3. SELECTED APPLICATION EXAMPLES OF GAS
3.1. Aroma Profile of Parmigiano-Reggiano Cheese
The flavor profile is one of the most significant parameters for the characterization of a food product. A representative example is the definition of the aroma profile of a typical Italian product such as the Parmigiano-Reggiano cheese. Characteris- tic features of this product are defined area of origin, strictly and traditionally defined production technology, and rules for the feeding of the animals.
A comprehensive study of the volatile fraction of this food has been carried out on a significant number of samples of certified origin and aging, coming from different geographical zones of the typical production area and representative of all seasonal productions [8,32]. For the characterization of an aromatic finger- print, sampling is a crucial step, which has to be accurately evaluated. To obtain a complete chemical definition of Parmigiano-Reggiano cheese aroma, two dif- ferent sampling techniques were used, namely DHS and SDE techniques. With the DHS procedure, the most volatile compounds are preferentially collected, whereas the use of SDE also provides interesting information about long-chain components of the cheese volatile fraction. These compounds greatly contribute to the flavor of the product.
Twenty-one samples of aged (24 mo) Parmigiano-Reggiano cheese were obtained from producers in different zones of the production area. Six samples were produced during the winter and five during each other season. The analyses were carried over a period of 1 yr, in such a way that all samples were at the same aging level. For each determination, samples were taken at the center of 1-kg cheese block.
The SDE procedure was performed using a micro-version of a Likens- Nickerson apparatus [33] in the configuration for heavier-than-water solvents.
The aroma components were extracted by steam distillation and the aqueous dis- tillate was simultaneously extracted with dichloromethane. The organic extracts were analyzed by GC–FID and GC–MS. The DHS technique was carried out with adsorption on Tenax traps and thermal desorption with cryofocusing of the volatile substances into the GC capillary column.
For the chromatographic separations, a J&W DB-Wax fused-silica capillary column was used. The identification was carried out by GC–MS in the EI mode.
The MS identifications were confirmed by comparison of the mass spectra obtained to mass spectra in the National Bureau of Standards (NBS) library and to those of authentic substances, where possible. The compounds identified in the DHS and SDE extracts are listed in Tables 2 and 3, respectively [8]. From the comparison of the GC patterns, noticeable differences in composition can be observed in the vola-
422 Careri and Mangia
Table 2 Volatile Constituents of Parmigiano-Reggiano Cheese (Headspace)
Peak no.a Compound IDb Occurrencec
1 n-Hexane MS, RT 21
2 2-Methylhexane MS 21
3 n-Heptane MS, RT 21
4 Methyl cyclohexane MS 18
5 Acetone MS, RT 21
6 2-Methylpropanal MS 21
7 n-Octane MS, RT 10
8 Methyl acetate MS, RT 8
9 1-Octene MS, RT 4
10 Tetrahydrofuran MS, RT 21
11 Tetrachloromethane MS 15
12 (?)-Octene MS 21
13 Ethyl acetate MS, RT 21
14 2-Butanone MS, RT 21
15 2-Methylbutanal MS 21
16 3-Methylbutanal MS, RT 21
17 3-Methyl-2-butanone MS, RT 21
18 Benzene MS, RT 21
19 Ethanol MS, RT 21
