Meat Science 100 (2015) 41–51

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Effect of immobilized Lactobacillus casei on the evolution of flavor compounds in probiotic dry-fermented sausages during ripening Marianthi Sidira a,b, Panagiotis Kandylis a, Maria Kanellaki a, Yiannis Kourkoutas b,⁎ a b

Food Biotechnology Group, Section of Analytical Environmental and Applied Chemistry, Department of Chemistry, University of Patras, GR-26500 Patras, Greece Applied Microbiology and Molecular Biotechnology Research Group, Department of Molecular Biology & Genetics, Democritus University of Thrace, Alexandroupolis 68100, Greece

a r t i c l e

i n f o

Article history: Received 5 February 2014 Received in revised form 8 August 2014 Accepted 15 September 2014 Available online 28 September 2014 Keywords: SPME GC/MS Volatiles Probiotic sausages Immobilized L. casei ATCC 393 Wheat

a b s t r a c t The effect of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds in probiotic dry-fermented sausages during ripening was investigated. For comparison reasons, sausages containing free L. casei cells or no starter culture were also included in the study. Samples were collected after 1, 28 and 45 days of ripening and subjected to SPME GC/MS analysis. Both the probiotic culture and the ripening process affected significantly the concentration of all volatile compounds. The significantly highest content of total volatiles, esters, alcohols and miscellaneous compounds was observed in sausages containing the highest amount of immobilized culture (300 g/kg of stuffing mixture) ripened for 45 days. Principal component analysis of the semi-quantitative data revealed that primarily the concentration of the immobilized probiotic culture affected the volatile composition. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Nowadays there is a growing interest in developing novel foods containing probiotic microorganisms, such as bifidobacteria and lactic acid bacteria (LAB). Such functional cultures may contribute to microbial safety and offer organoleptic, technological and nutritional advantages, but more importantly confer a health benefit on the host (Deshpande, Rao, & Patole, 2011; Mitropoulou, Nedovic, Goyal, & Kourkoutas, 2013). To deliver the health benefits, probiotics need to contain an adequate amount of live bacteria (at least 10 6 –10 7 cfu/g) (Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004), able to survive the acidic conditions of the upper gastro-intestinal (GI) tract and proliferate in the intestine, a requirement that is not always fulfilled (Boylston et al., 2004). In general, the food industry has adopted the recommended level of 106 cfu/g of probiotic bacteria at the time of consumption. Thus, a daily intake of at least 10 8 –10 9 viable cells, which could be achieved with a daily consumption of at least 100 g of probiotic food, has been suggested as the minimum intake to provide a probiotic effect. Incorporating probiotics into a food matrix presents a fully new challenge, not only because of their interactions with other constituents, but also because of the severe conditions often employed during food ⁎ Corresponding author at: Applied Biotechnology Research Group, Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis GR_68100, Greece. Tel.: +30 25510 30633; fax: +30 25510 30624. E-mail address: [email protected] (Y. Kourkoutas).

http://dx.doi.org/10.1016/j.meatsci.2014.09.011 0309-1740/© 2014 Elsevier Ltd. All rights reserved.

processing and storage, which might lead to important losses in viability. To overcome such deficiencies, immobilization techniques are usually applied in order to maintain cell viability, activity and functionality. Consequently, many studies have focused on immobilization of probiotic bacteria in various supports, such as starch (MattilaSandholm et al., 2002), fruit pieces (Kourkoutas, Xolias, Kallis, Bezirtzoglou, & Kanellaki, 2005; Kourkoutas et al., 2006), casein (Dimitrellou, Kourkoutas, Koutinas, & Kanellaki, 2009) and wheat grains (Bosnea et al., 2009), aimed at stabilization of cells and formulation of new types of foods fortified with immobilized health-promoting bacteria that are only released upon reaching the human gut. Dry-fermented sausages are typical Mediterranean meat-products, the acceptability of which is strongly influenced by their quality. The meat industry is seeking for functional starter cultures that meet health promoting, food safety, shelf-life, technological effectiveness and economic feasibility criteria (Ammor & Mayo, 2007). Among LAB, Lactobacillus casei ATCC 393 strain has been extensively added into food products (Kourkoutas et al., 2005, 2006) to confer probiotic properties (Choi et al., 2006; Saxami et al., 2012; Sidira et al., 2010). Recently, Sidira, Karapetsas, Galanis, Kanellaki, and Kourkoutas (2014) investigated the effect of cell immobilization and the concentration of immobilized cells on wheat on the effective survival of probiotic L. casei ATCC 393 cells during ripening and during heat treatment of probiotic dry-fermented sausages. The presence of the probiotic strain at levels above the minimum concentration for conferring a probiotic effect (≥6 log cfu/g) at the end of the ripening process and after mild heat treatment was confirmed by an efficient molecular tool (Karapetsas, Vavoulidis, Galanis, Sandaltzopoulos, & Kourkoutas, 2010;

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M. Sidira et al. / Meat Science 100 (2015) 41–51

Sidira et al., 2014). Noticeably, the probiotic properties of free and immobilized L. casei ATCC 393 were previously assessed by documenting maintenance of cell viability after transit through the GI tract, adhesion at the large intestine and regulation of the intestinal microbial flora in rats (Saxami et al., 2012; Sidira et al., 2010). Apart from a beneficial to human health product, the development of a unique aromatic profile is an undeniable aim for the food industry. The aroma development in dry-fermented sausages is related to the high number of changes that occur during fermentation, maturation and drying. The correlation between the starter culture and the flavor of fermented sausages has been well established (Flores, Durá, Marco, & Toldrá, 2004). There are a number of potential precursors responsible for the flavor and odor, which may be produced by lipid hydrolysis and autoxidation, proteolysis and transformation of amino acids to aromatic compounds and carbohydrate metabolism. In addition, the contribution of spices and other condiments by directly affecting flavor and odor and by the modulation of autoxidative reactions add characteristic notes to the final flavor of the sausage. Therefore, several studies have been focused on the improvement of nutritional profile and sensory characteristics of fermented sausages (Di Cagno et al., 2008; Flores et al., 2004; Marco, Navarro, & Flores, 2004, 2006; Olivares, Navarro, & Flores, 2009). Hence, the objective of the present study was to investigate the generation of volatile compounds during ripening of probiotic dryfermented sausages containing free or immobilized L. casei ATCC 393. The strategy adopted was to assess the effect of concentration of immobilized L. casei ATCC 393 on the volatile composition. For comparison reasons, sausages containing free L. casei cells or no starter culture were also included. 2. Materials & methods 2.1. Bacterial strain and culture conditions L. casei ATCC 393 (DSMZ, Germany) was grown at 37 °C for 72 h on MRS Broth. Pressed wet cells (≈ 0.5–1.0 g dry weight) were prepared and used directly for production of probiotic dryfermented sausages. 2.2. Preparation of support and cell immobilization Wheat grains were boiled and sterilized at 130 °C for 15 min. Cell immobilization was carried out as described recently (Bosnea et al., 2009). In brief, 500 g of wheat grains along with 8 g (wet weight) of L. casei cells were introduced in 2 L of MRS broth. The mixture was allowed to ferment at 37 °C for 48 h without agitation. When immobilization was complete, the fermented liquid was decanted and the immobilized biocatalyst was washed twice with sterile 1/4 strength Ringer's solution. The immobilization process is shown in Fig. 1.

Fig. 1. Process of cell immobilization on wheat grains and production of probiotic dryfermented sausages.

2.3. Production of probiotic dry fermented sausages Dry-fermented sausages were prepared using traditional techniques, as described recently (Sidira et al., 2014). In brief, a batch consisting of ground pork meat (2.0 kg), lard (0.5 kg), ground orange peel (25.0 g), ground leek (412.5 g), sodium chloride (50.0 g), white pepper (3.75 g), red pepper (3.75 g), cumin (3.75 g), ground garlic (1.25 g), oregano (10.0 g), sucrose (15.0 g) and lactose (5.0 g) was inoculated with immobilized L. casei on wheat (wheat grains containing the immobilized cells were added to the above mixture). After mixing, the stuffing of natural casings produced fresh sausages (Fig. 1). Hence, probiotic dry-fermented sausages were produced containing 300 (sample I-300), 100 (sample I-100) or 30 g (wet weight) (sample I-30) of immobilized cells/kg of the above stuffing mixture. For comparison reasons, dry-fermented sausages containing free L. casei (1.0 g wet

weight/kg of stuffing mixture) (sample Fr) and with no starter (sample NC) were also produced, as described above. The initial cell counts of L. casei ATCC 393 in the probiotic products ranged ≥6 log cfu/g in all cases (when incorporated in either immobilized or free form). Ripening was carried out at room temperature (19–23 °C with a relative humidity between 40 and 85%) for 10–12 days and then the temperature was decreased to 4–6 °C at a rate of 2–4 °C/day with a relative humidity between 50 and 75% for up to 45 days. All experiments were carried out in triplicate (3 independent batches of sausages were prepared). Samples were collected after 1, 28 and 45 days of ripening and subjected to SPME GC/MS analysis to determine the volatile composition.

