Key Aroma Compounds in Smoked Cooked Loin - Journal of

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Cite This: J. Agric. Food Chem. 2018, 66, 3683−3690

Key Aroma Compounds in Smoked Cooked Loin Monika Kosowska,*,†,‡ Małgorzata A. Majcher,§ Henryk H. Jeleń,§ and Teresa Fortuna† †

Department of Food Analysis and Quality Assessment, Faculty of Food Technology, University of Agriculture in Krakow, ulica Balicka 122, 30-149 Kraków, Poland ‡ Regis, Limited, ulica Sławka 3a, 30-633 Kraków, Poland § Faculty of Food Science and Nutrition, Poznań University of Life Sciences, ulica Wojska Polskiego 31, 60-624 Poznań, Poland ABSTRACT: Smoked cooked loin is one of the most popular meat products in Poland. In this study, key volatile compounds in this traditional Polish meat product were determined using gas chromatography−olfactometry and aroma extract dilution analysis (AEDA). In total, 27 odor-active volatile compounds were identified, with flavor dilution (FD) factors ranging from 4 to 1024, with the highest FD factors noted for 2-methoxyphenol, 2-methoxy-4-(prop-2-enyl)phenol, and 2-methoxy-4-(E)-(prop-1-en-1yl)phenol. Results of the quantitative analyses based on determinations with stable isotope dilution assays (SIDAs) and standard addition (SA), followed by calculations of the odor activity value (OAV), enabled identifying 24 of the volatile compounds responsible for flavor development in the analyzed smoked cooked loin. The highest OAVs were obtained for 2-methoxyphenol, 2-methyl-3-furanthiol, 1-octen-3-one, and 2-methyl-3-(methyldithio)furan. KEYWORDS: smoked cooked loin, aroma-active compounds, AEDA, OAV, GC × GC

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INTRODUCTION Meat and meat products are important sources of proteins and many minerals and vitamins indispensable in a human’s diet. Taken globally, the most often consumed meat is pork (15.6 kg capita−1 year−1), followed by poultry (13.6 kg capita−1 year−1) and beef (9.6 kg capita−1 year−1 ). 1 In Poland, pork consumption reaches as much as 35.5 kg capita−1 year−1.2 Among meat products, highly valued are smoked products, including, in particular, smoked cooked loin made of the longissimus muscle (m. longissimus dorsi). The quality and consumption attractiveness of meat products, including the smoked products, are largely determined by their flavor being a resultant of taste and olfactory impressions and sensations. Consumers eagerly buy meat products with desirable tenderness and natural taste and odor.3 In this respect, preferred are smoked products with a gentle salty flavor and characteristic odor of cured meat. In the case of smoked meat products, the smoky flavor may vary from mild to very strong.4 Raw pork is characterized by low aromaticity; however, it constitutes a matrix rich in non-volatile precursors of volatile compounds responsible for aroma development in meat products (amino acids, peptides, saccharides, inorganic salts, and organic acids).5 Heat treatment initiates a series of reactions that result in the development of a characteristic flavor of meat. The mechanism of volatile synthesis in meat is multi-oriented and involves Maillard reactions, oxidation of lipids, interactions between lipid oxidation products and Maillard reaction products, and thiamine degradation.5−8 Heat treatment results in the formation of thousands of volatile compounds representing various chemical classes, i.e., hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids, esters, lactones, furans, pyrans, pyrroles, pyrazines, pyridines, phenols, thiophenes, thiazoles, thiazolines, oxazoles, and other nitrogen or sulfur compounds. © 2018 American Chemical Society

