Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages

Food Microbiology 33 (2013) 61e68

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Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages Aida Cachaldora, Sonia Fonseca, Inmaculada Franco, Javier Carballo* Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 April 2012 Received in revised form 29 August 2012 Accepted 31 August 2012 Available online 13 September 2012

The aim of this study was to determine the technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase and enterotoxigenic activities) of 38 strains of Staphylococcaceae (11 of Staphylococcus epidermidis, 15 of Staphylococcus equorum, 5 of Staphylococcus pasteuri and 7 of Staphylococcus saprophyticus) isolated from Androlla and Botillo, two Spanish traditional sausages, in order to evaluate their suitability as potential starter cultures in the manufacture of these sausages. Most strains were able to grow at 10
C, in the presence of 10% and 15% NaCl and at pH values of 5.5 and 5.0, except for S. equorum strains, growth of which was reduced at these pH values. The proteolytic activity assessed by the agar plate method showed that 89.5% and 52.6% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were not confirmed by electrophoretic assays as only 47.2% of the strains changed the SDS-PAGE profile of actin, myosin and/or sarcoplasmic protein extracts. The assessment of the lipolytic activity by titration showed that only 21.0% of the strains can hydrolyze pork fat to any extent; whereas the profiles of the freed fatty acids were different in the different strains. Most of the strains showed decarboxylase activity against histidine, lysine, ornithine and tyrosine, but the quantities of biogenic amines produced were in most cases <25 ppm and <5 ppm for putrescine and cadaverine, respectively. Only four strains (10.5%), of S. epidermidis, produced enterotoxin C. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Staphylococcaceae Technological properties Safety properties Proteolysis Lipolysis Biogenic amines

1. Introduction Androlla and Botillo are two traditional raw, cured sausages manufactured in Galicia (NW of Spain). These sausages are manufactured using traditional methods, without the addition of starter cultures and additives. With such methods, the safety and quality of the final products are not guaranteed. During fermentation and ripening of sausages, proteolysis and lipolysis are largely responsible for the organoleptic and texture characteristics of the final products. Numerous studies suggested that proteolysis is due mainly to endogenous enzymes. However, in several studies there was evidence that the proteolytic activity of microoganisms were involved in the ripening process, with effects on mainly the sarcoplasmic proteins (Casquete et al., 2011a; Hugas and Monfort, 1997; Molly et al., 1997; Lizaso et al., 1999; Sanz et al., 1999). Similarly, some authors have concluded that tissue lipases are primarily responsible for lipid degradation during fermentation

* Corresponding author. Tel.: þ34 988 387052; fax: þ34 988 387001. E-mail address: [email protected] (J. Carballo). 0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2012.08.013

(Molly et al., 1997; Kenneally et al., 1998; Galgano et al., 2003), but numerous studies over the last decade have found lipolytic bacteria, especially staphylococci, involved in sausage fermentation (Hugas and Monfort, 1997; Montel et al., 1998; Mauriello et al., 2004; Casaburi et al., 2008). Lactic acid bacteria and staphylococci are the microorganisms most commonly used in starter cultures for fermented meats (Casquete et al., 2011b). The use of starter cultures containing carefully selected indigenous microbiota, adapted to meat environment, with lipolytic and proteolytic activities, and able to generate large amounts of aroma compounds, could allow development of improved sensory qualities in sausages; and guarantee products with reproducible organoleptic and hygienic properties in a shorter ripening time (Leroy et al., 2006; Casaburi et al., 2008). To date, several studies have been carried out in order to evaluate the proteolytic and lipolytic activities of Staphylococcus strains isolated from sausages (Mauriello et al., 2004; Martín et al., 2007; Bonomo et al., 2009). These studies have been mostly qualitative and do not give detailed information about the proteins that are hydrolyzed and their degradation products, and/or the profiles of the fatty acid released by these strains after a specific period of fermentation.

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Moreover, as some staphylococci have amino acid decarboxylase and enterotoxigenic activities that could adversely affect the safety of the products, the absence of these activities would seem to be an essential selection criterion for starter cultures (Casaburi et al., 2005). The aim of this study was to determine the technological properties and safety characteristics of 38 strains of Staphylococcaceae isolated from Androlla and Botillo sausages that might be used in starter cultures for the manufacture of these sausages. 2. Materials and methods 2.1. Staphylococci strains and preparation of cell suspension The staphylococci strains used in this study are listed in Table 1. They were isolated and identified as previously reported (GarcíaFontán et al., 2007a,b). Their identity was confirmed by sequencing the 16S rRNA gene and comparing the sequences obtained with those available in GenBank (National Center for Biotechnology Information, Bethesda, MD, USA). Subsequently, their genomic DNA was characterized by (GTG)5-PCR fingerprinting to confirm that strains of the same species were different. Strains were stored at 80
C in Brain Heart Infusion broth (BHI; Oxoid, Basingstoke, UK) with 20% (v/v) glycerol as a cryoprotective agent. Before use, they were grown overnight at 37
C in BHI broth. The correlation between the log cfu/mL and the absorbance at 650 nm (A650) of cultures was established for each strain. Samples Table 1 Strains used in this study. Strains

Identitya

Source

SA03 SA04 SA05 SA07 SA10 SA11 SA14 SA15 SA16 SA19 SA20 SA24 SA25 SA27 SA34 SA41 SA47 SA48 SA49 SA51 SA52 SA54 SB02 SB03 SB04 SB08 SB11 SB12 SB19 SB21 SB22 SB23 SB24 SB25 SB27 SB29 SB32 SB34

S. pasteuri S. equorum S. equorum S. equorum S. equorum S. equorum S. equorum S. equorum S. epidermidis S. equorum S. equorum S. equorum S. equorum S. pasteuri S. epidermidis S. epidermidis S. epidermidis S. epidermidis S. epidermidis S. pasteuri S. saprophyticus S. equorum S. saprophyticus S. pasteuri S. saprophyticus S. saprophyticus S. saprophyticus S. saprophyticus S. pasteuri S. saprophyticus S. equorum S. equorum S. epidermidis S. epidermidis S. epidermidis S. equorum S. epidermidis S. epidermidis

Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo

a Identity of the strains was determined as previously reported (García-Fontán et al., 2007a,b).

of BHI broth cultures were collected after 24 h of incubation and the A650 was measured. The cultures were centrifuged at 12,000  g for 10 min. Pellets were washed twice with 20 mM phosphate buffer, pH 7.0, and resuspended in the same buffer to obtain inocula containing approximately 109 cfu mL1. 2.2. Effect of pH, temperature and NaCl on microbial growth Ripening of Androlla and Botillo sausages takes place at 10e 12
C. The NaCl concentrations in these sausages can reach 3 or 4%, and the pH values are around 5.5. Consequently, each strain was tested for their abilities to grow at 10
C in BHI broth, pH 7.0; in BHI broth adjusted to pH 5 and 5.5 by addition of lactic acid; and in BHI broth supplemented with 10% and 15% NaCl. Ten microliters of an overnight culture of each strain were inoculated into 5 mL of these various media and the growth was scored as positive or negative after incubation at 37
C for 72 h. 2.3. Detection of nitrate reductase activity Nitrate reductase activity was determined according to Harrigan and McCance (1976). Ten microliters of an overnight culture of each strain was inoculated into 5 mL of peptone water supplemented with KNO3 and were incubated at 37
C for 7 days. After incubation, 1 mL of each of GriesseIlosvay reagents 1 and 2 (reagent 1, 1 g of sulphanilic acid in 100 mL of 5 N acetic acid; reagent 2, 1 g of alpha naphthol in 100 mL of 5 N acetic acid) were added. The rapid development of a red color indicated the presence of nitrate reductase activity. 2.4. Proteolytic activity 2.4.1. Qualitative assessment by agar plate method Sarcoplasmic and myofibrillar proteins were extracted according to the method of Fadda et al. (1999). Sarcoplasmic proteins were sterilized by filtration through a polyvinylidene fluoride filter (0.22 mm, Millipore, Billerica, MA, USA); myofibrillar proteins were extracted under sterile conditions. The sarcoplasmic and myofibrillar proteins were added at concentrations of 0.5 and 0.2 mg mL1 respectively, to sterile medium containing 0.25% yeast extract, 0.1% glucose and 1.5% agar. The medium was poured into Petri dishes and, after solidification, three wells were bored in the agar in each plate. Forty microliters of cell suspension was pipetted into each well. After incubation at 37
C for 48 h, the agar disc was removed from each dish and stained for 30 min in 0.05% (w/v) Coomassie blue R-250 (SigmaeAldrich Chemie GmbH, Taufkirchen, Germany) in methanol:acetic acid:water (50:10:40) and destained in 20% (v/v) methanol:ethanol:acetic acid:water (20:10:5:65). The diameters of clear zones surrounding inoculated wells, which indicated proteolytic activity, were measured. 2.4.2. Quantitative assessment by electrophoretic methods Strains that have exhibited proteolytic activity by agar plate method were then tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. Actin and myosin were extracted and quantified with the methods described by Pérez-Juan et al. (2007) and Abdel-Mohsen et al. (2003) with modifications concerning the centrifugation times and composition of the buffers for resuspension. Volumes of 0.2 mL of cell suspension were inoculated in 1 mL of sarcoplasmic protein, actin and myosin extract (0.15 mg mL1) supplemented with 1% of glucose, and incubated at 37
C for 72 h in a shaken bath. Uninoculated protein extracts were used as control and incubated under the same conditions. After incubation, samples were taken to determine proteolytic activity. Protein degradation was assayed by SDS-PAGE as described by

A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

Laemmli (1970) on 12% polyacrylamide gels. Ten microliters of each protein preparation was mixed with 19 mL of Laemmli buffer (BioRad, Hercules, CA, USA) and 1 mL of b-mercaptoethanol. Thirty microliters of each preparation, or 10 mL of SDS-PAGE Molecular Weight Standard Low Range (Bio-Rad) in the case of the standard, were placed in different wells in the gel. Electrophoresis was carried out at 220 V for about 45 min. Gels were soaked in a fixing solution of methanol:water:acetic acid (50:43:7) for 15 min, stained in 0.05% (w/v) Coomassie blue R-250 in methanol:acetic acid:water (45:10:45) for 2 h and destained until the background was clear. The molecular weights of the products of proteolysis were estimated by reference to the relative mobilities of standard proteins. The results corresponding to each band were expressed as a percentage of total absorbance at 550 nm. Presumptive identities for the proteins were determined from their molecular weights.

63

a saline (0.85 g NaCl L1) solution and mixed with 25 mL of the latex reagents sensitized with antisera SEA to SED. After that, plates were kept at room temperature for 24 h without shaking. Agglutination of latex particles resulted in the formation of a lattice structure, and a diffused layer was formed indicating a positive result. 2.8. Statistical analysis All statistical analyses were performed using the computer programme StatisticaÓ 8.0 for Windows (Statsoft Inc., Tulsa, OK, USA). Significant differences between strain groups regarding their lipolytic activity were determined using one-way analysis of variance (ANOVA). Differences between mean values were established using Duncan’s test and were considered significant when P < 0.05. 3. Results and discussion

