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Volatile compounds in ground beef subjected to high pressure processing: A comparison of dynamic headspace and solid-phase microextraction
Food Chemistry 124 (2011) 1201–1207
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Analytical Methods
Volatile compounds in ground beef subjected to high pressure processing: A comparison of dynamic headspace and solid-phase microextraction Ana Rivas-Cañedo, Cristina Juez-Ojeda, Manuel Nuñez, Estrella Fernández-García * Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña, km. 7, Madrid 28040, Spain
a r t i c l e
i n f o
Article history: Received 17 August 2009 Received in revised form 3 February 2010 Accepted 18 July 2010
Keywords: High pressure processing Volatile compounds Beef SPME Dynamic headspace extraction
a b s t r a c t The effect on the volatile profile of cooked beef meat, previously subjected to high pressure (400 MPa, 10 min at 12 °C) followed by a 3-d refrigerated storage, was investigated by comparing two extraction techniques i.e. dynamic headspace and solid-phase microextraction. Dynamic headspace was more efficient in extracting 2,3-butanedione and secondary alcohols. Solid-phase microextraction, being more efficient in extracting substances such as 1-alcanols, ethyl esters and acids, permitted to better categorize the effects caused in the volatile fraction by refrigerated storage and high pressure processing. The volatile fraction of cooked control beef meat contained high amounts of diketones and low amounts of methyl ketones, secondary alcohols, aldehydes and fatty acids. While diketones nearly disappeared after the 3-d refrigerated storage, the other compounds together with ethanol and ethyl esters increased significantly. Pressurized beef samples underwent fewer changes than non-pressurized samples during refrigerated storage, leading to a volatile profile closer to that of control beef. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Due to its physicochemical characteristics, meat is one of the most perishable foods. The concern of achieving microbiologically safe meats and meat products, with an extended shelf-life, increases the interest in non-thermal preservation technologies such as high pressure processing (HPP). Pressure treatments yield high quality products due to their ability to kill spoilage and pathogenic microorganisms as well as to inactivate some key food enzymes (Hogan, Kelly, & Sun, 2005; Patterson, 2005). The effects of HPP on spoilage and pathogenic bacteria in meats have been previously reported (Carlez, Rosec, Richard, & Cheftel, 1993; Morales, Calzada, Ávila, & Nuñez, 2008). One of main advantages of HPP is that supposedly neither modifications in the taste and flavour characteristics nor changes in the nutritional value and vitamin content occur (Aymerich, Picouet, & Monfort, 2008). Nevertheless, HPP can modify the texture, appearance and colour of meats (Cheftel & Culioli, 1997). Volatile compounds released from foods are closely related to their aroma and can be determined to monitor their quality and safety. Raw meat has little aroma (Shahidi, Rubin, & D’Souza, 1986). However, during cooking, thermally induced reactions occur resulting in the characteristic aroma of meat. According to Mottram (1998), over 1000 volatile compounds have been identified in meat, which can be grouped into chemical families such * Corresponding author. Fax: +34 913572293. E-mail address: [email protected] (E. Fernández-García). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.045
as hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids and esters, as well as heterocycles, aromatic and sulphur compounds. Lipid degradation and the Maillard reaction, together with the interaction between some products of those, are likely the main chemical reactions taking place that contribute to meat aroma. To our knowledge, in spite of the importance of the volatile fraction for quality assessment, the effects of HPP on the volatile composition of meats and meat products have not been deeply studied yet. Rivas-Cañedo, Fernández-García, and Nuñez (2009a) reported changes in the volatile fraction of beef and chicken meat after a 400 MPa for 10 min at 12 °C treatment, most of which were associated with the alteration of the metabolism and growth of microorganisms commonly found in meats. Regarding extraction techniques, the methods usually employed to analyze the volatile fraction of beef include dynamic headspace (Elmore et al., 2004), simultaneous distillation–extraction (Raes et al., 2003), and solid-phase microextraction (Stetzer, Cadwallader, Singh, Mckeith, & Brewer, 2008). It is generally accepted that there is no ideal method for the extraction of volatile compounds, and that the use of different extraction techniques leads to different volatile profiles of the same food product (Mallia, Fernández-García, & Bosset, 2005). For those reasons, comparative studies may be a necessary step in order to select the technique providing a better understanding of the effect of any treatment on the volatile profile, which has been one of the objectives of this study. Dynamic headspace extraction (DHE), often referred to as ‘‘purge and trap”, is an extraction and pre-concentration technique
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in which the volatile compounds of the gas phase are continuously absorbed into an inert material or concentrated in a trap. Solidphase microextraction (SPME) is an equilibrium method which uses a retractable fibre coated with a sorbent exposed to the sample headspace (Yang & Peppard, 1994). The aim of the present work was to compare these two techniques for studying the effect of HPP on the volatile profile of beef. 2. Materials and methods 2.1. Meat samples and HPP Minced beef meat was purchased at a local supermarket, divided into three batches of six 50 g samples, wrapped in aluminium foil and vacuum packed in two multilayer plastic bags (HT 3050, Cryovac Sealed Air Corporation, Milano, Italy). Six samples (control batch) were immediately frozen at -35 °C. Another six samples (HPP batch) were subjected the day after to high pressure processing at 400 MPa for 10 min at 12 °C (come-up and comedown times 90 s and 1 s respectively) in a 100 L capacity discontinuous isostatic press at NC Hyperbaric (Burgos, Spain). The last six samples were kept under untreated (non-pressurized, untreated batch). Pressurized and untreated samples were stored at 4 °C during three days and then frozen until analysis. 2.2. Volatile compound analysis Before analysis, meat samples were thawed overnight at 5 °C and then cooked in an oven (UT6 model, Thermo, Langenselbold, Germany) to an inner temperature of 60 °C (100 °C, 10 min) in a stainless steel lidded dish as previously described (Rivas-Cañedo et al., 2009a). Volatile compounds were extracted in triplicate by DHE and SPME and analyzed by gas chromatography-mass spectrometry (GC–MS) (HP-MSD HP 5973, Agilent). 2.2.1. Dynamic headspace extraction (DHE) Ten grams of cooked meat were homogenized in a mechanical grinder (IKA Labortechnik, Staufen, Germany) with 20 g of anhydrous sodium sulfate (Na2SO4) and 20 lL of an aqueous solution of 670 mg/L cyclohexanone (Sigma–Aldrich, Alcobendas, Spain) as internal standard (Rivas-Cañedo et al., 2009a). An aliquot of the mixture (3.5 g) was subjected to volatile extraction in an automatic dynamic headspace apparatus (Purge and Trap, HP 7695, Agilent), coupled to GC–MS, for 20 min at 45 °C using helium (45 mL/min) with 10 min of previous equilibration. Volatile compounds were concentrated in a Vocarb 4000 trap, (Carbopack B/C and Carboxen 1000/1001; Tekmar, Manson, OH), maintained at 35 °C, with 4 min dry purge, and desorbed during 2 min at 260 °C through a transfer line heated at 200 °C, directly into the injection port at 220 °C with a split ratio of 20:1 and 1.4 mL/min helium flow. 2.2.2. Solid-phase microextraction (SPME) Fifteen grams of cooked meat were homogenized in a mechanical grinder with 15 g of Na2SO4 and 30 lL of an aqueous solution of 670 mg/L cyclohexanone. Twelve grams of the mixture were weighed in a 40 mL headspace glass vial sealed with a PTFE faced silicone septum (Supelco, Bellefonte, PA, USA). Vials were submerged in a thermostatic bath at 45 °C (D3 model, HAAKE, Berlin, Germany) for both equilibration and extraction phases (1 h each). An SPME manual holder equipped with a 2 cm 50/30 lm StableFlex Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/ PDMS) coated fibre (Supelco, Bellefonte, PA, USA) was inserted through the PTFE septum for headspace extraction, after which it was inserted into the GC injection port for desorption (260 °C/ 10 min in splitless mode). Before use, the fibre was conditioned