20 2-Ethylfuran MS, RT 21
21 Ethyl propanoate MS, RT 5
22 2-Pentanone MS, RT 21
23 Pentanal MS 1
24 Methyl butanoate MS, RT 21
25 2,2-Dimethyldecane MS 5
26 Chloroform MS 21
27 Isobutylacetate MS, RT 1
28 Toluene MS, RT 21
29 (R)-2-butanol MS, RT 21
30 Ethyl butanoate MS, RT 21
31 1-Propanol MS, RT 18
32 Dimethyl disulfide MS 21
33 Isopropenyl acetate MS, RT 1
34 Butyl acetate MS 1
35 Hexanal MS, RT 16
36 2-Hexanone MS, RT 21
37 2-Methyl-1-propanol MS, RT 21
38 Ethylbenzene MS, RT 12
39 Propyl butanoate MS, RT 17
40 Ethyl pentanoate MS, RT 20
41 Isopropylbenzene MS 13
42 p-Xylene MS, RT 19
Analysis of Flavors and Fragrances 423
Table 2 Continued
Peak no.a Compound IDb Occurrencec
43 2-Pentanol MS, RT 21
44 m-Xylene MS, RT 17
45 1-Butanol MS, RT 21
46 o-Xylene MS, RT 5
47 2-Heptanone MS, RT 21
48 Methyl hexanoate MS, RT 19
49 Limonene MS, RT 16
50 4-Methyl-1-hexene MS 7
51 3-Methyl-1-butanol MS, RT 21
52 Butyl butanoate MS, RT 13
53 Ethyl hexanoate MS, RT 21
54 3-Methyl-3-buten-1-ol MS, RT 21
55 1-Pentanol MS, RT 18
56 4-Hydroxy-3-propyl-2-hexanone MS 3
57 Acetoin MS, RT 21
58 1-Hydroxy-2-propanone MS, RT 16
59 1-Methylvinylbenzene MS 19
60 Propyl hexanoate MS 13
61 2,6-Dimethylpyrazine MS, RT 21
62 2-Heptanol MS, RT 19
63 Ethyl heptanoate MS, RT 18
64 1,3-Butanediol MS 10
65 1-Hexanol MS, RT 20
66 Dimethyl trisulfide MS 21
67 2-Nonanone MS, RT 21
68 Nonanal MS, RT 9
69 Methyl octanoate MS, RT 5
70 2-Butoxyethanol MS 20
71 Ethyl octanoate MS, RT 21
72 Acetic acid MS, RT 21
73 2-Ethyl-1-hexanol MS 12
74 Benzaldehyde MS, RT 21
75 Tetramethylurea MS 4
76 Propanoic acid MS, RT 17
77 1-Octanol MS 2
78 Ethyl nonanoate MS 1
79 2-Methylpropanoic acid MS 8
80 2,3-Butanediol MS 6
81 Benzonitrile MS 10
82 2-Propoxyethanol MS 2
83 1-Methoxy-2-propanol MS 6
84 Methyl decanoate MS 3
424 Careri and Mangia
Table 2 Continued
Peak no.a Compound IDb Occurrencec
85 2-Undecanone MS, RT 21
86 1,2-Propanediol MS 3
87 Butanoic acid MS, RT 21
88 2-Hydroxybenzaldehyde MS 3
89 Acetophenone MS, RT 20
90 Ethyl decanoate MS, RT 21
91 Furfuryl alcohol MS, RT 21
92 3-Methylbutanoic acid MS 21
93 Dimethyl tetrasulfide MS 16
94 Naphthalene MS 18
95 3-Methyl-2-(5H)-furanone MS 6
96 Pentanoic acid MS, RT 21
97 2-Phenyl-2-propanol MS 4
98 Acetamide MS 11
99 2-(2-Butoxy)-ethoxyethanol MS 9
100 1-methylnaphthalene MS 3
101 Hexanoic acid MS, RT 21
102 Geranyl acetone MS 9
103 Tetramethyl thiourea MS 9
104 2-Ethylhexanoic acid MS 5
105 Benzothiazole MS 15
106 Heptanoic acid MS, RT 21
107 1-Dodecanol MS, RT 10
108 Phenol MS 19
109 Octanoic acid MS, RT 17
110 Nonanoic acid MS, RT 18
aFor peak sequences see Ref. 8.
bMS, Mass spectrum of unknown identical to that in literature; RT, agreement of retention time with authentic compound.
cOccurrence of identification in 21 samples.
Source: Modified from Ref. 8.
tile fraction collected using the two techniques [8]. As expected, DHS sampling enables the detection of the most volatile compounds, such as acetone, ethyl acetate, ethanol, and ethyl propionate. In contrast, the chromatograms of the SDE extracts contained abundant signals corresponding to the less volatile compounds. Among the 110 compounds identified in the DHS fraction, hydrocarbons, alcohols, esters, and ketones predominated, while free fatty acids were the most abundant constit- uents of the aroma SDE extracts, in which 105 components were identified.