2.5. Lipolytic activity 3.1. Technological properties Lipolytic activity was assessed using the method described by Vignolo et al. (1988) with some modifications. One milliliter of a cell suspension of each strain was inoculated into 30 mL of a sterile broth containing 1% (w/v) peptone, 0.5% (w/v) yeast extract, 0.5 g (w/v) meat extract, 3% (w/v) NaCl, pH 7.0, supplemented with 15 g of pork fat. After incubation at 37
C for 72 h with shaking, the lipolytic activity was measured by titration. The fat was extracted according to the method of Folch et al. (1957) and the fatty acids were titrated following the procedure of Vignolo et al. (1988). Free fatty acid content was expressed as oleic acid (%) (Mauriello et al., 2004). The medium described above was also used to determine the ability of the strains to release free fatty acids from triglycerides. After 72 h of incubation, the fat of each culture was extracted as before and the free fatty acids were separated from the triglycerides in columns of NH2-aminopropyl following the procedure described by Kaluzny et al. (1985). Methyl esters of free fatty acids were prepared by the method of Shehata et al. (1970) and were quantified by Gas Chromatography following the procedure described by Franco et al. (2006) using an internal standard (C13:0) at a concentration of 4000 ppm. The free fatty acid content was expressed as mg fatty acid per 100 g of fat. 2.6. Decarboxylase activity Decarboxylase activity was initially tested by an agar plate method as described by Lorenzo et al. (2010). The plates were incubated at 37
C and examined after 72 h of incubation. A purple halo around a colony indicated amine production. The strains positive for decarboxylase activity by the agar plate method were further studied. A 0.1 mL aliquot of cell suspension was inoculated into 5 mL of the culture medium described by Joosten and Northolt (1989), without agar or bromocresol purple, supplemented with ornithine (2%) or lysine (2%). After incubation at 37
C for 72 h with shaking, the biogenic amines in preparation were determined by HPLC (Lorenzo et al., 2010). The quantity of each biogenic amine was expressed in ppm. 2.7. Detection of enterotoxin production Staphylococcal enterotoxins (SEs) A, B, C and D were detected using the Staphylococcal enterotoxin test e reversed passive latex agglutination (SET-RPLA) kit (Oxoid) and following the manufacturer’s instructions. In brief, each strain was cultured in Tryptone Soya Broth (TSB) at 37
C for 18e24 h with shaking and then filtered through a PVDF filter (0.22 mm, Millipore). Aliquots of 25 mL of the filtrate were placed in microtitration plate wells, diluted 1/2 with

Most of the strains (84.2%) were able to grow at 10
C, in the presence of 10% and 15% salt (92.1% and 86.8%, respectively), as well as at pH values of 5.0 and 5.5, except for most Staphylococcus equorum strains which did not grow at these pH values (Table 2). These results are in agreement with those obtained by others (Mauriello et al., 2004). All strains of S. equorum and 63.6% of the strains of Staphylococcus epidermidis reduced nitrate, but most strains of Staphylococcus saprophyticus and Staphylococcus pasteuri did not (Table 2). Similar results have been reported by other authors (Mauriello et al., 2004; Martín et al., 2007). The ability to reduce nitrates to nitrites and the ability to grow at different conditions of temperature, pH values and salt concentrations are considered to be important for starter cultures selection, since starters should contribute to the development of the color of the sausage and be able to persist throughout the maturation process (Liepe, 1983). However, strains with low nitrate reductase activity could be used in artisanal sausages made without addition of nitrate (Mauriello et al., 2004). Acid tolerance is important in the starters, because the batter becomes acid as it is fermented and, moreover, acid conditions are necessary to prevent pathogens development during fermented sausages production (Liepe, 1983; García-Varona et al., 2000). The contribution of staphylococci to protein degradation in fermented sausages remains unclear (Fadda et al., 1999; Ammor and Mayo, 2007). The results obtained by the agar plate method showed that 89.5% of the strains hydrolyzed sarcoplasmic proteins and 52.6% of them hydrolyzed myofibrillar proteins (Table 2). Bonomo et al. (2009) obtained a similar result with various species of Staphylococcus, whereas other authors (Mauriello et al., 2002, 2004; Casaburi et al., 2005; Drosinos et al., 2007) found that Staphylococcus spp. isolated from fermented sausages were more proteolytic for myofibrillar than for sarcoplasmic proteins. The proteolytic activity of the strains was then assessed by SDSPAGE. The sarcoplasmic protein extract used in the test (Fig. 1a, lane A) contained several proteins of approximately 102, 66, 60, 55 (glucose phosphate isomerase), 48 (enolase), 45 (creatine phosphate kinase), 42 (aldolase), 37 (glyceraldehyde phosphate dehydrogenase), 35, 25 and 24 kDa. Analysis of control samples reflected no changes in the protein profile (Fig. 1a, lane B). Of 36 strains tested, 2 of S. equorum (SA07 and SB22) were able to hydrolyze all the proteins present in the sarcoplasmic protein extract, showing a complete disappearance of all the bands and the generation of a new peptide of approximately 44 kDa in the case of SB22 (Fig. 1a, lane C). The strain SA52 (S. saprophyticus) caused a complete disappearance of proteins of approximately 66, 60 and 24 kDa

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A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

Table 2 Numbers of strains of four species of Staphylococcus tested for their growth and metabolic properties and numbers of strains positive for each test.

Number of strains Growth At pH 5.0 At pH 5.5 At 10
C In 10% NaCl In 15% NaCl Nitrate reduction Proteolytic activitya Sarcoplasmic proteins Myofibrillar proteins Decarboxylase activityb Histidine Tyrosine Ornithine Lysine a b

S. equorum

S. epidermidis

S. saprophyticus

S. pasteuri

15

11

7

5

1 2 14 15 14 15

10 11 8 9 8 7

6 7 6 7 7 1

5 5 4 4 4 1

13 (12 halo > 25 mm; 1 halo < 25 mm) 4 (4 halo < 25 mm)

11 (6 halo > 25 mm; 5 halo < 25 mm) 6 (2 halo > 25 mm; 4 halo < 25 mm)

5 2 5 2

7 14 14 15

9 8 10 10

6 7 7 7

(3 halo > 25 mm; halo < 25 mm) (3 halo > 25 mm; halo < 25 mm)

5 2 5 3

(3 halo > 25 mm; halo < 25 mm) (2 halo > 25 mm; halo < 25 mm)

4 5 4 4

Proteolytic activity by agar plate method. Decarboxylase activity by agar plate method.