in the injection port of the GC (270 °C/1 h) as recommended by manufacturer. After each run, the fibre was cleaned up to avoid carry-over problems and periodically, fibre sensitivity was tested with an aqueous solution of our internal standard. All analyses were run using the same fibre unit. 2.2.3. Gas chromatography–mass spectrometry Chromatographic separation was carried out in a Zebron 100% polyethylene glycol capillary column (60 m long; 0.25 mm i.d; 0.50 lm film thickness; ZB-WAX plus, Phenomenex, Torrance, CA) with 1 mL/min helium flow. Injection port temperature was 220 °C for DHE, as described earlier (Rivas-Cañedo et al., 2009a), and 260 °C for SPME analysis, to assure a complete desorption of the volatile compounds from the fibre. Part of the temperature program was common to both extraction techniques: 16 min at 45 °C, first ramp 4 °C/min to 110 °C, 9 min at 110 °C, second ramp at 15 °C/min to 230 °C and 3 min at 230 °C. A final ramp to 250 °C at 10 °C/min and 2 min at 250 °C was added to the method used for SPME samples in order to allow the elution of the higher boiling point compounds. Detection was performed with electron impact ionization, with 70 eV ionization energy operating in the full-scan mode from 33 to 280 amu at 2.97 scans/s. Interface, source and quadrupole temperatures were 280, 230 and 150 °C, respectively. Compound identification was carried out by injection of commercial standards, by spectra comparison using the Wiley7Nist05 Library (Wiley & Sons Inc., Germany), and/or by calculation of linear retention indexes (LRI) relative to a series of alkanes (C5-C20). The sums of abundances of up to four characteristic ions per compound were used for semi-quantitative determination. The abundances have been referred to the internal standard (compound peak area multiplied by 103 and divided by the internal standard peak area). 2.3. Statistical analysis Statistics was performed with the SPSS Win 12.0 software (SPSS Inc., Chicago, IL). Sums of abundances by chemical families were calculated, namely linear and branched-chain hydrocarbons, linear and branched-chain aldehydes, methyl ketones, diketones, miscellaneous ketones, primary alcohols (except ethanol), secondary alcohols, enols, branched-chain alcohols, miscellaneous alcohols, benzene compounds, sulphur compounds, ethyl esters, terpenoids, heterocycles, carboxylic acids and miscellaneous compounds, in order to make the results more manageable and ease comprehension. Abundances of volatile compounds were subjected to analysis of variance (ANOVA) using treatment and headspace extraction method as main effects. In a subsequent step, and due to the very significant differences found depending on the technique used, the effect of the treatment (control, refrigerated untreated and refrigerated HPP) on the volatile profile of cooked beef was studied by means of one-way ANOVA for each extraction method. Tukey’s test was used for mean comparison. In all cases, significance was assigned at P < 0.05. 3. Results Table 1 lists the 65 compounds detected by means of the two extraction techniques, in the volatile fraction of control, refrigerated untreated and HPP-treated beef after cooking, ordered by chemical family and together with their chromatographic indices, the ions used for quantitation, the method of extraction, and the significance of the effects, i.e. method of extraction (ME) and treatment (T). DHE allowed the detection of 33 volatile compounds, whereas 62 compounds were detected in samples extracted by
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Table 1 Compounds identified in the volatile fraction of cooked control and refrigerated (untreated and pressurized) beef meat extracted by dynamic headspace (DHE) and solid-phase microextraction (SPME). Volatile compound
LRIa
QIb
IDc
Hydrocarbons Pentane Hexane Heptane Octane 2,2,4,6,6-Pentamethylheptane
500 600 700 800 945
43, 57, 43, 43, 57,
42, 41, 71, 57, 56,
41, 43, 57, 85, 71,
Aldehydes Ethanal Pentanal Hexanal Heptanal Octanal Nonanal 2-Methylbutanal 3-Methylbutanal
708 986 1096 1201 1303 1413 917 920
44, 58, 56, 70, 84, 57, 57, 44,
43, 57 44, 44, 56, 56, 58, 58,
42, 41 57, 57, 57 43, 41, 41,
Ketones 2-Propanone 2-Butanone 2,3-Butanedione 2,3-Pentanedione 3-Hydroxy-2-butanone
819 908 985 1072 1306
58, 43, 43, 43, 45,
43, 72, 86, 57, 43,
Alcohols Ethanol 1-Propanol 1-Butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octanol 1-Dodecanol 2-Propanol 2-Butanol 2-Pentanol 3-Methyl-1-butanol 1-Penten-3-ol 1-Octen-3-ol 2,6-Dimethyl-7-octen-2-ol 1-Methoxy-2-propanol 2-Butoxyethanol 2-Ethyl-1-hexanol 2,3-Butanediol
944 1059 1169 1264 1364 1476 1576 1984 935 1045 1137 1220 1181 1470 1485 1146 1430 1504 1597
45, 59, 56, 42, 56, 70, 55, 55, 45, 45, 45, 55, 57, 57, 59, 45, 57, 57, 45,
46, 60, 43 55, 43, 56, 56, 43, 43, 59, 55, 70, 41 72, 55, 47, 45, 43, 43,
Esters Ethyl acetate Ethyl butanoate Ethyl lactate
896 1050 1355
Benzene compounds Toluene 1,3-Bis(1,1-dimethylethyl)benzene Benzaldehyde Phenylethanal Benzyl alcohol Phenol
57 56 41 71 85
ST, ST, ST, ST, ST,
Method of extraction
MS MS MS MS MS
DHE, DHE, DHE DHE, DHE,
SPME SPME
DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME SPME SPME
42, 39 57, 42 42, 87 100, 42 88, 42
MS ST, MS ST, MS MS MS
DHE, SPME DHE, SPME DHE, SPME SPME DHE, SPME
43, 42 42, 41
ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS MS MS MS ST, MS MS ST, MS MS MS MS MS MS
43, 45, 61, 70 71, 43, 88, 60 45, 75, 43
1055 1450 1560 1683 1923 2045
Heterocycles Pyridine Pyrazine 2-Pentylfuran Pyrrol
55 86 43
ME
T
***
ns ns ns ns
ns *
SPME SPME
MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS
72 55
Significance of effectsd
* ***
*
***
***
***
**
***
***
*
ns ns ns
ns * ***
**
***
***
ns
**
***
**
***
***
***
***
***
***
DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME SPME DHE, SPME DHE, SPME SPME SPME DHE,SPME DHE, SPME SPME SPME DHE, SPME DHE, SPME SPME
***
***
MS MS MS
91, 92, 65, 93 175, 57, 190, 176 105, 106, 77, 51 91, 65, 92, 120 79, 108, 107, 77 94, 66, 65, 39
1202 1228 1240 1540
79, 80, 81, 67,
52, 53, 82, 39,
Sulphur compounds Carbon disulphide Dimethyl sulphide Methyl thioacetate Dimethyl sulfoxide Dimethyl sulphone
734 753 1062 1621 1967
76, 62, 43, 63, 79,
78, 47, 90, 78, 94,
Terpene derivatives Exobornyl acetate Terpene derivative
1625 1670
Fatty acids Acetic acid
1470
41, 41, 41, 69, 69, 41, 43, 43 42, 85, 43, 75, 41, 41, 57
70 55 43 70 41 59 41 43 55 83 42 87 70
***
***
***
ns
***
***
***
**
***
ns ns ns ns
ns *** ** ***
***
***
*
***
***
***
***
***
***
***
ns
***
***
***
*
**
ns
***
***
SPME SPME SPME
***
***
***
***
***
***
MS MS MS ST, MS MS MS
DHE, SPME DHE, SPME DHE, SPME SPME SPME SPME
**
ns
***
**
***
***
***
***
***
**
***
*
51, 78 52, 51 138, 53 41
MS MS MS MS
SPME SPME SPME SPME
***
ns
***
***
77 46, 47, 45, 91,
MS MS MS MS MS
95, 136, 121, 93 71, 81, 95, 55 43, 45, 60, 42
61 45 46 92
***
**
***
ns
DHE DHE, SPME SPME SPME SPME
***
ns
***
***
MS MS
SPME SPME
***
***
***
ns
MS
SPME
***
***
***
**
***
ns
***
***
(continued on next page)
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Table 1 (continued) Volatile compound
LRIa
QIb
Propanoic acid 2-Methylpropanoic acid Butanoic acid Hexanoic acid Octanoic acid Nonanoic acid
1555 1583 1644 1861 2076 2182
74, 41, 60, 60, 60, 60,
Miscellaneous Acetonitrile c-Butyrolactone Naphthalene
1016 1678 1809
41, 40, 39 42, 86, 41, 56 128, 127, 129
45, 43, 73, 73, 73, 73,
57, 79, 41, 41, 43, 41,
56 88 45 87 41 57
IDc
Method of extraction
Significance of effectsd ME
T
MS MS ST, MS ST, MS ST, MS MS
SPME SPME SPME SPME SPME SPME
***
***
***
***
***
***
MS MS MS
DHE SPME DHE, SPME
***
***
*
***
ns ns
**
***
*
ns ***
ns
a
LRI: linear retention indices, calculated in relation to the retention time of n-alkane (C5–C20) series. QI: ions used for quantification. PI: peak identification: ST, comparison of spectra and retention time with commercial standards; MS, tentatively identified by spectra comparison using the Wiley Library. d Significance of effects (ME, method of extraction; T, treatment): ns, non significant. * P < 0.05. ** P < 0.01. *** P < 0.001. b
c
SPME. Sixty-one compounds were significantly affected by the extraction technique and 41 compounds were affected by the treatment, i.e. refrigeration and/or HPP. In order to highlight the differences between the extraction techniques in a simple and immediate way, Fig. 1 depicts the comparison of the relative percentages of the main chemical families present in beef samples. DHE extracted 2,3-butanedione (diacetyl), low MW alkanols and ethanal more efficiently than SPME whereas the latter extracted aldehydes, alcohols, sulphur- and benzene compounds, ethyl esters and fatty acids more efficiently. The 12 compounds affected by treatment, as extracted by DHE (Table 2), are also to be found in Table 3 together with another 29 compounds affected by treatment, as extracted by SPME. Twenty-three compounds were found to significantly increase during refrigerated storage as compared with control (day 0) samples, especially when using SPME as the extraction technique (Table 3). Most of these compounds did not increase significantly during refrigerated storage in samples subjected to HPP. This includes aldehydes like ethanal and phenylethanal, alcohols like ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-methyl-1-butanol, 1-methoxy-2-propanol, esters, some low molecular weight fatty acids,
and some miscellaneous compounds like benzaldehyde, benzyl alcohol and c-butyrolactone. Only five out of the 23 compounds, ethanal (by DHE, Table 2) and 3-methylbutanal, 1-propanol, pyrazine and dimethyl sulphone (in SPME, Table 3), increased significantly during refrigerated storage of both HPP treated and untreated. Ten compounds significantly decreased during refrigerated storage, including dimethyl sulphide as determined by DHE (Table 2). Among them, diacetyl, 3-hydroxy-2-butanone (acetoin) and 2butanone (all of them metabolically related compounds), decreased to a lesser extent when samples had been subjected to HPP. On the other hand, compounds like hexanal, 2,3-pentanedione, 1-pentanol, 1-penten-3-ol, and 1-octen-3-ol tended to decrease more in HPP samples than in refrigerated samples, although differences were not statistically significant. Only three compounds, 2-methylbutanal, 2,3-butanediol and dimethyl sulphide (as extracted by SPME) were found to significantly increase during refrigerated storage only in samples subjected to HPP, while some compounds like pentane, pentanal, 1-hexanol, 2pentylfuran, and butanoic and hexanoic acids, significantly decreased only in HPP samples as compared with control samples.
Reelattivee % of vollatille com c mpo ound ds
DHE
SPME
100%
100%
80%
80%
60%
60%
40%
40%
20%
20%
0%
0%
Control
Untreated HPP-treated
Control
Untreated HPP-treated
Fig. 1. Comparison of the volatile fractions of control, untreated and HPP-treated beef meat, as extracted by DHE and SPME. Bars represent the relative percentages of the main chemical families: linear alkanes, linear aldehydes, methyl ketones, diketones and related compounds, primary alcohols (except ethanol), secondary alcohols, benzene compounds, sulphur compounds, ethyl esters, and fatty acids.
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Untreated Mean ± SD
Table 3 Mean abundancesA (±SD) of the cooked beef volatile compounds significantly affected by treatment, as extracted by solid-phase microextraction.