M. Sidira et al. / Meat Science 100 (2015) 41–51

2.4. Solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis The SPME GC/MS analysis was carried out as described recently (Di Cagno et al., 2008) with some modifications. In brief, grated samples (≈ 6 g each) were placed into a 20 mL headspace vial fitted with a Teflon-lined septum sealed with an aluminum crimp seal, through which the SPME syringe needle (bearing a 2 cm fiber coated with 50/ 30 mm Divinylbenzene/Carboxen on poly-dimethyl-siloxane bonded to a flexible fused silica core, Supelco, Sigma-Aldrich, Poole, UK) was introduced. The container was then thermostated at 35 °C for 30–35 min. The absorbed volatile analytes were then analyzed by GC/MS (Shimadzu GC-17A, MS QP5050, capillary column Supelco CO Wax-10 60 m, 0.32 mm i.d., 0.25 μm film thickness). Helium was used as the carrier gas (linear velocity of 1.8 mL/min). Oven temperature was set at 40 °C for 2 min, followed by a temperature gradient of 10 °C/min to 200 °C, and then 15 °C/min to 250 °C. A final extension was applied at 250 °C for 5 min (Di Cagno et al., 2008). The injector was operated in splitless mode. Injector and detector temperatures were 280 °C and 250 °C, respectively. The mass spectrometer was operated in the electron impact mode with the electron energy set at 70 eV. The identification was carried out by comparing the retention times and mass spectra of volatiles to those of authentic compounds generated in the laboratory, by mass spectra obtained from NIST107, NIST21 and SZTERP libraries, and by determining Kovats retention indexes and comparing them with those reported in the literature (Kandylis, Goula, & Koutinas, 2008; Kandylis & Koutinas, 2008; Kandylis et al., 2011; Vichi, Guadayol, Caixach, López-Tamames, & Buxaderas, 2007a; Vichi, Guadayol, Caixach, López-Tamames, & Buxaderas, 2006; Vichi et al., 2007b; Vichi et al., 2003). Kovats retention indexes were determined by injection of a standard mixture containing the homologous series of normal alkanes (C8–C24) in pure hexane under exactly the same experimental conditions, as described above. All authentic compounds used were obtained from Sigma-Aldrich. Methyl octanoate (SigmaAldrich) diluted in pure hexane was used as an internal standard (IS) at various concentrations (1.25, 12.5, 125 and 1250 μg/kg of sausage). The volatile compounds were semi-quantified by dividing the peak areas of the compounds of interest by the peak area of the IS and multiplying this ratio by the initial concentration of the IS (expressed as μg/kg). The peak areas were measured from the full scan chromatograph using total ion current (TIC). Each experiment was carried out in triplicate and the mean data are presented (standard deviation for all values was about ± 10% in most cases). 2.5. Experimental design and statistical analysis All treatments were carried out in triplicate (3 independent batches of sausages were prepared). The experiments were designed and analyzed statistically by ANOVA. A two-way ANOVA was applied to study the effect of the starter culture and ripening time on the evolution of volatiles and the three independent replicates were included as parameters. Duncan's multiple range test was used to determine significant differences among results (coefficients, ANOVA tables and significance (P b 0.05) were computed using Statistica v.5.0). Principal component analysis (PCA) of data was computed using SPSS (v. 15.0). 3. Results and discussion 3.1. SPME GC/MS analysis For the evaluation of the aromatic profile during ripening, the dryfermented sausages were analyzed using an SPME GC/MS technique. Semi-quantitative results of the volatile compounds are presented in Tables 1–4. In total, 146 compounds were detected. Esters, organic acids, alcohols and carbonyl compounds are generally considered as the most important compounds usually identified

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by SPME GC/MS technique in dry fermented sausages. Nevertheless, aroma perception in meat products depends not only on the concentration and odor thresholds of volatile compounds, but also on their interactions with other food components and among volatile compounds. From a quantitative point of view, both the probiotic culture and the ripening time affected significantly (P b 0.05) the concentration of all volatile compounds. In general, addition of free or immobilized probiotic culture and the ripening time resulted in increased concentration of all volatiles. Thus, the significantly (P b 0.05) highest content of total volatiles, esters, alcohols and miscellaneous compounds was observed in I-300 sample ripened for 45 days (Table 4). Likewise, significantly (P b 0.05) higher concentration of organic acids and carbonyl compounds were detected both in I-300 and I-100 samples ripened for 45 days. Esters are very fragrant compounds with very low odor detection thresholds and they provide fruity notes to the dry-fermented sausage flavor (Marco et al., 2006). Noticeably, the generation of esters was very important during ripening and was significantly (P b 0.05) higher in sausages containing increased amounts of immobilized probiotic cells (I-300 and I-100 samples). Many of the esters identified were ethyl esters that are considered essential for obtaining the proper fermented sausage aroma by adding a fruity note and masking rancid odors (Flores et al., 2004). Ethyl 3-methyl-butanoate has low threshold values and has a high impact on sausage aroma (Flores et al., 2004). This compound along with ethyl butanoate, ethyl hexanoate and ethyl octanoate derive from staphylococci esterase activity (Marco et al., 2006; Olivares et al., 2009). The esterase enzyme of staphylococci acts by esterifying acids and alcohols until the esters arrive at a maximum concentration above which the enzyme action is reversed and ester hydrolysis occurs (Marco et al., 2006). Acids were among the predominant group of volatile compounds. Long (C14–C18) and medium (C6–C12) chain fatty acids come from the hydrolysis of triglycerides and phospholipids and from degradation of lipids. They can act as precursors of compounds affecting taste or aroma, but they are not directly responsible for sensory improvements in meat products. They basically originate from the lipolytic activity of micrococci (Ansorena, Astiasarián, & Bello, 2000). Short chain fatty acids (C b 6) with greater implications in flavor development, due to the very strong odors and to their lower threshold values, were detected in most samples. Acetic acid detected in all sausages ripened for at least 28 days is released from fermentation pathways. Many metabolic pathways are involved in the biosynthesis of alcohols that are encountered in fermented meat products, such as methyl ketone reduction, amino acid metabolism and lipid oxidation. The concentration of alcohols showed a significant increase (P b 0.05) during ripening and was higher (P b 0.05) in samples with high content of immobilized L. casei cells (I-300 and I-100 samples). Ethanol identified in all cases was among the most abundant compounds and is a product of carbohydrate metabolism. Methyl branched alcohols, such as 2-methyl-1-propanol, may derive from transformation of certain amino acids (Val) which are mediated by the LAB and yeast present in the sausages. Thus, they can be decarboxylated to the corresponding aldehydes and, at the same time, these aldehydes can be converted to the corresponding alcohols by the activity of alcohol dehydrogenase or transformed to the corresponding acid by an aldehyde dehydrogenase (Flores et al., 2004). In yeast, the final products produced from amino acids are generally the corresponding methyl branched alcohols (Flores et al., 2004). 1-Octen-3-ol present in all samples derives from lipid oxidation and has a characteristic odor of mushroom and a very low odor threshold (Ansorena, Gimeno, Astiasarián, & Bello, 2001). 2,3-Butanediol is produced by the reduction of methyl-ketones from the a-oxidation of fatty acids (Marco et al., 2006). 2-phenylethanol present in I-30 sample ripened for 28 days and in Fr and NC sausages ripened for 28 and 45 days was considered among the most important odorants in Spanish sausages (Schmidt & Berger, 1998).

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Table 1 Effect of immobilized and free L. casei on aroma related compounds (μg/kg) isolated by probiotic dry-fermented sausages after ripening for 1 day using SPME GC/MS analysis. Identification method

KI

KIref

I-300

I-100

I-30

Fr

NC

Potential origin

RT, KI, MS MS RT, KI, MS MS MS

1437 1476 1825 2390 2482

1449a 1845a

16.0 15.5 21.5 13.2 Nd

0.8 0.9 1.2 0.8

Nd 6.3 ± 0.5 26.9 ± 1.4 19.2 ± 1.1 Nd

Nd Nd Nd Nd Nd

Nd Nd Nd Nd Nd

Nd Nd 4.2 ± 0.5 Nd 5.2 ± 0.6

Staphylococci esterase activity Unknown Unknown Unknown Unknown

Organic acids Octanoic acid Nonanoic acid Decanoic acid

RT, KI, MS KI, MS RT, KI, MS

2116 2211 2319

2152a 2213b 2336b

71.5 ± 3.6 Nd 52.5 ± 3.2

84.7 ± 4.7 44.3 ± 2.6 48.1 ± 2.5

Nd Nd Nd

Nd Nd Nd

Nd Nd Nd

Lipid autooxidation Lipid autooxidation Lipolysis

RT, KI, MS MS RT, KI, MS RT, KI, MS MS RT, MS RT, KI, MS RT, KI, MS RT, KI, MS KI, MS MS RT, KI, MS MS

961 1107 1142 1354 1403 1406 1451 1549 1555 1622 1631 1704 1707

935a

1159.6 ± 58.2 Nd 9.4±1.8 18.4 ± 1.9 Nd 7.5 ± 1.5 19.6 ± 1.2 171.0 ± 8.6 100.7 ± 5.3 18.7 ± 1.9 11.9 ± 1.6 35.4 ± 1.8 16.4 ± 1.9

1545.6 ± 82.5 7.4 ± 1.4 8.7±1.5 19.3 ± 1.9 Nd 10.2 ± 1.3 20.1 ± 2.3 102.3 ± 6.1 43.5 ± 2.3 32.0 ± 1.6 Nd 28.7 ± 1.5 13.4 ± 1.7

242.4 ± 12.3 Nd Nd 11.6 ± 1.2 33.1 ± 1.7 2.9 ± 1.2 13.7 ± 1.5 212.3 ± 11.4 52.4 ± 2.7 Nd Nd 36.5 ± 1.9 7.4 ± 1.5

376.7 ± 19.2 Nd Nd 10.3 ± 1.4 25.8 ± 1.3 Nd 8.0 ± 1.4 77.3 ± 3.9 26.4 ± 1.4 Nd Nd 20.5 ± 1.1 7.5 ± 1.4

267.2 ± 14.3 Nd Nd 8.3 ± 1.6 20.9 ± 1.2 13.7 ± 1.8 8.2 ± 1.4 136.4 ± 6.9 47.0 ± 2.6 Nd Nd 22.8 ± 1.8 5.4 ± 1.3

Carbohydrate fermentation Unknown origin Lipid autooxidation Lipid autooxidation Unknown Unknown Lipid β-oxidation, seasonings Seasonings Lipid autooxidation Unknown Unknown Seasonings Seasonings

Nd 15.6 15.2 18.1 Nd Nd 17.6 92.5

± 1.1 ± 4.5

Nd 11.8 ± 1.8 13.8 ± 2.0 Nd 9.5 ± 1.4 10.9 ± 1.5 20.5 ± 1.3 81.1 ± 5.2

23.0 ± 1.2 31.0 ± 1.7 19.7 ± 1.9 22.1 ± 1.2 9.7 ± 1.6 17.3 ± 1.9 15.7 ± 1.8 11.1 ± 1.6

16.9 ± 1.9 17.2 ± 1.9 9.4 ± 1.5 12.6 ± 1.7 4.4 ± 1.2 8.2 ± 1.4 8.6 ± 1.4 3.9 ± 1.2

13.7 ± 1.9 10.4 ± 1.5 10.7 ± 1.5 15.7 ± 1.9 5.8 ± 1.4 9.5 ± 1.8 11.0 ± 1.9 5.3 ± 1.3

Lipid oxidation Seasonings Seasonings Seasonings Unknown Seasonings Unknown Seasonings

20.5 ± 1.3 Nd 30.9 ± 1.6 Nd 95.8 ± 5.7 Nd

Nd Nd 29.9 ± 1.7 Nd 75.7 ± 4.7 Nd

Nd Nd 14.1 ± 1.7 11.1 ± 1.6 19.4 ± 1.9 Nd

7.6 ± 1.4 Nd 10.2 ± 1.5 7.1 ± 1.4 16.0 ± 1.9 402.4 ± 22.3

11.0 ± 1.5 4.1 ± 1.1 5.6 ± 1.2 7.0 ± 1.4 14.1 ± 1.9 Nd

Orange peel, limonene unknown Aminoacid catabolism Seasonings Seasonings Seasonings Seasonings

