone 88 a 0 b 8 Toluene 358 a 0 b 11 Ethyl benzene 57 b 892 a 23 1,3-Dimethyl benzene 188 a 0 b 10 Octane 0 b 79 a 2 Xylene 0 b 79 a 3 Odor characteristics of irradiated samples Hospital odor, alcohol, solvent, wet dog a,b Means with no common superscript differ significantly (p < 0.05), n = 4. *Contains acidic, aliphatic, aliphatic hydroxyl, amide, aromatic, and basic amino acid groups. similar to those of the reference (Tables 1 and 4). Among the volatiles of nonirradiated liposome that contained the all amino acid homopolymers but amide group ( without amide group ), 1-methyl pyrrole was the only volatile not found in the reference. Dimethyl tetrasulfide was the only new volatile that was not detected in irradiated reference, and 1,4-dioxane was still remaining in the liposome without amide group after irradiation (Tables 1 and 5). The odor characteristics of liposome without amide group sample were similar to those of the reference (Tables 1 and 5). The difference between the volatiles of the reference and the volatiles of liposome that contained the all amino acid homopolymers but aromatic group ( without aromatic group ) was very small. In nonirradiated samples, hexane and 4-methyl-3- hexanol were the 2 volatiles found only in 1 of the 2 samples (the reference and the without aromatic group ), and dimethyl tetrasulfide was the only volatile not detected in the irradiated reference (Tables 1 and 6). Dimethyl tetrasulfide could be formed in irradiated liposomes only when no aromatic and amide but all other amino acid side chain groups were present. The irradiated liposome without aromatic group also had odor characteristics similar to those of the reference (Tables 1 and 6). The difference between the volatiles of the reference and those of liposome that contained all amino acid homopolymer but basic group ( without basic group ) was also very small. In nonirradiated samples, 4-methyl-3-hexanol and 1,4-dioxane were the 2 volatiles found only in the reference and without basic group, but little difference in volatile profiles and odor characteristics were detected in irradiated samples (Tables 1 and 7). This shows that 1,4-dioxane is derived from the basic amino acid side chain group. The volatile profiles and odor characteristics between the reference sample and the all amino acid homopolymers but sulfur amino acid group ( without sulfur group ) were totally different. Both the irradiated and nonirradiated liposomes without sulfur group produced no sulfur volatiles, and odor was characterized as a hospital odor, alcohol, solvent, or wet dog, and the intensity (data not shown) was very weak compared to that of other sulfur amino acid-containing liposomes (Table 8). In the nonirradiated liposome without sulfur group, 4-methyl 3-hexanol not found in the reference was detected, but butanal and ethyl acetate found in the reference were not (Tables 1 and 8). In the irradiated liposome without sulfur group, 1,1-dimethyl cyclopropane, pentane, and butanal were the new volatiles not found in the irradiated reference. Within the liposomes without sulfur group, irradiation newly produced 1,1-dimethyl cyclopropane, pentane, 2-methyl propanal, 3-methyl furan, butanal, benzene, octane, and xylene. However, many volatiles present in nonirradiated samples such as 1,1-dimethylethyl, 2- ethoxy butane, 2-pentene, 4-methoxy-3-hexanol, 1,4-dioxane, 3,3-dimethyl-2-butanone, toluene, and 1,3-dimethyl benzene disappeared after irradiation (Table 8). The volatile profiles of liposomes with amino acid homopolymers (Tables 1 through 8) clearly show that many aldehydes and hydrocarbons could be produced from amino acid side chains, and sensory characteristics of liposomes explained why irradiation odor in meat was different from lipid oxidation odor (Ahn and others 1997, 1998b, 1999). Patterson and Stevenson (1995) identified dimethyl trisulfide and bis(methylthio-)methane as the most potent off-odor sulfur compounds in irradiated Vol. 67, Nr. 7, 2002 JOURNAL OF FOOD SCIENCE 2663