(Fig. 1a, lane D) and the strain SB03 (S. pasteuri) was able to hydrolyze the protein of 60 kDa (Fig. 1a, lane E). The action of the strain SB02 (S. saprophyticus) caused the disappearance of proteins of about 102, 60, 42 (aldolase) and 35 kDa, and the appearance of some polypeptides with molecular weight less than 20 kDa (Fig. 1a, lane F). The rest of strains did not show proteolytic activity against the sarcoplasmic proteins. Similar results were obtained previously by other authors (Mauriello et al., 2002; Casaburi et al., 2005). The protein profile of the extract of actin consisted of an intense band at approximately 45 kDa (actin) and many other polypeptides at 66, 55 (desmin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain) and 24 kDa (troponin T) (Fig. 1b, lane A). The strains studied showed a highly variable activity against actin. The strain SA47 (S. epidermidis) hydrolyzed all the proteins present in the extract (data not shown). The strains SA16 and SA49 (S. epidermidis) caused a complete disappearance of actin band (45 kDa) (Fig. 1b, lane C) whereas the strains SA34, SB24 and SB34 (S. epidermidis) caused a reduction of intensity of this protein band (Fig. 1b, lane D). In contrast, the strain SA41 (S. epidermidis) hydrolyzed all the proteins of the extract except the actin (Fig. 1b, lane E). No proteolytic changes were observed after the action of the rest of the strains, as occurred in the uninoculated control. SDS-PAGE profiles of myosin extracts are shown in Fig. 1c. This fraction consisted of an intense band at approximately 200 kDa (myosin heavy chain) and many other proteins of 102 (alpha-actinin), 55 (desmin), 45 (actin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), 20 kDa (troponin C) and 12 kDa (Fig. 1c, lane A). The activity of the strains studied against myosin was highly variable. Of 36 strains tested, only the strain SA10 (S. equorum) was able to hydrolyze totally the myosin heavy chain (200 kDa), causing the appearance of a new band at about 42 kDa (Fig. 1c, lane C). The actions of the strains SA03 (S. pasteuri), SA11 (S. equorum), SA41, SA47, SA49, SB27, SB32 and SB34 (S. epidermidis) resulted in a partial hydrolysis of the myosin heavy chain (200 kDa) (Fig. 1c, lane D) and the generation of new peptides (at about 180, 160, 66 and 53 kDa) in some cases. The remaining strains did not hydrolyze myosin. The degradation of myosin (200 kDa), actin (45 kDa) and other myofibrillar proteins by Staphylococcus strains isolated from sausages has already been observed (Mauriello et al., 2002; Drosinos et al., 2007; Casquete et al., 2012). The results for proteolytic activities obtained by the agar plate method were not in line with those obtained by electrophoretic assays. This could be due to the different composition of the

substrates used for each determination (Mauriello et al., 2002), since the liquid medium used in the electrophoretic study contains meat proteins as the only source of nitrogen, while the medium used for the agar plate method also contained yeast extract as nitrogen source and vitamins. The titration method (Table 3) showed that only 8 strains have an appreciable lipolytic activity (values from 1.4 to 28.9% of oleic acid). These values are in line with those reported by several authors (Mauriello et al., 2004; Villani et al., 2007; Essid et al., 2007; Casaburi et al., 2008). On the contrary, others (Drosinos et al., 2007; Bonomo et al., 2009) did not detect lipolytic activity in staphylococci isolates. The strains that showed the highest lipolytic activity by the titration method also showed the highest values in total free fatty acids, resulting in a significant positive correlation index (r ¼ 0.92; P < 0.001) between values of % of oleic acid and total free fatty acid contents. Table 4 shows the amounts (expressed as mg fatty acid/100 g of fat) of free fatty acids released by the staphylococci strains after 72 h of incubation. The strains with low lipolytic activity (n ¼ 30) gave similar profiles of free fatty acid; oleic acid (C18:1 n9) and palmitic acid (C16:0) were the most abundant, followed by stearic (C18:0) and linoleic (C18:2 n6) acids. In addition, saturated fatty acids (SFA) and unsaturated fatty acids (UFA) were present in similar percentages, and within the UFA the monounsaturated fatty acids (MUFA) were predominant. When comparing low lipolytic vs. high lipolytic activity strains, significant differences (P < 0.001) were found for SFA, MUFA, PUFA and total free fatty acids. Concerning the strains with high lipolytic activity, three different behaviors were observed. In the first, shown by strains SA47, SA48 (S. epidermidis) and SA51 (S. pasteuri), the total free fatty acid contents ranged between 1152.4 and 1795.0 mg of fatty acid/ 100 g of fat and the UFA concentrations were higher than those of SFA. Oleic acid was the main fatty acid (approximately 37% of total fatty acids), followed by linoleic and palmitic acids. The second group, of strains SA27 (S. pasteuri), SA34 and SA49 (S. epidermidis) showed the same fatty acid profile as the low lipolytic activity strains. The third group, of strains SB12 (S. saprophyticus) and SB32 (S. epidermidis), showed concentrations of 2100.0 and 2919.4 mg of free fatty acid/100 g of fat; the SFA were the main fractions and palmitic acid was the main fatty acid (30%), followed by oleic, estearic and linoleic acids. The relatively large amounts of estearic,

A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

a

65

Table 3 Lipolytic activity, by titration method, of Staphylococcus strains. Strain

Species

SA03 SA04 SA05 SA07 SA10 SA11 SA14 SA15 SA16 SA19 SA20 SA24 SA25 SA27 SA34 SA41 SA47 SA48 SA49 SA51 SA52 SA54 SB02 SB03 SB04 SB08 SB11 SB12 SB19 SB21 SB22 SB23 SB24 SB25 SB27 SB29 SB32 SB34