Nd 43.0 ± 2.3 Nd 5.5 ± 0.4 42.4 ± 2.6 57.4 ± 3.2 31.6 ± 1.9

Nd 70.2 ± Nd 9.1± 50.5 ± 57.6 ± 44.4 ±

180.0 ± 11.2 4.8 ± 0.7 4.9 ± 0.5 Nd 26.3 ± 1.6 106.3 ± 5.8 30.4 ± 2.1

Nd 7.9 ± 0.8 Nd Nd 11.8 ± 1.6 20.0 ± 2.3 18.4 ± 2.9

Nd Nd Nd Nd 27.6 ± 2.1 23.5 ± 3.2 21.0 ± 2.5

Strecker degradation, microbial metabolism Lipid autooxidation Lipid autooxidation, seasonings Lipid autooxidation Lipid autooxidation Carbohydrate fermentation Lipid autooxidation

Alcohols Ethanol 2-Ethyl-cyclobutanol 1-Butanol 1-Hexanol 2-Butoxy-ethanol 2-Hexenol 1-Octen-3-ol Beta-linalool 1-Octanol 2-(2-Ethoxyethoxy)-ethanol p-Mentha-2,8-dienol Alpha-terpineol endo-1,7,7-Trimethyl-bicyclo[2.2.1]heptan-2-ol (Borneol) Decanol beta-Citronellol trans-Carveol Geraniol p-Cymen-8-ol cis-Carveol Benzyl alcohol 2-Phenyl-ethanol 4-Isopropenyl-1-cyclohexenyl) methanol (Perillol) Phenol 4-[1-Methylethyl]-benzenemethanol (cumic alcohol) Eugenol m-Thymol o-Thymol

KI, MS RT, MS RT, MS RT, MS MS KI, MS KI, MS RT, KI, MS

1759 1764 1838 1846 1854 1873 1884 1923

MS KI, MS MS MS RT, MS RT, MS

2002 2017 2104 2178 2190 2220

Carbonyl compounds 3-Methyl-butanal Hexanal 2-Methyl-2-butenal Heptanal Octanal Acetoin Nonanal

RT, MS RT, KI, MS MS RT, KI, MS RT, KI, MS RT, MS RT, KI, MS

961 1074 1091 1189 1294 1295 1398

1179c 1357d

1455d 1556b 1562d 1636a 1715b

1783c

1879e 1883d 1926f

2020f

1074d 1190f 1296f 1399a

± ± ± ±

± 1.8 ± 1.6 ± 1.9

4.5

2.6 3.2 2.5

M. Sidira et al. / Meat Science 100 (2015) 41–51

Compound Esters Ethyl octanoate n-Octyl acetate 2-Phenylethyl-acetate 2-Propenyl octadecanoate Ethyl citrate

Furfural Citronellal Decanal Benzaldehyde 4-(1-Methylethyl)-1-cyclohexene-1-carboxaldehyde p-Menth-8-en-2-one [E]-2-Decenal 3,7-Dimethyl-2,6-octadienal Dodecanal alpha-Citral [S]-2-Methyl-5-[1-methylethenyl]-2-cyclohexen-1-one 2-Undecenal 4-[1-Methylethyl]-benzaldehyde (cuminal) N,N-Dibutyl-formamide 2-Caren-10-al Nonanophenone Piperonal

1473 1483 1502 1534 1578 1619 1653 1691 1713 1738 1746 1757 1774 1777 1805 2018 2248

KI, MS

1017 1021 1066 1084 1096 1115 1121 1129 1137 1141 1164 1177 1187 1194 1205 1248 1251 1256 1265 1273 1283 1324 1330 1382 1466 1491 1608 1682 1727 1733

MS MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS RT, MS KI, MS KI, MS MS MS KI, MS KI, MS KI, MS MS MS MS MS MS KI, MS MS MS MS

1533b

1657f

1776e

1017e 1022f

1099h 1115h 1119d 1133d 1142f 1141e 1163h 1187f 1194c 1204e 1248h 1250d 1265d 1273h 1283e

1608h

11.2 ± 0.8 11.5 ± 0.9 310.5 ± 16.7 94.1 ± 5.4 Nd 7.0 ± 0.8 21.2 ± 1.4 14.7 ± 1.1 21.3 ± 1.8 15.3 ± 1.1 26.1 ± 1.7 32.8 ± 1.9 537.0 ± 17.4 Nd 74.5 ± 4.2 Nd Nd

Nd 10.2 ± 1.1 109.0 ± 6.4 54.5 ± 2.8 Nd 6.3 ± 0.5 35.4 ± 1.9 7.0 ± 0.8 17.8 ± 0.9 14.9 ± 1.5 20.5 ± 1.7 32.45 ± 1.9 589.3 ± 43.2 Nd 95.1 ± 6.3 20.3 ± 2.1 Nd

Nd 27.2 ± 2.4 196.2 ± 11.4 11.4 ± 1.6 23.0 ± 1.9 7.1 ± 2.5 Nd 23.0 ± 2.8 28.9 ± 2.1 26.1 ± 1.9 27.0 ± 1.8 Nd 794.3 ± 45.2 Nd 188.5 ± 11.5 Nd Nd

Nd 6.3 ± 1.1 85.5 ± 4.8 7.2 ± 2.5 36.4 ± 2.8 2.2 ± 2.1 Nd Nd 15.1 ± 4.1 12.8 ± 3.2 15.5 ± 2.1 Nd 815.0 ± 44.2 7.6 ± 1.4 325.7 ± 18.4 Nd 10.7 ± 1.6

Nd 8.6 ± 1.1 125.8 ± 7.2 5.7 ± 0.9 13.3 ± 0.8 5.8 ± 2.1 Nd 9.4 ± 1.6 Nd 17.9 ± 1.9 13.7 ± 1.8 Nd 456.9 ± 25.3 Nd 75.8 ± 4.2 Nd Nd

Carbohydrate fermentation Seasonings Lipid autooxidation Amino acid catabolism Unknown seasonings Lipid oxidation Unknown Unknown Seasonings Unknown Lipid autooxidation Seasonings Unknown Seasonings Unknown Seasonings

518.4 ± 27.9 330.7 ± 18.6 13.8 ± 1.7 Nd 75.3 ± 4.7 53.7 ± 3.4 Nd Nd Nd 35.7 ± 2.8 483.1 ± 27.2 19.3 ± 2.0 Nd 15364.4 ± 872.9 268.3 ± 15.4 188.4 ± 10.4 28.5 ± 2.5 11.8 ± 2.6 22.8 ± 2.1 318.2 ± 17.2 11.7 ± 1.6 23.6 ± 1.4 10.7 ± 0.9 145.0 ± 8.3 23.6 ± 2.3 9.3 ± 1.5 251.4 ± 15.2 32.6 ± 2.6 36.9 ± 2.9 Nd

Nd Nd 28.1 ± 2.4 Nd 75.2 ± 4.8 37.4 ± 2.9 Nd Nd Nd 28.6 ± 2.4 356.8 ± 29.8 Nd Nd 12946.6 ± 753.8 128.7 ± 6.7 123.8 ± 7.2 29.2 ± 2.5 15.6 ± 1.9 31.7 ± 1.8 251.6 ± 15.2 8.1 ± 1.4 16.4 ± 1.4 Nd 192.2 ± 10.7 12.1 ± 1.7 Nd 179.0 ± 9.2 35.1 ± 2.8 Nd Nd

179.5 ± 11.8 125.7 ± 7.2 5.2 ± 1.3 1.8 ± 0.4 31.5 ± 2.6 33.4 ± 2.7 13.3 ± 1.7 10.8 ± 1.5 19.4 ± 2.0 27.1 ± 2.4 412.2 ± 30.7 15.5 ± 1.8 15.3 ± 1.8 8972.9 ± 543.9 31.9 ± 2.6 94.5 ± 5.8 20.8 ± 2.0 9.8 ± 1.5 16.3 ± 1.9 118.2 ± 6.9 20.9 ± 1.3 Nd Nd 203.3 ± 20.2 20.5 ± 2.3 Nd 166.2 ± 9.3 75.1 ± 4.8 Nd Nd

99.1 ± 6.0 53.7 ± 3.7 Nd Nd 33.7 ± 2.7 11.9 ± 1.6 6.2 ± 1.3 5.1 ± 1.1 8.1 ± 1.4 14.2 ± 1.8 148.7 ± 8.5 6.2 ± 1.3 5.1 ± 1.2 3404.1 ± 190.7 10.6 ± 1.4 80.0 ± 4.0 3.8 ± 0.9 5.6 ± 1.3 14.8 ± 1.8 112.2 ± 5.6 9.2 ± 1.5 Nd Nd 57.9 ± 3.9 7.5 ± 1.5 Nd 144.3 ± 8.2 23.6 ± 2.2 Nd 7.4 ± 1.5

125.0 ± 7.3 72.4 ± 4.7 Nd Nd 26.3 ± 2.4 20.3 ± 2.0 7.4 ± 1.4 6.0 ± 1.3 10.9 ± 1.6 22.4 ± 2.2 273.5 ± 15.7 11.1 ± 1.6 6.6 ± 1.3 5598.4 ± 299.2 21.0 ± 2.0 86.8 ± 5.4 11.5 ± 1.6 4.4 ± 1.4 14.7 ± 1.8 115.2 ± 6.7 14.3 ± 1.8 Nd Nd 120.1 ± 7.0 9.5 ± 1.6 Nd 147.5 ± 8.4 42.4 ± 3.2 8.0 ± 1.4 Nd

Seasonings Seasonings Amino acid catabolism Unknown Seasonings Seasonings Flavor processed meat Unknown origin, meat or food contaminant Unknown origin, meat or food contaminants Seasonings Seasonings Seasonings Unknown origin, meat or food contaminants Orange peel Seasonings Seasonings Seasonings and/or orange peel Unknown Unknown origin, meat or food contaminants Seasonings Seasonings Seasonings Seasonings Unknown Orange peel Seasonings Seasonings Unknown Seasonings Seasonings

M. Sidira et al. / Meat Science 100 (2015) 41–51

Miscellaneous compounds alpha-Pinene Origanene Dimethyl-disulfide 3-Ethyl-4-methyl-hexane beta-Pinene Sabinene Ethyl-benzene m-Xylene p-Xylene 3-Carene beta-Myrcene 4-Carene o-Xylene delta-Limonene beta-Phellandrene gamma-Terpinene Ocimene 2,4-Dimethyl-thiophene Styrene p-Cymene Terpinolene 2,5-Dimethyl-pyrazine 2,6-Dimethyl-pyrazine Dipropyl disulfide Limonene oxide Allyl disulfide β-Caryophyllene Dipropyl trisulfide Valencene beta-Selinene

RT, MS MS MS RT, KI, MS MS MS KI, MS MS MS MS MS MS KI, MS MS MS MS MS

I-300: sausages containing 300 g immobilized L. casei/kg of stuffing mixture, I-100: sausages containing 100 g immobilized L. casei/kg of stuffing mixture, I-30: sausages containing 30 g immobilized L. casei/kg of stuffing mixture, Fr: sausages produced with free L. casei, NC: sausages produced by no adjunct culture. RT: Positive identification by retention times that agree with authentic compounds and by mass spectra of authentic compounds generated in the laboratory. KI: Tentative identification by Kovats retention index. MS: Positive identification by mass spectra obtained from NIST107. NIST21 and SZTERP libraries. Nd: Not detected. a: Kandylis et al., 2011, b: Kandylis & Koutinas, 2008, c: Kandylis et al., 2008, d: Vichi et al., 2003, e: Vichi et al., 2007b, f: Vichi et al., 2007a, h: Vichi et al., 2006.