Analysis of Flavors and Fragrances 425
Table 3 Volatile Constituents of Parmigiano-Reggiano Cheese (SDE)
Peak no.a Compound IDb Occurrencec
1 2-Pentanone MS, RT 21
2 Diacetyl MS 21
3 Methyl butanoate MS, RT 21
4 Chloroform MS 21
5 Toluene MS, RT 21
6 (E)-2-butenal MS, RT 21
7 Ethyl butanoate MS, RT 21
8 (Z)-2-butenal MS 19
9 1-Propanol MS, RT 18
10 3-Hexanone MS, RT 20
11 Dimethyl disulfide MS 21
12 Hexanal MS, RT 16
13 2-Hexanone MS, RT 21
14 2-Methyl-1-propanol MS, RT 21
15 3-Penten-2-one MS, RT 16
16 Ethyl benzene MS, RT 12
17 Propyl butanoate MS, RT 17
18 2-Pentanol MS, RT 21
19 m-Xylene MS, RT 17
20 1-Butanol MS, RT 21
21 3-Penten-2-ol MS, RT 15
22 2-Heptanone MS, RT 21
23 Methyl hexanoate MS, RT 19
24 2-Pentenal MS, RT 19
25 Limonene MS, RT 16
26 3-Methyl-1-butanol MS, RT 21
27 Butyl butanoate MS, RT 13
28 Ethyl hexanoate MS, RT 21
29 3-Methyl-3-buten-1-ol MS, RT 21
30 1-Pentanol MS, RT 18
31 2-Vinyl-2-butenal MS 10
32 Acetoin MS, RT 21
33 2-Octanone MS, RT 21
34 Methyl heptanoate MS, RT 6
35 1-Hydroxy-2-propanone MS, RT 21
36 (Z)-2-heptenal MS, RT 6
37 Propyl hexanoate MS 13
38 2,6-Dimethylpyrazine MS, RT 21
39 2-Heptanol MS, RT 19
40 Ethyl heptanoate MS, RT 18
41 1,3-Butanediol MS 17
42 2-(Chloromethyl)furan MS 5
426 Careri and Mangia
Table 3 Continued
Peak no.a Compound IDb Occurrencec
43 1-Hexanol MS, RT 20
44 Dimethyl trisulfide MS 21
45 2-Nonanone MS, RT 21
46 Nonanal MS, RT 9
47 2,4-Hexandienal MS, RT 6
48 Isobutyl hexanoate MS 6
49 Ethyl octanoate MS, RT 21
50 3-Ethyl-2,5-dimethylpyrazine MS 6
51 3-(Methyltiopropanal) MS 21
52 Acetic acid MS, RT 21
53 Furfural MS, RT 20
54 Benzaldehyde MS, RT 21
55 Propanoic acid MS, RT 17
56 1-Hepten-4-ol MS 14
57 2-Undecanone MS, RT 21
58 Butanoic acid MS, RT 21
59 Phenylacetaldehyde MS, RT 21
60 Ethyl decanoate MS, RT 21
61 Furfuryl alcohol MS, RT 21
62 3-Methyl butanoic acid MS 21
63 n-Heptadecane MS, RT 21
64 (?-Dimethylbutanoic acid) MS 21
65 Pentanoic acid MS, RT 21
66 Unknown MS 3
67 (2,6,10,14-Tetramethyl hexadecane) MS 10
68 1-Decanal MS 19
69 n-Octadecane MS, RT 18
70 2-Tridecanone MS, RT 21
71 Hexanoic acid MS, RT 21
72 Long-chain carbonyl compound MS 21
73 1-Undecanol MS, RT 21
74 2,6-Bis(t-butyl)-4-methylphenol MS 12
75 Tetradecanal M, RT 17
76 Heptanoic acid MS, RT 21
77 δ-Octalactone MS 11
78 Long-chain carbonyl compound MS 15
79 Long-chain carbonyl compound MS 17
80 2-Pentadecanone MS 21
81 Pentadecanal MS 14
82 Ethyl tetradecanoate MS, RT 18
83 Octanoic acid MS, RT 21
84 Hexadecanal MS 14
Analysis of Flavors and Fragrances 427
Table 3 Continued
Peak no.a Compound IDb Occurrencec
85 γ-Decalactone MS, RT 11
86 Nonanoic acid MS, RT 21
87 δ-Decalactone MS, RT 21
88 Ethyl hexadecanoate MS 18
89 Decanoic acid MS, RT 21
90 9-Decenoic acid MS 21
91 γ-Dodecalactone MS, RT 21
92 1-Hexadecanol MS, RT 12
93 Undecanoic acid MS 20
94 δ-Dodecalactone MS, RT 21
95 Dodecanoic acid MS, RT 21
96 Tridecanoic acid MS 21
97 (?-Methyltridecanoic acid) MS 18
98 δ-Tetradecalactone MS 21
99 Tetradecanoic acid MS, RT 21
100 9-Tetradecenoic acid MS 21
101 (?-Methyltetradecanoic acid) MS 17
102 (?-Methyltetradecanoic acid) MS 21
103 Pentadecanoic acid MS, RT 21
104 Hexadecanoic acid MS, RT 21
105 9-Hexadecenoic acid MS 20
aFor peak sequence see Ref. 8.
bMS, Mass spectrum of unknown identical to that in literature; RT, agreement of retention time with authentic compound.
cOccurrence of identification in 21 samples. Tentative identification in parentheses.
Source: Modified from Ref. 8.
With regard to the different classes of substances, in the DHS extracts, hydrocarbons, which are secondary products of lipid auto-oxidation, ranging from n-hexane to 1-methylnaphthalene were identified. Linear long-chain C17and C18
hydrocarbons were found in the samples obtained using the SDE method.
