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Pervasiveness of Staphylococcus carnosus over Staphylococcus xylosus is affected by the level of acidification within a conventional meat starter culture set-up
International Journal of Food Microbiology xxx (xxxx) xxx–xxx
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Pervasiveness of Staphylococcus carnosus over Staphylococcus xylosus is affected by the level of acidification within a conventional meat starter culture set-up Despoina Angeliki Stavropouloua, Hannelore De Maereb, Alberto Berardoc, Bente Janssensa, ⁎ Panagiota Filippoua, Luc De Vuysta, Stefaan De Smetc, Frédéric Leroya, a
Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Research Group of Technology and Quality of Animal Products, KU Leuven Technology Campus, Ghent, Belgium c Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Ghent, Belgium b
A R T I C LE I N FO
A B S T R A C T
Keywords: Staphylococcus carnosus Staphylococcus xylosus pH Meat fermentation Starter culture Coagulase-negative staphylococci
Staphylococcus carnosus and Staphylococcus xylosus are commonly used, individually or in combination, within conventional starter cultures for the purposes of colour and flavour development during meat fermentation. Yet, little is known about the relative importance of both species under different processing conditions. The present study aimed at investigating the competitiveness of S. carnosus within a meat starter culture under different acidification profiles. The experimental set-up involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mincebased meat fermentation models, to fermented sausage production on pilot-scale level. In general, S. carnosus gained a fitness advantage over S. xylosus in the most acidified variants of each set-up. In contrast, increasing persistence of S. xylosus was seen at the mildest acidification profiles, especially when approximating actual meat fermentation practices. Under such conditions, S. carnosus was reduced to co-prevalence in the mincebased meat fermentation models and was fully outcompeted on pilot-scale level. The latter was even the case when no S. xylosus starter culture was added, whereby S. carnosus was overpowered by staphylococci that originated from the meat background (mostly S. xylosus strains). The results of the present study suggested that conventional starter cultures behave differently when applied in different technological set-ups or using different recipes, with possible repercussions on fermented meat product quality.
1. Introduction The industrial use of starter cultures to produce fermented sausages is widespread as a strategy to improve process control, thereby also standardising the end-product and contributing to biosafety and quality (Ravyts et al., 2012; Talon and Leroy, 2011). Conventional meat starter cultures consist mainly of selected strains of lactic acid bacteria (LAB) for acidification, besides coagulase-negative staphylococci (CNS) for colour and flavour development (Leroy et al., 2006). In European-type fermented sausages, the LAB fraction most often consists of strains of Lactobacillus sakei, whereas strains of Staphylococcus carnosus and/or Staphylococcus xylosus are usually representing the CNS group (Ravyts et al., 2012). The use of S. carnosus may be somewhat surprising, as this CNS species is rarely isolated from spontaneously fermented meat products (García-Fontan et al., 2007; Talon et al., 2007). In contrast, S.
⁎
xylosus seems to commonly dominate such artisan-type fermented meat products prepared without starter culture (Aquilanti et al., 2016; Cocolin et al., 2001; García-Varona et al., 2000). Nonetheless, S. carnosus is known to be very effective during meat fermentation when added as a starter culture (Leroy et al., 2006). Indeed, S. carnosus harbours genes that both reflect adaptation to the meat fermentation process and are relevant to technologically important properties, among others the potential for nitrate and nitrite reduction, carbohydrate degradation, and catalase activity (Sánchez Mainar et al., 2017). Also, genes that are related to pathogenicity and virulence seem to be lacking (Rosenstein et al., 2009), which meets food safety expectations (EFSA, 2005). The genome of S. carnosus is one of the smallest among the staphylococcal genomes available, suggesting that this CNS species may have become adapted to domesticated use in meat processing environments (Rosenstein and Götz, 2010).
Corresponding author. E-mail address: [email protected] (F. Leroy).
https://doi.org/10.1016/j.ijfoodmicro.2018.03.006 Received 26 December 2017; Received in revised form 27 February 2018; Accepted 7 March 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Stavropoulou, D.A., International Journal of Food Microbiology (2018), https://doi.org/10.1016/j.ijfoodmicro.2018.03.006
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Currently, it is not well known to which degree S. carnosus and S. xylosus prevail at an individual level under different meat processing conditions. This is not trivial, as both CNS species may not only have different metabolic impacts on fermented meat quality (Sánchez Mainar et al., 2017), but also since fermented meats are produced according to a very wide spectrum of different recipes and processing conditions (Leroy et al., 2015). In general, S. carnosus seems to be mostly present in acidic sausages (Aymerich et al., 2003), being a rather acid-tolerant CNS species (Janssens et al., 2012; Ravyts et al., 2010; Stahnke et al., 2002). This acid tolerance contrasts with the behaviour of S. xylosus, being more at ease under mildly acidified conditions (Aquilanti et al., 2016; Blaiotta et al., 2004; Fonseca et al., 2013; Pisacane et al., 2015). One should not overlook the fact that the meat starter culture also needs to compete with the background microbiota of the raw meat, of which the community dynamics depend on the applied practices too (Cocconcelli and Fontana, 2014; Janssens et al., 2012, 2013; Leroy et al., 2014; Stahnke et al., 2002). More insights into the relationship between the technological fitness of conventional starter culture CNS species during meat fermentation and the major processing factors is required, with the level of acidification being a key component of process variation. Heavily acidified fermented meats are representative for the Northern-European type of sausage fermentation, whereas mild acidification is more common in Mediterranean-type variants. Therefore, the aim of the present study was to investigate how the competitive behaviour of S. carnosus is affected by acidification in the presence or absence of S. xylosus. The methodology involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mince-based meat fermentation models, to fermented sausage production on pilot scale.
Table 1 Acidification profiles used for the liquid (temperature and pH) and mince-based meat fermentation model experiments (temperature). Profile
Strong acidification
Mild acidification
Time (days)
Temperature (°C)
pH
Temperature (°C)
pH
0 1 2 3 4 5 6 7
25 25 25 20 18 16 14 14
5.80 5.60 5.20 5.00 4.90 4.80 4.80 4.80
20 20 20 18 16 14 14 14
5.80 5.80 5.60 5.30 5.25 5.20 5.20 5.20
2.2. Liquid meat fermentation models All fermentation experiments were performed with a co-culture consisting of L. sakei CTC 494, S. carnosus 833, and S. xylosus 3PA6. Inoculation levels added to MSM were set at about 6 log of colonyforming units (cfu) per ml for L. sakei CTC 494 and 5 log cfu per ml for both CNS strains. The fermentations were carried out in 15-litre computer-controlled Biostat C fermentors (Sartorius, Melsungen, Germany) connected to a cryostat (Frigomix 2000; Sartorius) to run them at low temperatures. The fermentors contained 10 l of MSM and were sterilised in situ at 121 °C for 20 min. Glucose was sterilised separately and was added aseptically to the fermentors. Moderate agitation (150 rpm; turbine impeller) was maintained to ensure homogeneity of the medium. Two different temperature and pH profiles were implied, representing strong or mild acidification profiles typical for Northern- and Southern-European meat fermentations, respectively (Table 1). The fermentations were followed for 7 days, whereby temperature and pH were controlled on-line with Micro MFCS for Windows NT software (Sartorius), as described previously (Leroy and De Vuyst, 1999). After each sampling at regular time intervals, the sampling valve was sterilised using an external steam generator (J. Strobel & Söhne, Munich, Germany) during 10 min. Every experiment was done in triplicate.