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

Lipolytic activitya % Oleic acid

b

c

a

pasteuri equorum equorum equorum equorum equorum equorum equorum epidermidis equorum equorum equorum equorum pasteuri epidermidis epidermidis epidermidis epidermidis epidermidis pasteuri saprophyticus equorum saprophyticus pasteuri saprophyticus saprophyticus saprophyticus saprophyticus pasteuri saprophyticus equorum equorum epidermidis epidermidis epidermidis equorum epidermidis epidermidis

0.46 0.88 0.50 0.75 0.36 0.44 0.58 0.99 0.46 0.49 0.53 0.45 0.79 9.27 1.43 0.62 21.45 26.15 26.34 13.09 0.27 0.59 0.17 0.81 0.39 0.11 0.40 12.47 0.39 0.50 0.40 0.29 0.04 0.10 0.33 0.04 28.96 0.24

(0.41e0.51) (0.83e0.93) (0.45e0.55) (0.73e0.77) (0.34e0.38) (0.42e0.46) (0.57e0.59) (0.98e1.0) (0.45e0.47) (0.46e0.52) (0.51e0.55) (0.43e0.47) (0.77e0.81) (9.24e9.30) (1.40e1.46) (0.60e0.64) (21.44e21.46) (26.14e26.16) (26.33e26.35) (13.05e13.13) (0.23e0.31) (0.55e0.63) (0.14e0.20) (0.78e0.84) (0.36e0.42) (0.08e0.14) (0.26e0.64) (12.32e12.62) (0.39e0.39) (0.48e0.52) (0.38e0.42) (0.27e0.31) (0.02e0.06) (0.08e0.12) (0.31e0.35) (0.04e0.04) (28.79e29.13) (0.19e0.29)

Mean values and ranges of two replicates.

3.2. Safety properties

Fig. 1. SDS-PAGE of protein hydrolysis by Staphylococcus strains. (a) Sarcoplasmic proteins. Lane A: purified sarcoplasmic proteins (GPI: glucose phosphate isomerase, E: enolase, CK: creatine phosphate kinase, A: aldolase. GH: glyceraldehyde phosphate dehydrogenase). Lane B: uninoculated control after 72 h of incubation. Lane CeF: samples containing different isolates after 72 h of incubation. Lane C: SB22. Lane D: SA52. Lane E: SB03. Lane F: SB02. Lane M: molecular weight standard. (b) Actin. Lane A: purified actin preparation (D: desmin, Act: actin, Tt: troponin T, Tr: tropomyosin, MLC: myosin light chain, Ti: troponin I). Lane B: uninoculated control after 72 h of incubation. Lane CeE: samples containing different isolates after 72 h of incubation. Lane C: SA16. Lane D: SB24. Lane E: SA41. Lane M: molecular weight standard. (c) Myosin. Lane A: purified myosin preparation (MHC: myosin heavy chain, a-act: alphaactinin, D: desmin, Act: actin, Tt: troponin T, Tr: tropomyosin, MLC: myosin light chain, Tc: troponin C). Lane B: uninoculated control after 72 h of incubation. Lane CeD: samples containing different isolates after 72 h of incubation. Lane C: SA10. Lane D: SB34. Lane M: molecular weight standard.

oleic and linoleic acids released by most of the strains may be due to the specificity of staphylococcal lipases for the positions sn1 and sn3 of the triglycerides, as these are the positions most frequently occupied by these fatty acids (Hierro et al., 1997). However, some strains released relatively large amounts of palmitic acid, found mainly in the position sn2, which indicates a different specificity of the enzymes of these strains.

The results of the decarboxylase activity tested, by the agar plate method, are shown in Table 2. Among the 38 staphylococci strains tested, 68.4% were able to decarboxylate histidine, 89.4% tyrosine, 92.1% ornitine, and 94.7% lysine. S. saprophyticus was the species with the highest decarboxylase activity. The biogenic amine content of fermented meat products is due to the result of a complex equilibrium between the abiotic sausage environment and the enzymatic activities of the microbial population (Bover-Cid et al., 2001). The decarboxylase activity of staphylococci strains isolated from sausages has been widely studied (Casaburi et al., 2005; Martín et al., 2007). Several staphylococci strains belonging to different species isolated from dry fermented sausages decarboxylated amino acids (Drosinos et al., 2007; Bonomo et al., 2009), which agrees with the results obtained in the present study. Table 5 shows the amounts of putrescine and cadaverine formed by the staphylococci strains. The amount of putrescine produced generally varied between 0.2 and 24.2 ppm, although one strain of S. epidermidis (SB27) and one of S. equorum (SB29) were able to produce 977.1 and 1415.0 ppm, respectively. The production of cadaverine was lower (<5.3 ppm), with the exception of one strain of S. epidermidis (SB27) that produced more than 36 ppm. The concentrations of putrescine and cadaverine observed in the present study are comparable with those reported by Martín et al.