45

46

Table 2 Effect of immobilized and free L. casei on aroma related compounds (μg/kg) isolated by probiotic dry-fermented sausages after ripening for 28 days using SPME GC/MS analysis. Identification method

KI

KIref

I-300

I-100

I-30

Fr

NC

Potential origin

Esters Ethyl acetate n-Propyl acetate Ethyl butanoate Ethyl 3-methyl-butanoate Isoamyl acetate Ethyl pentanoate Ethyl hexanoate Ethyl lactate Ethyl octanoate Ethyl decanoate 2-Phenylethyl-acetate 2-Propenyl octadecanoate

RT, KI, MS MS RT, KI, MS MS KI, MS MS RT, KI, MS KI, MS RT, KI, MS KI, MS RT, KI, MS MS

895 1023 1024 1060 1118 1123 1255 1348 1437 1641 1825 2390

895a

1177.9 ± 59.1 Nd Nd 45.5 ± 2.4 Nd Nd Nd 213.0 ± 10.8 121.7 ± 6.1 135.3 ± 6.9 53.0 ± 2.6 30.6 ± 1.7

1052.0 ± 52.6 Nd Nd Nd Nd Nd Nd 61.4 ± 3.3 78.3 ± 4.2 111.6 ± 5.6 57.7 ± 3.1 32.3 ± 1.7

939.0 ± 47.2 7.3 ± 0.5 32.4 ± 1.7 Nd 0.8 ± 0.4 Nd Nd 18.8 ± 1.1 43.4 ± 2.5 32.9 ± 1.9 16.0 ± 1.8 Nd

398.3 ± 21.7 1.8 ± 0.5 17.4 ± 1.2 Nd 1.8 ± 1.0 3.5 ± 0.4 26.4 ± 1.5 18.8 ± ±1.4 27.0 ± 1.7 18.2 ± 1.4 7.4 ± 0.7 Nd

719.3 ± 36.2 Nd Nd Nd Nd Nd 22.6 ± 1.2 4.9 ± 0.5 Nd 34.5 ± 1.8 11.7 ± 0.7 Nd

Staphylococci esterase activity Staphylococci esterase activity Staphylococci esterase activity Staphylococci esterase activity Microbial proteolytic activity, leucine catabolism Microbial metabolism activity Staphylococci esterase activity Staphylococci esterase activity Staphylococci esterase activity Microbial enzymatic activity Unknown Unknown

Organic acids Acetic acid Hexanoic acid Heptanoic acid Octanoic acid Nonanoic acid Decanoic acid Hexadecanoic acid

RT, KI, MS RT, KI, MS KI, MS RT, KI, MS KI, MS RT, KI, MS MS

1460 1874 1911 2116 2211 2319 2440

1448c 1841c 1962c 2152a 2213a 2336d

10398.4 ± 523.2 Nd Nd 238.5 ± 12.3 Nd 153.4 ± 8.9 Nd

12840.1 ± 456.9 Nd Nd 303.5 ± 14.5 Nd 200.2 ± 11.3 310.3 ± 16.7

338.0 ± 16.9 Nd 19.7 ± 1.9 Nd Nd Nd Nd

198.8 ± 11.9 Nd 21.9 ± 2.1 Nd 25.4 ± 2.3 Nd Nd

492.9 ± 32.4 39.8 ± 2.3 54.8 ± 2.9 Nd Nd Nd Nd

Carbohydrate fermentation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipolysis Enzymatic activity in meat

RT, KI, MS MS RT, KI, MS RT, MS MS MS KI, MS MS RT, KI, MS MS RT, KI, MS RT, KI, MS RT, KI, MS RT, KI, MS RT, KI, MS MS KI, MS MS RT, KI, MS MS

961 1088 1142 1250 1303 1321 1322 1340 1354 1403 1451 1456 1545 1549 1555 1613 1622 1631 1704 1707

935a

8728.0 ± 436.4 Nd 13.6 ± 1.9 Nd 6.9 ± 1.3 Nd Nd Nd Nd Nd 53.7 ± 3.6 12.2 ± 2.6 481.3 ± 25.2 591.2 ± 31.2 218.4 ± 11.9 Nd 90.5 ± 5.4 52.7 ± 3.6 68.4 ± 3.9 23.2 ± 2.2

3270.9 ± 172.0 Nd 9.5 ± 1.6 43.8 ± 3.2 12.2 ± 2.6 16.8 ± 2.8 Nd Nd 18.1 ± 1.9 Nd 81.1 ± 4.3 Nd 255.6 ± 13.2 466.1 ± 25.1 231.2 ± 11.6 47.0 ± 3.4 105.7 ± 5.3 73.7 ± 4.2 68.9 ± 3.7 30.7 ± 1.9

702.3 ± 37.1 Nd Nd Nd Nd Nd 6.3 ± 1.4 10.8 ± 1.5 16.5 ± 1.9 14.8 ± 1.7 28.1 ± 1.5 Nd 38.5 ± 2.1 161.8 ± 8.9 48.1 ± 2.5 12.9 ± 1.6 Nd 22.8 ± 1.2 39.3 ± 2.1 11.3 ± 1.6

433.4 ± 22.8 2.5 ± 1.1 Nd Nd Nd 2.8 ± 1.1 Nd 3.5 ± 1.6 4.9 ± 1.3 4.6 ± 1.5 7.6 ± 1.4 Nd 92.2 ± 5.2 102.6 ± 6.1 17.0 ± 1.9 6.1 ± 1.5 Nd 13.1 ± 1.9 17.5 ± 1.9 7.3 ± 2.1

577.3 ± 32.9 Nd Nd 13.3 ± 1.9 Nd Nd Nd Nd 41.1 ± 2.1 9.6 ± 1.6 50.2 ± 3.2 8.8 ± 1.7 Nd 106.3 ± 5.4 39.7 ± 2.1 Nd Nd Nd 21.8 ± 2.1 Nd

Carbohydrate fermentation Amino acid catabolism Lipid autooxidation Lipid autooxidation Unknown Seasonings Lipid oxidation Lipid oxidation Lipid autooxidation Unknown Lipid β-oxidation, seasonings Lipid autooxidation Carbohydrate fermentation Seasonings Lipid autooxidation Unknown Unknown Unknown Seasonings Seasonings

KI, MS RT, MS RT, MS RT, MS KI, MS KI, MS RT, KI, MS MS KI, MS MS RT, MS RT, MS

1759 1764 1838 1846 1873 1884 1923 1966 2017 2104 2190 2220

60.9 ± 3.7 Nd 20.9 ± 1.5 23.7 ± 2.3 Nd 62.7 ± 3.5 Nd Nd Nd 164.7 ± 8.9 365.0 ± 20.1 Nd

67.8 ± 3.8 Nd 23.7 ± 1.9 Nd Nd 111.3 ± 5.7 Nd Nd Nd 228.4 ± 11.6 444.0 ± 23.1 Nd

25.5 ± 1.3 8.9 ± 1.6 13.0 ± 1.7 16.2 ± 1.9 10.7 ± 1.6 23.0 ± 1.3 88.5 ± 4.7 5.2 ± 1.2 10.9 ± 1.6 146.0 ± 7.3 46.4 ± 2.5 Nd

Nd Nd 9.5 ± 1.7 Nd 5.7 ± 1.4 9.9 ± 1.7 31.3 ± 1.9 Nd Nd 75.0 ± 4.1 4.5 ± 1.5 89.3 ± 4.9

Nd Nd 15.0 ± 1.9 Nd 13.9 ± 1.9 20.4 ± 2.1 64.3 ± 3.6 Nd Nd 142.8 ± 8.1 Nd 110.9 ± 5.9

Lipid oxidation Seasonings Seasonings Seasonings Seasonings Unknown Seasonings Lipid oxidation Amino acid catabolism Seasonings Seasonings Seasonings

Alcohols Ethanol 2-Methyl-1-propanol 1-Butanol 1-Pentanol 5-Ethyl-2-heptanol 3-Methyl- 2-buten-1-ol 2-Penten-1-ol 1-Butoxy-2-propanol 1-Hexanol 2-Butoxy ethanol 1-Octen-3-ol 1-Heptanol 2,3-Butanediol beta-Linalool 1-Octanol [E]-2-Octen-1-ol 2-(2-Ethoxyethoxy)-ethanol p-Mentha-2,8-dienol alpha-Terpineol endo-1,7,7-Trimethyl-bicyclo[2.2.1]heptan-2-ol (borneol) Decanol beta-Citronellol trans-Carveol Geraniol cis-Carveol Benzyl alcohol 2-Phenyl-ethanol Dodecanol Phenol 4-[1-Methylethyl]-benzenemethanol (cumic alcohol) m-Thymol o-Thymol

1040b 1120c 1260a,1258b 1386d 1449a 1647a 1845a

1179b

1320c 1357c 1455c 1462e 1559b 1556d 1562c 1636a 1715d

1783b

1879f 1883c 1926e 2020e

M. Sidira et al. / Meat Science 100 (2015) 41–51

Compound

Miscellaneous compounds alpha-Pinene Origanene Dimethyl disulfide beta-Pinene Sabinene 3-Carene beta-Myrcene alpha-Terpinene 4-Carene delta-Limonene beta-Phellandrene Methyl propyl disulfide gamma-Terpinene Ocimene 2,4-Dimethyl-thiophene Styrene p-Cymene Terpinolene 2,5-Dimethyl-pyrazine Dipropyl disulfide Limonene-oxide β-Caryophyllene Dipropyl trisulfide Valencene Benzothiazole