2. Materials and methods 2.1. Bacterial strains, media, and inoculum build-up Strains of Lactobacillus sakei (CTC 494) as well as S. carnosus (833) and S. xylosus (3PA6 or 2S7–2) were used in the present study, either alone or in combination. All strains were present in the collection of the research group of Industrial Microbiology and Food Biotechnology (Vrije Universiteit Brussel, Brussels, Belgium). The LAB and CNS strains were stored at −80 °C in glycerol-containing (25%, v/v) de ManRogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, Hampshire, UK) and brain heart infusion (BHI) medium (Oxoid), respectively. For the respective enumeration of LAB and CNS, MRS agar medium (Oxoid) and mannitol salt-phenol-red agar medium (MSA; VWR International, Darmstadt, Germany) were used. The liquid meat fermentation models were based on a meat simulation medium (MSM), reflecting high contents of salt, lactate and meatderived peptides, and serving as an approximation of the water phase of fermented sausages (Leroy and De Vuyst, 2005). This medium contained (per litre): 40.0 g of sodium chloride (VWR International), 13.0 g of glucose (VWR International), 11.0 g of bacteriological peptone (Oxoid), 8.8 g of meat extract (Lab Lemco; Oxoid), 6.1 g of calcium lactate.5H2O (VWR International), 2.2 g of yeast extract (VWR International), 0.2 g of ascorbic acid (Sigma-Aldrich, Saint-Louis, MO, USA), 0.2 g of sodium nitrate (VWR International), 0.038 g of MnSO4.5H2O (VWR International), and 1 ml of Tween 80 (VWR International). For the inoculum build-up, the strains were propagated twice in the appropriate medium and incubated at 30 °C for 12 h. The precultures were then inoculated (1%, v/v) into BHI medium (CNS) or MRS medium (LAB) to obtain the final inoculum by propagation at 30 °C for 12 h. The cell pellets were collected by centrifugation (8041 ×g at 4 °C for 20 min) and used as inoculum for the meat fermentation models and/or sausage preparation after their re-suspension in saline (0.85%, m/v, NaCl; VWR International).
2.3. Solid mince-based meat fermentation models To validate the findings of the liquid meat fermentation models, a more realistic approach was adopted using solid mince-based meat fermentation models. For the preparation of the meat batter, fresh pork (3 kg) mince was supplemented with 2.5% of sodium chloride (m/m; Merck Millipore, Darmstadt, Germany), 500 ppm of ascorbic acid (Sigma-Aldrich), 200 ppm of sodium nitrate (VWR International), and 200 ppm of MnSO4.4H2O (VWR International). Two different batches were prepared by adding different glucose (VWR International) concentrations to achieve a strong (0.7%, m/m) or mild (0.1%, m/m) pH decrease during the fermentation courses. As was the case for the liquid meat fermentation models, the two batches had a different temperature profile (Table 1). The starter culture consisting of L. sakei CTC 494, S. carnosus 833, and S. xylosus 3PA6 was inoculated into the meat batter at a level of approximately 6 log cfu/g. The meat mixture of each batch was then stuffed into 60-ml plastic containers (approximately 100 g per container; VWR International) to enable fermentation in the absence of air. The containers were placed in a water bath coupled to a cryostat (Frigomix 3000T, Sartorius) and the fermentations were followed for 7 days. Bacterial counts and pH measurements (see below) were performed at days 1, 2, 3, 4, and 7. For each time point, three randomly selected containers were selected for the analysis. 2.4. Pilot-scale production of dry fermented sausages Fermented sausages were produced in a pilot-scale plant at the 2
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Technology Campus Ghent (KU Leuven, Ghent, Belgium). For their preparation, the following basic ingredients were mixed (in %, m/m): cutter lean pork (70.5), pork backfat (27.0), sodium chloride (2.5), sodium ascorbate (0.05), and sodium nitrate (0.015). The total sausage batter was around 30 kg while the initial weight of the dry fermented sausages was approximately 200 g. In a first experimental set-up, a fermentation was carried out using a starter culture composed of L. sakei CTC 494 and S. carnosus 833, whether or not accompanied by S. xylosus 2S7–2. This fermentation was performed without adding carbohydrates to the meat batter, representative of low-acidified, artisantype fermented meat products, as to meet the conditions under which S. carnosus has most difficulties with respect to competitiveness (see Results). In a second experiment, the meat batter was inoculated with L. sakei CTC 494 and S. carnosus 833 solely, using three different final carbohydrate concentrations (0.00, 0.25, and 0.70% of glucose, m/m), to be able to test the impact of the acidification level. The meat batter of each batch was stuffed into collagen casings of 50 mm diameter (Naturin, Weinheim, Germany). The sausages were ripened in a climate chamber (Kerres Anlagensysteme, Backnang, Germany) for 28 days. The fermentation step lasted 2 or 4 days for the experiments without or with carbohydrates added, respectively, before the pH started to level off. The fermentation step was performed at 24 °C and a relative humidity of 94%. For the ripening process, the temperature was decreased to 12 °C and the relative humidity was adjusted to 80% for the last 10 days. Samples were taken after inoculation (day 0), at the end of the fermentation (day 2 or 4) and at the end of the ripening. Three sausages were analysed per batch and per time point for each experimental setup.
containing the appropriate media and 25% (v/v) of glycerol and used for DNA extraction. For the evaluation of the background microbiota of the meat used for both the mince-based meat fermentation models and the pilot-scale sausage production experiments, additional isolates were obtained before inoculation.
2.5. pH and water activity measurements
3.1. Liquid meat fermentation models
The pH of each sample of the mince-based and pilot-scale meat fermentation experiments was measured using a DY-P10 pH meter (Sartorius). Regarding the mince-based meat fermentation model samples, one randomly selected container was used and triplicate measurements were performed. For the pilot-scale sausage production samples, the pH measurements were performed for three randomly selected sausages per experimental variant. Additionally, water activity (aw) of the fermented sausages was also measured, with triplicate measurements for each sample, using a Hydropalm 23 aw meter (Rotronic, New York, NY, USA).