One of the most difficult tasks in the analysis of volatile compounds is their isolation from a sample to accurately reproduce the aroma of the product and not change the profile of odor-active compounds. In the case of meat products, the most commonly applied methods of volatile isolation include solvent-assisted flavor evaporation (SAFE),9 dynamic headspace analysis (DHSA), and solid-phase microextraction (SPME). Distillation in the SAFE apparatus is claimed to be the method that enables preparing an extract that accurately reproduces the aroma of a product it had been obtained from.7 It has been applied for the isolation of volatiles from multiple food products, including such meat products as salami,10 ham,11,12 fried duck livers,13 and gravies.14 Volatile compounds are also isolated from meat products using a DHSA, also referred to as the “purge and trap” technique, usually with the use of Tenax sorption traps. Despite various methodological difficulties posed by its applications, many research works have appeared that addressed optimization of DHSA use in the analysis of volatiles in meat products.15,16 Another and, simultaneously, the most commonly applied method for volatile isolation from the headspace is the SPME technique developed by Pawliszyn. It has been frequently applied to isolate volatile compounds from meat products, including cooked hams, rawripening loins and hams, and smoked bacons.15,17−19 Aroma of a meat product is a mixture of hundreds of volatile compounds with different flavor notes; however, only a minority of them imparts the characteristic flavor of a food product. It is estimated that barely 3% out of ca. 1000 identified volatiles impart flavors to food products.20 Hence, it is of outmost importance to separate odor-active compounds from Received: Revised: Accepted: Published: 3683

January 17, 2018 March 23, 2018 March 24, 2018 March 24, 2018 DOI: 10.1021/acs.jafc.7b05996 J. Agric. Food Chem. 2018, 66, 3683−3690

Article

Journal of Agricultural and Food Chemistry

Table 1. Method of Quantitation, Quantitation Ions, Response Factor, and Regression Coefficient of Calibration Curves Used for Concentration Calculations of Key Odorants Present in the Smoked Cooked Loin compound 2,3-butanedione hexanal octanal 1-octen-3-one 2-methyl-3-furanthiol dimethyl trisulfide nonanal 3-(methylthio)propanal 2-ethyl-3,5-dimethylpyrazine 2,3-diethyl-5-methylpyrazine (E)-2-nonenal 3-isobutyl-2-methoxypyrazine butanoic acid 2-methyl-3-(methyldithio)furan 2-acetyl-2-thiazoline 2-methoxyphenol 2-phenylethanol 2,6-dimethylphenol 5-methyl-2-methoxyphenol 4-methyl-2-methoxyphenol 4-ethyl-2-methoxyphenol 4-methylphenol 3-methylphenol 2-methoxy-4-(2-propenyl)phenol 4-ethylphenol 3-ethylphenol 2-methoxy-4-vinylphenol 2,6-dimethoxyphenol 2-methoxy-4-(E)-(1-propenyl)phenol

method of quantitationa

quantitation ionb

SA SIDA SIDA SIDA SA SA SIDA SIDA SIDA SIDA SA SA SIDA SA SA SIDA SIDA SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3 SIDA/2H3

86 56 57 70 114 126 57 104 135 150 83 124 73 113 60 124 91 107 123 138 137 108 108 164 107 107 150 154 164

ion ISc

labeled standard

Rf/r2d 2

[2H2]-hexanal [2H2]-hexanal [2H3]-1-octen-3-one

58 58 73

[2H2]-hexanal [2H2]-3-(methylthio)propanal [2H6]-2-ethyl-3,5-dimethylpyrazine [2H7]-2,3-diethyl-5-methylpyrazine

[2H7]- butanoic acid

58 107 141 157

77

[2H3]-2-methoxy-4-vinylphenol [2H10]-1-phenylethanol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol [2H3]-2-methoxy-4-vinylphenol

153 113 153 153 153 153 153 153 153 153 153 153 153 153

r = 1.3 1.5 1.1 r2 = r2 = 1.4 1.2 0.9 0.9 r2 = r2 = 1.2 r2 = r2 = 2.2 1.3 1.0 2.5 2.5 1.4 1.1 0.9 4.6 0.4 0.4 1.2 2.4 3.5

1.0

0.99 0.99

0.99 0.99 0.99 0.98

a SIDA, stable isotope dilution assay; SA, standard addition. bIons of analytes were used for quantitation. cIons of internal standard (IS, labeled isotope) were used for quantitation. dRf, response factor between the analyzed compound and its IS (labeled isotope); r2, regression coefficient.