Table 9 Summary of volatile changes in liposome containing amino acid homopolymers by irradiation No No No aliphatic No No No No Volatile compounds All acidic aliphatic OH amide aromatic basic sulfur B * A ** C *** B A C B A C B A C B A C B A C B A C B A C Sulfur dioxide Nd 1 D 2 N 3 Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd Nd 1-Butene D D Ô 4 D D Ô D D Ô D D Ô D D Ô D D Ô D D Ô D D Ô 1,1-Dimethyl Nd Nd 5 Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd D N cyclopropane Methane thiol Nd Nd Nd D N Nd D N Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Pentane Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd D N 1-Methyl pyrrole Nd Nd Nd Nd Nd Nd D Nd 0 D Nd 0 Nd Nd Nd Nd Nd Nd 1,1'-Oxybis ethane D D Ô D D Ô D D Ô Nd Nd D D Ô D D Ô D D Ô D D Ô 2-Propanone D D Ô D D Ô D D Ô D D Ô D D Ô D D Ô D D Ô D D Ô Dimethyl sulfide Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd Nd Carbon disulfide D D Ó 6 D D Ó D D Ó D D Ó D D Ó D D Ó D D Ó Nd Nd Methyl thiirane D Nd 0 7 Nd Nd D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 Nd Nd 2-Methyl-2-propanol Nd D N Nd Nd Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N 1,1-Dimethylethyl D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 2-Ethoxy butane D Nd 0 Nd Nd D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 2-Methyl propanal Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Hexane D D Ô Nd D N Nd D N Nd D N D D Nd D N D D Ó D D Ó Methylfuran Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd D N Butanal D D Ô D D D Nd 0 D D Ô Nd D N D D Ô D D Ô Nd D N 2-Pentene D Nd 0 D Nd 0 Nd Nd D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 Methylthio ethane Nd D N Nd D N Nd D N Nd Nd Nd D N Nd D N Nd D N Nd Nd Dimethyl tetrasulfide Nd Nd Nd Nd Nd D N Nd Nd Nd D N Nd D N Nd Nd Nd Nd 1-Methoxy-1-propene Nd Nd Nd D N Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd 4-Methyl-3-hexanol Nd Nd Nd Nd Nd Nd D Nd 0 Nd Nd D Nd 0 D Nd 0 D Nd 0 Ethyl acetate D Nd 0 Nd Nd Nd Nd D Nd 0 D Nd 0 D Nd 0 D Nd 0 Nd Nd Benzene Nd D N Nd D N Nd Nd Nd D N Nd D N Nd D N Nd D N Nd D N 3-Methyl-butanal D D D D Ó Nd Nd D D Ó D D D D Ó D D Ó D D Ô 1,4-Dioxane D Nd 0 D D D Nd 0 D Nd 0 D D Ó D Nd 0 Nd Nd D Nd 0 3,3-Dimethyl-2-butanone D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 Dimethyl disulfide D D Ó D D Ó D D Ó D D Ó D D - D D Ó D D Ó Nd Nd Toluene D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 D Nd 0 Ethyl benzene D D Ó D D Ó Nd D N D D Ó D D Ó D D Ó D D Ó D D Ó 1,3-Dimethyl-benzene D D Ô D D Ô Nd D N D D Ô D D Ô D D Ô D D Ô D Nd 0 Methyl ethyl disulfide Nd D N Nd D N Nd D N Nd Nd Nd D N Nd D N Nd D N Nd Nd Octane Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd D N Xylene Nd D N Nd D N Nd Nd Nd D N Nd D N Nd D N Nd D N Nd D N Dimethyl trisulfide Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd D N Nd Nd *B: before irradiation, **A: after irradiation, ***C: change of volatiles by irradiation 1 Nd: not detected, 2 D: detected, 3 N: newly generated, 4 Ô: decreased, 5 : no change, 6 Ó: increased, 7 0: disappeared. chicken meat, but our data indicated that many other sulfur compounds could be produced from methionine and cysteine (Table 7). Table 9 summarizes changes in volatiles of liposomes containing various amino acid homopolymer mixtures. Detailed explanations on the changes of volatiles in each amino acid homopolymer mixture can be found in the text. Conclusions THE PRODUCTION OF MANY NEW VOLATILES FROM AMINO ACIDS BY application of irradiation indicated that more than 1 site in amino acid side chains was susceptible to free radical attack, and many volatiles can apparently be produced by secondary chemical