During both the mild and strong acidified meat fermentation set-up in MSM, initial growth by L. sakei CTC 494 followed roughly the same pattern, increasing from 6.34 ± 0.08 to 9.07 ± 0.13 log cfu/ml after 24 h of fermentation (Fig. 1). This was followed by a steep decrease only in the strong acidified meat fermentation set-up, whereas under the mild conditions the population remained constant. Likewise, S. carnosus 833 displayed similar growth during both acidification scenarios, with a population increase from 5.57 ± 0.06 to 7.83 ± 0.17 log cfu/ml (mild acidification) or 7.85 ± 0.08 log cfu/ml (strong acidification) after 30 h of fermentation, and only underwent a subsequent decrease (to about 6.00 log cfu/ml) when the acidification profile was steep. When the pH of the fermentation did not drop below 5.3, S. xylosus increased to 7.05 ± 0.25 log cfu/ml after 24 h and then decreased slowly to 5.25 ± 0.08 log cfu/ml by the end of the fermentation. When the pH dropped below 5.3, however, the S. xylosus population started to decline after a small initial increase, fell back to 3.10 ± 0.14 log cfu/g after 2 days of fermentation, and subsequently decreased below the detection limit. For both acidification profiles, S. carnosus thus predominated as the major fraction within the CNS population (Fig. 1b,d).
2.7. Classification and identification of bacterial isolates through (GTG)5PCR fingerprinting of genomic DNA Genomic DNA extraction from cell pellets obtained by microcentrifugation at 13,000 rpm of 1.5 ml of an overnight LAB and CNS culture was performed with a Nucleospin 96 tissue kit (MachereyNagel, Düren, Germany), according to the manufacturer's instructions. Prior to extraction, the cell pellets were washed with Tris-ethylene diaminotetraacetic acid (EDTA)-sucrose buffer [TES buffer; 50 mM Tris base (Calbiochem, Darmstadt, Germany), 1 mM EDTA (Sigma-Aldrich), and 6.7% (m/v) sucrose (VWR International), pH 8.0]. Subsequently, (GTG)5-PCR fingerprints of the genomic DNA were obtained, followed by image analysis, as described previously (Braem et al., 2011). Numerical analysis of the fingerprints obtained was performed with BioNumerics 5.1 software (Applied Maths, Sint-Martens-Latem, Belgium). Confirmation of the species identity assigned to each LAB and CNS cluster was done by sequencing of the 16S rRNA and rpoB gene of representative isolates, respectively, as described previously (Braem et al., 2011). 3. Results
2.6. Enumeration and isolation of microorganisms For the liquid meat fermentation models, LAB and CNS counts were determined by plating the appropriate sample dilutions on MRS agar and MSA (VWR International), respectively. Differentiation of the CNS species in either S. carnosus or S. xylosus populations was done based on differences in colony shape, the former giving smooth and the latter serrated colony types [which was validated by (GTG)5-PCR fingerprinting of genomic DNA, see below]. For the meat systems, either 12 g of the mince-based meat fermentation model samples or 25 g of the pilot-scale sausage production samples were aseptically transferred into a stomacher bag (Seward, Worthing, West Sussex, UK) together with 108 ml or 225 ml of maximum recovery diluents [sterile solution of 0.85% (m/v) NaCl (VWR International) and 0.1% (m/v) bacteriological peptone (Oxoid)], respectively. This mixture was homogenised at high speed for 2 min in a stomacher (Stomacher 400; Seward). Appropriate decimal dilutions in saline were prepared and spread on MRS agar and MSA media. The agar media were incubated at 30 °C for 48–72 h and counts were determined from agar media containing 30–300 colonies. Next, 10–30% of the colonies were randomly picked up to analyse the LAB and CNS communities. The MSA- and MRS agar media-derived colonies were transferred into BHI and MRS media, respectively, and incubated overnight at 30 °C. The cultures obtained were stored at −80 °C in cryovials
3.2. Mince-based meat fermentation models As for the above-mentioned liquid meat fermentation model experiments, two different acidification profiles were imposed during the mince-based meat fermentation model systems (Fig. 2). In the experimental variant with the mild acidification profile, the pH dropped to around 5.3 after 2 days of fermentation and then remained constant. In parallel, the LAB population increased from 6.19 ± 0.11 to 8.81 ± 0.15 log cfu/g after day 2 and then remained stable during the next 5 days of fermentation. The MSA counts increased from 5.38 ± 0.20 log cfu/g after inoculation to their maximum of 7.46 ± 0.22 log cfu/g after 2 days of fermentation. Afterwards, they declined to 6.59 ± 0.13 log cfu/g. During the strong acidification setup, the pH dropped from 5.80 ± 0.08 to 4.67 ± 0.12 after 4 days of 3
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Bacterial counts (log cfu/g)
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Fig. 1. Counts of Lactobacillus sakei CTC 494 (grey squares), Staphylococcus carnosus 833 (white diamonds), and S. xylosus 3PA6 (black diamonds), as well as the pH profiles (grey circles) of fermentations in liquid meat simulation medium (MSM) with a mild (a, b) or strong (c, d) acidification profile. Percentages of counts of S. carnosus 833 (white bars) and S. xylosus 3PA6 (black bars) were determined based on colony morphology.
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Calculated based on colony morphology
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3.3. Pilot-scale dry-fermented sausage production
fermentation and remained constant till day 7. The LAB counts increased from 5.89 ± 0.10 to 9.14 ± 0.13 log cfu/g after 3 days of fermentation and then stabilized around this value. The initial level of CNS at 5.92 ± 0.23 log cfu/g increased to 7.24 ± 0.11 log cfu/g after 1 day of fermentation and then slowly decreased to 6.29 ± 0.09 log cfu/g after 7 days. With respect to the species diversity of CNS in the meat fermentation models, only S. carnosus and S. xylosus were isolated, outperforming other CNS species from the background microbiota. As compared to the experiments in MSM, S. xylosus generally displayed an enhanced competitiveness (Fig. 2b,d). At the mild acidification profiles (Fig. 2b), S. xylosus even was the most prevalent CNS species till day 3, followed by a co-prevalence with S. carnosus after day 7. In contrast, when the pH dropped below 5.00 (Fig. 2d), the relative abundance of the S. xylosus isolates declined from 70% (day 2) to 25% (day 3). By the end of the experiment, S. carnosus was the only CNS species isolated.