other odorless compounds.21 It may be performed with olfactometric methods that merge separation of compounds on chromatographic columns and detections of odors by a human organ of olfaction. The best known and most often applied method is aroma extract dilution analysis (AEDA). It was developed by Grosch and consists of the analysis of aroma of subsequent dilutions of the analyzed sample until the moment of aroma disappearance. The outcome of this analysis is determination of the so-called flavor dilution (FD) factor for each odor-emitting substance. A more precise analysis of odoractive compounds is based on the quantitation of the identified compound and determination of its odor detection threshold. In this case, use is made of the concept of odor activity value (OAV) elaborated by a research group of Rothe and Thomas in 1967 and defined as the ratio of the volatile concentration to its odor detection threshold. Studies aimed to characterize meat products based on AEDA and OAV were conducted for, e.g., salami and pork and beef gravies.10,14 In the case of salami, the highest FD values were obtained for 2-methoxyphenol (4096), followed by 2-methoxy-4-(prop-2-enyl)phenol, 2-methoxy-4(E)-(prop-1-en-1-yl)phenol, and acetic acid (2048), and the highest OAVs were obtained for acetic acid (8830) and acetic aldehyde (1610). In the case of gravies, the highest OVAs were calculated for 3-mercapto-2-methylpentane-1-ol (4800−4700) and 12-methyltridecanal (3600 but only in beef gravy).

The objective of this study was to identify key volatile compounds in smoked cooked loin being a typical cured meat product on the Polish market.

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MATERIALS AND METHODS

Smoked Cooked Loin. Smoked cooked loin was produced in a semi-technical scale by the Regis Sp. z o.o. company in three replications. The raw material, namely, the longissimus muscle (m. longissimus dorsi) was cured, then smoked using beech and alder wood chips at a temperature of 60 °C for 20 min, and finally cooked at a temperature of 76 °C until a temperature of 70 °C has been reached inside the product. The resultant products were characterized by the final yield of 97.26 ± 2.33%. Chemical Standards. Methylene chloride, methanol, and sodium sulfate were obtained from Sigma-Aldrich (Poznań, Poland). The following reference aroma compounds were also purchased from Sigma-Aldrich: hexanal, 2-heptanone, 2-methyl-3-furanthiol, octanal, (E)-2-nonenal, dimethyl trisulfide, nonanal, 2-furfurylthiol, 2,3butanedione, dimethyl disulfide, 3-(methylthio)propanal, dimethyltrisulfide, 1-octen-3-one, 2-ethyl-3,5-dimethylpyrazine, 2-phenylethanol, 5-methyl-2,3-diethylpyrazine, 3-isobutyl-2-methoxypyrazine, butyric acid, 2-acetyl-2-thiazoline, 2-methoxyphenol, 2-phenylethanol, 2,6dimethylphenol, 4-methyl-2-methoxyphenol, 3-methylphenol, 2-methoxy-4-(2-propenyl)phenol, 4-ethylphenol, 2-methoxy-4-(E)-(1propenyl)phenol, 2-methoxy-4-vinylphenol, and [2H10]-1-phenylethanol. The following compounds were purchased from AromaLAB AG (Freising, Germany): [2H6]-phenol, [2H2]-hexanal, [2H7]-butanoic acid, [2H6]-dimethyldisulfide, [2H2]-3-(methylthio)propanal, [2H3]-1octen-3-one, [2H6]-2-ethyl-3,5-dimethylpyrazine, [7H2]-5-methyl-2,33684

DOI: 10.1021/acs.jafc.7b05996 J. Agric. Food Chem. 2018, 66, 3683−3690

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were added to the analyzed samples at the first stage of their preparation, and then volatiles were isolated. Quantitation was carried out in a GC × GC−TOFMS apparatus, through monitoring selected ions, provided in Table 1. Before the quantitative analysis, a detector response coefficient (Rf) was calculated as a ratio of the analyzed compound to the isotopically labeled compound for each compound assayed with the SIDA method. When no direct isotopomers of the analyzed compounds were available, such as, e.g., 2-methoxyphenol and other phenols, we used 2-methoxy-4-vinylphenol isotopomer, which in our opinion is the most similar compound to the analyzed substances, and then correction was applied in the form of the response factor (Rf). In the case of the remaining 7 compounds, for which the isotopically labeled standards were unavailable, use was made of a method of IS addition to the sample (SA). To this end, the analyzed compound was added in five concentrations to the samples. On the basis of areas of peaks obtained for particular concentrations of the standard compounds, standard curves were plotted and their regression coefficients were calculated and presented in Table 1. In the case of both methods, computations were made with ChromaTOF software (version 3.34). Descriptive Sensory Evaluation. The flavor profiling method was used to compare results of instrumental analyses to those of the sensory evaluation. The evaluation was conducted by a nine person panel of selected evaluators and experts. Each assessment was carried out in three replications. During each assessment, the panelists received two slices of the smoked cooked loin. The samples were described with three-digit codes and presented to the panelists at room temperature. The list of attributes was prepared on the basis of guidelines of the American Meat Science Association27 and completed with attributes provided in the AromaSphere database.28 The first stage involved determination of the number of attributes and order of their appearance. Then, intensity of each identified attribute was evaluated on a 10 cm line scale anchored at non-perceptible attribute− very perceptible attribute. The analysis provided the flavor profile of the analyzed product, which was presented in the form of a polar diagram (Figure 1).