RT, MS RT, KI, MS RT, KI, MS RT, KI, MS MS RT, KI, MS RT, MS MS KI, MS RT, KI, MS MS KI, MS KI, MS MS RT, KI, MS KI, MS MS KI, MS MS MS MS MS KI, MS MS MS MS MS KI, MS MS

961 1074 1189 1224 1257 1294 1295 1305 1330 1398 1411 1436 1496 1502 1534 1545 1619 1653 1663 1711 1738 1746 1774 1777 1805 1819 1919 1966 2133

KI, MS

1017 1021 1066 1096 1115 1141 1164 1171 1177 1194 1205 1236 1248 1251 1256 1265 1273 1283 1324 1382 1466 1608 1682 1727 1977

MS KI, MS KI, MS KI, MS KI, MS MS KI, MS RT, MS KI, MS MS KI, MS MS MS KI, MS KI, MS KI, MS MS MS MS KI, MS MS MS MS

1074c 1190e 1228e 1296e

1330a 1399a 1425c 1506e 1533d 1548e 1657e

1776f

1978f

1017f 1022f 1099h 1115h 1141f 1163h

1189b 1204f 1248h 1250c 1265c 1273h 1283f

1608h

Nd 85.1 ± 4.4 Nd Nd 9.3 ± 0.9 13.7 ± 0.8 41.4 ± 3.2 Nd 28.8 ± 2.4 39.4 ± 3.0 Nd Nd Nd Nd 323.8 ± 17.1 Nd 42.3 ± 3.2 62.7 ± 4.1 Nd Nd Nd 49.1 ± 3.5 366.2 ± 21.2 Nd 119.0 ± 7.2 Nd 416.4 ± 22.8 Nd 46.5 ± 2.9

Nd 110.7 ± 6.4 16.1 ± 1.8 Nd 13.6 ± 1.7 34.9 ± 1.9 35.0 ± 2.8 Nd 71.7 ± 4.5 46.7 ± 3.2 Nd Nd Nd Nd 369.0 ± 21.9 Nd Nd 84.8 ± 5.4 Nd Nd Nd 51.0 ± 3.6 614.5 ± 43.2 Nd 136.2 ± 7.9 Nd 455.3 ± 27.1 Nd 52.8 ± 3.7

2.6 ± 0.9 43.2 ± 3.2 Nd Nd Nd 8.0 ± 1.4 Nd Nd 16.5 ± 1.9 19.3 ± 2.1 Nd Nd 4.0 ± 0.9 260.2 ± 14.2 106.5 ± 5.4 Nd 12.4 ± 1.7 Nd 88.8 ± 5.4 Nd 6.1 ± 1.4 23.3 ± 2.1 347.6 ± 19.4 11.7 ± 1.6 74.8 ± 3.8 Nd 58.6 ± 3.9 1.8 ± 0.5 7.9 ± 1.4

Nd 29.0 ± 2.5 Nd Nd Nd Nd 8.0 ± 0.9 2.0 ± 0.3 6.5 ± 1.1 12.0 ± 0.6 Nd Nd Nd Nd 43.9 ± 2.3 Nd Nd Nd 26.9 ± 1.6 Nd Nd 19.7 ± 1.9 126.7 ± 7.3 7.8 ± 1.5 30.7 ± 2.5 Nd Nd 4.8 ± 1.2 4.9 ± 1.4

Nd 16.5 ± 0.9 Nd 3.4 ± 0.5 Nd Nd Nd Nd 26.4 ± 1.7 19.0 ± 2.1 11.0 ± 1.8 44.1 ± 3.2 Nd Nd 85.3 ± 5.4 38.0 ± 1.9 Nd 17.4 ± 1.1 31.7 ± 2.6 18.7 ± 1.9 Nd 46.2 ± 3.2 160.0 ± 8.0 25.4 ± 2.2 18.0 ± 0.9 16.1 ± 1.8 Nd Nd Nd

Strecker degradation, microbial metabolism Lipid autooxidation Lipid autooxidation Lipid autooxidation Seasonings Lipid autooxidation Carbohydrate fermentation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Amino acid catabolism Lipid autooxidation Seasonings Lipid oxidation Unknown Lipid autoxidation Seasonings Unknown Seasonings Unknown Seasonings Lipid oxidation Unknown Unknown Raw meat

564.3 ± 20.2 253.8 ± 22.7 43.0 ± 3.2 135.2 ± 7.8 86.4 ± 5.3 79.0 ± 4.0 1198.3 ± 61.9 Nd Nd 37605.7 ± 2220.3 493.5 ± 25.8 100.3 ± 6.0 478.1 ± 24.9 12.2 ± 1.6 Nd 79.4 ± 4.0 634.5 ± 33.4 41.5 ± 3.1 35.3 ± 1.9 344.6 ± 19.2 Nd 424.8 ± 25.2 Nd 95.9 ± 5.8 Nd

568.6 ± 31.4 228.6 ± 14.5 Nd 144.7 ± 8.2 70.7 ± 4.3 70.7 ± 3.9 1004.0 ± 50.2 Nd Nd 32679.4 ± 1637.3 64.9 ± 4.3 72.8 ± 4.6 420.3 ± 23.2 27.7 ± 2.5 Nd 50.4 ± 3.5 664.6 ± 35.4 35.1 ± 2.8 Nd 449.5 ± 24.9 Nd 421.1 ± 23.1 91.3 ± 5.5 Nd Nd

92.8 ± 5.6 69.0 ± 4.4 11.0 ± 1.6 64.0 ± 3.2 42.7 ± 2.4 93.3 ± 5.6 427.5 ± 23.9 Nd 9.2 ± 1.5 7984.3 ± 405.7 120.5 ± 6.2 86.3 ± 5.3 275.0 ± 17.8 12.4 ± 1.7 8.0 ± 1.4 68.0 ± 4.3 335.6 ± 19.8 36.6 ± 2.8 Nd 366.2 ± 21.4 13.3 ± 1.7 128.9 ± 7.5 40.0 ± 2.0 28.9 ± 2.5 7.5 ± 1.5

52.1 ± 3.8 28.7 ± 2.5 Nd 74.7 ± 4.8 20.2 ± 2.0 60.3 ± 3.2 234.5 ± 15.8 2.7 ± 2.2 2.5 ± 2.1 4107.3 ± 207.6 77.0 ± 4.9 Nd 240.1 ± 13.0 Nd Nd 30.0 ± 2.5 294.2 ± 16.7 294.2 ± 15.9 Nd 79.4 ± 4.0 6.8 ± 1.4 64.9 ± 4.3 Nd 16.9 ± 1.9 Nd

56.5 ± 3.8 51.7 ± 3.6 Nd 56.1 ± 3.9 Nd 34.8 ± 2.8 151.2 ± 8.6 Nd Nd 2646.7 ± 143.8 3.7 ± 1.1 Nd 46.8 ± 3.3 Nd Nd 35.5 ± 2.8 373.6 ± 21.7 Nd Nd 50.1 ± 3.5 6.9 ± 1.4 43.9 ± 3.2 Nd Nd Nd

Seasonings Seasonings Amino acid catabolism Seasonings Seasonings Seasonings Seasonings Seasonings Seasonings Orange peel Seasonings Unknown Seasonings Seasonings and/or orange peel Unknown Unknown origin, meat or food contaminants Seasonings Seasonings Seasonings Unknown Orange peel Seasonings Unknown Seasonings Unknown

M. Sidira et al. / Meat Science 100 (2015) 41–51

Carbonyl compounds 3-Methyl-butanal Hexanal Heptanal [E]-2-Hexenal 3-Octanone Octanal Acetoin 1-Octen-3-one [E]-2-Heptenal Nonanal [E,E]-2,4-Hexadienal [E]-2-Octenal [E,E]-2,4-Heptadienal Decanal Benzaldehyde [E]-2-Nonenal p-Menth-8-en-2-one [E]-2-Decenal 2-Methyl benzaldehyde 2,4-Nonadienal alpha-Citral [S]-2-Methyl-5-[1-methylethenyl]-2-cyclohexen-1-one 4-[1-Methylethyl]-benzaldehyde (cuminal) N,N-Dibutyl-formamide 2-Caren-10-al 2,4-Dodecadienal 3-Methyl-butyramide 2-Methyl-3-phenyl-2-propenal Tetradecanal

I-300: sausages containing 300 g immobilized L. casei/kg of stuffing mixture, I-100: sausages containing 100 g immobilized L. casei/kg of stuffing mixture, I-30: sausages containing 30 g immobilized L. casei/kg of stuffing mixture, Fr: sausages produced with free L. casei, NC: sausages produced by no adjunct culture. RT: Positive identification by retention times that agree with authentic compounds and by mass spectra of authentic compounds generated in the laboratory. KI: Tentative identification by Kovats retention index. MS: Positive identification by mass spectra obtained from NIST107. NIST21 and SZTERP libraries. Nd: Not detected. a: Kandylis et al., 2011, b: Kandylis et al., 2008, c: Vichi et al., 2003, d: Kandylis & Koutinas, 2008, e: Vichi et al., 2007a, f: Vichi et al., 2007b, h: Vichi et al., 2006. 47

48

Table 3 Effect of immobilized and free L. casei on aroma related compounds (μg/kg) isolated by probiotic dry-fermented sausages after ripening for 45 days using SPME GC/MS analysis. Identification method

KI

KIref

I-300

I-100

I-30

Fr

NC

Potential origin

Esters Ethyl acetate Ethyl propanoate N-propyl acetate Ethyl butyrate Ethyl 3-methyl-butanoate Ethyl pentanoate Ethyl hexanoate Ethyl lactate Ethyl octanoate Ethyl decanoate 2-phenylethyl-acetate

RT, KI, MS MS MS MS MS MS RT, KI, MS KI, MS RT, KI, MS KI, MS RT, KI, MS

895 1013 1023 1033 1061 1130 1255 1349 1437 1642 1825

895a

1260a,1258b 1386c 1449a 1647a 1845a

1387.1 ± 70.1 Nd Nd 326.7 ± 16.8 105.1 ± 5.5 18.6 ± 1.1 Nd 290.5 ± 15.1 192.6 ± 9.8 203.6 ± 10.2 90.1 ± 5.7

1788.9 ± 91.3 Nd Nd Nd Nd 21.2 ± 1.2 Nd 82.3 ± 4.5 97.3 ± 5.3 167.9 ± 9.8 80.5 ± 6.2

312.8 ± 15.9 6.9 ± 0.9 4.4 ± 0.6 79.4 ± 4.3 7.7 ± 1.2 19.4 ± 1.4 Nd 45.2 ± 2.5 79.3 ± 4.2 66.2 ± 3.9 13.4 ± 1.2