Bacterial counts (log cfu/g)
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% N = 13
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Fermented sausages were produced to investigate how S. carnosus behaved at different acidification levels and how its fitness was affected by the presence or absence of S. xylosus in conventional starter cultures for sausage fermentation (Fig. 3). The aw values were similar for all investigated batches, decreasing from an initial value of 0.965 to 0.936 after acidification and 0.897 at the end of maturation. To evaluate the competitiveness of S. carnosus at its most unfavourable conditions of high pH, a trial in slightly acidified sausages with no carbohydrates added was carried out during a conventional starter culture set-up with (Fig. 3a,b) or without (Fig. 3c,d) the addition of S. xylosus. In general, the pH showed a similar decrease for both experimental variants, decreasing from pH 5.74 to pH 5.51 after 2 days of fermentation, followed by an increase to pH 5.81–5.92 after 28 days. Also, the patterns of the MRS and MSA counts as well as the pH evolution were comparable for both variants. In all cases, whether or not S. xylosus was added as a co-starter culture, the conditions were
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Fig. 2. Counts of lactic acid bacteria (grey squares) and coagulase-negative staphylococci (black triangles), as well as the pH profiles (grey circles) and the species diversity of the staphylococcal communities of mince-based meat fermentation models inoculated with a starter culture of S. carnosus 833, S. xylosus 3PA6, and L. sakei CTC 494, with a mild (a, b) or strong (c, d) acidification profile. Species were identified as S. carnosus (white bars) and S. xylosus (black bars) by gene-sequencing.
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Bacterial counts (log cfu/g)
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Fig. 3. Counts of lactic acid bacteria (grey squares) and coagulase-negative staphylococci (black triangles), as well as the pH profiles (grey circles) and the species diversity of the staphylococcal communities of fermented sausages produced on pilot-scale with (a, b) no carbohydrates added and inoculated with S. carnosus 833, S. xylosus 2S7–2, and L. sakei CTC 494; (c, d) no carbohydrates added and inoculated with S. carnosus 833 and L. sakei CTC 494; (e, f) glucose added (final concentration of 0.25%, m/m) and inoculated with S. carnosus 833 and L. sakei CTC 494; and (g, h) glucose added (final concentration of 0.75%, m/m) and inoculated with S. carnosus 833 and L. sakei CTC 494. Species were identified as S. carnosus (white bars), S. xylosus (black bars), S. equorum (light grey bars), and S. saprophyticus (dark grey bars) by gene-sequencing.
d
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Time (days) decreasing experimental control but increasing realism were adopted to investigate the competitive behaviour of S. carnosus in the presence or absence of S. xylosus and to verify how this was affected by acidification. A general finding was that acidification favoured S. carnosus over S. xylosus, although the latter CNS species could take over when the pH drop was small. These results agreed with previous findings that have demonstrated the prevalence of S. xylosus in mildly acidified fermented sausages (Blaiotta et al., 2004; Cocolin et al., 2001; Iacumin et al., 2006). The results of the three experimental designs differed with respect to the fitness of S. xylosus, which seemed more at ease in the meatbased fermentation model systems. The latter suggested that, in contrast to the liquid simulation approach, the effect of a solid meat matrix gave a selective advantage to S. xylosus, which is known to be welladapted to meat and animal skin environments (Leroy et al., 2017; Vanderhaeghen et al., 2015; Vermassen et al., 2014, 2016). This CNS species possesses the metabolic flexibility to adapt to various substrates and environmental niches (Dordet-Frisoni et al., 2007; Nagase et al., 2002; Shale et al., 2005) and may enhance its persistence by the formation of biofilms (Planchon et al., 2006). Staphylococcus carnosus is of major technological interest for meat fermentation, being safe to use and generally having good nitrate reductase activity for proper colour development (Sánchez Mainar et al., 2017). Yet, this CNS species is only rarely recovered from spontaneously fermented meats, which seems at odds with its common use as the main staphylococcal component within conventional meat starter
unfavourable for S. carnosus that was outcompeted quickly. In contrast, S. xylosus became the most prevalent CNS species by the end of the fermentation (day 2), whether or not added as a starter culture. In the latter case, Staphylococcus equorum was also present but in low numbers and only at day 28. In the acidified experimental variants, S. xylosus lost its competitive benefit, giving rise to prevalence by S. carnosus (Fig. 3e-h). When sausages were prepared with a low concentration of carbohydrates added (0.25%, m/m), the pH dropped to 5.25 ± 0.11 at day 4, whereas at the high level of carbohydrates added (0.75%, m/m), the pH dropped to pH 4.90 ± 0.05. During both experimental variants, the initial decrease in pH values was followed by a rise. The initial counts of presumable CNS started at around 6.41 ± 0.26 log cfu/g for both experimental variants, amounting to about 7.01 ± 0.31 log cfu/g after 4 days of fermentation. The LAB counts started at 6.44 ± 0.12 log cfu/ g, reaching their highest value of 7.92 ± 0.22 log cfu/g after 4 days of fermentation. At all time points investigated, S. carnosus dominated the fermentations, especially at the low pH scenario. Isolates belonging to S. xylosus and Staphylococcus saprophyticus species were found too, but only at the intermediate acidification profiles and in numbers lower than 25% (Fig. 3f).
4. Discussion During the present study, three different experimental designs of 5
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Acknowledgements
cultures, whether or not in combination with S. xylosus (Leroy et al., 2006). This apparently contradicts both the suggestion that the genome of S. carnosus provides excellent adaptability to the fermented meat matrix (Rosenstein et al., 2009) and its more pronounced durability in acidified environments compared to other CNS species, including S. xylosus (Ravyts et al., 2010). Acid tolerance may be ascribed to the finding that strains of this species usually possess several acid stresscoping strategies, such as arginine deiminase (Sánchez Mainar et al., 2014) and amino acid decarboxylation activities (Stavropoulou et al., 2015). The results obtained during the present study underlined that the performance of S. carnosus was enhanced in environments where acidification was pronounced and when other CNS species were outcompeted. In Northern-European fermented sausages, which are generally of a more acidic nature than Southern-European ones, S. carnosus starter cultures are therefore generally found to be robust, steering the meat fermentation process relatively easily (Janssens et al., 2012; Ravyts et al., 2010; Stahnke et al., 2002). The reason for the apparently paradoxal absence of S. carnosus in spontaneously fermented sausages remains unclear. It may be that S. carnosus is only occasionally encountered on raw meat (Janssens et al., 2012). As such, a very low initial presence would be insufficient to generate a meaningful population of S. carnosus during fermentation, despite the competitive features that characterize this CNS species. The reason for its scarceness in fresh meat may relate to a poor adaptation to animal skin compared to other CNS species, resulting in low contamination levels after slaughter (Vanderhaeghen et al., 2015). Alternatively, some of the specific ecological determinants within unfermented meat may not be supportive for its growth, including the relatively high pH (usually around 5.6–5.8). From the present study, it became clear that dominance of the CNS communities by S. carnosus is indeed not to be taken for granted in low-acidic environments, so that other CNS species may take over the lead even when S. carnosus is added as the sole CNS starter culture. In fermentations without carbohydrates added, the technological fitness of S. carnosus was so low that it was outperformed by strains of CNS species that were originating from the background of the meat, mostly belonging to S. xylosus and to a lesser degree S. equorum. In fermented sausages with a low concentration of carbohydrates added, S. saprophyticus emerged from the meat background besides S. xylosus, but only in low proportions. The latter three species are indeed among the CNS species that are most commonly found in spontaneously fermented sausages with a low to mild acidification profile (Drosinos et al., 2005; Fonseca et al., 2013; García-Varona et al., 2000; Greppi et al., 2015; Papamanoli et al., 2002; Pisacane et al., 2015). The findings of the present study also met the finding that S. xylosus is the dominant CNS species isolated from slightly fermented sausages, whereas S. carnosus has been found only in more acidic sausages (Aymerich et al., 2003). In conclusion, it seemed that the inclusion of both S. carnosus and S. xylosus in conventional meat starter cultures offers more technological flexibility than when only one of these CNS species would be used. This is particularly the case since different types of fermented sausage may rely on different degrees of acidification. The results obtained during the present study indeed suggested that conventional starter cultures may behave differently when applied in dissimilar technological set-ups or using different recipes, with possible repercussions on product quality. Further studies should therefore explore how conventional starter cultures behave during meat fermentation if they are supplemented with other species of CNS, as to evaluate the impact of starter culture innovation on CNS ecology and its technological implications. Although most of the differences in competitiveness of CNS in meat seem to be situated at species level, further in-depth exploration of strain variability will have to indicate to which degree the latter can also have some influence on the overall community patterns.