Aldehydes account for about 2% of the relative chromatographic peak area in both series of extracts [32]. Short-chain aldehydes can be formed from amino acids during cheese ripening via Strecker degradation [34]. In DHS samples, linear and branched-chain saturated aldehydes as well as two aromatic derivatives were identified. Unsaturated aldehydes were isolated in the SDE samples, to- gether with long-chain compounds, such as tetradecanal, pentadecanal, and hexa- decanal. Long-chain aldehydes derive from fatty acids by anα-oxidation mecha- nism [35]. In the SDE extracts of all 21 samples analyzed, mass spectral data
428 Careri and Mangia
evidenced the presence of a sulfur-containing aldehyde, 3-(methylthio)propanal, which had not previously been reported as a component of Parmigiano-Reggiano cheese aroma.
Ketones are the most abundant constituents of the Parmigiano-Reggiano aroma, accounting for 26% of the total chromatographic peak area of the DHS fraction [32]. 2-Alkanones are the most frequently found. Among them, 2-penta- none and 2-heptanone are the most abundant. Methyl ketones were isolated with both sampling methods used. These compounds, which can be formed as second- ary products of theβ-oxidation of fatty acids during lipolysis [36], play an impor- tant role in defining the flavor of this product.
Fatty acids were the most important components of the SDE extracts, ac- counting for 81.4% of the total chromatographic peak area (10.7% in the DHS extracts) [32]. In particular, long-chain fatty acids (C10 : 0to C16 : 1) were isolated.
Free fatty acids can derive from three biochemical pathways during the ripening of cheese: lipolysis, proteolysis, and lactose fermentation [37]. Free fatty acids deriving from lipolysis are the linear-chain homologues from C4to C18. In addi- tion to the even-numbered straight-chain acids, which are by far the most abun- dant acids in the fatty acid class, small amounts of odd-numbered branched-chain acids, such as isobutanoic and isovaleric acids, were found. These compounds derive from deamination of amino acids caused by proteolytic enzymes. Short- chain organic acids, e.g., acetic, propionic, and butanoic acids, found in different amounts using the two sampling techniques, derive from lactose fermentation.
Free fatty acids are considered to greatly contribute to the flavor of aged cheese.
In the samples examined, significant differences in the content of some acids were observed according to the season of production [32].
Among esters, ethyl acetate was the most abundant component. Ethyl esters of odd-numbered acids from C2to C16predominated with respect to the methyl and butyl derivatives. Ethyl esters are considered to contribute to the typical fruity note of the cheese.
A number of lactones were isolated in the SDE samples, whereas, owing to their scarce volatility, they were not found using the DHS method. These compounds are known to contribute to the cheese flavor, being recognized as contributors to the pleasant aroma of butter [38]. Among lactones, the long-chain homologues (δ-decalactone andδ-dodecalactone) are quantitatively predominant.
Long-chain lactones derive from the corresponding hydroxy acids (C8to C16) by the loss of water [39].
Some sulfur compounds were also found, in particular dimethyl disulfide in the DHS fraction. These compounds, which derive from sulfur-containing amino acids by Strecker degradation, can be considered important contributors to the aroma of the product.
Finally, some heterocyclic compounds, such as alkylpyrazines and furans, were identified. These substances are important trace components of flavor of
Analysis of Flavors and Fragrances 429
unprocessed and heated foods, as natural components or deriving from nonenzy- matic Maillard reaction [40,41].
From the results obtained in this study, it can be inferred that the use of GC–MS in combination with adequate aroma sampling techniques can solve ana- lytical problems of aroma characterization, even in the case of very complex matrices. The combined use of the two extraction procedures proved helpful in providing a complete fingerprint of the aged Parmigiano-Reggiano cheese aroma, which can be used as a useful reference for the characterization of the product.
Further, the results obtained enabled the correlation of the composition of the volatile fraction of Parmigiano-Reggiano cheese with sensory attributes [42].
This subsequent investigation evidences the significance of the flavor in defining the quality of a food product and, in addition, the contribution of the different volatile compound classes or of the individual substances to the sensory attri- butes. For example, esters, particularly methyl butanoate, ethyl hexanoate, and isobutyl acetate, are found to be positively related to the ‘‘fragrant’’ and ‘‘fruity’’
notes of aged cheese. Short-chain methyl-branched alcohols, secondary products of proteolysis, contribute to a positive ‘‘maturation’’ component of the aroma, in opposition to the sharp stimuli of free fatty acids (Fig. 6).
Figure 6 Principal components analysis of volatile, nonvolatile, and sensory data of Parmigiano-Reggiano cheese. Variable loadings on the plane of the first two components.
(From Ref. 42.)
430 Careri and Mangia