diethylpyrazine, [2H2]-2-isobutyl-3-methoxypyrazine, [2H3]-2-methoxy-4-vinylphenol, and 2-acetyl-1-pyrroline. The purity of solvents and reference standards was a minimum of 99 and 97%, respectively. Isolation Method. Volatile compounds were isolated from smoked cooked loin. To this end, 500 mL of saturated NaCl solution was added to 500 g of a ground sample. Next, volatiles were extracted with 300 mL of dichloromethane for 2 h, and phases were separated via centrifugation (4000 rpm). The volatile compounds were isolated through distillation with the SAFE method. The resultant extract was dried with anhydrous sodium sulfate and concentrated in a KudernaDanish concentrator at 40 °C to the volume of ca. 500 μL. Gas Chromatography−Olfactometry (GC−O). The volatile compounds were separated on a HP 5890 gas chromatograph using two columns of different polarities: SPB-5 (30 m × 0.53 mm × 1.5 μm) and Supelcowax 10 (30 m × 0.53 mm × 1 μm). The chromatograph was equipped in the “Y”-type splitter, dividing the isolated volatile compounds to an olfactometric port and flame ionization detector (FID) (1:1). Parameters of the separation process were as follows for the SPB-5 column: initial temperature of the oven at 40 °C for 1 min, then temperature increment with the rate of 6 °C/ min to 180 °C, and next temperature increment at 25 °C/min to 260 °C, whereas parameters of the separation process were as follows for the Supelcowax 10 column: initial temperature at 40 °C for 1 min, followed by a temperature increase of 8 °C/min to 240 °C maintained for 10 min. For all flavor notes recorded at specific retention times, retention indices were calculated to compare the results to those obtained by gas chromatography−mass spectrometry (GC−MS) and literature data. Retention indices were calculated for each compound using a series of n-alkanes, C7−C24, and the Kovats formula.22 Gas Chromatography−Mass Spectrometry (GC−MS and GC × GC−TOFMS). The volatiles were identified using two apparatuses: an Agilent Technologies 7890A gas chromatograph coupled with an Agilent Technologies 5975C mass spectrometer (GC−MS) equipped with a Supelcowax-10 column (30 m × 0.25 mm × 0.25 μm) and a Leco Pegasus IV gas chromatograph operating in the mode of a twodimensional gas chromatograph coupled with a time-of-flight (TOF) mass spectrometer (GC × GC−TOFMS). Chromatographic separations were conducted on two columns: primary, SPB-5 (30 m × 0.32 mm × 0.25 μm), and secondary, Supelcowax 10 (1 m × 0.1 mm × 0.1 μm). Operating conditions were as follows: helium flow rate of 0.8 mL/s, temperature of the injection port at 220 °C, and injection mode split of 10:1. Oven parameters for GC−MS were the same as for GC− O on the Supelcowax-10 column, whereas oven parameters for GC × GC/TOFMS were the same as for GC−O on the SPB-5 column, with the temperature increase by +15 °C in the case of the second oven. In both cases, spectra were collected in the mass range of m/z 33−333, temperature of the transfer line was 280 °C, and temperature of the ion source was 250 °C. In the case of GC × GC/TOFMS analysis, mass spectra were collected at the rate of 150 spectra/min and modulation time was 3 s. The volatiles were identified by comparing their mass spectra, flavor notes, and retention indices to those of standard compounds, National Institute of Standards and Technology (NIST) 05 library of mass spectra, and literature data. In addition, to compare retention indices and flavor notes, use was made of earlier works.10,12,13,23−26 and databases: www.flavornet.org, www.odour.org. uk, and www.pherobase.com. AEDA.21 The analysis with the AEDA method was conducted by injecting 2 μL of the concentrated extract obtained with the SAFE method onto a column of the chromatograph. The analysis of flavor notes appearing during separation was carried out by two trained persons at the O-port. Next, the extract was diluted with dichloromethane and subjected to further analyses. The analysis was found complete when aroma was no longer perceptible. This allowed for computation of FD factors for each compound as the highest dilution in which the compound was perceptible for the last time. Quantitation by Stable Isotope Dilution Assays (SIDAs) and Standard Addition (SA) Method. For quantitation of volatiles, the method with an isotopically labeled internal standard (SIDA) was applied for 22 compounds, and the method with an internal standard (IS) was applied for 7 compounds (Table 1). Solutions of standards