The authors acknowledge the financial support of the Research Council of the Vrije Universiteit Brussel (SRP7, IRP2, HOA, and IOF342 projects), the Hercules Foundation (projects UABR 09/004 and UAB 13/002), and the Research Foundation Flanders (project FWOAL632). This work is dedicated to the memory of Maarten Janssens, who also participated in this study. References Aquilanti, L., Garofalo, C., Osimani, A., Clementi, F., 2016. Ecology of lactic acid bacteria and coagulase-negative cocci in fermented dry sausages manufactured in Italy and other Mediterranean countries: an overview. Int. Food Res. J. 23, 429–445. Aymerich, T., Martín, B., Garriga, M., Hugas, M., 2003. Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic staphylococci from artisanal low-acid sausages. Appl. Environ. Microbiol. 69, 4583–4594. Blaiotta, G., Pennacchia, C., Villani, F., Ricciardi, A., Tofalo, R., Parente, E., 2004. Diversity and dynamics of communities of coagulase-negative staphylococci in traditional fermented sausages. J. Appl. Microbiol. 97, 271–284. Braem, G., De Vliegher, S., Supré, K., Haesebrouck, F., Leroy, F., De Vuyst, L., 2011. (GTG)5-PCR fingerprinting for the classification and identification of coagulase-negative Staphylococcus species from bovine milk and teat apices: a comparison of type strains and field isolates. Vet. Microbiol. 147, 67–74. Cocconcelli, P.S., Fontana, C., 2014. Bacteria. In: Toldrá, F. (Ed.), Handbook of Fermented Meat and Poultry, Second edition. John Wiley & Sons, Chichester, UK, pp. 117–128. Cocolin, L., Manzano, M., Cantoni, C., Comi, G., 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl. Environ. Microbiol. 67, 5113–5121. Dordet-Frisoni, E., Dorchies, G., De Araujo, C., Talon, R., Leroy, S., 2007. Genomic diversity in Staphylococcus xylosus. Appl. Environ. Microbiol. 73, 7199–7209. Drosinos, E.H., Mataragas, M., Xiraphi, N., Moschonas, G., Gaitis, F., Metaxopoulos, J., 2005. Characterization of the microbial flora from a traditional Greek fermented sausage. Meat Sci. 69, 307–317. EFSA, 2005. Opinion of the scientific panel on additives and products or substances used in animal feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance. EFSA J. 223, 1–12. Fonseca, S., Cachaldora, A., Gómez, M., Franco, I., Carballo, J., 2013. Monitoring the bacterial population dynamics during the ripening of Galician chorizo, a traditional dry fermented Spanish sausage. Food Microbiol. 33, 77–84. García-Fontan, M.C., Lorenzo, J.M., Parada, A., Franco, I., Carballo, J., 2007. Microbiological characteristics of “androlla”, a Spanish traditional pork sausage. Food Microbiol. 24, 52–58. 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Community dynamics of coagulasenegative staphylococci during spontaneous artisan-type meat fermentations differ between smoking and moulding treatments. Int. J. Food Microbiol. 166, 168–175. Leroy, F., De Vuyst, L., 1999. Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K. Appl. Environ. Microbiol. 65, 974–981. Leroy, F., De Vuyst, L., 2005. Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain. Int. J. Food Microbiol. 100, 141–152. Leroy, F., Verluyten, J., De Vuyst, L., 2006. Functional meat starter cultures for improved sausage fermentation. Int. J. Food Microbiol. 106, 270–285. Leroy, F., Goudman, T., De Vuyst, L., 2014. The influence of processing parameters on starter culture performance. In: Toldrá, F. (Ed.), Handbook of Fermented Meat and Poultry, Second Edition. John Wiley & Sons, Chichester, UK, pp. 169–175. Leroy, F., Scholliers, P., Amilien, V., 2015. Elements of innovation and tradition in meat fermentation: conflicts and synergies. Int. J. Food Microbiol. 212, 2–8. Leroy, S., Vermassen, A., Ras, G., Talon, R., 2017. Insight into the genome of Staphylococcus xylosus, a ubiquitous species well adapted to meat products. Microorganisms 5, 52. Nagase, N., Sasaki, A., Yamashita, K., Shimizu, A., Wakita, Y., Kitai, S., Kawano, J., 2002. Isolation and species distribution of staphylococci from animal and human skin. J. Vet. Med. Sci. 64, 245–250. Papamanoli, E., Kotzekidou, P., Tzanetakis, N., Litopoulou-Tzanetaki, E., 2002. Characterization of Micrococcaceae isolated from dry fermented sausage. Food Microbiol. 19, 441–449. Pisacane, V., Callegari, M.L., Puglisi, E., Dallolio, G., Rebecchi, A., 2015. Microbial
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Shale, K., Lues, J.F.R., Venter, P., Buys, E.M., 2005. The distribution of Staphylococcus sp. on bovine meat from abattoir deboning rooms. Food Microbiol. 22, 433–438. Stahnke, L.H., Holck, A., Jensen, A., Nilsen, A., Zanardi, E., 2002. Maturity acceleration of Italian dried sausage by Staphylococcus carnosus - relationship between maturity and flavor compounds. J. Food Sci. 67, 1914–1921. Stavropoulou, D.A., Borremans, W., De Vuyst, L., De Smet, S., Leroy, F., 2015. Amino acid conversions by coagulase-negative staphylococci in a rich medium: assessment of inter- and intraspecies heterogeneity. Int. J. Food Microbiol. 212, 34–40. Talon, R., Leroy, S., 2011. Diversity and safety hazards of bacteria involved in meat fermentation. Meat Sci. 89, 303–309. Talon, R., Leroy, S., Lebert, I., 2007. Microbial ecosystems of traditional fermented meat products: the importance of indigenous starters. Meat Sci. 77, 55–62. Vanderhaeghen, W., Piepers, S., Leroy, F., Van Coillie, E., Haesebrouck, F., De Vliegher, S., 2015. Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants. Vet. J. 203, 44–51. Vermassen, A., de la Foye, A., Loux, V., Talon, R., Leroy, S., 2014. Transcriptomic analysis of Staphylococcus xylosus in the presence of nitrate and nitrite in meat reveals its response to nitrosative stress. Front. Microbiol. 5, 691. Vermassen, A., Dordet-Frisoni, E., de La Foye, A., Micheau, P., Laroute, V., Leroy, S., Talon, R., 2016. Adaptation of Staphylococcus xylosus to nutrients and osmotic stress in a salted meat model. Front. Microbiol. 7, 87.
