Effect of commercial starter cultures on volatile ...

Research Article Received: 2 December 2014

Revised: 27 March 2015

Accepted article published: 8 April 2015

Published online in Wiley Online Library: 27 April 2015

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7203

Effect of commercial starter cultures on volatile compound profile and sensory characteristics of dry-cured foal sausage José M Lorenzo,* María Gómez, Laura Purriños and Sonia Fonseca Abstract BACKGROUND: The present work deals with the evaluation of the effect of three different commercial starter cultures (Chr. Hansen, Hørsholm, Denmark) on the volatile compound profile and sensory properties, as well as some important physicochemical parameters, of dry-fermented foal sausages at the end of ripening in order to select the most suitable starter culture for this elaboration. The sausage batches were named as follows: CO (non-inoculated control), FS (Lactobacillus sakei + Staphylococcus carnosus), SM (L. sakei + S. carnosus + Staphylococcus xylosus + Pediococcus pentosaceus + Debaryomyces hansenii) and TR (L. sakei + S. carnosus +S. xylosus). RESULTS: The pH values differed significantly among batches, with the highest values corresponding to CO followed by TR, SM and FS. The highest amounts of volatile compounds were found in FS batch. Hexanal was the most abundant compound, especially in FS and SM batches. These batches also showed higher levels of compounds derived from carbohydrate fermentation and amino acid catabolism. Sensory results showed that acid taste was significantly lower in CO batch than in inoculated batches. CONCLUSION: According to most parameters, batches inoculated with FS and SM starters showed marked acidity compared with TR and CO batches, as expected from the manufacturer’s indications. Therefore the most suitable starter culture for use in the manufacture of foal sausages in Mediterranean countries such as Spain with a preference for low-acidity products was found to be TR culture. © 2015 Society of Chemical Industry Keywords: dry-cured foal sausage; physicochemical parameters; sensory analysis; starter cultures; volatile compounds

INTRODUCTION

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Traditional fermented meat products, especially fermented sausages, are produced and consumed in many countries. The safety of such products depends mainly on a fall in pH and a decrease in water activity, allowing the control of pathogen growth using the ‘hurdle technology’ concept.1 However, fermented meat products have usually been manufactured following empirical methods by means of spontaneous fermentation, and the quality and safety of final products are not guaranteed, being deficient in some cases. Nowadays, the use of starter cultures, generally consisting of a combination of lactic acid bacteria (LAB), Staphylococcus species and in some cases yeasts, has become common in the manufacture of several types of fermented products in order to ensure safety by restraining the development of wild microbiota, thus reducing the risk of pathogenic and spoilage bacteria, as well as to contribute to colour and flavour development and extend shelf-life, maintaining the typical characteristics obtained in artisanal productions.2 LAB in dry-fermented meat products are responsible for the rapid fermentation of carbohydrates added to the mixture, leading to a decrease in pH and subsequent development of a tangy flavour, whose intensity will depend essentially on the starter culture applied and the carbohydrate substrates present.3 Staphylococci participate in the development and stability of good J Sci Food Agric 2016; 96: 1194–1201

red colour by nitrate reductase activity that leads to the formation of nitrosomyoglobin. Several Staphylococcus species, as well as some selected yeasts, also have an important role in aroma development through their protease and lipase activities.4 Protein hydrolysis during ripening yields peptides and free amino acids, while hydrolysis of triglycerides involves the liberation of fatty acids, which undergo later enzymatic processes to yield, as final products, low-molecular-weight compounds that affect taste and flavour development.5 Nevertheless, the characteristic aroma of fermented and ripened sausages is conferred by many different non-volatile and volatile compounds,6 resulting not only from the activity of some bacterial groups but also from spices and other condiments and the activity of endogenous meat enzymes.7 Therefore a sensory analysis performed by a panel of experts is required in order to complement and qualify the results obtained when studying volatile compounds. Consumption of horse or foal meat has increased in recent years, but it is still not comparable to the consumption of other

∗

Correspondence to: José M Lorenzo, Centro Tecnológico de la Carne de Galicia, Rúa Galicia N∘ 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, E-32900 Ourense, Spain. E-mail: [email protected] Centro Tecnológico de la Carne de Galicia, Rúa Galicia N∘ 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, E-32900 Ourense, Spain

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Effect of commercial starter cultures on dry-cured foal sausage characteristics

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Table 1. Effect of commercial starter cultures on chemical composition and pH of dry-cured foal sausage Batcha Parameter Moisture (g kg−1 ) Protein (g kg−1 DM) Fat (g kg−1 DM) pH

CO 313.07 ± 13.17 480.25 ± 15.84 317.94 ± 20.92 5.43 ± 0.07a

FS 308.28 ± 14.44 460.79 ± 18.97 327.56 ± 28.53 5.03 ± 0.05b

SM 302.98 ± 8.16 455.89 ± 12.92 330.31 ± 24.94 5.08 ± 0.04b

TR 302.48 ± 19.05 457.89 ± 34.04 329.11 ± 48.71 5.30 ± 0.10ab

SEM

Sign.b

2.56 4.15 5.60 0.03

NS NS NS ***

Values are mean ± standard deviation of eight replicates. Means in the same row not followed by a common letter differ significantly (P < 0.05). a Batches: CO, control without starter culture; FS, with F-SC-111 Bactoferm (L. sakei + S. carnosus); SM, with SM-194 (L. sakei + S. carnosus + S. xylosus + P. pentosaceus + D. hansenii); TR, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus). b Significance: NS, not significant; *** P < 0.001.