Pressurized Mean ± SD
Compounds significantly increasing with refrigerated storage Ethanal 46.15c ± 3.07 164.9a ± 18.46 10596a ± 1829 Ethanol 1018b ± 313 1-Propanol 0c ± 0 10.67a ± 0.60 2-Butanol 11.65b ± 0.68 18.16a ± 0.76 Benzaldehyde 2.27b ± 0.29 5.62a ± 0.71
89.64b ± 16.23 2423b ± 287 1.78b ± 1.01 14.18ab ± 3.80 2.90b ± 0.43
Compounds significantly decreasing with refrigerated storage 30.13b ± 2.29 2-Butanone 47.81a ± 3.88 2,3-Butanedione 2609a ± 116 25.49c ± 5.03 0b ± 0 3-Hydroxy-2-butanone 38.06a ± 27.16 Pentanal 1.39a ± 0.06 0.59ab ± 0.65 15.65b ± 2.64 1-Pentanol 26.48a ± 0.74 1-Penten-3-ol 3.14a ± 0.07 1.50b ± 0.36 Dimethyl sulphide 11.15a ± 0.92 5.52b ± 0.56
32.19b ± 6.20 310.4b ± 64.24 2.31b ± 2.05 0.33b ± 0.28 13.47b ± 4.26 1.33b ± 0.59 11.48a ± 0.81
Control Mean ± SD
Compounds significantly increasing with treatments Ethanal 20.26b ± 1.61 29.24a ± 2.24 2.46b ± 0.16 2.54b ± 0.59 2-MethylbutanalB 3-Methylbutanal 5.83b ± 0.54 8.63a ± 1.18 Ethanol 61.86b ± 23.59 621.3a ± 67.47 1-Propanol 0.37c ± 0.13 5.06a ± 0.22 2-Propanol 11.24b ± 1.25 14.88a ± 0.72 2-Butanol 6.42b ± 0.39 12.68a ± 1.03 2-Pentanol 0.78b ± 0.48 1.88a ± 0.49 3-Methyl-1-butanol 1.66b ± 0.08 4.69a ± 0.48 1-Methoxy-2-propanol 1.73b ± 0.22 6.32a ± 1.10 2,3-Butanediol B 54.12b ± 3.03 67.47ab ± 3.93 Ethyl acetate 6.05b ± 1.26 105.8a ± 25.43 Ethyl butanoate 0.06b ± 0.10 8.43a ± 2.38 Ethyl lactate 1.48b ± 0.89 20.17a ± 6.41 Methyl thioacetate 1.79b ± 0.58 5.40a ± 1.82 Exobornyl acetate 0.82b ± 0.20 2.22 a ± 0.65 Acetic acid 94.18b ± 4.82 307.6a ± 37.57 6.39a ± 0.29 Propanoic acid 5.08b ± 0.40 2-Methylpropanoic 4.75b ± 0.26 7.04a ± 0.35 acid 64.84a ± 7.81 Benzaldehyde 22.56b ± 0.32 Phenylethanal 0.81b ± 0.70 113.1a ± 35.01 Benzyl alcohol 2.80b ± 0.27 4.91a ± 0.89 Pyrazine 1.38b ± 0.06 2.67a ± 0.26 Dimethyl sulphone 1.46c ± 0.16 5.25a ± 0.55 Dimethyl sulphide B 30.80b ± 6.40 23.14b ± 0.68 c-Butyrolactone 23.66b ± 1.16 48.19a ± 7.95
A Calculated as the sum of abundances of up to four characteristic ions and referred to the IS (compound area 1000/IS area). Means of triplicate experiments. abc Means followed by different letters differ significantly (P < 0.05).
4. Discussion 4.1. Comparison of extraction techniques The results of this study show in the first place that the volatile profile of cooked beef cannot be established with certainty since it has been proven strongly dependent on the method of extraction. The susceptibility of the compounds to extraction depends on the chemical nature, especially its polarity and volatility (Mallia et al., 2005). Although, to our knowledge, the literature dealing with the comparison of different extraction methods of volatile compounds in meat and meat products is scarce (Elmore, Papantoniou, & Mottram, 2001; Garcia-Esteban, Ansorena, Astiasaran, Martin, & Ruiz, 2004), the existing literature can provide evidence that the volatile profile depends on the employed methodology. As an example, the general volatile fraction of beef consisted of aldehydes, ketones, pyrazines and furans, if using simultaneous distillation-extraction, (Raes et al., 2003), or composed of aldehydes, ketones, hydrocarbons and sulphur compounds if using DHE (Insausti, Goñi, Petri, Gorraiz, & Beriain, 2005). Within the same extraction method, the working conditions (such as temperature or trapping material) may also influence the volatile profile, which can further complicate drawing of conclusions and comparison of the results with bibliographic data. Consequently with all this, the estimation of the effect of any technological treatment (HPP in our case) on the evolution of the volatile fraction of meat can diverge when considering data obtained by different methods and must always be taken with caution. Undoubtedly, in the conditions of our study, SPME proved to be a more sensitive tool than DHE to detect the changes underwent by the beef meat samples subjected to the different treatments. When the DHE method was used, the volatile fraction of control samples (neither stored nor pressurized) mainly consisted of approximately 70% of diketones, 20% of methyl ketones and 6% of secondary alcohols (Fig. 1). The volatile fraction of control samples when extracted by SPME was comparable but somehow more equilibrated, composed of 50% of diketones, 25% of methyl ketones, 9% of linear aldehydes and 9% of fatty acids. As it will be further discussed, the volatile profile of beef changed during refrigerated storage, but the estimation, both quantitative and qualitative, of the degree of change, necessarily depended
Untreated Mean ± SD
Compounds significantly decreasing with treatments PentaneC 2.76a ± 0.33 2.07ab ± 0.46 PentanalC 19.47a ± 2.76 13.16ab ± 6.15 Hexanal 238.3a ± 21.58 119.4b ± 48.33 2-Propanone 665.43a ± 83.19 448.1b ± 16.52 2-Butanone 205.0a ± 20.49 133.7b ± 11.86 2,3-Butanodione 292.7a ± 8.17 18.81c ± 1.25 2,3-Pentanodione 3.06a ± 0.43 1.60b ± 0.68 3-Hydroxy-2-butanone 1223a ± 63 13.91c ± 1.39 1-Pentanol 73.61a ± 9.04 42.60b ± 11.44 1-Penten-3-ol 7.15a ± 1.07 3.73b ± 0.74 1-Octen-3-ol 18.88a ± 0.45 11.51b ± 1.52 1-HexanolC 8.13a ± 0.52 6.71ab ± 2.13 2-PentylfuranC 1.22a ± 0.14 0.96ab ± 0.36 Butanoic acidC 87.73a ± 8.84 96.00a ± 2.13 Hexanoic acidC 21.19a ± 4.65 20.55ab ± 3.41 A B C abc
Pressurized Mean ± SD 19.16b ± 6.53 4.54a ± 0.91 10.43a ± 1.24 95.96b ± 77.00 0.94b ± 0.19 14.45ab ± 1.73 8.29b ± 0.86 0.78b ± 0.27 2.12b ± 0.25 2.65b ± 0.63 76.01a ± 8.20 5.53b ± 3.46 0.60b ± 0.46 1.39 b ± 0.69 1.36b ± 0.87 0.74b ± 0.12 100.1b ± 16.80 3.88c ± 0.23 4.16b ± 0.48 22.93b ± 5.20 0.60b ± 0.27 3.77ab ± 0.16 2.13a ± 0.30 3.55b ± 0.98 41.59a ± 0.83 33.19b ± 4.01 1.37b ± 0.52 7.26b ± 1.16 80.28b ± 22.39 626.8 a ± 56.31 194.2ab ± 36.95 116.6b ± 40.79 1.09b ± 0.27 381.4b ± 178.5 28.27b ± 3.95 3.11b ± 0.50 8.96b ± 2.15 4.06b ± 0.48 0.40b ± 0.10 60.30b ± 6.86 12.17b ± 1.87
Means of triplicate experiments. Compounds increasing significantly only in HPP samples. Compounds decreasing significantly only in HPP samples. Means followed by different letters differ significantly (P < 0.05).