417.2 ± 21.4 6.6 ± 0.4 4.1 ± 0.3 66.8 ± 3.5 9.0 ± 1.1 19.6 ± 1.4 115.3 ± 5.9 70.6 ± 3.8 80.1 ± 4.2 60.4 ± 3.6 11.3 ± 0.9

239.0 ± 12.1 7.4 ± 0.7 Nd 31.6 ± 1.7 Nd Nd 69.3 ± 4.3 20.7 ± 1.3 Nd 88.1 ± 4.7 63.2 ± 3.4

Staphylococci esterase activity Staphylococci esterase activity Staphylococci esterase activity Esterification of alcohols and acids Staphylococci esterase activity Microbial metabolism activity Staphylococci esterase activity Staphylococci esterase activity Staphylococci esterase activity Microbial enzymatic activity Microbial activity

Organic acids Acetic acid Hexanoic acid Heptanoic acid Octanoic acid Decanoic acid

RT, KI, MS RT, KI, MS KI, MS RT, KI, MS RT, KI, MS

1460 1874 1911 2116 2319

1448d 1841d 1962d 2152a 2336c

17905.6 ± 895.3 12.5 ± 0.9 Nd 379.6 ± 21.3 286.9 ± 15.8

19082.4 ± 963.8 Nd Nd 294.0 ± 14.7 155.5 ± 7.8

1471.5 ± 75.4 Nd 76.0 ± 3.8 74.6 ± 3.9 Nd

1286.5 ± 65.1 Nd 47.7 ± 2.6 Nd Nd

129.4 ± 6.7 Nd 177.7 ± 8.9 Nd Nd

Carbohydrate fermentation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipolysis

Alcohols Ethanol 3-Methyl-2-butanol 1-Penten-3-ol 2-Penten-1-OL 1-Butoxy-2-propanol 1-Hexanol 2-Butoxy-ethanol 1-Octen-3-ol 2,3-Butanediol beta-Linalool 1-Octanol 1,3-Butanediol [E]-2-Octen-1-ol 2-(2-Ethoxyethoxy)-ethanol p-Mentha-2,8-dienol alpha-Terpineol endo-1,7,7-Trimethyl-bicyclo[2.2.1]heptan-2-ol (borneol) Decanol beta-Citronellol Dihydrocarveol trans-Carveol cis-Carveol Benzyl alcohol 2-Phenyl-ethanol Phenol 4-[1-Methylethyl]-benzenemethanol (cumic alcohol) Z-2-Octadecen-1-ol m-Thymol o-Thymol

RT, KI, MS RT, MS MS KI, MS MS RT, KI, MS MS RT, KI, MS RT, KI, MS RT, KI, MS RT, KI, MS MS MS KI, MS MS KI, MS MS KI, MS RT, MS MS RT, MS KI, MS KI, MS RT, KI, MS KI, MS MS MS RT, MS RT, MS

961 1115 1158 1321 1341 1354 1403 1451 1545 1549 1555 1565 1616 1622 1631 1704 1707 1759 1764 1802 1838 1873 1884 1923 2017 2104 2133 2190 2220

935a

9357.4 ± 568.2 Nd Nd Nd Nd Nd Nd 63.5 ± 3.3 445.7 ± 24.6 886.3 ± 47.1 190.1 ± 9.9 125.0 ± 6.7 Nd 123.3 ± 6.3 76.4 ± 4.1 99.6 ± 5.0 35.0 ± 2.5 96.7 ± 5.1 Nd Nd 50.2 ± 3.7 Nd 80.7 ± 4.6 Nd Nd 240.6 ± 13.1 77.4 ± 5.2 581.1 ± 31.9 1693.6 ± 87.9

4473.2 ± 235.9 Nd Nd Nd Nd 23.4 ± 2.1 Nd 111.9 ± 6.4 Nd 516.9 ± 32.1 161.4 ± 9.5 85.0 ± 5.2 54.1 ± 3.1 120.1 ± 7.2 91.2 ± 4.7 68.4 ± 3.6 36.3 ± 2.1 90.2 ± 4.7 Nd Nd 25.2 ± 2.1 Nd 100.0 ± 5.7 Nd Nd 184.6 ± 9.5 Nd 364.2 ± 21.2 Nd

1930.4 ± 98.7 Nd 6.6 ± 1.1 8.9 ± 1.4 11.7 ± 1.5 22.9 ± 1.3 20.6 ± 1.1 37.8 ± 1.9 Nd 288.9 ± 17.1 81.9 ± 4.9 90.6 ± 4.8 34.1 ± 1.9 Nd 62.3 ± 3.2 67.4 ± 3.8 22.8 ± 1.6 37.0 ± 2.1 13.9 ± 1.9 Nd 53.4 ± 2.8 27.7 ± 1.7 33.1 ± 1.9 Nd 13.0 ± 1.7 67.1 ± 3.8 Nd 76.6 ± 3.9 Nd

1401.6 ± 78.2 Nd 8.7 ± 1.4 Nd 11.2 ± 1.4 18.9 ± 1.9 21.7 ± 2.1 56.8 ± 2.9 193.5 ± 10.1 338.2 ± 17.0 51.9 ± 3.5 66.3 ± 3.9 30.2 ± 2.5 Nd 64.8 ± 4.5 56.8 ± 5.2 21.0 ± 1.9 Nd Nd Nd 44.5 ± 2.4 27.5 ± 1.9 21.6 ± 2.1 50.3 ± 3.5 8.8 ± 1.4 72.4 ± 4.6 Nd 7.3 ± 1.3 Nd

1881.7 ± 104.7 4.6 ± 1.7 22.4 ± 2.1 Nd 13.8 ± 1.8 70.0 ± 4.5 20.2 ± 1.4 169.6 ± 9.6 Nd 365.1 ± 19.2 44.2 ± 3.2 37.0 ± 1.9 Nd Nd 92.0 ± 4.6 32.6 ± 2.6 43.0 ± 2.9 Nd Nd 25.7 ± 1.4 67.4 ± 4.4 51.8 ± 3.6 42.9 ± 3.5 140.1 ± 7.0 Nd 57.1 ± 3.9 Nd 8.1 ± 1.5 129.9 ± 6.7

Carbohydrate fermentation Strecker degradation, microbial metabolism Lipid β-oxidation Lipid oxidation Unknown Lipid autooxidation Unknown Lipid β-oxidation, seasonings Carbohydrate fermentation Seasonings Lipid autooxidation Carbohydrate fermentation Unknown Unknown Seasonings Seasonings Seasonings Lipid oxidation Seasonings

Carbonyl compounds 3-Methyl-butanal Pentanal 2-Pentanone 2,3-Pentanedione Hexanal 2-Methyl-2-butenal

RT, MS KI, MS MS MS RT, KI, MS MS

961 1020 1025 1055 1074 1091

Nd Nd Nd 64.3 ± 3.4 202.2 ± 11.2 Nd

Nd Nd Nd Nd 215.8 ± 11.9 Nd

78.9 ± 4.9 9.8 ± 1.5 Nd 12.3 ± 1.6 126.5 ± 7.3 Nd

47.4 ± 3.3 24.8 ± 2.2 Nd 14.1 ± 1.8 271.5 ± 15.2 11.2 ± 2.6

105.8 ± 6.3 13.6 ± 2.7 11.1 ± 1.6 Nd 222.0 ± 21.2 Nd

Strecker degradation, microbial metabolism Lipid autooxidation Lipid β-oxidation Bacterial metabolism Lipid autooxidation Lipid autooxidation, seasonings

1163a 1320d 1357d 1455d 1559b 1556c 1562d 1590c 1636a 1715c 1783b

1879e 1883d 1926f 2020f

1002d

1074d

Seasonings Seasonings Unknown Seasonings Amino acid catabolism Seasonings Seasonings Seasonings

M. Sidira et al. / Meat Science 100 (2015) 41–51

Compound

Miscellaneous compounds 3-Methyl-pentane alpha-Pinene Origanene Methyl benzene Dimethyl disulfide beta-Pinene Sabinene Ethyl benzene p-Xylene 3-Carene beta-Myrcene 4-Carene o-Xylene delta-Limonene beta-Phellandrene 2-Pentyl-furan Methyl propyl disulfide gamma-Terpinene Ocimene Styrene p-Cymene Terpinolene Dipropyl disulfide Limonene oxide Allyl disulfide β-Caryophyllene Dipropyl trisulfide Valencene

KI, MS RT, KI, MS RT, KI, MS MS KI, MS RT, KI, MS RT, MS KI, MS RT, KI, MS KI, MS KI, MS KI, MS KI, MS MS MS KI, MS MS MS MS MS KI, MS MS MS MS MS

1184 1189 1224 1257 1289 1294 1295 1330 1398 1436 1496 1534 1545 1619 1645 1653 1691 1711 1719 1746 1774 1777 1805 1919 2133

MS KI, MS

1015 1017 1021 1037 1066 1096 1115 1121 1137 1141 1164 1177 1187 1194 1205 1234 1236 1248 1251 1265 1273 1283 1382 1466 1491 1608 1682 1727

MS MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS KI, MS RT, MS KI, MS MS MS KI, MS MS KI, MS KI, MS KI, MS MS MS MS KI, MS MS KI, MS

1181d 1190f 1228f 1292f 1296f 1330a 1399a 1425d 1506f 1533c 1548f

1657f

1776e

1017e 1022e

1099h 1115h 1119d 1142f 1141e 1163h 1187f 1189b 1204e

1248h 1250d 1265d 1273h 1283e

1608h

Nd Nd Nd Nd Nd 18.8 ± 1.9 Nd 92.2 ± 5.6 66.7 ± 4.3 Nd Nd 457.7 ± 31.2 Nd Nd Nd 95.3 ± 5.7 Nd Nd Nd 85.3 ± 5.4 638.2 ± 33.7 Nd 178.8 ± 9.9 893.0 ± 43.2 77.4 ± 4.9

Nd Nd Nd Nd Nd 50.7 ± 3.5 Nd 69.9 ± 4.4 74.8 ± 4.8 Nd Nd 496.1 ± 26.9 Nd Nd Nd 113.3 ± 6.6 Nd Nd Nd 55.6 ± 3.8 722.7 ± 39.2 Nd 166.7 ± 9.3 932.0 ± 47.1 55.5 ± 3.8

Nd Nd Nd Nd Nd 7.1 ± 1.4 Nd 27.5 ± 2.4 36.8 ± 2.9 Nd 10.3 ± 1.5 228.9 ± 12.4 Nd 26.0 ± 1.3 Nd Nd 61.2 ± 4.1 31.2 ± 2.6 Nd 80.4 ± 5.2 583.3 ± 31.1 23.4 ± 2.2 107.2 ± 6.4 130.7 ± 7.5 15.6 ± 1.8