analyses of traditional Italian salami reveal microorganisms transfer from the natural casing to the meat matrix. Int. J. Food Microbiol. 207, 57–65. Planchon, S., Gaillard-Martinie, B., Dordet-Frisoni, E., Bellon-Fontaine, M.N., Leroy, S., Labadie, J., Hébraud, M., Talon, R., 2006. Formation of biofilm by Staphylococcus xylosus. Int. J. Food Microbiol. 109, 88–96. Ravyts, F., Steen, L., Goemaere, O., Paelinck, H., De Vuyst, L., Leroy, F., 2010. The application of staphylococci with flavour-generating potential is affected by acidification in fermented dry sausages. Food Microbiol. 27, 945–954. Ravyts, F., De Vuyst, L., Leroy, F., 2012. Bacterial diversity and functionalities in food fermentations. Eng. Life Sci. 12, 356–367. Rosenstein, R., Götz, F., 2010. Genomic differences between the food-grade Staphylococcus carnosus and pathogenic staphylococcal species. Int. J. Med. Microbiol. 300, 104–108. Rosenstein, R., Nerz, C., Biswas, L., Resch, A., Raddat, G., Schuster, S.C., Götz, F., 2009. Genome analysis of the meat starter culture bacterium Staphylococcus carnosus TM300. Appl. Environ. Microbiol. 75, 811–822. Sánchez Mainar, M., Weckx, S., Leroy, F., 2014. Coagulase-negative staphylococci favor conversion of arginine into ornithine despite a widespread genetic potential for nitric oxide synthase activity. Appl. Environ. Microbiol. 80, 7741–7751. Sánchez Mainar, M., Stavropoulou, D.A., Leroy, F., 2017. Exploring the metabolic potential of coagulase-negative staphylococci to improve the quality and safety of fermented meats: a review. Int. J. Food Microbiol. 247, 24–37.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Pervasiveness of Staphylococcus carnosus over Staphylococcus xylosus is affected by the level of acidification within a conventional meat starter culture set-up Despoina Angeliki Stavropouloua, Hannelore De Maereb, Alberto Berardoc, Bente Janssensa, ⁎ Panagiota Filippoua, Luc De Vuysta, Stefaan De Smetc, Frédéric Leroya, a
Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Research Group of Technology and Quality of Animal Products, KU Leuven Technology Campus, Ghent, Belgium c Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Ghent, Belgium b
A R T I C LE I N FO
A B S T R A C T
Keywords: Staphylococcus carnosus Staphylococcus xylosus pH Meat fermentation Starter culture Coagulase-negative staphylococci
Staphylococcus carnosus and Staphylococcus xylosus are commonly used, individually or in combination, within conventional starter cultures for the purposes of colour and flavour development during meat fermentation. Yet, little is known about the relative importance of both species under different processing conditions. The present study aimed at investigating the competitiveness of S. carnosus within a meat starter culture under different acidification profiles. The experimental set-up involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mincebased meat fermentation models, to fermented sausage production on pilot-scale level. In general, S. carnosus gained a fitness advantage over S. xylosus in the most acidified variants of each set-up. In contrast, increasing persistence of S. xylosus was seen at the mildest acidification profiles, especially when approximating actual meat fermentation practices. Under such conditions, S. carnosus was reduced to co-prevalence in the mincebased meat fermentation models and was fully outcompeted on pilot-scale level. The latter was even the case when no S. xylosus starter culture was added, whereby S. carnosus was overpowered by staphylococci that originated from the meat background (mostly S. xylosus strains). The results of the present study suggested that conventional starter cultures behave differently when applied in different technological set-ups or using different recipes, with possible repercussions on fermented meat product quality.
1. Introduction The industrial use of starter cultures to produce fermented sausages is widespread as a strategy to improve process control, thereby also standardising the end-product and contributing to biosafety and quality (Ravyts et al., 2012; Talon and Leroy, 2011). Conventional meat starter cultures consist mainly of selected strains of lactic acid bacteria (LAB) for acidification, besides coagulase-negative staphylococci (CNS) for colour and flavour development (Leroy et al., 2006). In European-type fermented sausages, the LAB fraction most often consists of strains of Lactobacillus sakei, whereas strains of Staphylococcus carnosus and/or Staphylococcus xylosus are usually representing the CNS group (Ravyts et al., 2012). The use of S. carnosus may be somewhat surprising, as this CNS species is rarely isolated from spontaneously fermented meat products (García-Fontan et al., 2007; Talon et al., 2007). In contrast, S.
⁎
xylosus seems to commonly dominate such artisan-type fermented meat products prepared without starter culture (Aquilanti et al., 2016; Cocolin et al., 2001; García-Varona et al., 2000). Nonetheless, S. carnosus is known to be very effective during meat fermentation when added as a starter culture (Leroy et al., 2006). Indeed, S. carnosus harbours genes that both reflect adaptation to the meat fermentation process and are relevant to technologically important properties, among others the potential for nitrate and nitrite reduction, carbohydrate degradation, and catalase activity (Sánchez Mainar et al., 2017). Also, genes that are related to pathogenicity and virulence seem to be lacking (Rosenstein et al., 2009), which meets food safety expectations (EFSA, 2005). The genome of S. carnosus is one of the smallest among the staphylococcal genomes available, suggesting that this CNS species may have become adapted to domesticated use in meat processing environments (Rosenstein and Götz, 2010).