meats such as beef, chicken and pork.8 This increase might be due to changes in attitude towards this type of meat and the interest of consumers in tasting new meat products.9 Horse meat is characterized by low cholesterol and fat contents, high levels of unsaturated fatty acids and high levels of Fe-haem and vitamin B.10 – 12 These nutritional characteristics mean that this type of meat may be considered as a good alternative to pork meat, both for consumption fresh and for manufacturing meat products. Although a number of studies on different aspects of spontaneously fermented foal sausages have been published,13 – 15 to our knowledge the present study is the first to investigate the effect of different starter cultures on the volatile compound profile and sensory properties of dry-fermented foal sausages. The results obtained in this study should be considered as the final step of a previous work16 in which the effect of the same commercial starter cultures on the physicochemical characteristics, microbial counts and free fatty acid composition of dry-fermented foal sausages was studied. Therefore the aim of this work was to evaluate the effect of different combined commercial starter cultures on the volatile compound profile and sensory properties of dry-fermented foal sausages at the end of ripening in order to select the most suitable starter culture for this elaboration.

MATERIAL AND METHODS

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Chemical composition and pH Moisture, fat and protein (Kjeldahl N × 6.25) were quantified according to ISO 1442:1997, ISO 1443:1973 and ISO 937:1978 respectively. Fat and protein contents were expressed as g kg−1 dry matter (DM). The pH of sausages was measured using a digital pH meter (Model 710 A+, Thermo Orion, Ely, UK) equipped with a penetration probe. Volatile compound profile Each sample (1 g) was minced and weighed into a 24 mL headspace vial and then sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA, USA). A solid phase microextraction (SPME) device (Supelco) containing a fused silica fibre (10 mm length) coated with a 50/30 μm layer of DVD/CAR/PDMS was used. Headspace SPME (HS-SPME) extraction and chromatography were carried out under the conditions described by Gómez and Lorenzo.17 Results were expressed as area units (AU) × 109 kg−1 sample. Sensory analysis Sensory analysis was conducted with ten panellists selected from the Meat Technology Centre of Galicia. The panellists were trained for 2 weeks according to ISO 8586:2012 on the attributes and scale to be used. Thirteen sensory traits of dry-cured foal sausages, grouped as appearance (fat distribution and colour intensity), odour (odour intensity, black pepper odour and mould odour), taste (acid taste and saltiness), texture (hardness, juiciness and pastosity) and flavour (flavour intensity, cured flavour and rancid flavour), were assessed. The casings were removed and the sausages were cut into slices approximately 4 mm thick and served at room temperature on white plastic dishes. The samples were individually labelled with three-digit random numbers. The intensity of each attribute was expressed on an unstructured scale from 0 (sensation not perceived) to 9 (maximum sensation). The samples were evaluated by


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Sausage manufacturing Four different batches of foal sausage were manufactured according to traditional techniques, one of them without starter culture and the other three with the addition of different commercial starter cultures (Chr. Hansen, Hørsholm, Denmark) in a proportion defined by the manufacturer in each case. The batches were named as follows: (i) CO batch, control without starter culture; (ii) FS batch, with F-SC-111 Bactoferm (Lactobacillus sakei + Staphylococcus carnosus); (iii) SM batch, with SM-194 (L. sakei + S. carnosus + Staphylococcus xylosus + Pediococcus pentosaceus + Debaryomyces hansenii); (iv) TR batch, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus). All four batches were manufactured with the same ingredients, formulation and technology on two separate occasions (February and March 2013). The foal sausage formulation included lean foal meat (850 g kg−1 ), pork back fat (150 g kg−1 ), NaCl (25 g kg−1 ), lactose (20 g kg−1 ), dextrin (20 g kg−1 ), sodium caseinate (20 g kg−1 ), glucose (7 g kg−1 ), black pepper (1.5 g kg−1 ), white pepper (1 g kg−1 ), sodium ascorbate (0.5 g kg−1 ), sodium nitrite (0.15 g kg−1 ) and potassium nitrate (0.15 g kg−1 ). All foals used in this study were reared in an extensive production system in freedom regimen.

The lean foal meat and the pork back fat were ground through a 10 mm diameter mincing plate and vacuum mixed together with the other ingredients for 3 min. The mix was maintained at 4 ∘ C for 24 h and then stuffed into natural casings with a diameter of 60 mm and a length of 40 cm. The sausages were fermented for 2 days at 20 ∘ C and 80% relative humidity (RH) and then transferred into a drying/ripening chamber where they were kept for a further 47 days at 11 ∘ C and 75% RH. Analyses were performed on samples taken at the end of ripening.