Figure 1. Sensory aroma profile of a smoked cooked loin.

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RESULTS AND DISCUSSION To determine the key volatile compounds responsible for the flavor of smoked cooked loin, the extract obtained with the SAFE method was subjected to GC−O analysis and ADEA. Results of analyses with the AEDA method in the smoked cooked loin were presented in the form of a chromatogram and an aromagram in Figure 2. The GC−O analysis demonstrated the presence of 27 odoractive compounds representing the following groups: organic acids, aldehydes, ketones, sulfur compounds (thiols and 3685


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Figure 2. Chromatogram and aromagram of volatiles isolated from a smoked cooked loin with their FD factors and retention indices (RIs) on a Supelcowax-10 column. Numbers refer to Table 2.

were determined for 2-methoxyphenol as well as in the case of smoked cooked loin analyzed in our study. Investigations have shown that methoxyphenols, such as 2-methoxyphenol, 4methyl-2-methoxyphenol, 2,6-dimethoxyphenol, 2-methoxy-4(prop-2-enyl)phenol, and 2-methoxy-4-(E)-(prop-1-en-1-yl)phenol, are the major components of smoke.30,31 Their concentration in food products depends upon the type of wood used for smoking, parameters of the smoking process, and type of food product being smoked. The other phenolic compounds identified in the loin included 3-methylphenol (FD of 512) with a phenolic odor, 5-methyl-2-methoxyphenol (FD of 512) with a smoky odor, 4-ethyl-2-methoxyphenol (FD of 256) with a pungent odor, 2,6-dimethoxyphenol (FD of 128) with a smoky odor, 2-methoxy-4-vinylphenol (FD of 128) with a odor of cloves, 4-ethylphenol, 3-ethylphenol, and 2,6dimethylphenol (FD of 32) with a odor flavor, 2-phenylethanol (FD of 4) with a sweet odor, and 4-methylphenol (FD of 4) with tallow odor. In turn, the identified sulfur compounds included 3-methylthiopropanal (methional) with a characteristic flavor of cooked potatoes as well as dimethyl trisulfide with a flavor of cooked cabbage, both with FD equal to 128. Sulfur

sulfides), nitrogen compounds (pyrazines and pyrroles), and phenols. The intensity of identified compounds was expressed with a FD factor and ranged from 4 to 1024 (Table 2). The highest FD values (1024) were obtained for three compounds with the following flavor notes: smoky and pungent, which were next identified as 2-methoxyphenol, 2-methoxy-4-(prop-2enyl)phenol, and 2-methoxy-4-(E)-(prop-1-en-1-yl)phenol. Phenols are compounds formed from phenolic acids and lignin upon thermal degradation or enzymatic degradation with microorganisms.29 In the analyzed meat products, a total of 14 odor-active phenolic compounds derived from the smoking process were quantified. A model study described in the literature demonstrated ferulic acid to be a precursor of phenolic compounds, including 2-methoxy-4-vinylphenol, the main product of pyrolysis, and secondary reaction products, such as, e.g., 4-ethyl-2-methoxyphenol, vanillin, or 2-methoxyphenol.29 The effect of identified volatile compounds on flavor development in smoked food products has already been reported but concerned such products as salami, oscypek (goat cheese), Frankfurter-type sausages, as well as the smoke.10,26,30 In the above-cited works, the highest FD values 3686