on the extraction technique. The comparable volatile profiles of control samples provided by both techniques turned completely different after the transformations occurred during storage, if they had been extracted by DHE or by SPME (Fig. 1, untreated). These transformations were likely due to microbial growth. DHE gave out a profile consisting of 50% of methyl ketones, 20% of linear aldehydes and 17% of secondary alcohols, while the SPME profile pointed to a deep microbial alteration, with 30% of methyl ketones, approximately 30% of low molecular fatty acids, 8% of esters, and low levels of benzene compounds. The very significant decrease in diacetyl and related compounds during storage was validated by both extraction techniques alike, DHE providing better efficiency in extracting diacetyl whereas acetoin was more efficiently extracted by SPME. This fact had been observed in cheese by Mallia et al. (2005). The volatile profile of HPP samples did not undergo the changes of untreated meat during storage at 4 °C, especially based on the SPME data. In fact, the volatile profile of control and stored HPP
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samples look very much alike when extracted by SPME (Fig. 1), with a 44% of methyl ketones, 22% of diketones and 13% of fatty acids. However, if extracted by DHE (Fig. 1), control and HPP samples differed considerably, yet mainly due to the decrease in diketones (down to 18%) and the increase in 2-alcanols (up to 37%) and methyl ketones (up to 32%). Although more information can be obtained with SPME, one of the main drawbacks of this technique is that fastidious artefact peaks can form. These artefacts may have different origins, such as thermal oxidation occurring during the desorption step (Lestremau, Andersson, & Desauziers, 2004) or siloxane formation (Perera, Marriot, & Galbally, 2002). In our opinion, the moisture of some food products can also affect the formation of siloxanes. However, to our knowledge no literature dealing with this particular effect of water has been published. To decrease artefact formation in high moisture samples as well as to enhance volatilization by salting out, the use of anhydrous salts for sample preparation (as carried out in the present study) is advisable. 4.2. Effect of high pressure processing Pressurization reduced the formation of some compounds, mainly as a result of alterations on the microbial balance. As expected, higher levels of many compounds resulting from microbial fermentations were observed in untreated samples after 3 days of storage (Tables 2 and 3). Ethanal, its reduction product ethanol, and the esterification products, ethyl esters, increased dramatically. Ethanol is mainly derived from carbohydrate fermentation by different microorganims, such as lactic acid bacteria (Kandler, 1983) and yeasts. The presence of large quantities of ethanol enhanced the formation of ethyl esters in untreated samples, possibly by pseudomonas (Morales, Fernández-García, & Nuñez, 2005), staphylococci (Talon, Chastagnac, Vergnais, Montel, & Berdagué, 1998) or yeasts (Olesen & Stahnke, 2000), all well known ethyl ester producers. Most of these microorganisms are pressure sensitive (Rubio, Martínez, García-Cachán, Rovira, & Jaime, 2007) which explains the reduced ethyl ester formation observed in HPP samples, although the possibility of an inhibition of microbial esterase activity by pressure should not be overlooked. In a previous work we reported lower levels of ethanol and ethyl esters, together with lower counts of bacteria, in particular Gram-negatives and coliforms, in beef and chicken breast samples treated at 400 MPa for 10 at 12 °C in comparison with untreated samples (Rivas-Cañedo et al., 2009a). The compounds presumably originating from amino acid catabolism followed two different trends. While 3-methyl-1-butanol, 2methylpropanoic acid, benzaldehyde and phenylacetaldehyde increased during the 3-day storage at 4 °C in untreated samples, 2methylbutanal and dimethyl sulphide increased only in HPP-treated samples. Branched-chain aldehydes 2- and 3-methylbutanal are generated from isoleucine and leucine by transamination or Strecker degradation (Christensen, Dudley, Pederson, & Steele, 1999; Montel, Masson, & Talon, 1998) and can be further reduced to 2- and 3-methyl-1-butanol through the activity of bacterial deshydrogenases or chemical reactions (Mollimard & Spinnler, 1996) or oxidised to acids. 2-Methylpropanoic acid can be produced by the same path from oxidation of valine and dimethyl sulphide results from methionine degradation (McSweeney & Sousa, 2000). Benzaldehyde and phenylethanal can result from the catabolism of phenylalanine by microbial activity, for example in pseudomonas, some lactic acid bacteria (Groot & de Bont, 1998) and moulds (McSweeney & Sousa, 2000). In a previous study, a decrease in benzaldehyde in HPP-treated dry-cured ham (400 MPa, 10 min, 12 °C) was observed (Rivas-Cañedo, Fernández-García, & Nuñez, 2009b). In agreement with these results, Campus, Flores, Martínez, and Toldrá (2008) reported a decrease in phenylethanal
in HPP-treated dry cured pork loin (300–400 MPa, 10 min, 20 °C) together with a decrease in the activity of aminopeptidases. Our results could be pointing to a discriminating effect of HPP on key enzymes or on different microbial groups with different amino acid metabolic activities. On the other hand, an effect of the state of oxidation of treated and non treated vacuum packed meat can not be discarded. The lower levels found after 3 days of refrigerated storage of compounds associated with lipid oxidation, such as 1-alcanals, 1alcohols, fatty acids and furans, as well as 1-penten-3-ol and 1-octen-3-ol, are difficult to explain (Tables 2 and 3). Levels were even lower in HPP samples. There seems to be a controversy in relation to the effect of HPP on lipid oxidation. On one hand, HPP is supposed to enhance lipid oxidation on muscle products. Thus, Cheah and Ledward (1996) reported increased oxidation in pork treated above 300 MPa for 20 min at ambient temperature. However, Orlien, Hansen, and Skibsted (2000) did not observe any significant increase in the levels of compounds supposedly resulting from lipid oxidation in chicken breast subjected to treatments up to 500 MPa. Besides lipo-oxidation, these compounds might also derive from the action of lipolytic microorganisms. For example, 1penten-3-ol may result from the oxidation of linolenic acid (Elmore et al., 2004) and 1-octen-3-ol might originate from linoleic acid and linoleate autooxidation (Ulrich & Grosch, 1987). The latter might also come from linoleic and linolenic acid catabolism by moulds (Mollimard & Spinnler, 1996). Nevertheless, higher levels of 1-octen-3-ol were found in beef meat inoculated with Serratia proteamaculans, Carnobacterium divergens and Pseudomonas fragi (Ercolini, Russo, Nasi, Ferranti, & Villani, 2009). HPP, in combination with vacuum storage (low oxygen content and therefore low redox potential), might affect the metabolism and growth of the microorganisms involved in lipid catabolism. Another possibility is that a phenomenon of scalping (adsorption of compounds into the packaging material) could be occurring in the samples packaged during a longer period. The behaviour of diacetyl and acetoin deserves a comment. A pronounced decrease of these compounds was observed during storage, together with an increase of their reduction products 2,3-butanediol and 2-butanol, the decrease being less steep in HPP samples. High amounts of these compounds have been previously found in meat (Raes et al., 2003) and are generally associated with lactose and citrate metabolism (Cogan, 1995; McSweeney & Sousa, 2000) by the action of different bacteria, especially lactic acid bacteria and staphylococci (Berdagué, Monteil, Montel, & Talon, 1993). Dainty, Edwards, Hibbard, and Marnewick (1989) reported the production of diacetyl and acetoin in meat inoculated with Enterobacteriaceae or Brochothrix thermosphacta. Other authors suggested the catabolism of aspartate (Kieronczyk, Skeie, Langsrud, Le Bars, & Yvon, 2004) or the action of meat enzymes (Stahnke, 1995), as alternate origins of diacetyl. The results observed in this study are consistent with a previous report (RivasCañedo et al., 2009a), where higher levels of diacetyl were observed in HPP meat samples in comparison with control samples. The increase of 2,3-butanediol and 2-butanol indicates that a reduction of diacetyl is occurring during storage, either by enzyme activity or by a modification of the redox potential.
5. Conclusions Both techniques DHE and SPME proved suitable for monitoring the changes in the volatile fraction caused by refrigeration and HPP, although SPME provides wider information. Storage caused changes in the volatile fraction of beef after cooking. Most of these changes might be related to the alteration of the microbiota or of the enzymatic activities. Although the volatile fraction of HPP
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samples underwent some changes, pressurization helped to maintain a volatile profile closer to that of fresh meat (not been previously stored). Acknowledgements This work was supported by projects CPE 03-012 (INIA), TEMINYSA S-0505/AGR0314 (Comunidad de Madrid) and CARNISENUSA CSD 2007-00016 (Consolider). The authors thank NC Hyperbaric (Burgos, Spain) for the processing of samples. INIA, for granting Ana Rivas-Cañedo with a predoctoral scholarship, and B. Rodríguez and M. de Paz, for their valuable technical assistance, are also acknowledged. References Aymerich, T., Picouet, P. A., & Monfort, J. M. (2008). Decontamination technologies for meat products. Meat Science, 78, 114–129. Berdagué, J. L., Monteil, P., Montel, M. C., & Talon, R. (1993). Effects of starter cultures on the formation of flavour compounds in dry sausage. Meat Science, 35, 275–287. Campus, M., Flores, M., Martínez, A., & Toldrá, F. (2008). Effect of high pressure treatment on colour, microbial and chemical characteristics of dry cured loin. Meat Science, 80, 1174–1181. Carlez, A., Rosec, J. P., Richard, N., & Cheftel, J. C. (1993). High pressure inactivation of Citrobacter freundii, Pseudomonas fluorescens and Listeria innocua in inoculated minced beef muscle. Lebensmittel Wissenschaft und Technologie, 26, 357–363. Cheah, P. B., & Ledward, D. A. (1996). High pressure effects on lipid oxidation in minced pork. Meat Science, 43, 123–134. Cheftel, J. C., & Culioli, J. (1997). Effects of high pressure on meat: A review. Meat Science, 46, 211–236. Christensen, J. E., Dudley, E. G., Pederson, J. A., & Steele, J. L. (1999). Peptidases and amino acid catabolism in lactic acid bacteria. Antonie van Leeuwenhoek, 76, 217–246. Cogan, T. M. (1995). Flavour production by dairy starter cultures. Journal of Applied Bacteriology, 79, 49S–64S. Dainty, R. H., Edwards, R. A., Hibbard, C. M., & Marnewick, J. J. (1989). Volatile compounds associated with microbial growth on normal and high pH beef stored at chill temperatures. Journal of Applied Bacteriology, 66, 281–289. Elmore, J. S., Papantoniou, E., & Mottram, D. S. (2001). A comparison of headspace entrainment on Tenax with solid phase microextraction for the analysis of the aroma volatiles of cooked beef. In R. L. Rouseff & K. R. Cadwallader (Eds.), Headspace analysis of foods and flavors: Theory and practice (pp. 125–132). New York: Kluwer Academic/Plenum Publishers. Elmore, J. S., Warren, H. E., Mottram, D. S., Scollan, N. D., Enser, M., Richardson, R. I., et al. (2004). A comparison of the aroma volatiles and fatty acid compositions of grilled beef muscle from Aberdeen Angus and Holstein-Friesian steers fed diets based on silage or concentrates. Meat Science, 68, 27–33. Ercolini, D., Russo, F., Nasi, A., Ferranti, P., & Villani, F. (2009). Mesophilic and psychrotrophic bacteria from meat and their spoilage potential in Vitro and in beef. Applied and Environmental Microbiology, 75, 1990–2001. Garcia-Esteban, M., Ansorena, D., Astiasaran, I., Martin, D., & Ruiz, J. (2004). Comparison of simultaneous distillation extraction (SDE) and solid-phase microextraction (SPME) for the analysis of volatile compounds in dry-cured ham. Journal of the Science of Food and Agriculture, 84, 1364–1370. Groot, M. N. N., & de Bont, J. A. M. (1998). Conversion of phenylalanine to benzaldehyde initiated by an aminotransferase in Lactobacillus plantarum. Applied and Environmental Microbiology, 64, 3009–3013. Hogan, E., Kelly, A. L., & Sun, D. W. (2005). High pressure processing of foods: An overview. In D. W. Sun (Ed.), Emerging technologies for food processing (pp. 3–31). London: Elsevier Academic Press. Insausti, K., Goñi, V., Petri, E., Gorraiz, C., & Beriain, M. J. (2005). Effect of weight at slaughter on the volatile compounds of cooked beef from Spanish cattle breeds. Meat Science, 70, 83–90.