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Table 2. Effect of commercial starter cultures on volatile compounds (AUa × 109 kg−1 sample) of dry-cured foal sausage Batchb Compound

KIc

Amino acid catabolism Benzeneacetaldehyde 1114 Benzaldehyde, 3-ethyl 1236 Carbohydrate fermentation Acetic acid 698 Butanoic acid 887 Lipid oxidation and microbial 𝛽-oxidation Heptane 700 Hexane, 3-methyl 754 Pentane, 2,3,3-trimethyl 759 Octane 800 Hexane, 2,2,5-trimethyl 787 Hexane, 2,3,5-trimethyl 812 1-Pentanol 819 Hexanal 838 Octane, 3,3-dimethyl 907 2-Hexenal 912 1-Hexanol 928 Heptanal 948 Heptane, 2,2,4,6,6-pentamethyl 1001 1-Octen-3-ol 1026 Hexanoic acid 1058 1,3-Hexadiene, 3-ethyl-2-methyl 1075 3,5-Octadien-2-ol 1096 2-Octenal 1118 3,5-Octadien-2-one 1134 2-Nonanone 1142 Nonanal 1152 Dodecane 1206 2-Nonenal 1225 Octanoic acid 1256 2,4-Nonadienal 1287 2,4-Decadienal 1382 Spices 𝛼-Thujene 944 𝛼-Pinene 951 𝛽-Pinene 997 3-Carene 1024 D-Limonene 1042 p-Cymene 1046 𝛾-Terpinene 1067 𝛼-Terpinolene 1104 4-Terpineol 1233 𝛿-Elemene 1359 𝛽-Elemene 1360 Humulene 1478 Unknown origin Benzyl alcohol 1121

RId

CO

FS

SM

M, K M, K

0.91 ± 0.24a 0.71 ± 0.26a

1.29 ± 0.26b 1.33 ± 0.12b

1.05 ± 0.22ab 1.32 ± 0.17b

M, K M, K

28.17 ± 2.20a 15.41 ± 1.89

51.90 ± 6.07d 16.59 ± 1.63

47.13 ± 2.87c 14.81 ± 1.30

M, S M M M, S M M M, K M, S M M, K M, K M, S M M, K M, K M M M, K M M, K M, S M, S M, K M, K M, K M, K

2.67 ± 0.63a 2.82 ± 0.22b 4.04 ± 1.78b 5.87 ± 2.37b 22.83 ± 8.85a 1.22 ± 0.37 7.30 ± 3.23ab 214.16 ± 37.42b 2.99 ± 0.77ab 1.53 ± 0.55a 4.88 ± 2.87b 10.20 ± 1.77ab 201.12 ± 33.41 16.31 ± 4.04a 12.13 ± 2.76ab 2.10 ± 1.01 NDa 2.30 ± 0.66a 1.91 ± 0.88a 2.10 ± 1.44 9.41 ± 1.52a 0.68 ± 0.22 0.93 ± 0.22a 1.21 ± 0.35a 0.40 ± 0.14a 0.28 ± 0.11a

4.72 ± 1.47b 1.48 ± 0.52a 2.02 ± 0.80a 3.73 ± 1.35a 46.32 ± 6.46b 0.91 ± 0.11 9.28 ± 3.72ab 286.94 ± 25.25c 3.26 ± 0.57b 1.97 ± 0.26ab 4.55 ± 1.68b 16.02 ± 3.44c 186.95 ± 36.26 17.48 ± 2.96a 15.66 ± 1.98c 2.67 ± 0.60 1.99 ± 0.17b 4.13 ± 0.84b 2.50 ± 0.62ab 1.30 ± 0.08 12.76 ± 2.44b 0.73 ± 0.10 1.51 ± 0.20b 1.86 ± 0.56b 0.84 ± 0.21b 0.77 ± 0.23c

M, K M, K M M, K M, K M M, K M, K M M M M, K

34.11 ± 8.31 73.85 ± 38.30 81.28 ± 32.56 105.82 ± 47.02 130.49 ± 43.34 29.38 ± 12.24 12.96 ± 7.76ab 4.77 ± 3.56 1.62 ± 0.81 1.43 ± 0.40a 0.27 ± 0.05a 0.47 ± 0.10a

M, K

9.80 ± 1.47a

TR

SEM

0.82 ± 0.28a 0.49 ± 0.29a

0.05 0.10

40.01 ± 5.25b 14.73 ± 2.12

1.77 0.32

5.51 ± 0.94b 1.57 ± 0.79a 2.73 ± 1.89ab 3.45 ± 1.01a 40.35 ± 7.29b 0.78 ± 0.36 10.88 ± 3.63b 255.91 ± 19.64c 2.85 ± 0.31a,b 2.26 ± 0.52b 5.65 ± 1.42b 12.49 ± 2.89b 157.73 ± 32.63 19.52 ± 1.82ab 14.32 ± 1.60bc 2.94 ± 0.69 1.85 ± 0.29b 4.30 ± 1.11b 3.13 ± 0.84b 0.68 ± 0.24 10.01 ± 1.81a 0.60 ± 0.11 1.34 ± 0.20ab 1.81 ± 0.54b 0.84 ± 0.29b 0.62 ± 0.12b