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Journal of Agricultural and Food Chemistry Table 2. Key Aroma Compounds Identified in Smoked Cooked Loin RIa odor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

b

buttery fresh, green grass green, orange peel mushroom cooked meat popcorn cooked cabbage fatty roasted coffee boiled potatoes earthy pepper chips fatty earthy, pepper cheesy boiled meat roasted smoky, meat sweet, flowery phenolic, burnt smoky, sweet sweet pungent fecal, animalic phenolic pungent phenolic phenolic, plastic clove-like smoky pungent

compound

c

2,3-butanedione hexanal octanal 1-octen-3-one 2-methyl-3-furanthiol 2-acetyl-1-pyrroline dimethyl trisulfide nonanal 2-furfurylthiol 3-(methylthio)propanal 2-ethyl-3,5-dimethylpyrazine 2,3-diethyl-5-methylpyrazine (E)-2-nonenal 3-isobutyl-2-methoxypyrazine butanoic acid 2-methyl-3-(methyldithio)furan 2-acetyl-2-thiazoline 2-methoxyphenol 2-phenylethanol 2,6-dimethylphenol 5-methyl-2-methoxyphenol 4-methyl-2-methoxyphenol 4-ethyl-2-methoxyphenol 4-methylphenol 3-methylphenol 2-methoxy-4-(2-propenyl)phenol 4-ethylphenol 3-ethylphenol 2-methoxy-4-vinylphenol 2,6-dimethoxyphenol 2-methoxy-4-(E)-(1-propenyl)phenol

Supelcowax-10

DB-5

FDd

982 1085 1297 1306 1311 1359 1371 1383 1431 1448 1469 1470 1486 1515 1591 1660 1793 1857 1898 1905 1930 1948 2014 2061 2066 2160 2170 2190 2248 2278 2353

595 796 1010 985 884 929 982

16 16 32 16 32 1 128 1 16 128 32 1 1 32 32 64 4 1024 4 32 512 4 256 4 512 1024 32 32 128 128 1024

913 909 1081 1159 1169 1194 835 1178 1098 1081 1105 1107 1200 1203 1283 1070 1075 1350 1160 1169 1320 1350 1410

a Retention indices. bOdor was percieved at the sniffing port (nomenclature according to databases AromaSphere, Flavornet, and American Meat Science Association). cCompounds were identified by comparing them to reference compounds on the basis of the following criteria: retention index (RI), mass spectra obtained by MS(EI), and odor quality at the sniffing port. dFlavor dilution factor on the Supelcowax-10 column.

conducted thus far that aimed to elucidate the mechanism of their synthesis. For example, the formation of odor-active sulfur compounds, including 2-methyl-3-(methyldithio)furan and 2methyl-3-furanthiol, was demonstrated in model studies in which thiamine and L-cysteine were heated in the presence of reducing sugars.36,37 According to the concept of sensomics, only those compounds that occur in the concentration exceeding their detection threshold affect aroma development in food products.38 Considering that, to obtain more precise data for key aroma compounds that impart the final aroma to loin, in the next stage of the study, we determined contents of all compounds identified with the GC−O technique. Two quantitative methods were used to this end: SIDA, in which an isotopomer of an individual compound is used as an IS, and SA, in which the standard is added to the assayed sample. Then, OAVs were calculated as a ratio of the concentration to odor detection threshold. Values of odor detection thresholds were derived from literature data10,35 and provided together with OAVs in Table 3. Calculations were made based on odor thresholds (OTs) of volatile compounds in water, because the loin contained 70.51 ± 1.28% of water and only 2.93 ± 0.31% of fat. An exception was 5-methyl-2-methoxyphenol, for which

compounds significantly contribute to the development of the characteristic flavor of food products, including both fermented products, such as, e.g., wines, beer, and cheeses, and heattreated products, such as, e.g., meat gravies, cured meat products, or roasted sesame seeds.14,29,32 Methional is one of the most frequently identified key aroma compounds. According to author of a review,20 it occurs in almost every second food product (45%). It was also identified many times in meat, including pork, beef, and poultry. It was determined as a key component of aromas in roasted livers (beef, pork, and duck livers),13 pork and beef gravies,14 and Hungarian salami.10 Methional is synthesized as a result of L-methionine reaction in the presence of a reducing sugar in Strecker’s degradation process. In turn, dimethyl trisulfide may be formed from Lmethionine upon enzymatic and chemical transformations. It has thus far been identified as a key aromatic compound in, i.e., pork33 and beef.34 The other identified compounds had lower FD values (64−1); they included compounds formed upon Maillard reaction, degradation of thiamines, and oxidation of lipids. In turn, 2-methyl-3-(methyldithio)furan and 2-methyl-3furanthiol are sulfur compounds that impart characteristic flavor notes of cooked meat. They have very low detection thresholds in the range of 0.004−0.007 μg/L of water and play a key role in meaty flavor development.35 Ample model studies have been 3687