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Kandler, O. (1983). Carbohydrate metabolism in lactic acid bacteria. Antonie van Leeuwenhoek, 49, 209–224. Kieronczyk, A., Skeie, S., Langsrud, T., Le Bars, D., & Yvon, M. (2004). The nature of aroma compounds produced in a cheese model by glutamate dehydrogenase positive Lactobacillus INF15D depends on its relative aminotransferase activities towards the different amino acids. International Dairy Journal, 14, 227–235. Lestremau, F., Andersson, F. A. T., & Desauziers (2004). Investigation of artefact formation during analysis of volatile sulphur compounds using solid phase microextraction (SPME). Chromatographia, 59, 607–613. Mallia, S., Fernández-García, E., & Bosset, J. O. (2005). Comparison of purge and trap and solid phase microextraction techniques for studying the volatile aroma compounds of three European PDO hard cheeses. International Dairy Journal, 15, 741–758. McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways for the production of flavour compounds in cheeses during ripening: A review. Lait, 80, 293–324. Mollimard, P., & Spinnler, H. E. (1996). Review: Compounds involved in the flavor of surface mold-ripened cheeses: Origins and properties. Journal of Dairy Science, 79, 169–184. Montel, M. C., Masson, F., & Talon (1998). Bacterial role in flavour development. Meat Science, 49, S111–S123. Morales, P., Calzada, J., Ávila, M., & Nuñez, M. (2008). Inactivation of Escherichia coli O157:H7 in ground beef by single-cycle and multiple-cycle high-pressure treatments. Journal of Food Protection, 71, 811–815. Morales, P., Fernández-García, E., & Nuñez, M. (2005). Production of volatile compounds in cheese by Pseudomonas fragi strains of dairy origin. Journal of Food Protection, 68, 1399–1407. Mottram, D. S. (1998). Flavour formation in meat and meat products: A review. Food Chemistry, 62, 415–424. Olesen, P. T., & Stahnke, L. H. (2000). The influence of Debaryomyces hansenii and Candida utilis on the aroma formation in garlic spiced fermented sausages and model minces. Meat Science, 56, 357–368. Orlien, V., Hansen, E., & Skibsted, L. H. (2000). Lipid oxidation in high pressure processed chicken breast muscle during chill storage: Critical working pressure in relation to oxidation mechanism. European Food Research and Technology, 211, 99–104. Patterson, M. F. (2005). A review. Microbiology of pressure-treated foods. Journal of Applied Microbiology, 98, 1400–1409. Perera, R. M. M., Marriot, P. J., & Galbally, I. E. (2002). Headspace solid-phase microextracion-comprehensive two-dimensional gas chromatography of wound induced plant volatile organic compound emissions. The Analyst, 127, 1601–1607. Raes, K., Balcaen, A., Dirinck, P., De Winne, A., Claeys, E., Demeyer, D., et al. (2003). Meat quality, fatty acid composition and flavour analysis in Belgian retail beef. Meat Science, 65, 1237–1246. Rivas-Cañedo, A., Fernández-García, E., & Nuñez, M. (2009a). Volatile compounds in fresh meats subjected to high pressure processing: Effect of the packaging material. Meat Science, 81, 321–328. Rivas-Cañedo, A., Fernández-García, E., & Nuñez, M. (2009b). Volatile compounds in dry-cured Serrano ham subjected to high pressure processing. Effect of the packaging material. Meat Science, 82, 162–169. Rubio, B., Martínez, B., García-Cachán, M. D., Rovira, J., & Jaime, I. (2007). Effect of high pressure preservation on the quality of dry cured beef ‘‘Cecina de León”. Innovative Food Science and Emerging Technologies, 8, 102–110. Shahidi, F., Rubin, L. J., & D’Souza, L. A. (1986). Meat flavour volatiles: A review of the composition, techniques of analysis, and sensory evaluation. CRC Critical Reviews in Food Science and Nutrition, 24, 141–243. Stahnke, L. H. (1995). Dried sausages fermented with Staphylococcus xylosus at different temperatures and with different ingredient levels. Part II. Volatile components. Meat Science, 41, 193–209. Stetzer, A. J., Cadwallader, K., Singh, T. K., Mckeith, F. K., & Brewer, M. S. (2008). Effect of enhancement and ageing on flavor and volatile compounds in various beef muscles. Meat Science, 79, 13–19. Talon, R., Chastagnac, C., Vergnais, L., Montel, M. C., & Berdagué, J. L. (1998). Production of esters by Staphylococci. International Journal of Food Microbiology, 45, 143–150. Ulrich, F., & Grosch, W. (1987). Identification of the most intense volatile flavour compounds formed during autoxidation of linoleic acid. Zeitschrift für Lebensmitteluntersuchung und Forschung A, 184, 277–282. Yang, X., & Peppard, T. (1994). Solid-phase microextraction for flavor analysis. Journal of Agricultural and Food Chemistry, 42, 1925–1930.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Volatile compounds in ground beef subjected to high pressure processing: A comparison of dynamic headspace and solid-phase microextraction Ana Rivas-Cañedo, Cristina Juez-Ojeda, Manuel Nuñez, Estrella Fernández-García * Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña, km. 7, Madrid 28040, Spain
a r t i c l e
i n f o
Article history: Received 17 August 2009 Received in revised form 3 February 2010 Accepted 18 July 2010
Keywords: High pressure processing Volatile compounds Beef SPME Dynamic headspace extraction
a b s t r a c t The effect on the volatile profile of cooked beef meat, previously subjected to high pressure (400 MPa, 10 min at 12 °C) followed by a 3-d refrigerated storage, was investigated by comparing two extraction techniques i.e. dynamic headspace and solid-phase microextraction. Dynamic headspace was more efficient in extracting 2,3-butanedione and secondary alcohols. Solid-phase microextraction, being more efficient in extracting substances such as 1-alcanols, ethyl esters and acids, permitted to better categorize the effects caused in the volatile fraction by refrigerated storage and high pressure processing. The volatile fraction of cooked control beef meat contained high amounts of diketones and low amounts of methyl ketones, secondary alcohols, aldehydes and fatty acids. While diketones nearly disappeared after the 3-d refrigerated storage, the other compounds together with ethanol and ethyl esters increased significantly. Pressurized beef samples underwent fewer changes than non-pressurized samples during refrigerated storage, leading to a volatile profile closer to that of control beef. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Due to its physicochemical characteristics, meat is one of the most perishable foods. The concern of achieving microbiologically safe meats and meat products, with an extended shelf-life, increases the interest in non-thermal preservation technologies such as high pressure processing (HPP). Pressure treatments yield high quality products due to their ability to kill spoilage and pathogenic microorganisms as well as to inactivate some key food enzymes (Hogan, Kelly, & Sun, 2005; Patterson, 2005). The effects of HPP on spoilage and pathogenic bacteria in meats have been previously reported (Carlez, Rosec, Richard, & Cheftel, 1993; Morales, Calzada, Ávila, & Nuñez, 2008). One of main advantages of HPP is that supposedly neither modifications in the taste and flavour characteristics nor changes in the nutritional value and vitamin content occur (Aymerich, Picouet, & Monfort, 2008). Nevertheless, HPP can modify the texture, appearance and colour of meats (Cheftel & Culioli, 1997). Volatile compounds released from foods are closely related to their aroma and can be determined to monitor their quality and safety. Raw meat has little aroma (Shahidi, Rubin, & D’Souza, 1986). However, during cooking, thermally induced reactions occur resulting in the characteristic aroma of meat. According to Mottram (1998), over 1000 volatile compounds have been identified in meat, which can be grouped into chemical families such * Corresponding author. Fax: +34 913572293. E-mail address: [email protected] (E. Fernández-García). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.045
as hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids and esters, as well as heterocycles, aromatic and sulphur compounds. Lipid degradation and the Maillard reaction, together with the interaction between some products of those, are likely the main chemical reactions taking place that contribute to meat aroma. To our knowledge, in spite of the importance of the volatile fraction for quality assessment, the effects of HPP on the volatile composition of meats and meat products have not been deeply studied yet. Rivas-Cañedo, Fernández-García, and Nuñez (2009a) reported changes in the volatile fraction of beef and chicken meat after a 400 MPa for 10 min at 12 °C treatment, most of which were associated with the alteration of the metabolism and growth of microorganisms commonly found in meats. Regarding extraction techniques, the methods usually employed to analyze the volatile fraction of beef include dynamic headspace (Elmore et al., 2004), simultaneous distillation–extraction (Raes et al., 2003), and solid-phase microextraction (Stetzer, Cadwallader, Singh, Mckeith, & Brewer, 2008). It is generally accepted that there is no ideal method for the extraction of volatile compounds, and that the use of different extraction techniques leads to different volatile profiles of the same food product (Mallia, Fernández-García, & Bosset, 2005). For those reasons, comparative studies may be a necessary step in order to select the technique providing a better understanding of the effect of any treatment on the volatile profile, which has been one of the objectives of this study. Dynamic headspace extraction (DHE), often referred to as ‘‘purge and trap”, is an extraction and pre-concentration technique