Corresponding author. E-mail address: [email protected] (F. Leroy).
https://doi.org/10.1016/j.ijfoodmicro.2018.03.006 Received 26 December 2017; Received in revised form 27 February 2018; Accepted 7 March 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Stavropoulou, D.A., International Journal of Food Microbiology (2018), https://doi.org/10.1016/j.ijfoodmicro.2018.03.006
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Currently, it is not well known to which degree S. carnosus and S. xylosus prevail at an individual level under different meat processing conditions. This is not trivial, as both CNS species may not only have different metabolic impacts on fermented meat quality (Sánchez Mainar et al., 2017), but also since fermented meats are produced according to a very wide spectrum of different recipes and processing conditions (Leroy et al., 2015). In general, S. carnosus seems to be mostly present in acidic sausages (Aymerich et al., 2003), being a rather acid-tolerant CNS species (Janssens et al., 2012; Ravyts et al., 2010; Stahnke et al., 2002). This acid tolerance contrasts with the behaviour of S. xylosus, being more at ease under mildly acidified conditions (Aquilanti et al., 2016; Blaiotta et al., 2004; Fonseca et al., 2013; Pisacane et al., 2015). One should not overlook the fact that the meat starter culture also needs to compete with the background microbiota of the raw meat, of which the community dynamics depend on the applied practices too (Cocconcelli and Fontana, 2014; Janssens et al., 2012, 2013; Leroy et al., 2014; Stahnke et al., 2002). More insights into the relationship between the technological fitness of conventional starter culture CNS species during meat fermentation and the major processing factors is required, with the level of acidification being a key component of process variation. Heavily acidified fermented meats are representative for the Northern-European type of sausage fermentation, whereas mild acidification is more common in Mediterranean-type variants. Therefore, the aim of the present study was to investigate how the competitive behaviour of S. carnosus is affected by acidification in the presence or absence of S. xylosus. The methodology involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mince-based meat fermentation models, to fermented sausage production on pilot scale.
Table 1 Acidification profiles used for the liquid (temperature and pH) and mince-based meat fermentation model experiments (temperature). Profile
Strong acidification
Mild acidification
Time (days)
Temperature (°C)
pH
Temperature (°C)
pH
0 1 2 3 4 5 6 7
25 25 25 20 18 16 14 14
5.80 5.60 5.20 5.00 4.90 4.80 4.80 4.80
20 20 20 18 16 14 14 14
5.80 5.80 5.60 5.30 5.25 5.20 5.20 5.20
2.2. Liquid meat fermentation models All fermentation experiments were performed with a co-culture consisting of L. sakei CTC 494, S. carnosus 833, and S. xylosus 3PA6. Inoculation levels added to MSM were set at about 6 log of colonyforming units (cfu) per ml for L. sakei CTC 494 and 5 log cfu per ml for both CNS strains. The fermentations were carried out in 15-litre computer-controlled Biostat C fermentors (Sartorius, Melsungen, Germany) connected to a cryostat (Frigomix 2000; Sartorius) to run them at low temperatures. The fermentors contained 10 l of MSM and were sterilised in situ at 121 °C for 20 min. Glucose was sterilised separately and was added aseptically to the fermentors. Moderate agitation (150 rpm; turbine impeller) was maintained to ensure homogeneity of the medium. Two different temperature and pH profiles were implied, representing strong or mild acidification profiles typical for Northern- and Southern-European meat fermentations, respectively (Table 1). The fermentations were followed for 7 days, whereby temperature and pH were controlled on-line with Micro MFCS for Windows NT software (Sartorius), as described previously (Leroy and De Vuyst, 1999). After each sampling at regular time intervals, the sampling valve was sterilised using an external steam generator (J. Strobel & Söhne, Munich, Germany) during 10 min. Every experiment was done in triplicate.
2. Materials and methods 2.1. Bacterial strains, media, and inoculum build-up Strains of Lactobacillus sakei (CTC 494) as well as S. carnosus (833) and S. xylosus (3PA6 or 2S7–2) were used in the present study, either alone or in combination. All strains were present in the collection of the research group of Industrial Microbiology and Food Biotechnology (Vrije Universiteit Brussel, Brussels, Belgium). The LAB and CNS strains were stored at −80 °C in glycerol-containing (25%, v/v) de ManRogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, Hampshire, UK) and brain heart infusion (BHI) medium (Oxoid), respectively. For the respective enumeration of LAB and CNS, MRS agar medium (Oxoid) and mannitol salt-phenol-red agar medium (MSA; VWR International, Darmstadt, Germany) were used. The liquid meat fermentation models were based on a meat simulation medium (MSM), reflecting high contents of salt, lactate and meatderived peptides, and serving as an approximation of the water phase of fermented sausages (Leroy and De Vuyst, 2005). This medium contained (per litre): 40.0 g of sodium chloride (VWR International), 13.0 g of glucose (VWR International), 11.0 g of bacteriological peptone (Oxoid), 8.8 g of meat extract (Lab Lemco; Oxoid), 6.1 g of calcium lactate.5H2O (VWR International), 2.2 g of yeast extract (VWR International), 0.2 g of ascorbic acid (Sigma-Aldrich, Saint-Louis, MO, USA), 0.2 g of sodium nitrate (VWR International), 0.038 g of MnSO4.5H2O (VWR International), and 1 ml of Tween 80 (VWR International). For the inoculum build-up, the strains were propagated twice in the appropriate medium and incubated at 30 °C for 12 h. The precultures were then inoculated (1%, v/v) into BHI medium (CNS) or MRS medium (LAB) to obtain the final inoculum by propagation at 30 °C for 12 h. The cell pellets were collected by centrifugation (8041 ×g at 4 °C for 20 min) and used as inoculum for the meat fermentation models and/or sausage preparation after their re-suspension in saline (0.85%, m/v, NaCl; VWR International).