Nd 18.6 ± 1.9 Nd Nd Nd 12.0 ± 2.6 27.5 ± 1.7 38.0 ± 1.9 48.2 ± 3.4 Nd Nd 180.5 ± 11.2 Nd 20.5 ± 2.1 36.5 ± 2.9 Nd 62.4 ± 4.1 57.6 ± 3.9 Nd 104.3 ± 6.2 454.4 ± 32.8 11.7 ± 1.6 101.6 ± 6.1 Nd 13.1 ± 2.8

18.1 ± 2.9 25.9 ± 1.5 11.2 ± 1.6 10.2 ± 1.5 24.2 ± 2.2 9.9 ± 1.5 Nd 81.5 ± 5.1 47.6 ± 3.3 186.8 ± 11.4 Nd 160.2 ± 9.1 92.1 ± 5.4 70.3 ± 4.5 Nd 48.0 ± 2.4 58.9 ± 3.9 71.7 ± 4.5 57.8 ± 3.9 70.0 ± 3.5 100.2 ± 6.0 21.6 ± 2.1 536.8 ± 28.8 Nd Nd

Lipid β-oxidation Lipid autooxidation Lipid autooxidation Seasonings Lipid β-oxidation Lipid autooxidation Carbohydrate fermentation Lipid autooxidation Lipid autooxidation Lipid autooxidation Lipid autooxidation Amino acid catabolism Lipid autooxidation Seasonings Lipid autooxidation Lipid oxidation Unknown Lipid autoxidation Unknown Unknown Seasonings Unknown Seasonings Unknown Raw meat

431.1 ± 23.6 772.7 ± 41.6 458.4 ± 25.9 Nd 74.5 ± 4.7 220.1 ± 21.0 147.0 ± 8.4 Nd Nd 140.3 ± 8.0 2162.2 ± 109.1 Nd Nd 59553.8 ± 3080.7 144.4 ± 8.3 Nd 131.7 ± 7.5 625.6 ± 34.4 59.6 ± 3.0 81.2 ± 5.2 976.7 ± 52.8 72.9 ± 4.6 587.7 ± 38.4 Nd Nd 568.3 ± 30.4 Nd 93.8 ± 5.7

409.1 ± 30.5 834.9 ± 44.8 385.7 ± 22.3 Nd Nd 190.8 ± 10.5 92.7 ± 5.6 Nd Nd 91.5 ± 5.5 967.6 ± 52.4 Nd Nd 33657.5 ± 1782.9 429.7 ± 24.5 Nd 113.7 ± 6.6 466.3 ± 28.3 84.9 ± 5.2 76.4 ± 4.8 851.8 ± 47.6 44.6 ± 3.3 471.3 ± 33.6 Nd Nd 533.6 ± 35.7 93.3 ± 5.7 49.3 ± 3.4

Nd 136.2 ± 7.8 108.2 ± 6.4 39.4 ± 3.0 8.3 ± 1.4 110.5 ± 6.5 80.5 ± 5.0 10.8 ± 1.5 10.8 ± 1.5 114.4 ± 6.7 676.3 ± 38.9 13.3 ± 1.7 13.8 ± 1.7 10941.6 ± 652.1 222.1 ± 14.1 Nd 138.2 ± 7.9 263.6 ± 18.2 Nd 146.0 ± 7.3 727.8 ± 46.4 32.6 ± 2.7 454.7 ± 32.9 29.5 ± 2.5 Nd 204.2 ± 20.2 74.7 ± 5.7 Nd

Nd 127.8 ± 7.4 70.3 ± 4.5 46.9 ± 3.3 Nd 228.5 ± 14.5 69.3 ± 4.4 14.0 ± 1.7 10.1 ± 1.6 169.0 ± 9.5 696.8 ± 36.9 6.9 ± 1.4 Nd 9894.4 ± 504.7 189.0 ± 11.5 Nd Nd 329.2 ± 18.6 Nd 250.2 ± 14.5 1097.5 ± 64.8 27.3 ± 2.4 191.1 ± 11.6 37.6 ± 3.9 Nd 209.4 ± 12.5 Nd Nd

Nd 61.2 ± 4.1 38.3 ± 2.9 38.8 ± 3.0 Nd 108.7 ± 6.4 28.0 ± 1.4 11.1 ± 1.6 14.5 ± 1.7 55.2 ± 3.7 186.2 ± 11.3 13.0 ± 1.7 Nd 5675.1 ± 383.8 Nd 22.1 ± 2.1 Nd 30.2 ± 2.5 Nd 154.9 ± 8.7 774.9 ± 43.7 10.2 ± 1.5 54.8 ± 3.8 42.7 ± 2.2 11.7 ± 1.6 120.4 ± 7.0 Nd Nd

Seasonings Seasonings Seasonings Amino acid catabolism Seasonings Seasonings Flavor processed meat Unknown origin, meat or food contaminants Seasonings Seasonings Seasonings Unknown origin, meat or food contaminants Orange peel Seasonings Lipid autooxidation Unknown Seasonings Seasonings and/or orange peel Unknown origin, meat or food contaminants Seasonings Seasonings Unknown Orange peel Seasonings Seasonings Unknown Seasonings

M. Sidira et al. / Meat Science 100 (2015) 41–51

2-Heptanone Heptanal [E]-2-Hexenal 3-Octanone 2-Octanone Octanal Acetoin [E]-2-Heptenal Nonanal [E]-2-Octenal [E,E]-2,4-Heptadienal Benzaldehyde [E]-2-Nonenal p-Menth-8-en-2-one Butyrolactone [E]-2-Decenal 3,7-Dimethyl-2,6-octadienal 2,4-Nonadienal Caprolactone [S]-2-Methyl-5-[1-methylethenyl]-2-cyclohexen-1-one 4-[1-Methylethyl]-benzaldehyde (cuminal) N,N-Dibutyl-formamide 2-Caren-10-al 3-Methyl-butyramide Tetradecanal

I-300: sausages containing 300 g immobilized L. casei/kg of stuffing mixture, I-100: sausages containing 100 g immobilized L. casei/kg of stuffing mixture, I-30: sausages containing 30 g immobilized L. casei/kg of stuffing mixture, Fr: sausages produced with free L. casei, NC: sausages produced by no adjunct culture. RT: Positive identification by retention times that agree with authentic compounds and by mass spectra of authentic compounds generated in the laboratory. KI: Tentative identification by Kovats retention index. MS: Positive identification by mass spectra obtained from NIST107. NIST21 and SZTERP libraries. Nd: Not detected. a: Kandylis et al., 2011, b: Kandylis et al., 2008, c: Kandylis & Koutinas, 2008, d: Vichi et al., 2003, e: Vichi et al., 2007b, f: Vichi et al., 2007a, h: Vichi et al., 2006.

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M. Sidira et al. / Meat Science 100 (2015) 41–51

Table 4 Sum of volatile compounds (μg/kg) isolated by probiotic dry-fermented sausages after ripening for 1, 28 and 45 days using SPME GC/MS analysis. Ripening time (days)

Samples

Sum of esters

Sum of organic acids

Sum of alcohols

Sum of carbonyl compounds

Sum of miscellaneous compounds

Total volatiles

0

I-300 I-100 I-30 Fr NC I-300 I-100 I-30 Fr NC I-300 I-100 I-30 Fr NC

66.2 ± 5.2 52.4 ± 4.2 Nd Nd 9.4 ± 1.6 1777.0 ± 126.7 1393.3 ± 99.7 1090.6 ± 80.8 520.6 ± 44.5 793.0 ± 57.1 2614.3 ± 189.9 2238.1 ± 167.3 634.7 ± 51.1 861.0 ± 65.8 519.3 ± 39.9

124.0 ± 9.6 177.1 ± 13.9 Nd Nd Nd 10790.3 ± 769.9 13654.1 ± 706.3 357.7 ± 26.6 246.1 ± 23.1 587.5 ± 53.2 18584.6 ± 1319.9 19531.9 ± 1394.8 1622.1 ± 117.5 1334.2 ± 95.7 307.1 ± 22.1

1874.8 2084.4 806.5 1077.0 653.8 11038.0 5606.5 1507.8 940.3 1235.4 14222.6 6506.1 3008.7 2574.0 3319.2

1357.1 1244.6 1705.4 1398.1 805.0 1643.7 2092.3 1093.3 322.9 577.2 2869.9 2953.1 1597.1 1555.9 2055.5

18277.2 14367.5 10641.1 4273.0 6775.7 42705.8 3764.4 10321.0 5686.5 3557.5 67302.0 39844.7 14557.5 13665.3 7452.0

21699.3 17926.0 13153.0 6748.1 8243.9 67954.8 59810.6 14370.4 7716.4 6750.6 105593.4 71073.9 21420.1 19990.4 13653.1

28

45

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

148.8 174.9 75.7 101.5 75.2 805.0 428.4 129.8 97.0 108.2 1172.7 502.2 232.1 217.4 271.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

92.8 119.9 151.3 135.6 77.4 136.8 191.1 100.7 35.5 55.4 226.8 228.1 138.7 156.0 193.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1482.1 1204.8 948.1 360.9 541.6 3530.3 2658.2 777.5 436.4 287.8 4968.6 3045.1 1261.6 1032.0 699.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1738.5 1517.7 1175.1 598.1 695.8 5368.6 4083.5 1115.4 636.5 561.7 7877.9 5337.5 1801.0 1566.8 1227.0

I-300: sausages containing 300 g immobilized L. casei/kg of stuffing mixture, I-100: sausages containing 100 g immobilized L. casei/kg of stuffing mixture, I-30: sausages containing 30 g immobilized L. casei/kg of stuffing mixture, Fr: sausages produced with free L. casei, NC: sausages produced by no adjunct culture. Nd: Not detected.