66

Table 4 Free fatty acid profile (mg fatty acid/100 g of fat) and standard error of the mean (SEM) of low lipolytic and high lipolytic staphylococci strains. Fatty acids

High lipolytic activity

n ¼ 30a

SEM

SA47

SA48

SA51

7.52 18.61 21.88 8.55 72.79 22.19 9.30 11.02 44.42 80.72 40.49 18.50 17.83 11.51 11.13 10.74 0.63 5.25 6.08 213.21 206.40 125.46 80.95 419.60

1.98 2.11 0.83 0.80 5.34 1.16 0.32 0.42 3.08 7.41 3.60 0.83 0.92 0.45 0.71 0.56 0.63 1.02 1.88 13.83 12.73 8.25 4.75 26.23

ND 25.71 25.17 11.37 210.15 55.21 16.71 18.84 186.80 632.70 352.60 38.05 21.63 28.17 28.01 13.52 ND 10.89 ND 525.54 1149.98 734.93 415.05 1675.52

ND 24.78 24.76 10.98 225.91 58.00 17.45 19.17 227.81 670.05 364.58 38.67 21.75 31.95 28.72 13.05 ND 17.41 ND 582.16 1212.87 779.17 433.70 1795.03

ND 24.12 22.50 10.66 145.46 41.14 15.02 16.29 106.79 405.61 237.23 31.67 19.31 15.12 16.67 11.66 ND 13.16 20.05 380.57 771.87 478.16 293.72 1152.45

SEM 0.46 0.83 0.21 24.61 5.22 0.72 0.91 35.54 82.63 40.60 2.24 0.79 5.10 3.90 0.56 1.91 6.68 60.03 137.72 93.84 43.88 197.37

Significance

SA27

SA34

SA49

SEM

SB12

SB32

SEM

L*H

H

23.66 26.44 42.20 11.99 440.51 59.48 19.29 17.27 236.85 482.64 155.29 29.65 20.26 17.90 15.49 12.01 19.21 261.28 30.80 886.70 1035.54 577.30 458.24 1922.24

22.64 24.92 47.40 11.37 628.04 64.95 22.28 17.34 319.14 579.20 142.32 26.95 19.17 16.62 13.76 10.33 18.48 127.88 28.14 1155.33 985.58 678.11 307.48 2140.92

25.36 28.17 37.83 13.00 495.42 91.11 30.18 27.10 657.44 885.32 571.56 49.32 26.94 40.97 39.62 14.80 20.40 29.51 23.81 1338.14 1709.69 1044.51 665.18 3107.85

0.79 0.94 2.77 0.48 55.66 9.76 3.25 3.27 128.69 121.38 140.97 7.05 2.43 7.91 8.35 1.30 0.56 67.16 2.04 131.10 233.49 141.95 103.68 364.27

27.27 31.12 69.17 14.74 738.83 53.94 44.93 21.54 248.12 463.31 208.60 25.60 26.06 21.78 20.83 13.23 20.82 19.15 31.00 1252.05 847.98 560.56 287.41 2100.03

ND 26.70 57.87 13.35 791.21 92.45 42.32 23.88 623.86 639.97 420.79 39.76 24.53 27.25 25.64 13.66 18.82 15.56 21.80 1620.46 1298.95 783.55 515.40 2919.41

13.64 2.21 5.65 0.70 26.19 19.26 1.31 1.17 187.87 88.33 106.10 7.08 0.76 2.74 2.41 0.22 1.00 1.80 4.60 184.21 225.49 111.50 114.00 409.69

ns ns *** * *** *** *** *** *** *** *** *** * *** *** ns *** ** ** *** *** *** *** ***

ns ns ** * ** ns ** ns ns ns ns ns ns ns ns ns *** ns * ** ns ns ns ns

SFA: sum of saturated fatty acids; UFA: sum of unsaturated fatty acids; MUFA: sum of monounsaturated fatty acids; PUFA: sum of polyunsaturated fatty acids. L*H: Significantly different values when comparing low lipolytic vs. high lipolytic activity strains *(P < 0.05); **(P < 0.01); ***(P < 0.001); ns: no significant difference; H: Significantly different values when comparing among them the three groups of strains of high lipolytic activity *(P < 0.05); **(P < 0.01); ***(P < 0.001); ns: no significant difference. a n ¼ SA03, SA04, SA05, SA07, SA10, SA11, SA14, SA15, SA16, SA19, SA20, SA24, SA25, SA41, SA52, SA54, SB02, SB03, SB04, SB08, SB11, SB19, SB21, SB22, SB23, SB24, SB25, SB27, SB29 and SB34.

A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

C10:0 C12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 n9 C18:2 n6 C18:3 n3 C20:0 C20:1 n9 C20:2 n6 C20:4 n6 C22:0 C22:2 n6 C24:0 SFA UFA MUFA PUFA Total

Low lipolytic activity

A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

Acknowledgments

Table 5 Biogenic amine production (ppm) of Staphylococcus strains. Strain

SA03 SA04 SA05 SA07 SA10 SA11 SA14 SA15 SA16 SA19 SA20 SA24 SA25 SA27 SA34 SA41 SA47 SA48 SA49 SA51 SA52 SA54 SB02 SB03 SB04 SB08 SB11 SB12 SB19 SB21 SB22 SB23 SB24 SB25 SB27 SB29 SB32 SB34

Species

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

pasteuri equorum equorum equorum equorum equorum equorum equorum epidermidis equorum equorum equorum equorum pasteuri epidermidis epidermidis epidermidis epidermidis epidermidis pasteuri saprophyticus equorum saprophyticus pasteuri saprophyticus saprophyticus saprophyticus saprophyticus pasteuri saprophyticus equorum equorum epidermidis epidermidis epidermidis equorum epidermidis epidermidis

Biogenic aminesa Putrescine

Cadaverine

12.39 16.98 21.87 24.22 11.00 8.82 21.27 13.89 14.75 17.51 9.86 6.01 7.91 1.19 4.56 1.70 0.98 1.57 1.53 2.23 1.63 1.48 0.95 2.08 0.83 1.91 1.16 0.43 ND 1.01 1.46 0.19 0.37 2.26 977.13 1415.05 1.03 ND

1.43 1.49 1.62 1.17 1.51 0.54 2.22 1.09 1.41 2.07 4.51 0.41 0.25 3.23 1.21 0.46 5.04 3.67 4.04 4.33 4.30 4.22 4.79 0.40 0.58 0.83 0.61 0.65 ND 3.04 5.31 0.63 1.11 0.72 36.52 0.69 4.14 ND