2.29 ± 0.73a 2.22 ± 0.77ab 3.47 ± 1.13ab 5.65 ± 0.94b 15.75 ± 4.85a 1.03 ± 0.31 5.37 ± 2.52a 166.63 ± 39.91a 2.12 ± 0.52a 1.88 ± 0.50ab 1.82 ± 0.47a 8.87 ± 1.94a 158.50 ± 50.14 23.64 ± 1.00a 11.13 ± 3.29a 1.89 ± 1.17 NDa 2.89 ± 0.99ab 1.56 ± 0.97a 1.17 ± 0.63 8.34 ± 1.71a 0.60 ± 0.25 1.27 ± 0.56ab 1.21 ± 0.44a 0.65 ± 0.30ab 0.41 ± 0.17ab

0.32 0.19 0.31 2.58 0.33 0.08 0.73 9.71 0.17 0.09 0.43 0.65 7.50 0.84 0.54 0.21 0.07 0.29 0.19 0.25 0.44 0.03 0.08 0.10 0.06 0.05

39.29 ± 20.84 90.95 ± 23.47 109.29 ± 39.83 146.55 ± 47.36 179.85 ± 54.84 36.49 ± 16.64 13.16 ± 4.16ab 5.14 ± 1.91 1.89 ± 0.67 2.16 ± 0.40b 0.63 ± 0.14b 0.71 ± 0.19b

31.24 ± 23.30 67.66 ± 18.72 83.94 ± 25.62 122.44 ± 59.33 138.54 ± 40.96 32.24 ± 14.51 18.40 ± 5.87b 4.63 ± 2.86 1.41 ± 0.60 1.81 ± 0.32ab 0.48 ± 0.14b 0.56 ± 0.19ab

28.68 ± 10.65 79.58 ± 19.74 93.65 ± 34.88 126.00 ± 50.82 131.64 ± 38.59 44.39 ± 6.83 10.41 ± 3.97a 4.57 ± 1.39 1.65 ± 0.58 1.42 ± 0.52a 0.39 ± 0.09a 0.58 ± 0.15ab

8.69 0.31 3.24 4.96 0.04 6.11 9.41 3.43 1.18 0.47 0.15 0.10

12.36 ± 1.33b

11.06 ± 1.49ab

10.05 ± 1.78a

0.03

Sign.e ** ***

***

NS *** *

NS *** **

NS * ***

NS * * ***

NS * **

NS *** * **

NS ***

NS NS * * ***

NS NS NS NS NS NS NS NS NS * ***

NS **

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Values are mean ± standard deviation of eight replicates (ND, not detected). Means in the same row not followed by a common letter differ significantly (P < 0.05). a Area units resulting from counting the total ion chromatogram (TIC) for each compound. b Batches: CO, control without starter culture; FS, with F-SC-111 Bactoferm (L. sakei + S. carnosus); SM, with SM-194 (L. sakei + S. carnosus + S. xylosus + P. pentosaceus + D. hansenii); TR, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus). c Kovats index calculated for a J&W Scientific DB-624 capillary column (30 m × 0.25 mm i.d., 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass-selective detector. d Reliability of identification: K, Kovats index in agreement with literature;21,22,33 – 37 M, mass spectrum agreeing with mass database (NIST 05); S, mass spectrum and retention time identical with an authentic standard. e Significance: NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.



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Figure 1. Total area of groups of volatile compounds, classified by most probable origin, of dry-cured foal sausage in control batch and in batches manufactured with different starter cultures. Batches: CO, control without starter culture; FS, with F-SC-111 Bactoferm (L. sakei + S. carnosus); SM, with SM-194 (L. sakei + S. carnosus + S. xylosus + P. pentosaceus + D. hansenii); TR, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus). Mean values corresponding to the same group of compounds not having a common letter differ significantly (P < 0.05).

panellists in two sessions (four samples per session). During sensory evaluation, the panellists were situated in separate cubicles illuminated with red light. Water to clean the palates and remove residual flavours was used at the beginning of the session and in between samples. Statistical analysis All statistical analysis was performed using IBM SPSS Statistics 19 software (IBM, Chicago, IL, USA). After verification of normal distribution and constant variance of the data, significant differences were determined using one-way analysis of variance (ANOVA). Duncan’s test was performed to compare mean values at a significance level of P < 0.05. To evaluate the relation between variables, factorial analysis of the volatile compounds with significant differences (P < 0.05) among the four groups was carried out. Principal component analysis (PCA) was used as extraction method and was performed on the correlation matrix.