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in which the volatile compounds of the gas phase are continuously absorbed into an inert material or concentrated in a trap. Solidphase microextraction (SPME) is an equilibrium method which uses a retractable fibre coated with a sorbent exposed to the sample headspace (Yang & Peppard, 1994). The aim of the present work was to compare these two techniques for studying the effect of HPP on the volatile profile of beef. 2. Materials and methods 2.1. Meat samples and HPP Minced beef meat was purchased at a local supermarket, divided into three batches of six 50 g samples, wrapped in aluminium foil and vacuum packed in two multilayer plastic bags (HT 3050, Cryovac Sealed Air Corporation, Milano, Italy). Six samples (control batch) were immediately frozen at -35 °C. Another six samples (HPP batch) were subjected the day after to high pressure processing at 400 MPa for 10 min at 12 °C (come-up and comedown times 90 s and 1 s respectively) in a 100 L capacity discontinuous isostatic press at NC Hyperbaric (Burgos, Spain). The last six samples were kept under untreated (non-pressurized, untreated batch). Pressurized and untreated samples were stored at 4 °C during three days and then frozen until analysis. 2.2. Volatile compound analysis Before analysis, meat samples were thawed overnight at 5 °C and then cooked in an oven (UT6 model, Thermo, Langenselbold, Germany) to an inner temperature of 60 °C (100 °C, 10 min) in a stainless steel lidded dish as previously described (Rivas-Cañedo et al., 2009a). Volatile compounds were extracted in triplicate by DHE and SPME and analyzed by gas chromatography-mass spectrometry (GC–MS) (HP-MSD HP 5973, Agilent). 2.2.1. Dynamic headspace extraction (DHE) Ten grams of cooked meat were homogenized in a mechanical grinder (IKA Labortechnik, Staufen, Germany) with 20 g of anhydrous sodium sulfate (Na2SO4) and 20 lL of an aqueous solution of 670 mg/L cyclohexanone (Sigma–Aldrich, Alcobendas, Spain) as internal standard (Rivas-Cañedo et al., 2009a). An aliquot of the mixture (3.5 g) was subjected to volatile extraction in an automatic dynamic headspace apparatus (Purge and Trap, HP 7695, Agilent), coupled to GC–MS, for 20 min at 45 °C using helium (45 mL/min) with 10 min of previous equilibration. Volatile compounds were concentrated in a Vocarb 4000 trap, (Carbopack B/C and Carboxen 1000/1001; Tekmar, Manson, OH), maintained at 35 °C, with 4 min dry purge, and desorbed during 2 min at 260 °C through a transfer line heated at 200 °C, directly into the injection port at 220 °C with a split ratio of 20:1 and 1.4 mL/min helium flow. 2.2.2. Solid-phase microextraction (SPME) Fifteen grams of cooked meat were homogenized in a mechanical grinder with 15 g of Na2SO4 and 30 lL of an aqueous solution of 670 mg/L cyclohexanone. Twelve grams of the mixture were weighed in a 40 mL headspace glass vial sealed with a PTFE faced silicone septum (Supelco, Bellefonte, PA, USA). Vials were submerged in a thermostatic bath at 45 °C (D3 model, HAAKE, Berlin, Germany) for both equilibration and extraction phases (1 h each). An SPME manual holder equipped with a 2 cm 50/30 lm StableFlex Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/ PDMS) coated fibre (Supelco, Bellefonte, PA, USA) was inserted through the PTFE septum for headspace extraction, after which it was inserted into the GC injection port for desorption (260 °C/ 10 min in splitless mode). Before use, the fibre was conditioned
in the injection port of the GC (270 °C/1 h) as recommended by manufacturer. After each run, the fibre was cleaned up to avoid carry-over problems and periodically, fibre sensitivity was tested with an aqueous solution of our internal standard. All analyses were run using the same fibre unit. 2.2.3. Gas chromatography–mass spectrometry Chromatographic separation was carried out in a Zebron 100% polyethylene glycol capillary column (60 m long; 0.25 mm i.d; 0.50 lm film thickness; ZB-WAX plus, Phenomenex, Torrance, CA) with 1 mL/min helium flow. Injection port temperature was 220 °C for DHE, as described earlier (Rivas-Cañedo et al., 2009a), and 260 °C for SPME analysis, to assure a complete desorption of the volatile compounds from the fibre. Part of the temperature program was common to both extraction techniques: 16 min at 45 °C, first ramp 4 °C/min to 110 °C, 9 min at 110 °C, second ramp at 15 °C/min to 230 °C and 3 min at 230 °C. A final ramp to 250 °C at 10 °C/min and 2 min at 250 °C was added to the method used for SPME samples in order to allow the elution of the higher boiling point compounds. Detection was performed with electron impact ionization, with 70 eV ionization energy operating in the full-scan mode from 33 to 280 amu at 2.97 scans/s. Interface, source and quadrupole temperatures were 280, 230 and 150 °C, respectively. Compound identification was carried out by injection of commercial standards, by spectra comparison using the Wiley7Nist05 Library (Wiley & Sons Inc., Germany), and/or by calculation of linear retention indexes (LRI) relative to a series of alkanes (C5-C20). The sums of abundances of up to four characteristic ions per compound were used for semi-quantitative determination. The abundances have been referred to the internal standard (compound peak area multiplied by 103 and divided by the internal standard peak area). 2.3. Statistical analysis Statistics was performed with the SPSS Win 12.0 software (SPSS Inc., Chicago, IL). Sums of abundances by chemical families were calculated, namely linear and branched-chain hydrocarbons, linear and branched-chain aldehydes, methyl ketones, diketones, miscellaneous ketones, primary alcohols (except ethanol), secondary alcohols, enols, branched-chain alcohols, miscellaneous alcohols, benzene compounds, sulphur compounds, ethyl esters, terpenoids, heterocycles, carboxylic acids and miscellaneous compounds, in order to make the results more manageable and ease comprehension. Abundances of volatile compounds were subjected to analysis of variance (ANOVA) using treatment and headspace extraction method as main effects. In a subsequent step, and due to the very significant differences found depending on the technique used, the effect of the treatment (control, refrigerated untreated and refrigerated HPP) on the volatile profile of cooked beef was studied by means of one-way ANOVA for each extraction method. Tukey’s test was used for mean comparison. In all cases, significance was assigned at P < 0.05. 3. Results Table 1 lists the 65 compounds detected by means of the two extraction techniques, in the volatile fraction of control, refrigerated untreated and HPP-treated beef after cooking, ordered by chemical family and together with their chromatographic indices, the ions used for quantitation, the method of extraction, and the significance of the effects, i.e. method of extraction (ME) and treatment (T). DHE allowed the detection of 33 volatile compounds, whereas 62 compounds were detected in samples extracted by
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Table 1 Compounds identified in the volatile fraction of cooked control and refrigerated (untreated and pressurized) beef meat extracted by dynamic headspace (DHE) and solid-phase microextraction (SPME). Volatile compound
LRIa
QIb
IDc
Hydrocarbons Pentane Hexane Heptane Octane 2,2,4,6,6-Pentamethylheptane
500 600 700 800 945
43, 57, 43, 43, 57,
42, 41, 71, 57, 56,
41, 43, 57, 85, 71,
Aldehydes Ethanal Pentanal Hexanal Heptanal Octanal Nonanal 2-Methylbutanal 3-Methylbutanal
708 986 1096 1201 1303 1413 917 920
44, 58, 56, 70, 84, 57, 57, 44,
43, 57 44, 44, 56, 56, 58, 58,
42, 41 57, 57, 57 43, 41, 41,
Ketones 2-Propanone 2-Butanone 2,3-Butanedione 2,3-Pentanedione 3-Hydroxy-2-butanone
819 908 985 1072 1306
58, 43, 43, 43, 45,
43, 72, 86, 57, 43,
Alcohols Ethanol 1-Propanol 1-Butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octanol 1-Dodecanol 2-Propanol 2-Butanol 2-Pentanol 3-Methyl-1-butanol 1-Penten-3-ol 1-Octen-3-ol 2,6-Dimethyl-7-octen-2-ol 1-Methoxy-2-propanol 2-Butoxyethanol 2-Ethyl-1-hexanol 2,3-Butanediol
944 1059 1169 1264 1364 1476 1576 1984 935 1045 1137 1220 1181 1470 1485 1146 1430 1504 1597
45, 59, 56, 42, 56, 70, 55, 55, 45, 45, 45, 55, 57, 57, 59, 45, 57, 57, 45,
46, 60, 43 55, 43, 56, 56, 43, 43, 59, 55, 70, 41 72, 55, 47, 45, 43, 43,
Esters Ethyl acetate Ethyl butanoate Ethyl lactate
896 1050 1355
Benzene compounds Toluene 1,3-Bis(1,1-dimethylethyl)benzene Benzaldehyde Phenylethanal Benzyl alcohol Phenol
57 56 41 71 85
ST, ST, ST, ST, ST,
Method of extraction
MS MS MS MS MS
DHE, DHE, DHE DHE, DHE,
SPME SPME
DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME SPME SPME
42, 39 57, 42 42, 87 100, 42 88, 42
MS ST, MS ST, MS MS MS
DHE, SPME DHE, SPME DHE, SPME SPME DHE, SPME
43, 42 42, 41
ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS MS MS MS ST, MS MS ST, MS MS MS MS MS MS
43, 45, 61, 70 71, 43, 88, 60 45, 75, 43
1055 1450 1560 1683 1923 2045
Heterocycles Pyridine Pyrazine 2-Pentylfuran Pyrrol
55 86 43
ME
T
***
ns ns ns ns
ns *
SPME SPME
MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS
72 55
Significance of effectsd
* ***
*
***
***
***
**
***
***
*
ns ns ns
ns * ***
**
***
***
ns
**
***
**
***
***
***
***
***
***
DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME DHE, SPME SPME DHE, SPME DHE, SPME SPME SPME DHE,SPME DHE, SPME SPME SPME DHE, SPME DHE, SPME SPME
***
***
MS MS MS
91, 92, 65, 93 175, 57, 190, 176 105, 106, 77, 51 91, 65, 92, 120 79, 108, 107, 77 94, 66, 65, 39
1202 1228 1240 1540
79, 80, 81, 67,
52, 53, 82, 39,
Sulphur compounds Carbon disulphide Dimethyl sulphide Methyl thioacetate Dimethyl sulfoxide Dimethyl sulphone
734 753 1062 1621 1967
76, 62, 43, 63, 79,
78, 47, 90, 78, 94,
Terpene derivatives Exobornyl acetate Terpene derivative
1625 1670
Fatty acids Acetic acid
1470
41, 41, 41, 69, 69, 41, 43, 43 42, 85, 43, 75, 41, 41, 57
70 55 43 70 41 59 41 43 55 83 42 87 70
***
***
***
ns
***
***
***
**
***
ns ns ns ns
ns *** ** ***
***
***
*
***
***
***
***
***
***
***
ns
***
***
***
*
**
ns
***
***
SPME SPME SPME
***
***
***
***
***
***
MS MS MS ST, MS MS MS
DHE, SPME DHE, SPME DHE, SPME SPME SPME SPME
**
ns
***
**
***
***
***
***
***
**
***
*
51, 78 52, 51 138, 53 41
MS MS MS MS
SPME SPME SPME SPME
***
ns
***
***
77 46, 47, 45, 91,
MS MS MS MS MS
95, 136, 121, 93 71, 81, 95, 55 43, 45, 60, 42
61 45 46 92
***
**
***
ns
DHE DHE, SPME SPME SPME SPME
***
ns
***
***
MS MS
SPME SPME
***
***
***
ns
MS
SPME
***
***
***
**
***
ns
***
***
(continued on next page)
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A. Rivas-Cañedo et al. / Food Chemistry 124 (2011) 1201–1207
Table 1 (continued) Volatile compound
LRIa
QIb
Propanoic acid 2-Methylpropanoic acid Butanoic acid Hexanoic acid Octanoic acid Nonanoic acid
1555 1583 1644 1861 2076 2182
74, 41, 60, 60, 60, 60,
Miscellaneous Acetonitrile c-Butyrolactone Naphthalene
1016 1678 1809
41, 40, 39 42, 86, 41, 56 128, 127, 129