(11.97e12.81) (11.53e22.43) (19.22e24.52) (20.90e27.54) (7.54e14.46) (8.68e8.96) (18.95e23.59) (5.45e22.33) (10.62e18.88) (13.55e21.47) (7.35e12.37) (5.65e6.37) (7.56e8.26) (1.19e1.19) (3.17e5.95) (1.64e1.76) (0.73e1.23) (1.57e1.57) (1.53e1.53) (2.23e2.23) (1.63e1.63) (1.48e1.48) (0.95e0.95) (1.98e2.18) (0.35e1.31) (1.58e2.24) (1.01e1.31) (0.32e0.54) (1.01e1.01) (1.46e1.46) (0.08e0.30) (0.22e0.52) (2.21e2.31) (977.13e977.13) (1296.71e1533.39) (1.03e1.03)

67

(1.01e1.85) (1.22e1.76) (1.57e1.67) (0.93e1.41) (1.28e1.74) (0.54-0.54) (2.21e2.23) (0.90e1.28) (1.15e1.67) (1.68e2.46) (3.92e5.10) (0.35e0.47) (0.14e0.36) (3.23e3.23) (0.27e2.15) (0.12e0.80) (5.0e5.08) (3.67e3.67) (4.04e4.04) (4.33e4.33) (4.30e4.30) (4.22e4.22) (4.79e4.79) (0.33e0.47) (0.51e0.65) (0.75e0.91) (0.36e0.86) (0.62e0.68) (3.04e3.04) (5.31e5.31) (0.54e0.72) (1.11e1.11) (0.62e0.82) (36.52e36.52) (0.64e0.74) (4.14e4.14)

ND: not detected. a Mean values and ranges of two replicates.

(2006) and differ from the results described by Bover-Cid et al. (2001) who did not find aminogenesis in staphylococci strains. The lack of concordance between authors could be explained by the strain-specific character of this property (Drosinos et al., 2007), although the detection method used can have some influence. Only four strains, belonging to S. epidermidis, were able to produce enterotoxin C. Enterotoxin production does not seem a very common characteristic among the coagulase-negative staphylococci strains isolated from sausages; however, different authors (Bautista et al., 1988; Valle et al., 1990; Martín et al., 2006) found that some strains can have this ability. For this reason, this character should be checked in the strains selected for use as starter cultures. 4. Conclusions Technological and safety characteristics of staphylococci were highly variable among strains, even within the same species. Most of the strains studied possessed nitrate reductase activity, and suitable characteristics with respect to temperature, pH and NaCl concentrations permitting growth and inability to form large amounts of biogenic amines or enterotoxins. These would make them eligible, according to their proteolytic and lipolytic activities, as good starter cultures in combination with lactic acid bacteria strains. Further studies are necessary to evaluate the effectiveness and the prevalence of the strains in experimental sausages.

This work was financially supported by Xunta de Galicia (The Regional Government) (project 07TAL021383 PR).

References Abdel-Mohsen, H.A., Nakamoto, M., Kim, J.B., Nogusa, Y., Gekko, K., Ishioroshi, M., Samejima, K., Tanabe, S., Nishimura, T., 2003. Changes in the properties of porcine myosin during postmortem aging. Food Science and Technology Research 9, 297e303. Ammor, 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, 138e146. Bautista, L., Gaya, P., Medina, M., Núñez, M., 1988. A quantitative study of enterotoxin production by sheep milk staphylococci. Applied and Environmental Microbiology 54, 566e569. Bonomo, M.G., Ricciardi, A., Zotta, T., Sico, M.A., Salzano, G., 2009. Technological and safety characterization of coagulase-negative staphylococci from traditionally fermented sausages of Basilicata region (Southern Italy). Meat Science 83, 15e 23. Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2001. Amino aciddecarboxylase activity of bacteria isolated from fermented pork sausages. International Journal of Food Microbiology 66, 185e189. Casaburi, A., Blaiotta, G., Mauriello, G., Pepe, O., Villani, F., 2005. Technological activities of Staphylococcus carnosus and Staphylococcus simulans strains isolated from fermented sausages. Meat Science 71, 643e650. Casaburi, A., Di Monaco, R., Cavella, S., Toldrá, F., Ercolini, D., Villani, F., 2008. Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits. Food Control 11, 41e47. Casquete, R., Benito, M.J., Martín, A., Ruiz-Moyano, S., Córdoba, J.J., Córdoba, M.G., 2011a. Role of an autochthonous starter culture and the protease EPc222 on the sensory and safety properties of an traditional Iberian dry-cured sausage “salchichón”. Food Microbiology 28, 1432e1440. Casquete, R., Benito, M.J., Martín, A., Ruiz-Moyano, S., Hernández, A., Córdoba, M.G., 2011b. Effect of autochthonous starter cultures in the production of “salchichón” a traditional Iberian dry-fermented sausage, with different ripening processes. LWT-Food Science and Technology 44, 1562e1571. Casquete, R., Benito, M.J., Martín, A., Ruiz-Moyano, S., Pérez-Nevado, F., Córdoba, M.G., 2012. Comparison of the effects of a commercial and an autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter culture on the sensory and safety properties of a traditional Iberian dry-fermented sausage ‘‘salchichón”. International Journal of Food Science and Technology 47, 1011e1019. Drosinos, E.H., Paramithiotis, S., Kolovos, G., Tsikouras, I., Metaxopoulos, I., 2007. Phenotypic and technological diversity of lactic acid bacteria and staphylococci isolated from traditionally fermented sausages in Southern Greece. Food Microbiology 24, 260e270. Essid, I., Ismail, H.B., Hadj Ahmed, S.B., Ghedamsi, R., Hassouna, M., 2007. Characterization and technological properties of Staphylococcus xylosus strains isolated from a Tunisian traditional salted meat. Meat Science 77, 204e212. Fadda, S., Sanz, Y., Vignolo, G., Aristoy, M.C., Oliver, G., Toldrá, F., 1999. Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Applied and Environmental Microbiology 65, 3540e 3546. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497e509. Franco, I., Escamilla, M.C., García, J., García-Fontán, M.C., Carballo, J., 2006. Fatty acid profile of the fat from Celta pig breed fattened using a traditional feed: effect of the location in the carcass. Journal of Food Composition and Analysis 19, 792e 799. Galgano, F., Favati, F., Schirone, M., Martuscelli, M., Crudele, M.A., 2003. Influence of indigenous starter cultures on the free fatty acids content during ripening in artisan sausages produced in the Basilicata region. Food Technology and Biotechnology 41, 253e258. García-Fontán, M.C., Lorenzo, J.M., Parada, A., Franco, I., Carballo, J., 2007a. Microbiological characteristics of "androlla", a Spanish traditional pork sausage. Food Microbiology 24, 52e58. García-Fontán, M.C., Lorenzo, J.M., Martínez, S., Franco, I., Carballo, J., 2007b. Microbiological characteristics of Botillo, a Spanish traditional pork sausage. LWT-Food Science and Technology 40, 1610e1622. García-Varona, M., Santos, E.M., Jaime, I., Rovira, J., 2000. Characterization of Micrococcaceae isolated from different varieties of chorizo. International Journal of Food Microbiology 54, 189e195. Harrigan, W.F., McCance, M.E., 1976. Laboratory Methods in Foods and Dairy Microbiology. Academic Press, London. Hierro, E., De la Hoz, L., Ordóñez, J.A., 1997. Contribution of microbial and meat endogenous enzymes to the lipolysis of dry fermented sausages. Journal of Agricultural and Food Chemistry 45, 2989e2995. Hugas, M., Monfort, J.M., 1997. Bacterial starter cultures for meat fermentation. Food Chemistry 59, 547e554.