RESULTS AND DISCUSSION

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Volatile compound profile The average contents of volatile compounds (expressed as AU × 109 kg−1 sample) extracted at the end of ripening of the sausages produced with or without starter culture are reported in Table 2. The SPME technique is not normally used for absolute quantification, but when exactly the same extraction methodology is utilized, this technique permit the comparison of relative amounts between samples. In total, 43 volatile compounds were identified and quantified, grouped according to their probable origins as from lipid oxidation and microbial 𝛽-oxidation, carbohydrate fermentation, amino acid catabolism and spices. The remaining volatile compounds were grouped as ‘unknown origin’. Most identified compounds have been previously reported in Spanish sausages.17,21,22 Volatile compounds were studied according to their most probable origin,14,22 from amino acid catabolism, carbohydrate fermentation, lipid oxidation and microbial 𝛽-oxidation, spices and unknown origin, in order to understand how generation pathways were affected by the starter cultures inoculated. FS batch showed the highest (P < 0.05) total amounts of volatile compounds (Fig. 1) with a mean value of 1268.25 AU × 109 kg−1 sample, though CO, SM and TR values were similar (958.46, 1055.60 and 893.52 AU × 109 kg−1 sample respectively). In the four studied batches, lipid oxidation- and microbial 𝛽-oxidation-derived compounds constituted around 48% of total detected compounds. Volatiles coming from spices were the second most abundant group (46%), while those originating from carbohydrate fermentation and amino acid catabolism represented the lowest amounts (5 and 0.17% respectively). Among amino acid catabolism-derived volatiles (Table 2), only two compounds were detected, benzeneacetaldehyde and benzaldehyde, 3-ethyl, which showed a significant effect of the addition of different starter cultures. FS and SM batches presented higher amounts of these compounds (P < 0.05). Benzeneacetaldehyde was reported to provide floral aroma and to derive from the deamination of 2-phenylethylamine.23 Benzaldehyde, 3-ethyl has not been reported as essential in the flavour development of dry-cured meat products.24 Two carbohydrate fermentation-derived volatile compounds, acetic acid and butanoic acid, were found. Significant differences were observed only for acetic acid when different starter cultures were compared (P < 0.001), the amounts being as follows: FS > SM > TR > CO. This order is in accordance with the pH values. Acetic acid contributes to dry sausage aroma and may be produced by LAB and staphylococci and also by fatty acid oxidation and alanine catabolism.25 Lipid oxidation and microbial 𝛽-oxidation produced the largest group of compounds (27), hexanal was the most representative with amounts around 200 AU × 109 kg−1 sample. These amounts were significantly (P < 0.001) affected by the addition of starter cultures, showing FS and SM batches the highest amounts. This outcome might be a consequence of the influence of the starter culture on lipolysis and autoxidation rates. Hexanal and, in general, the aliphatic aldehydes derived from lipid metabolism give fatty, rancid or floral notes depending on their concentrations.23 They seem to be contributors to the loss of desirable flavour in meats because of their rate of formation during lipid oxidation and their low flavour thresholds.26 Except for 2-nonenal, significant differences (P < 0.05) were obtained for aliphatic aldehydes when comparing samples with different starter cultures, with higher amounts in FS and SM batches. As regards alcohols, these were significantly (P < 0.05) affected by the type of starter culture



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Chemical composition and pH Values corresponding to chemical composition and pH of dry-cured foal sausages are reported in Table 1. As expected, the compositional parameters evaluated in this study (moisture, protein and fat contents) did not show significant differences when comparing batches, since the same raw materials and formulation, except for the starter cultures, were used for their manufacture. The pH values, however, differed significantly (P < 0.001) among batches, which shows the important influence of starter cultures on sausages acidity. The highest pH values corresponded to CO (5.43 ± 0.07) followed by TR (5.30 ± 0.10), SM (5.08 ± 0.04) and FS (5.03 ± 0.05). These results indicate that the inoculation of starter cultures resulted in stronger acidification of the sausages, especially in SM and FS batches inoculated with those described as ‘fast cultures’ by the manufacturer. These findings are in agreement with those reported by other authors,18 – 20 who observed lower pH values in inoculated sausages than in non-inoculated control sausages.

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JM Lorenzo et al.

Fat distribution 6

Rancid flavour

b ab ab a

5 4

Colour intensity

Cured flavour

Odour intensity 3 2 1

Flavour intensity

Black pepper odour

0

Pastosity

Mould odour a

Juiciness

a ab ab b

Hardness

CO

b b b

Saltiness Acid taste

FS SM TR

Figure 2. Effect of commercial starter cultures on sensory characteristics of dry-cured foal sausage. Batches: CO, control without starter culture; FS, with F-SC-111 Bactoferm (L. sakei + S. carnosus); SM, with SM-194 (L. sakei + S. carnosus + S. xylosus + P. pentosaceus + D. hansenii); TR, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus). Mean values corresponding to the parameter not having a common letter differ significantly (P < 0.05).

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and most of them were more abundant in SM and FS batches, but 1-octen-3-ol appeared in greater amount in TR batch. This compound has a marked mushroom odour and a very low odour threshold21 that is related to mould inoculation.27 Alcohols normally present in fermented products, including dry-fermented sausages, are mainly generated from the reduction of aldehydes.28 Batches containing lower amounts of aldehydes also presented low alcohol quantities, as occurred for samples from CO and TR batches in our study. Nonetheless, the flavour of alcohols in general, owing to their relatively high threshold values, was considered unimportant compared with that of other compounds.29 Of the two detected ketones, only 3,5-octadien-2-one was affected by the starter culture, showing higher contents in sausages from SM batch. This compound was described as woody, mushroom, hay and fresh.30 Finally, the acids related to lipid oxidation and microbial 𝛽-oxidation, as occurred for most of the volatile compounds found in the present study, were significantly higher (P < 0.05) in FS and SM batches. Some hydrocarbons were also affected by the type of microorganisms added to the sausages, but some of them showed a different trend with respect to the previously studied compounds, with higher amounts in CO and TR batches (hexane, 3-methyl and octane). Like alcohols, aliphatic hydrocarbons have high threshold values and thus do not contribute significantly to the aroma of dry-ripened meat products.31 Regarding compounds derived from spices, a large number (12) were detected. All compounds in this group are classified as terpenes and most of them were previously identified in black pepper and white pepper, the spices employed in the present sausages.32 D-Limonene was also described to be related to pig diet.21