reactions after the primary radiolytic degradation of side chains. Only sulfur-containing volatiles, however, produced strong off-odor that was similar to irradiation odor of meat. The perception of odor from samples containing sulfur volatiles changed somewhat depending on the composition of other volatiles in the sample. Although some volatiles produced from nonsulfur amino acid homopolymers interacted with sulfur compounds, their roles in the odor characteristics of irradiated liposomes can be considered minor. References Ahn DU. 2002. Production of volatiles from amino acid homopolymers by irradiation. J Food Sci. Forthcoming. Ahn DU, Sell JL, Jeffery M, Jo C, Chen X, Wu C, Lee JI. 1997. Dietary vitamin E affects lipid oxidation and total volatiles of irradiated raw turkey meat. J Food Sci 62(5):954-959. Ahn DU, Olson DG, Lee JI, Jo C, Wu C, Chen X. 1998a. Packaging and irradiation effects on lipid oxidation and volatiles in pork patties. J Food Sci 63(1):15-19. Ahn DU, Olson DG, Jo C, Chen X, Wu C, Lee JI. 1998b. Effect of muscle type, packaging, and irradiation on lipid oxidation, volatile production and color in raw pork patties. Meat Sci 49(1):27-39. Ahn DU, Olson DG, Jo C, Love J, Jin SK. 1999. Volatiles production and lipid oxidation of irradiated cooked sausage with different packaging during storage. J Food Sci 64(2):226-229. Ahn DU, Jo C, Olson DG. 2000. Analysis of volatile components and the sensory characteristics of irradiated raw pork. Meat Sci 54:209-215. Ahn DU, Nam KC, Du M, Jo C. 2001. Volatile production of irradiated normal, pale soft exudative (PSE) and dark firm dry (DFD) pork with different packaging and storage. Meat Sci 57:419-426. Diehl JF. 1995. Chemical effects of ionizing radiation. In: JF Diehl, editor. Ch. 3. Safety of irradiated foods. 2 nd ed New York: Marcel Dekker Inc. P. 43-87. Du M, Nam KC, Hur SJ, Ismail H, Ahn DU. 2001a. Effect of dietary conjugated linoleic acid, irradiation and packaging conditions on the quality characteristics of raw broiler breast fillets. Meat Sci 60(1):9-15. Du M, Nam KC, Hur SJ, Ismail H, Ahn DU. 2001b. Volatiles, color, and lipid oxida- 2664 JOURNAL OF FOOD SCIENCE Vol. 67, Nr. 7, 2002

tion of broiler breast fillets irradiated before and after cooking. Poultry Sci Forthcoming. Hashim IB, Resurreccion AVA, MacWatters KH. 1995. Disruptive sensory analysis of irradiated frozen or refrigerated chicken. J Food Sci 60(4):664-666. Heath JL, Owens SL, Tesch S, Hannah KW. 1990. Effect of high-energy electron irradiation of chicken on thiobarbituric acid values, shear values, odor, and cook yield. Poultry Sci 69:313-319. Jo C, Ahn DU. 2000. Production of volatiles from irradiated oil emulsion systems prepared with amino acids and lipids. J Food Sci 65(4):612-616. Lee EJ Ahn DU. 2002. Production of off-odor volatiles from fatty acids and oils by irradiation. J Food Sci Submitted. Merritt C Jr, Angelini P, Wierbicki E, Shuts GW. 1975. Chemical changes associated with flavor in irradiated meat. J Agric Food Chem 23:1037-1043. Merritt C Jr, Angelini P, Graham RA. 1978. Effect of radiation parameters on the formation of radiolysis products in meat and meat substances. J Agric Food Chem 26:29-31. Patterson RLS, Stevenson MH. 1995. Irradiation-induced off-odor in chicken and its possible control. Br Poultry Sci 36:425-441. SAS 1989. SAS user s guide. Cary, NC: SAS Institute Inc. MS 20020084 Submitted 2/8/02, Accepted 5/12/02, Received 5/12/02 Journal paper number J 19734 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011-3150. Project No. 6523, and supported by the National Research Initiative Competitive Grant/USDA. The authors are with the Department of Animal Science, Iowa State University, Ames, Iowa 50011-3150. Direct inquiries to author Ahn (E-mail: duahn@iastate.edu). Vol. 67, Nr. 7, 2002 JOURNAL OF FOOD SCIENCE 2665