45, 43, 73, 73, 73, 73,
57, 79, 41, 41, 43, 41,
56 88 45 87 41 57
IDc
Method of extraction
Significance of effectsd ME
T
MS MS ST, MS ST, MS ST, MS MS
SPME SPME SPME SPME SPME SPME
***
***
***
***
***
***
MS MS MS
DHE SPME DHE, SPME
***
***
*
***
ns ns
**
***
*
ns ***
ns
a
LRI: linear retention indices, calculated in relation to the retention time of n-alkane (C5–C20) series. QI: ions used for quantification. PI: peak identification: ST, comparison of spectra and retention time with commercial standards; MS, tentatively identified by spectra comparison using the Wiley Library. d Significance of effects (ME, method of extraction; T, treatment): ns, non significant. * P < 0.05. ** P < 0.01. *** P < 0.001. b
c
SPME. Sixty-one compounds were significantly affected by the extraction technique and 41 compounds were affected by the treatment, i.e. refrigeration and/or HPP. In order to highlight the differences between the extraction techniques in a simple and immediate way, Fig. 1 depicts the comparison of the relative percentages of the main chemical families present in beef samples. DHE extracted 2,3-butanedione (diacetyl), low MW alkanols and ethanal more efficiently than SPME whereas the latter extracted aldehydes, alcohols, sulphur- and benzene compounds, ethyl esters and fatty acids more efficiently. The 12 compounds affected by treatment, as extracted by DHE (Table 2), are also to be found in Table 3 together with another 29 compounds affected by treatment, as extracted by SPME. Twenty-three compounds were found to significantly increase during refrigerated storage as compared with control (day 0) samples, especially when using SPME as the extraction technique (Table 3). Most of these compounds did not increase significantly during refrigerated storage in samples subjected to HPP. This includes aldehydes like ethanal and phenylethanal, alcohols like ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-methyl-1-butanol, 1-methoxy-2-propanol, esters, some low molecular weight fatty acids,
and some miscellaneous compounds like benzaldehyde, benzyl alcohol and c-butyrolactone. Only five out of the 23 compounds, ethanal (by DHE, Table 2) and 3-methylbutanal, 1-propanol, pyrazine and dimethyl sulphone (in SPME, Table 3), increased significantly during refrigerated storage of both HPP treated and untreated. Ten compounds significantly decreased during refrigerated storage, including dimethyl sulphide as determined by DHE (Table 2). Among them, diacetyl, 3-hydroxy-2-butanone (acetoin) and 2butanone (all of them metabolically related compounds), decreased to a lesser extent when samples had been subjected to HPP. On the other hand, compounds like hexanal, 2,3-pentanedione, 1-pentanol, 1-penten-3-ol, and 1-octen-3-ol tended to decrease more in HPP samples than in refrigerated samples, although differences were not statistically significant. Only three compounds, 2-methylbutanal, 2,3-butanediol and dimethyl sulphide (as extracted by SPME) were found to significantly increase during refrigerated storage only in samples subjected to HPP, while some compounds like pentane, pentanal, 1-hexanol, 2pentylfuran, and butanoic and hexanoic acids, significantly decreased only in HPP samples as compared with control samples.
Reelattivee % of vollatille com c mpo ound ds
DHE
SPME
100%
100%
80%
80%
60%
60%
40%
40%
20%
20%
0%
0%
Control
Untreated HPP-treated
Control
Untreated HPP-treated
Fig. 1. Comparison of the volatile fractions of control, untreated and HPP-treated beef meat, as extracted by DHE and SPME. Bars represent the relative percentages of the main chemical families: linear alkanes, linear aldehydes, methyl ketones, diketones and related compounds, primary alcohols (except ethanol), secondary alcohols, benzene compounds, sulphur compounds, ethyl esters, and fatty acids.
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A. Rivas-Cañedo et al. / Food Chemistry 124 (2011) 1201–1207 Table 2 Mean abundancesA (±SD) of the cooked beef volatile compounds significantly affected by treatment as extracted by dynamic headspace extraction. Control Mean ± SD
Untreated Mean ± SD
Table 3 Mean abundancesA (±SD) of the cooked beef volatile compounds significantly affected by treatment, as extracted by solid-phase microextraction.
Pressurized Mean ± SD
Compounds significantly increasing with refrigerated storage Ethanal 46.15c ± 3.07 164.9a ± 18.46 10596a ± 1829 Ethanol 1018b ± 313 1-Propanol 0c ± 0 10.67a ± 0.60 2-Butanol 11.65b ± 0.68 18.16a ± 0.76 Benzaldehyde 2.27b ± 0.29 5.62a ± 0.71
89.64b ± 16.23 2423b ± 287 1.78b ± 1.01 14.18ab ± 3.80 2.90b ± 0.43
Compounds significantly decreasing with refrigerated storage 30.13b ± 2.29 2-Butanone 47.81a ± 3.88 2,3-Butanedione 2609a ± 116 25.49c ± 5.03 0b ± 0 3-Hydroxy-2-butanone 38.06a ± 27.16 Pentanal 1.39a ± 0.06 0.59ab ± 0.65 15.65b ± 2.64 1-Pentanol 26.48a ± 0.74 1-Penten-3-ol 3.14a ± 0.07 1.50b ± 0.36 Dimethyl sulphide 11.15a ± 0.92 5.52b ± 0.56
32.19b ± 6.20 310.4b ± 64.24 2.31b ± 2.05 0.33b ± 0.28 13.47b ± 4.26 1.33b ± 0.59 11.48a ± 0.81
Control Mean ± SD
Compounds significantly increasing with treatments Ethanal 20.26b ± 1.61 29.24a ± 2.24 2.46b ± 0.16 2.54b ± 0.59 2-MethylbutanalB 3-Methylbutanal 5.83b ± 0.54 8.63a ± 1.18 Ethanol 61.86b ± 23.59 621.3a ± 67.47 1-Propanol 0.37c ± 0.13 5.06a ± 0.22 2-Propanol 11.24b ± 1.25 14.88a ± 0.72 2-Butanol 6.42b ± 0.39 12.68a ± 1.03 2-Pentanol 0.78b ± 0.48 1.88a ± 0.49 3-Methyl-1-butanol 1.66b ± 0.08 4.69a ± 0.48 1-Methoxy-2-propanol 1.73b ± 0.22 6.32a ± 1.10 2,3-Butanediol B 54.12b ± 3.03 67.47ab ± 3.93 Ethyl acetate 6.05b ± 1.26 105.8a ± 25.43 Ethyl butanoate 0.06b ± 0.10 8.43a ± 2.38 Ethyl lactate 1.48b ± 0.89 20.17a ± 6.41 Methyl thioacetate 1.79b ± 0.58 5.40a ± 1.82 Exobornyl acetate 0.82b ± 0.20 2.22 a ± 0.65 Acetic acid 94.18b ± 4.82 307.6a ± 37.57 6.39a ± 0.29 Propanoic acid 5.08b ± 0.40 2-Methylpropanoic 4.75b ± 0.26 7.04a ± 0.35 acid 64.84a ± 7.81 Benzaldehyde 22.56b ± 0.32 Phenylethanal 0.81b ± 0.70 113.1a ± 35.01 Benzyl alcohol 2.80b ± 0.27 4.91a ± 0.89 Pyrazine 1.38b ± 0.06 2.67a ± 0.26 Dimethyl sulphone 1.46c ± 0.16 5.25a ± 0.55 Dimethyl sulphide B 30.80b ± 6.40 23.14b ± 0.68 c-Butyrolactone 23.66b ± 1.16 48.19a ± 7.95
A Calculated as the sum of abundances of up to four characteristic ions and referred to the IS (compound area 1000/IS area). Means of triplicate experiments. abc Means followed by different letters differ significantly (P < 0.05).
4. Discussion 4.1. Comparison of extraction techniques The results of this study show in the first place that the volatile profile of cooked beef cannot be established with certainty since it has been proven strongly dependent on the method of extraction. The susceptibility of the compounds to extraction depends on the chemical nature, especially its polarity and volatility (Mallia et al., 2005). Although, to our knowledge, the literature dealing with the comparison of different extraction methods of volatile compounds in meat and meat products is scarce (Elmore, Papantoniou, & Mottram, 2001; Garcia-Esteban, Ansorena, Astiasaran, Martin, & Ruiz, 2004), the existing literature can provide evidence that the volatile profile depends on the employed methodology. As an example, the general volatile fraction of beef consisted of aldehydes, ketones, pyrazines and furans, if using simultaneous distillation-extraction, (Raes et al., 2003), or composed of aldehydes, ketones, hydrocarbons and sulphur compounds if using DHE (Insausti, Goñi, Petri, Gorraiz, & Beriain, 2005). Within the same extraction method, the working conditions (such as temperature or trapping material) may also influence the volatile profile, which can further complicate drawing of conclusions and comparison of the results with bibliographic data. Consequently with all this, the estimation of the effect of any technological treatment (HPP in our case) on the evolution of the volatile fraction of meat can diverge when considering data obtained by different methods and must always be taken with caution. Undoubtedly, in the conditions of our study, SPME proved to be a more sensitive tool than DHE to detect the changes underwent by the beef meat samples subjected to the different treatments. When the DHE method was used, the volatile fraction of control samples (neither stored nor pressurized) mainly consisted of approximately 70% of diketones, 20% of methyl ketones and 6% of secondary alcohols (Fig. 1). The volatile fraction of control samples when extracted by SPME was comparable but somehow more equilibrated, composed of 50% of diketones, 25% of methyl ketones, 9% of linear aldehydes and 9% of fatty acids. As it will be further discussed, the volatile profile of beef changed during refrigerated storage, but the estimation, both quantitative and qualitative, of the degree of change, necessarily depended
Untreated Mean ± SD
Compounds significantly decreasing with treatments PentaneC 2.76a ± 0.33 2.07ab ± 0.46 PentanalC 19.47a ± 2.76 13.16ab ± 6.15 Hexanal 238.3a ± 21.58 119.4b ± 48.33 2-Propanone 665.43a ± 83.19 448.1b ± 16.52 2-Butanone 205.0a ± 20.49 133.7b ± 11.86 2,3-Butanodione 292.7a ± 8.17 18.81c ± 1.25 2,3-Pentanodione 3.06a ± 0.43 1.60b ± 0.68 3-Hydroxy-2-butanone 1223a ± 63 13.91c ± 1.39 1-Pentanol 73.61a ± 9.04 42.60b ± 11.44 1-Penten-3-ol 7.15a ± 1.07 3.73b ± 0.74 1-Octen-3-ol 18.88a ± 0.45 11.51b ± 1.52 1-HexanolC 8.13a ± 0.52 6.71ab ± 2.13 2-PentylfuranC 1.22a ± 0.14 0.96ab ± 0.36 Butanoic acidC 87.73a ± 8.84 96.00a ± 2.13 Hexanoic acidC 21.19a ± 4.65 20.55ab ± 3.41 A B C abc
Pressurized Mean ± SD 19.16b ± 6.53 4.54a ± 0.91 10.43a ± 1.24 95.96b ± 77.00 0.94b ± 0.19 14.45ab ± 1.73 8.29b ± 0.86 0.78b ± 0.27 2.12b ± 0.25 2.65b ± 0.63 76.01a ± 8.20 5.53b ± 3.46 0.60b ± 0.46 1.39 b ± 0.69 1.36b ± 0.87 0.74b ± 0.12 100.1b ± 16.80 3.88c ± 0.23 4.16b ± 0.48 22.93b ± 5.20 0.60b ± 0.27 3.77ab ± 0.16 2.13a ± 0.30 3.55b ± 0.98 41.59a ± 0.83 33.19b ± 4.01 1.37b ± 0.52 7.26b ± 1.16 80.28b ± 22.39 626.8 a ± 56.31 194.2ab ± 36.95 116.6b ± 40.79 1.09b ± 0.27 381.4b ± 178.5 28.27b ± 3.95 3.11b ± 0.50 8.96b ± 2.15 4.06b ± 0.48 0.40b ± 0.10 60.30b ± 6.86 12.17b ± 1.87
Means of triplicate experiments. Compounds increasing significantly only in HPP samples. Compounds decreasing significantly only in HPP samples. Means followed by different letters differ significantly (P < 0.05).