Sensory analysis The sensory attributes evaluated in the dry-cured foal sausages were fat distribution, colour intensity, odour intensity, black pepper odour, mould odour, saltiness, acid taste, hardness, juiciness, pastosity, flavour intensity, cured flavour and rancid flavour (Fig. 2). Although Duncan’s test revealed differences among batches when comparing acid taste, colour intensity and hardness mean values, the ANOVA performed on the sensory results showed that only acid taste was significantly (P < 0.01) affected by the addition of commercial starter cultures, with the lowest values corresponding to control sausages. These findings are in agreement with those reported by Selgas et al.,38 who did not find significant (P > 0.05) differences among non-inoculated and inoculated dry-cured fermented sausages evaluated by trained judges. In this regard, Cano-García et al.39 only detected significant (P < 0.001) differences in appearance among non-inoculated and D. hansenii strain-inoculated dry-cured fermented sausages, the inoculated sausages being preferred by the consumer sensory panel. Higher colour and odour intensity scores were obtained fot inoculated dry-cured foal sausages, but the results were not significant. These findings are in agreement with those reported by Essid and Hassouna,20 who found that inoculated sausages presented a more pronounced red colour when compared with control sausages; this is related to the nitrate reductase activity of strains of S. xylosus inoculated into the sausages.40 In this regard, Gotterup et al.41 observed that the red colour intensity of fermented sausages was significantly (P < 0.05) affected by the intensity of nitrate reductase activity of strains used as starters. As commented above, inoculated sausages obtained higher acid taste scores compared with control sausages, and this fact could be related to the


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A Butanoic acid Hexanoic acid Benzeneacetaldehyde 1,3-Hexadiene-3-ethyl-2-methyl TR 2-Hexenal 2,4-Decadienal Nonanal Acetic acid

1

PC2 (30.15%)

Heptanal

SM

0

Humulene D-Limonene

3,5-Octadien-2-one 1-Pentanol

OctaneHexanal

FS

pH

α-Pinene β-Pinene

CO -1

-1

0 PC1 (30.91%)

1

B SM 1

3,5-Octadien-2-one1-Pentanol

Octane

PC3 (30.12%)

Hexanal Acetic acid 2-Hexenal 1,3-Hexadiene-3-ethyl-2-methyl 2,4-Decadienal Hexanoic acid

0

Butanoic acid

FS

Heptanal α-Pinene β-Pinene D-Limonene Nonanal

Benzeneacetaldehyde

Humulene

CO pH

TR

-1

-1

0 PC1 (30.91%)

1

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Figure 3. Relationships among types of dry-cured foal sausage and volatile compound profile and physicochemical properties obtained by PCA: A, projection of variables and dry-cured foal sausage groups in plane defined by PC1 and PC2; B, projection in plane defined by PC1 and PC3. Batches: CO, control without starter culture; FS, with F-SC-111 Bactoferm (L. sakei + S. carnosus); SM, with SM-194 (L. sakei + S. carnosus + S. xylosus + P. pentosaceus + D. hansenii); TR, with TRADI 302 Bactoferm (L. sakei + S. carnosus + S. xylosus).

www.soci.org acidifying activity of L. sakei strains included in the commercial starter cultures. This result is consistent with the data obtained for pH values, where inoculated sausages showed significantly (P < 0.001) lower pH values than control sausages (Table 1). Hardness of dry-cured foal sausages was higher in non-inoculated batches compared with inoculated batches, but these differences were not significant. This outcome is in disagreement with that observed by Essid and Hassouna,20 who found significant (P < 0.05) differences between control and inoculated sausages. In fact, hardness could arise from microbiological and physicochemical processes such as enhanced acidification and proteolysis as a result of L. sakei, S. xylosus and S. carnosus activities. Finally, inoculated sausages obtained higher flavour intensity and cured flavour scores compared with control sausages. These attributes are related to compounds (fatty acids, amino acids, aldehydes, esters, etc.) released by microbial and endogenous proteases and lipases throughout ripening of sausages.20 Therefore the proteolytic and lipolytic activities of the commercial starter cultures used are mainly responsible for the differences in flavour intensity and cured flavour among batches. Principal component analysis To establish which characteristics of foal sausages were affected by the addition of different starter cultures, a PCA was carried out using 18 parameters selected from a previous factorial analysis. The results of the first three principal components (PCs) are plotted in Figs 3A and 3B. The PCA showed that about 61.06% of the variability was explained by the first two PCs. PC1 was positively related to acetic acid, octane, 1-pentanol, hexanal, heptanal, 𝛼-pinene, 𝛽-pinene, D-limonene, hexanoic acid, 1,3-hexadiene, 3-ethyl-2-methyl, benzeneacetaldehyde, nonanal, 2,4-decadienal and humulene. PC1 was inversely related to butanoic acid, 2-hexenal, 3,5-octadien-2-one and pH. Sausages from FS batch were on the positive PC1 axis, while sausages from CO, SM and TR batches were on the negative PC1 axis. PC2 was only inversely related to 𝛼-pinene, 𝛽-pinene and pH. Sausages from FS and TR batches were on the positive PC2 axis, while sausages from CO and SM batches were on the negative PC2 axis. Finally, PC3 was only inversely related to butanoic acid, benzeneacetaldehyde, humulene and pH. Sausages from FS and SM batches were on the positive PC3 axis, while sausages from CO and TR batches were on the negative PC3 axis. PC1 shows a clear difference between FS batch and the other batches, as it obtained higher scores for most of the attributes analysed. These differences were also observed in the results for physicochemical parameters (Table 1) and volatile compounds (Table 2). On the one hand, PC2 seems to distinguish between sausages without inoculation (CO batch) and those with inoculation (FS, TR and SM batches), in spite of SM batch values being located on the negative PC2 axis, with values close to zero. On the other hand, PC3 allows separation of the batches according to their acidifying activity, discriminating FS and SM batches (‘fast cultures’ as described by the manufacturer) from CO and TR batches.