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Table 3. Concentration, Odor Thresholds, and Odor Activity Values of Aroma-Active Compounds of Smoked Cooked Loin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

compound

OTa (μg/L of water)

concentration (μg/kg)

OAVb

2-methoxyphenol 2-methyl-3-furanthiol 1-octen-3-one 2-methyl-3-(methyldithio)furan 2-methoxy-4-(2-propenyl)phenol dimethyl trisulfide 3-ethylphenol nonanal 3-isobutyl-2-methoxypyrazine 4-methyl-2-methoxyphenol 3-(methylthio)propanal 5-methyl-2-methoxyphenol 4-ethyl-2-methoxyphenol 2-methoxy-4-vinylphenol 3-methylphenol 2-acetyl-2-thiazoline (E)-2-nonenal octanal 4-ethylphenol 2-phenylethanol hexanal 4-methylphenol 2,3-butanedione 2-methoxy-4-(E)-(1-propenyl)phenol

1 0.007 0.005 0.004 1 0.01 1.7 8 0.002 30 0.2 13c 16 5 31 1 1 6.9 21 0.39 73 55 4 100

12710 19.9 14.1 10.7 1529 13.6 597 2360 0.44 6098 36.3 2266 2704 615 3529 60.9 59.4 295 726 11.5 1681 1054 78.8 1250

12710 2849 2816 2685 1529 1356 351 295 220 203 182 174 169 123 114 61 59 43 35 29 23 20 19.7 12.5

a

Odor thresholds in water.35 bOdor activity values were calculated by dividing the concentration of the analyte by its odor threshold value. cOdor thresholds in oil.10

thermal degradation of thiamine. A recent study by Thomas et al.15 indicates thiamine to be the major source of these compounds in cooked ham. Rapidly evaporating meaty flavors are associated with the presence of volatile 2-methyl-3furanthiol, whereas more stable meaty flavors are associated with the presence of a less volatile compound, namely, 2methyl-3-(methyldithio)furan, which is synthesized as a result of the 2-methyl-3-furanthiol reaction with methanthiol.39 The OAV determined for dimethyl trisulfide was high and reached OAV = 1356. This compound has a significant impact on the development of a characteristic flavor of cooked meat, although its own aroma is not a pleasant aroma as it exhibits sulfur notes typical of cooked cabbage. As reported by Kubec et al.,40 it may be generated from S-methylcysteine sulfoxide. Research reports indicated its presence as an odor-active compound in such meat products as Jinhua ham,11 American country ham,12 dry-cured Iberian loin,17 and pork and beef gravies.23 1-Octen-3-one, whose OAV was calculated at 2816, is a compound synthesized as a result of thermal degradation of unsaturated fatty acids, more specifically linoleic acid.29 It has a mushroom-like flavor and a low odor detection threshold, accounting for 0.005 μg/kg of water, which makes it a highly odor-active carbonyl compound derived from oxidative processes. It occurs in heat-treated meat products originating from various animal species (beef, pork, and poultry) and also in processing meaty flavors.41 Pyrazines are compounds formed as a result of heat treatment of food products or upon the activity of microorganisms. Several mechanisms of pyrazine synthesis have been identified thus far, of which the most thoroughly described mechanism is carbonyl−amine condensation of two amine− ketones (synthesized during Strecker’s degradation), resulting