on the extraction technique. The comparable volatile profiles of control samples provided by both techniques turned completely different after the transformations occurred during storage, if they had been extracted by DHE or by SPME (Fig. 1, untreated). These transformations were likely due to microbial growth. DHE gave out a profile consisting of 50% of methyl ketones, 20% of linear aldehydes and 17% of secondary alcohols, while the SPME profile pointed to a deep microbial alteration, with 30% of methyl ketones, approximately 30% of low molecular fatty acids, 8% of esters, and low levels of benzene compounds. The very significant decrease in diacetyl and related compounds during storage was validated by both extraction techniques alike, DHE providing better efficiency in extracting diacetyl whereas acetoin was more efficiently extracted by SPME. This fact had been observed in cheese by Mallia et al. (2005). The volatile profile of HPP samples did not undergo the changes of untreated meat during storage at 4 °C, especially based on the SPME data. In fact, the volatile profile of control and stored HPP
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A. Rivas-Cañedo et al. / Food Chemistry 124 (2011) 1201–1207
samples look very much alike when extracted by SPME (Fig. 1), with a 44% of methyl ketones, 22% of diketones and 13% of fatty acids. However, if extracted by DHE (Fig. 1), control and HPP samples differed considerably, yet mainly due to the decrease in diketones (down to 18%) and the increase in 2-alcanols (up to 37%) and methyl ketones (up to 32%). Although more information can be obtained with SPME, one of the main drawbacks of this technique is that fastidious artefact peaks can form. These artefacts may have different origins, such as thermal oxidation occurring during the desorption step (Lestremau, Andersson, & Desauziers, 2004) or siloxane formation (Perera, Marriot, & Galbally, 2002). In our opinion, the moisture of some food products can also affect the formation of siloxanes. However, to our knowledge no literature dealing with this particular effect of water has been published. To decrease artefact formation in high moisture samples as well as to enhance volatilization by salting out, the use of anhydrous salts for sample preparation (as carried out in the present study) is advisable. 4.2. Effect of high pressure processing Pressurization reduced the formation of some compounds, mainly as a result of alterations on the microbial balance. As expected, higher levels of many compounds resulting from microbial fermentations were observed in untreated samples after 3 days of storage (Tables 2 and 3). Ethanal, its reduction product ethanol, and the esterification products, ethyl esters, increased dramatically. Ethanol is mainly derived from carbohydrate fermentation by different microorganims, such as lactic acid bacteria (Kandler, 1983) and yeasts. The presence of large quantities of ethanol enhanced the formation of ethyl esters in untreated samples, possibly by pseudomonas (Morales, Fernández-García, & Nuñez, 2005), staphylococci (Talon, Chastagnac, Vergnais, Montel, & Berdagué, 1998) or yeasts (Olesen & Stahnke, 2000), all well known ethyl ester producers. Most of these microorganisms are pressure sensitive (Rubio, Martínez, García-Cachán, Rovira, & Jaime, 2007) which explains the reduced ethyl ester formation observed in HPP samples, although the possibility of an inhibition of microbial esterase activity by pressure should not be overlooked. In a previous work we reported lower levels of ethanol and ethyl esters, together with lower counts of bacteria, in particular Gram-negatives and coliforms, in beef and chicken breast samples treated at 400 MPa for 10 at 12 °C in comparison with untreated samples (Rivas-Cañedo et al., 2009a). The compounds presumably originating from amino acid catabolism followed two different trends. While 3-methyl-1-butanol, 2methylpropanoic acid, benzaldehyde and phenylacetaldehyde increased during the 3-day storage at 4 °C in untreated samples, 2methylbutanal and dimethyl sulphide increased only in HPP-treated samples. Branched-chain aldehydes 2- and 3-methylbutanal are generated from isoleucine and leucine by transamination or Strecker degradation (Christensen, Dudley, Pederson, & Steele, 1999; Montel, Masson, & Talon, 1998) and can be further reduced to 2- and 3-methyl-1-butanol through the activity of bacterial deshydrogenases or chemical reactions (Mollimard & Spinnler, 1996) or oxidised to acids. 2-Methylpropanoic acid can be produced by the same path from oxidation of valine and dimethyl sulphide results from methionine degradation (McSweeney & Sousa, 2000). Benzaldehyde and phenylethanal can result from the catabolism of phenylalanine by microbial activity, for example in pseudomonas, some lactic acid bacteria (Groot & de Bont, 1998) and moulds (McSweeney & Sousa, 2000). In a previous study, a decrease in benzaldehyde in HPP-treated dry-cured ham (400 MPa, 10 min, 12 °C) was observed (Rivas-Cañedo, Fernández-García, & Nuñez, 2009b). In agreement with these results, Campus, Flores, Martínez, and Toldrá (2008) reported a decrease in phenylethanal
in HPP-treated dry cured pork loin (300–400 MPa, 10 min, 20 °C) together with a decrease in the activity of aminopeptidases. Our results could be pointing to a discriminating effect of HPP on key enzymes or on different microbial groups with different amino acid metabolic activities. On the other hand, an effect of the state of oxidation of treated and non treated vacuum packed meat can not be discarded. The lower levels found after 3 days of refrigerated storage of compounds associated with lipid oxidation, such as 1-alcanals, 1alcohols, fatty acids and furans, as well as 1-penten-3-ol and 1-octen-3-ol, are difficult to explain (Tables 2 and 3). Levels were even lower in HPP samples. There seems to be a controversy in relation to the effect of HPP on lipid oxidation. On one hand, HPP is supposed to enhance lipid oxidation on muscle products. Thus, Cheah and Ledward (1996) reported increased oxidation in pork treated above 300 MPa for 20 min at ambient temperature. However, Orlien, Hansen, and Skibsted (2000) did not observe any significant increase in the levels of compounds supposedly resulting from lipid oxidation in chicken breast subjected to treatments up to 500 MPa. Besides lipo-oxidation, these compounds might also derive from the action of lipolytic microorganisms. For example, 1penten-3-ol may result from the oxidation of linolenic acid (Elmore et al., 2004) and 1-octen-3-ol might originate from linoleic acid and linoleate autooxidation (Ulrich & Grosch, 1987). The latter might also come from linoleic and linolenic acid catabolism by moulds (Mollimard & Spinnler, 1996). Nevertheless, higher levels of 1-octen-3-ol were found in beef meat inoculated with Serratia proteamaculans, Carnobacterium divergens and Pseudomonas fragi (Ercolini, Russo, Nasi, Ferranti, & Villani, 2009). HPP, in combination with vacuum storage (low oxygen content and therefore low redox potential), might affect the metabolism and growth of the microorganisms involved in lipid catabolism. Another possibility is that a phenomenon of scalping (adsorption of compounds into the packaging material) could be occurring in the samples packaged during a longer period. The behaviour of diacetyl and acetoin deserves a comment. A pronounced decrease of these compounds was observed during storage, together with an increase of their reduction products 2,3-butanediol and 2-butanol, the decrease being less steep in HPP samples. High amounts of these compounds have been previously found in meat (Raes et al., 2003) and are generally associated with lactose and citrate metabolism (Cogan, 1995; McSweeney & Sousa, 2000) by the action of different bacteria, especially lactic acid bacteria and staphylococci (Berdagué, Monteil, Montel, & Talon, 1993). Dainty, Edwards, Hibbard, and Marnewick (1989) reported the production of diacetyl and acetoin in meat inoculated with Enterobacteriaceae or Brochothrix thermosphacta. Other authors suggested the catabolism of aspartate (Kieronczyk, Skeie, Langsrud, Le Bars, & Yvon, 2004) or the action of meat enzymes (Stahnke, 1995), as alternate origins of diacetyl. The results observed in this study are consistent with a previous report (RivasCañedo et al., 2009a), where higher levels of diacetyl were observed in HPP meat samples in comparison with control samples. The increase of 2,3-butanediol and 2-butanol indicates that a reduction of diacetyl is occurring during storage, either by enzyme activity or by a modification of the redox potential.
5. Conclusions Both techniques DHE and SPME proved suitable for monitoring the changes in the volatile fraction caused by refrigeration and HPP, although SPME provides wider information. Storage caused changes in the volatile fraction of beef after cooking. Most of these changes might be related to the alteration of the microbiota or of the enzymatic activities. Although the volatile fraction of HPP
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