CONCLUSIONS

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According to the results obtained in this study, the use of different commercial starter cultures from Chr. Hansen for the manufacture of dry-cured foal sausages led to significantly different effects on their acidity (as reflected by pH), volatile compounds derived from carbohydrate fermentation and acid taste from sensory analysis.


JM Lorenzo et al.

According to these parameters, batches inoculated with FS and SM starters showed marked acidity compared with TR and CO batches, as expected from the manufacturer’s indications. Therefore the selection of the most suitable starter culture for the manufacture of foal sausages will depend on the type of product demanded by the consumer in different parts of the world. In Mediterranean countries such as Spain, a low-acidity product is preferred as obtained by the inoculation of TR culture.

ACKNOWLEDGEMENTS The authors are grateful to Xunta de Galicia (FEADER 2013/42) for financial support. Special thanks are given to Monte Cabalar (A Estrada, Pontevedra) for supplying the foal samples for this research.

REFERENCES 1 Barbuti S and Parolari G, Validation of manufacturing process to control pathogenic bacteria in typical dry fermented products. Meat Sci 62:323–329 (2002). 2 Casaburi A, Di Monaco R, Cavella S, Toldrá F, Ercolini D and Villani F, Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits. Food Microbiol 25:335–347 (2008). 3 Demeyer D, Todorov N, Nevel CV and Vets J, 𝛼 –𝜀 Diaminopimelic acid (DAPA) in a sheep rumen infused with a synthetic diet of sugars and urea: evidence for degradation of bacteria. Z Tierphysiol Tierer 48:21–32 (1982). 4 Talon R, Walter D, Chartier S, Barriere C and Montel MC, Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci. Int J Food Microbiol 52:47–56 (1999). 5 Fernández M, Ordóñez JA, Bruna JM, Herranz B and de la Hoz L, Accelerated ripening of dry fermented sausages. Trends Food Sci Technol 11:201–209 (2000). 6 Stahnke LH, Aroma components from dried sausages fermented with Staphylococcus xylosus. Meat Sci 38:39–53 (1994). 7 Hierro E, de la Hoz L and Ordóñez JA, Contribution of microbial and meat endogenous enzymes to the lipolysis of dry fermented sausages. J Agric Food Chem 45:2989–2995 (1997). 8 Franco D, Rodríguez E, Purriños L, Crecente S, Bermúdez R and Lorenzo JM, Meat quality of ‘Galician Mountain’ foals breed. Effect of sex, slaughter age and livestock production system. Meat Sci 88:292–298 (2011). 9 Sarriés MV, Murray BE, Troy D and Beriain MJ, Intramuscular and subcutaneous lipid fatty acid profile composition in male and female foals. Meat Sci 72:475–485 (2006). 10 Lorenzo JM, Pateiro M and Franco D, Influence of muscle type on physicochemical and sensory properties of foal meat. Meat Sci 94:77–83 (2013). 11 Lorenzo JM and Pateiro M, Influence of type of muscles on nutritional value of foal meat. Meat Sci 93:630–638 (2013). 12 Lorenzo JM, Sarriés MV, Tateo A, Polidori P, Franco D and Lanza M, Carcass characteristics, meat quality and nutritional value of horsemeat: a review. Meat Sci 96:1478–1488 (2014). 13 Lorenzo JM and Franco D, Fat effect on physico-chemical, microbial and textural changes through the manufacture of dry-cured foal sausage. Lipolysis, proteolysis and sensory properties. Meat Sci 92:704–714 (2012). 14 Lorenzo JM, Montes R, Purriños L and Franco D, Effect of pork fat addition on the volatile compounds of foal dry-cured sausage. Meat Sci 91:506–512 (2012). 15 Lorenzo JM, Temperán S, Bermúdez R, Cobas N and Purriños L, Changes in physico-chemical, microbiological, textural and sensory attributes during ripening of dry-cured foal salchichón. Meat Sci 90:194–198 (2012). 16 Lorenzo JM, Gómez M and Fonseca S, Effect of commercial starter cultures on physicochemical characteristics, microbial counts and free fatty acid composition of dry-cured foal sausage. Food Control 46:382–389 (2014). 17 Gómez M and Lorenzo JM, Effect of fat level on physicochemical, volatile compounds and sensory characteristics of dry-ripened ‘chorizo’ from Celta pig breed. Meat Sci 95:658–666 (2013).