we assumed the OT in oil because data for its OT in water were unavailable. Results obtained indicate differences in contents of individual volatiles ranging from 12 710 μg/kg for 2-methoxyphenol (the highest assayed value) to 0.44 μg/kg for 3-isobutyl-2methoxypyrazine (the lowest assayed value). The highest OAV (i.e., OAV = 12 710) was determined for 2-methoxyphenol, which was probably developed in loin during smoking with the use of beech−alder wood chips. This compound was also identified in Frankfurter-type sausages smoked using both beech and alder wood.30 Also, Majcher and Jeleń,26 who investigated the aroma of oscypek cheese, determined the highest OAV for 2-methoxyphenol (OAV = 1280) as well as Sollner and Schieberle10 in Hungarian salami smoked using beech wood. Study results demonstrate that 2methoxyphenol together with 2-methoxy-4-(prop-2-enyl)phenol and other phenolics impart a characteristic smoky flavor note to loin. These results were confirmed in the sensory analysis when the panelists indicated the cured/smoky odor to be the prevailing odor (Figure 1). High OAVs were also calculated for sulfur compounds, including 2-methyl-3-furanthiol (OAV = 2849), 2-methyl-3(methyldithio)furan (OAV = 2685), and dimethyl trisulfide (OAV = 1356). The first two compounds have very low odor detection thresholds (0.007 and 0.004 μg/L of water, respectively); hence, high OAVs were calculated for them, despite their low contents (19.9 and 10.7 μg/kg, respectively). Both of these compounds display the aroma of cooked meat and, thus, are the key volatiles responsible for imparting the meaty flavor to loin, which was also reflected in results of the sensory evaluation when the meaty flavor was assessed as intensive. 2-Methyl-3-furanthiol is formed upon thermal reaction proceeding between cysteine and ribose or upon 3688



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in the formation of dihydropyrazine, followed by its oxidation. More substituted pyrazines, especially these with low odor detection thresholds, are generated in an alternative way (one of the substituents is an aldehyde). Three pyrazines were identified in the analyzed loin, i.e., 2-ethyl-3,5-dimethylpyrazine, 2,3-diethyl-5-methylpyrazine, and 3-isobutyl-2-methoxypyrazine. However, only 3-isobutyl-2-methoxypyrazine achieved OAV above 1. Despite its low concentration in the loin (below 0.5 μg/kg), its odor activity value reached 220 as a result of its very low odor detection threshold, accounting for 0.002 μg/kg of water. 3-Isobutyl-2-methoxypyrazine has an earth-like, paprika-like flavor and, thus far, has been identified as the key volatile in Hungarian salami,10 gravies,23 cereal coffee,42 and paprika and string bean.29 Methoxypyrazines are highly odoractive compounds generated as a result of metabolic reactions of microorganisms. In addition, they show high thermal stability, allowing them to survive high temperatures during, e.g., coffee seed roasting. The group of odor-active volatiles of the analyzed loin also included aldehydes: hexanal, octanal, nonanal, and (E)-2nonenal, whose OAVs ranged from 23 to 295. Hexanal and (E)-2-nonenal are products of linoleic acid oxidation, whereas octanal and nonanal are formed upon oxidative processes of oleic acid.7 Aldehydes have been identified many times in meat products made of pork, beef, or poultry meat. They were determined in Hungarian salami,10 gravies,23 dry-cured loin,17 American country ham,12 and Jihua ham.11 Analyses conducted in this study enabled identifying 24 odor-active volatile compounds. The greatest impact on flavor development of the analyzed smoked cooked loin was confirmed for 2-methoxyphenol, 2-methyl-3-furanthiol, 1octen-3-one, 2-methyl-3-(methyldithio)furane, 2-methoxy-4(prop-2-enyl)phenol, dimethyl trisulfide, 3-ethylphenol, nonanal, 3-isobutyl-2-methoxypyrazine, 4-methyl-2-methoxyphenol, methional, 5-methyl-2-methoxyphenol, 4-ethyl-2-methoxyphenol, 3-methylphenol, and 2-acetyl-2-thiazoline. The identified volatiles are substances typical of the smoking process (phenols) and compounds formed as a result of reactions characteristic for thermal transitions in meat (aldehydes, ketones, and sulfur and nitrogen compounds). Compounds determined in smoked cooked loin should be considered as potential key odorants. Definitive conclusions should be obtained through recombination experiments in a smoked cooked loin-like matrix.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +48126624779. E-mail: [email protected]. pl. ORCID

Monika Kosowska: 0000-0002-9750-2730 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the REGIS Sp. z o.o. company for the preparation of experimental material and purchase of selected reagents. Special thanks are extended to Dr. Werner Schroll for his help and valuable remarks during research. 3689


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Key Aroma Compounds in Smoked Cooked Loin - Journal of

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