68

A. Cachaldora et al. / Food Microbiology 33 (2013) 61e68

Joosten, H.M.L.J., Northolt, M.D., 1989. Detection, growth and amine-producing capacity of lactobacilli in cheese. Applied and Environmental Microbiology 55, 2356e2359. Kaluzny, M.A., Duncan, L.A., Merritt, M.V., Epps, D.E., 1985. Rapid separation of lipids classes in high yield and purity using bonded phase columns. Journal of Lipid Research 26, 135e140. Kenneally, P.M., Leuschner, R.G., Arendt, E.K., 1998. Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes. Journal of Applied Microbiology 84, 839e846. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680e685. Leroy, F., Verluyten, J., De Vuyst, L., 2006. Functional meat starter cultures for improved sausage fermentation. International Journal of Food Microbiology 106, 270e285. Liepe, H.U., 1983. Starter cultures in meat production. In: Rehm, H.J., Reeds, G. (Eds.), Biotechnology. Verlag-Chemie, Weinheim, Germany, pp. 400e423. Lizaso, G., Chasco, J., Beriain, M.J., 1999. Microbiological and biochemical changes during ripening of Salchichón, a Spanish dry cured sausage. Food Microbiology 16, 219e228. Lorenzo, J.M., Cachaldora, A., Fonseca, S., Gómez, M., Franco, I., Carballo, J., 2010. Production of biogenic amines “in vitro” in relation to the growth phase by Enterobacteriaceae species isolated from traditional sausages. Meat Science 86, 684e691. Martín, A., Colín, B., Aranda, E., Benito, M.J., Córdoba, M.G., 2007. Characterization of Micrococcaceae isolated from Iberian dry-cured sausages. Meat Science 75, 696e708. Martín, B., Jofré, A., Garriga, M., Pla, M., Aymerich, T., 2006. Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR. Applied and Environmental Microbiology 72, 6040e6048. Mauriello, G., Casaburi, A., Villani, F., 2002. Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmatic proteins and use of

selected strains in the production of “Naples type” salami. Journal of Applied Microbiology 92, 482e490. Mauriello, G., Casaburi, A., Blaiotta, G., Villani, F., 2004. Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy. Meat Science 67, 149e158. Molly, K., Demeyer, D., Johansson, G., Raemaekers, M., Ghistelinck, M., Geenen, I., 1997. The importance of meat enzymes in ripening and flavour generation in dry fermented sausages. First results of a European project. Food Chemistry 59, 539e545. Montel, M.C., Masson, F., Talon, R., 1998. Bacterial role in flavour development. Meat Science 49 (Suppl. 1), S111eS123. Pérez-Juan, M., Flores, M., Toldrá, F., 2007. Simultaneous process to isolate actomyosin and actin from post-rigor porcine skeletal muscle. Food Chemistry 101, 1005e1011. Sanz, Y., Fadda, S., Vignolo, G., Aristoy, M.C., Oliver, G., Toldrá, F., 1999. Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins. Journal of Agricultural and Food Chemistry 47, 3441e3448. Shehata, A.J., De Man, J.M., Alexander, J.C., 1970. A simple and rapid method for the preparation of methyl esters of fats in milligram amounts for gas chromatography. Canadian Institute of Food Science and Technology Journal 3, 85e89. Valle, J.E., Gómez-Lucía, S., Piriz, J., Goyache, J.A., Vadillo, S., 1990. Enterotoxin production by staphylococci isolated from healthy goats. Applied and Environmental Microbiology 56, 1323e1326. Vignolo, G.M., Ruiz Holgado, A.P., Oliver, G., 1988. Acid production and proteolytic activity of Lactobacillus strains isolated from dry sausages. Journal of Food Protection 51, 481e484. Villani, F., Casaburi, A., Pennacchia, C., Filosa, L., Russo, F., Ercolini, D., 2007. The microbial ecology of the soppressata of Vallo di Diano, a traditional dry fermented sausage from southern Italy, and in vitro and in situ selection of autochthonous starter cultures. Applied and Environmental Microbiology 73, 5453e5463.