J Sci Food Agric 2016; 96: 1194–1201

Effect of commercial starter cultures on dry-cured foal sausage characteristics 18 Baka AM, Papavergou EJ, Pragalaki T, Bloukas JG and Kotzekidou P, Effect of selected autochthonous starter cultures on processing and quality characteristics of Greek fermented sausages. LWT – Food Sci Technol 44:54–61 (2011). 19 Ciuciu Simion AM, Vizireanu C, Alexe P, Franco I and Carballo J, Effect of the use of selected starter cultures on some quality, safety and sensorial properties of Dacia sausage, a traditional Romanian dry-sausage variety. Food Control 35:123–131 (2014). 20 Essid I and Hassouna M, Effect of inoculation of selected Staphylococcus xylosus and Lactobacillus plantarum strains on biochemical, microbiological and textural characteristics of a Tunisian dry fermented sausage. Food Control 32:707–714 (2013). 21 Ansorena D, Gimeno O, Astiasarán I and Bello J, Analysis of volatile compounds by GC–MS of a dry fermented sausage: Chorizo de Pamplona. Food Res Int 34:67–75 (2001). 22 Fonseca S, Cachaldora A, Gómez M, Franco I and Carballo J, Effect of different autochthonous starter cultures on the volatile compounds profile and sensory properties of Galician chorizo, a traditional Spanish dry fermented sausage. Food Control 33:6–14 (2013). 23 Tabanelli G, Coloretti F, Chiavari C, Grazia L, Lanciotti R and Gardini F, Effects of starter cultures and fermentation climate on the properties of two types of typical Italian dry fermented sausages produced under industrial conditions. Food Control 26:416–426 (2012). 24 Andrade JM, Córdoba JJ, Sánchez B, Casado EM and Rodríguez M, Evaluation and selection of yeasts isolated from dry-cured Iberian ham by their volatile compound production. Food Chem 113:457–463 (2009). 25 Montel MC, Masson F and Talon R, Bacterial role in flavour development. Meat Sci 49:S111–S123 (1998). 26 Gray JI, Gomaa EA and Buckley DJ, Oxidative quality and shelf life of meats. Meat Sci 43:111–123 (1996). 27 Sunesen LO, Trihaas J and Stahnke LH, Volatiles in a sausage surface model – influence of Penicillium nalgiovense, Pediococcus pentosaceus, ascorbate, nitrate and temperature. Meat Sci 66:447–456 (2004). 28 Flores M, Dura M, Marco A and Toldrá F, Effect of Debaryomyces spp. on aroma formation and sensory quality of dry-fermented sausages. Meat Sci 68:439–446 (2004). 29 Drumm TD and Spanier AM, Changes in the content of lipid autoxidation and sulfur-containing compounds in cooked beef during storage. J Agric Food Chem 39:336–343 (1991).

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30 Giri A, Osako K, Okamoto A and Ohshima T, Olfactometric characterization of aroma active compounds in fermented fish paste in comparison with fish sauce, fermented soy paste and sauce products. Food Res Int 43:1027–1040 (2010). 31 Ramírez R and Cava R, Volatile profiles of dry-cured meat products from three different Iberian × Duroc genotypes. J Agric Food Chem 55:1923–1931 (2007). 32 Plessi M, Bertelli D and Miglietta F, Effect of microwaves on volatile compounds in white and black pepper. LWT – Food Sci Technol 35:260–264 (2002). 33 Lorenzo JM, Bedia M and Bañón S, Relationship between flavour deterioration and the volatile compound profile of semi-ripened sausage. Meat Sci 93:614–620 (2013). 34 Lorenzo JM, Influence of the type of fiber coating and extraction time on foal dry-cured loin volatile compounds extracted by solid-phase microextraction (SPME). Meat Sci 96:179–186 (2014). 35 Lorenzo JM, Franco D and Carballo J, Effect of the inclusion of chestnut in the finishing diet on volatile compounds during the manufacture of dry-cured ‘Lacón’ from Celta pig breed. Meat Sci 96:211–223 (2014). 36 Purriños L, Franco D, Carballo J and Lorenzo JM, Influence of the salting time on volatile compounds during the manufacture of dry-cured pork shoulder ‘lacón’. Meat Sci 92:627–634 (2012). 37 Purriños L, Carballo J and Lorenzo JM, The influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured ‘lacón’. Meat Sci 93:344–350 (2013). 38 Selgas MD, Ros J and García ML, Effect of selected yeast strains on the sensory properties of dry fermented sausages. Eur Food Res Technol 217:475–480 (2003). 39 Cano-García L, Belloch C and Flores M, Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages. Meat Sci 96:1469–1477 (2014). 40 Essid I, Ben Ismail H, Ben Hadj Ahmed S, Ghedamsi R and Mnasser H, Characterization and technological properties of Staphylococcus xylosus strains isolated from a Tunisian traditional salted meat. Meat Sci 77:204–212 (2007). 41 Gotterup J, Olsen K, Knochel S, Tjener K, Stahnke LH and Moller JKS, Colour formation in fermented sausages by meat-associated staphylococci with different nitrite and nitrate-reductase activities. Meat Sci 78:492–501 (2008).

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