Carbonyl compounds identified included mainly aldehydes, ketones and lactones. As mentioned above, methyl branched aldehydes, such as 2- and 3-methyl-butanal, derive from amino acid catabolism (Flores et al., 2004). However, most of aldehydes, come from lipid oxidation (pentanal, hexanal, 2-hexenal, 2,4-hexadienal, heptanal, 2-heptenal, 2,4-heptadienal, octanal, 2-octenal, nonanal, 2-nonenal 2,4-nonadienal, decanal, 2-decenal and 2-undecenal) (Ansorena et al., 2001; Marco et al., 2006). Hexanal detected in all samples ripened for at least 28 days, is the most abundant product of lipid oxidation in meats and therefore it has often been chosen as an index of the level of oxidation (Marco et al., 2006). High molecular aldehydes, such as tetradecanal present in all samples ripened for 28 and 45 days except in NC sausage, are not considered major contributors to the aroma, but their importance to flavor development is only due to the fact that they can act as precursors of lower molecular alkanals and alkenals (Ansorena et al., 2001). The formation of methyl ketones, such as 2-pentanone, 2heptanone and 2-octanone identified in NC sample ripened for 45 days can be explained by the presence of an incomplete βoxidation pathway in staphylococci (Leroy, Verluyten, & De Vuyst, 2006). Benzaldehyde detected in all samples, has been associated with almond flavor (Ansorena et al., 2001). Acetoin is a byproducts of the metabolism of LAB and can be formed through the chemical oxidation of 2,3-butanediol (Marco et al., 2006). A number of miscellaneous compounds were also detected, including terpenes, sulfur compounds, pyrazines and hydrocarbons. Terpenes may derive by spices, although some of them (a-terpinene and limonene) have been found in meat as a consequence of their presence in animal feedstuffs (Ansorena et al., 2001), while some sulfur compounds identified may come from garlic. 2-pentyl-furan identified in NC sample ripened for 45 days originates from lipid oxidation (Marco et al., 2006). Most pyrazines and hydrocarbons isolated in the present study have also been detected in previous studies (Ansorena et al., 2001; Meynier, Novelli, Chizzolini, Zanardi, & Gandemer, 1999). Hydrocarbons isolated in the present study, such as 3-methyl-pentane, 3-ethyl-4-methylhexane, ethyl benzene, m-, p- and o-xylene and styrene are not important contributors to the meat odor.

Fr sausages ripened for 45 days. The third group contained all sausages ripened for 1 day and samples I-30, Fr and NC ripened for 28 days. A fourth group consisting of NC sample ripened for 45 days was also evident. Although the results of PCA indicated that primarily the concentration of the immobilized probiotic cultures affected the volatile composition of dry-fermented sausages, it is yet unknown and difficult to interpret the relation between the microbial associations which are involved in the biochemical changes that occur during ripening of fermented sausages and the chemical compounds, due to the high complexity of the food ecosystem. 4. Conclusions The concentration of immobilized L. casei on wheat grains primarily affected the composition of volatiles generated during ripening of probiotic dry-fermented sausages. An improved profile of aroma-related compounds in sausages containing high amounts of the immobilized starter culture at the end of ripening process was observed. However, more research is required to better understand the microbial

3.2. Chemometrics Principal component analysis (PCA) is used in exploratory analysis, as it gives graphical representations of inter-sample and inter-variable relationships and provides a way to reduce the complexity of the data. The application of the PCA algorithm to data showed four distinctive groups (Fig. 2). The first group was composed by I-300 and I-100 samples ripened for 28 and 45 days, while the second group by I-30 and

Fig. 2. PCA plot of volatiles generated during ripening of probiotic dry-fermented sausages. I-300: sausages containing 300 g immobilized L. casei/kg of stuffing mixture, I-100: sausages containing 100 g immobilized L. casei/kg of stuffing mixture, I-30: sausages containing 30 g immobilized L. casei/kg of stuffing mixture, Fr: sausages produced with free L. casei, NC: sausages produced by no adjunct culture. The ripening time is indicated after the sample code.

M. Sidira et al. / Meat Science 100 (2015) 41–51

interactions which are responsible for the biochemical changes that occur during the ripening process of dry-fermented sausages. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research has been co-financed by the European Union (European Social Fund — ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) — Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. References Ammor, M. S., & Mayo, B. (2007). Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: An update. Meat Science, 76, 138–146. Ansorena, D., Astiasarián, I., & Bello, J. (2000). Changes in volatile compounds during ripening of chorizo de Pamplona elaborated with Lactobacillus plantarum and Staphylococcus carnosus. Food Science and Technology International, 6, 439–447. Ansorena, D., Gimeno, O., Astiasarián, I., & Bello, J. (2001). Analysis of volatile compounds by GC–MS of a dry-fermented sausage: Chorizo de Pamplona. Food Research International, 34, 67–75. Bosnea, L., Kourkoutas, Y., Albantaki, N., Tzia, C., Koutinas, A. A., & Kanellaki, M. (2009). Functionality of freeze-dried L. casei cells immobilized on wheat grains. LWT- Food Science and Technology, 42, 1696–1702. Boylston, T. D., Vinderola, C. G., Ghoddusi, H. B., & Reinheimer, J. A. (2004). Incorporation of bifidobacteria into cheeses: Challenges and rewards. International Dairy Journal, 19, 315–387. Choi, S. S., Kim, Y., Han, K. S., You, S., Oh, S., & Kim, S. H. (2006). Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro. Letters in Applied Microbiology, 42, 452–458. Deshpande, G., Rao, S., & Patole, S. (2011). Progress in the field of probiotics: Year 2011. Current Opinion in Gastroenterology, 27, 13–18. Di Cagno, R., Lòpez, C. C., Tofalo, R., Gallo, G., De Angelis, M., Paparella, A., Hammes, W. P., & Gobbetti, M. (2008). Comparison of the compositional, microbiological, biochemical and volatile profile characteristics of three Italian PDO fermented sausages. Meat Science, 78, 224–235. Dimitrellou, D., Kourkoutas, Y., Koutinas, A. A., & Kanellaki, M. (2009). Thermally-dried immobilized kefir on casein as starter culture in dried whey cheese production. Food Microbiology, 26, 809–820. Flores, M., Durá, M.A., Marco, A., & Toldrá, F. (2004). Effect of Debaryomyces spp. on aroma formation and sensory quality of dry-fermented sausages. Meat Science, 68, 439–446. Kandylis, P., Goula, A., & Koutinas, A. A. (2008). Corn starch gel for yeast cell entrapment. A view for catalysis of wine fermentation. Journal of Agricultural and Food Chemistry, 56, 12037–12045. Kandylis, P., & Koutinas, A. A. (2008). Extremely low temperature fermentations of grape must by potato-supported yeast, strain AXAZ-1. A contribution is performed for catalysis of alcoholic fermentation. Journal of Agricultural and Food Chemistry, 56, 3317–3327. Kandylis, P., Vekiari, A. S., Kanellaki, M., Kamoun, N. G., Msallem, M., & Kourkoutas, Y. (2011). Comparative study of extra virgin olive oil flavor profile of Koroneiki variety

51

(Olea europaea var. Microcarpa alba) cultivated in Greece and Tunisia during one period of harvesting. LWT - Food Science and Technology, 44, 1333–1341. Karapetsas, A., Vavoulidis, E., Galanis, A., Sandaltzopoulos, R., & Kourkoutas, Y. (2010). Rapid detection and identification of probiotic Lactobacillus casei ATCC 393 by multiplex PCR. Journal of Molecular Microbiology and Biotechnology, 18, 156–161. Kourkoutas, Y., Bosnea, L., Taboukos, S., Baras, C., Lambrou, D., & Kanellaki, M. (2006). Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces. Journal of Dairy Science, 89, 1431–1451. Kourkoutas, Y., Xolias, V., Kallis, M., Bezirtzoglou, E., & Kanellaki, M. (2005). Lactobacillus casei immobilization on fruit pieces for probiotic additive, fermented milk and lactic acid production. Process Biochemistry, 40, 411–416. Leroy, F., Verluyten, J., & De Vuyst, L. (2006). Functional meat starter cultures for improved sausage fermentation. International Journal of Food Microbiology, 106, 270–285. Marco, A., Navarro, J. L., & Flores, M. (2004). Volatile compounds of dry-fermented sausages as affected by solid-phase microextraction (SPME). Food Chemistry, 84, 633–641. Marco, A., Navarro, J. L., & Flores, M. (2006). The influence of nitrite and nitrate on microbial, chemical and sensory parameters of slow dry fermented sausage. Meat Science, 73, 660–673. Mattila-Sandholm, T., Myllärinen, P., Crittenden, R., Mogensen, G., Fondén, R., & Saarela, M. (2002). Technological challenges for future probiotic foods. International Dairy Journal, 12, 173–182. Meynier, A., Novelli, E., Chizzolini, R., Zanardi, E., & Gandemer, G. (1999). Volatile compounds of commercial Milano salami. Meat Science, 51, 175–183. Mitropoulou, G., Nedovic, V., Goyal, A., & Kourkoutas, Y. (2013). Immobilization technologies in probiotic food production. Journal of Nutrition and Metabolism (http://dx.doi. org/10.1155/2013/716861). Olivares, A., Navarro, J. L., & Flores, M. (2009). Establishment of the contribution of volatile compounds to the aroma of fermented sausages at different stages of processing and storage. Food Chemistry, 115, 1464–1472. Saxami, G., Ypsilantis, P., Sidira, M., Simopoulos, C., Kourkoutas, Y., & Galanis, A. (2012). Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat intestinal mucosa. Anaerobe, 18, 417–420. Schmidt, D., & Berger, R. G. (1998). Aroma compounds in fermented sausages of different origins. Lebensmittel Wissenschaft und Technologie, 31, 559–567. Sidira, M., Galanis, A., Ypsilantis, P., Karapetsas, A., Progaki, Z., Simopoulos, C., & Kourkoutas, Y. (2010). Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora. Journal of Molecular Microbiology and Biotechnology, 19, 224–230. Sidira, M., Karapetsas, A., Galanis, A., Kanellaki, M., & Kourkoutas, Y. (2014). Effective survival of immobilized Lactobacillus casei during ripening and heat treatment of probiotic dry-fermented sausages and investigation of microbial dynamics. Meat Science, 96, 948–955. Vichi, S., Castellote, A. I., Pizzale, L., Conte, L. S., Buxaderas, S., & López-Tamames, E. (2003). Analysis of virgin olive oil volatile compounds by headspace solid-phase microextraction coupled to gas chromatography with mass spectrometric and flame ionization detection. Journal of Chromatograph A, 983, 19–33. Vichi, S., Guadayol, J. M., Caixach, J., López-Tamames, E., & Buxaderas, S. (2006). Monoterpene and sesquiterpene hydrocarbons of virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry. Journal of Chromatography A, 1125, 117–123. Vichi, S., Guadayol, J. M., Caixach, J., López-Tamames, E., & Buxaderas, S. (2007a). Comparative study of different extraction techniques for the analysis of virgin olive oil aroma. Food Chemistry, 105, 1171–1178. Vichi, S., Riu-Aumatell, M., Mora-Pons, M., Guadayol, J. M., Buxaderas, S., & LópezTamames, E. (2007b). HS-SPME coupled to GC/MS for quality control of Juniperus communis L. berries used for gin aromatization. Food Chemistry, 105, 1748–1754.

Effect of immobilized Lactobacillus casei on the evolution of flavor compounds in probiotic dry-fermented sausages during ripening.

The effect of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds in probiotic dry-fermented sausages dur...
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