2.3. Solid mince-based meat fermentation models To validate the findings of the liquid meat fermentation models, a more realistic approach was adopted using solid mince-based meat fermentation models. For the preparation of the meat batter, fresh pork (3 kg) mince was supplemented with 2.5% of sodium chloride (m/m; Merck Millipore, Darmstadt, Germany), 500 ppm of ascorbic acid (Sigma-Aldrich), 200 ppm of sodium nitrate (VWR International), and 200 ppm of MnSO4.4H2O (VWR International). Two different batches were prepared by adding different glucose (VWR International) concentrations to achieve a strong (0.7%, m/m) or mild (0.1%, m/m) pH decrease during the fermentation courses. As was the case for the liquid meat fermentation models, the two batches had a different temperature profile (Table 1). The starter culture consisting of L. sakei CTC 494, S. carnosus 833, and S. xylosus 3PA6 was inoculated into the meat batter at a level of approximately 6 log cfu/g. The meat mixture of each batch was then stuffed into 60-ml plastic containers (approximately 100 g per container; VWR International) to enable fermentation in the absence of air. The containers were placed in a water bath coupled to a cryostat (Frigomix 3000T, Sartorius) and the fermentations were followed for 7 days. Bacterial counts and pH measurements (see below) were performed at days 1, 2, 3, 4, and 7. For each time point, three randomly selected containers were selected for the analysis. 2.4. Pilot-scale production of dry fermented sausages Fermented sausages were produced in a pilot-scale plant at the 2
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Technology Campus Ghent (KU Leuven, Ghent, Belgium). For their preparation, the following basic ingredients were mixed (in %, m/m): cutter lean pork (70.5), pork backfat (27.0), sodium chloride (2.5), sodium ascorbate (0.05), and sodium nitrate (0.015). The total sausage batter was around 30 kg while the initial weight of the dry fermented sausages was approximately 200 g. In a first experimental set-up, a fermentation was carried out using a starter culture composed of L. sakei CTC 494 and S. carnosus 833, whether or not accompanied by S. xylosus 2S7–2. This fermentation was performed without adding carbohydrates to the meat batter, representative of low-acidified, artisantype fermented meat products, as to meet the conditions under which S. carnosus has most difficulties with respect to competitiveness (see Results). In a second experiment, the meat batter was inoculated with L. sakei CTC 494 and S. carnosus 833 solely, using three different final carbohydrate concentrations (0.00, 0.25, and 0.70% of glucose, m/m), to be able to test the impact of the acidification level. The meat batter of each batch was stuffed into collagen casings of 50 mm diameter (Naturin, Weinheim, Germany). The sausages were ripened in a climate chamber (Kerres Anlagensysteme, Backnang, Germany) for 28 days. The fermentation step lasted 2 or 4 days for the experiments without or with carbohydrates added, respectively, before the pH started to level off. The fermentation step was performed at 24 °C and a relative humidity of 94%. For the ripening process, the temperature was decreased to 12 °C and the relative humidity was adjusted to 80% for the last 10 days. Samples were taken after inoculation (day 0), at the end of the fermentation (day 2 or 4) and at the end of the ripening. Three sausages were analysed per batch and per time point for each experimental setup.
containing the appropriate media and 25% (v/v) of glycerol and used for DNA extraction. For the evaluation of the background microbiota of the meat used for both the mince-based meat fermentation models and the pilot-scale sausage production experiments, additional isolates were obtained before inoculation.
2.5. pH and water activity measurements
3.1. Liquid meat fermentation models
The pH of each sample of the mince-based and pilot-scale meat fermentation experiments was measured using a DY-P10 pH meter (Sartorius). Regarding the mince-based meat fermentation model samples, one randomly selected container was used and triplicate measurements were performed. For the pilot-scale sausage production samples, the pH measurements were performed for three randomly selected sausages per experimental variant. Additionally, water activity (aw) of the fermented sausages was also measured, with triplicate measurements for each sample, using a Hydropalm 23 aw meter (Rotronic, New York, NY, USA).
During both the mild and strong acidified meat fermentation set-up in MSM, initial growth by L. sakei CTC 494 followed roughly the same pattern, increasing from 6.34 ± 0.08 to 9.07 ± 0.13 log cfu/ml after 24 h of fermentation (Fig. 1). This was followed by a steep decrease only in the strong acidified meat fermentation set-up, whereas under the mild conditions the population remained constant. Likewise, S. carnosus 833 displayed similar growth during both acidification scenarios, with a population increase from 5.57 ± 0.06 to 7.83 ± 0.17 log cfu/ml (mild acidification) or 7.85 ± 0.08 log cfu/ml (strong acidification) after 30 h of fermentation, and only underwent a subsequent decrease (to about 6.00 log cfu/ml) when the acidification profile was steep. When the pH of the fermentation did not drop below 5.3, S. xylosus increased to 7.05 ± 0.25 log cfu/ml after 24 h and then decreased slowly to 5.25 ± 0.08 log cfu/ml by the end of the fermentation. When the pH dropped below 5.3, however, the S. xylosus population started to decline after a small initial increase, fell back to 3.10 ± 0.14 log cfu/g after 2 days of fermentation, and subsequently decreased below the detection limit. For both acidification profiles, S. carnosus thus predominated as the major fraction within the CNS population (Fig. 1b,d).
2.7. Classification and identification of bacterial isolates through (GTG)5PCR fingerprinting of genomic DNA Genomic DNA extraction from cell pellets obtained by microcentrifugation at 13,000 rpm of 1.5 ml of an overnight LAB and CNS culture was performed with a Nucleospin 96 tissue kit (MachereyNagel, Düren, Germany), according to the manufacturer's instructions. Prior to extraction, the cell pellets were washed with Tris-ethylene diaminotetraacetic acid (EDTA)-sucrose buffer [TES buffer; 50 mM Tris base (Calbiochem, Darmstadt, Germany), 1 mM EDTA (Sigma-Aldrich), and 6.7% (m/v) sucrose (VWR International), pH 8.0]. Subsequently, (GTG)5-PCR fingerprints of the genomic DNA were obtained, followed by image analysis, as described previously (Braem et al., 2011). Numerical analysis of the fingerprints obtained was performed with BioNumerics 5.1 software (Applied Maths, Sint-Martens-Latem, Belgium). Confirmation of the species identity assigned to each LAB and CNS cluster was done by sequencing of the 16S rRNA and rpoB gene of representative isolates, respectively, as described previously (Braem et al., 2011). 3. Results
2.6. Enumeration and isolation of microorganisms For the liquid meat fermentation models, LAB and CNS counts were determined by plating the appropriate sample dilutions on MRS agar and MSA (VWR International), respectively. Differentiation of the CNS species in either S. carnosus or S. xylosus populations was done based on differences in colony shape, the former giving smooth and the latter serrated colony types [which was validated by (GTG)5-PCR fingerprinting of genomic DNA, see below]. For the meat systems, either 12 g of the mince-based meat fermentation model samples or 25 g of the pilot-scale sausage production samples were aseptically transferred into a stomacher bag (Seward, Worthing, West Sussex, UK) together with 108 ml or 225 ml of maximum recovery diluents [sterile solution of 0.85% (m/v) NaCl (VWR International) and 0.1% (m/v) bacteriological peptone (Oxoid)], respectively. This mixture was homogenised at high speed for 2 min in a stomacher (Stomacher 400; Seward). Appropriate decimal dilutions in saline were prepared and spread on MRS agar and MSA media. The agar media were incubated at 30 °C for 48–72 h and counts were determined from agar media containing 30–300 colonies. Next, 10–30% of the colonies were randomly picked up to analyse the LAB and CNS communities. The MSA- and MRS agar media-derived colonies were transferred into BHI and MRS media, respectively, and incubated overnight at 30 °C. The cultures obtained were stored at −80 °C in cryovials
3.2. Mince-based meat fermentation models As for the above-mentioned liquid meat fermentation model experiments, two different acidification profiles were imposed during the mince-based meat fermentation model systems (Fig. 2). In the experimental variant with the mild acidification profile, the pH dropped to around 5.3 after 2 days of fermentation and then remained constant. In parallel, the LAB population increased from 6.19 ± 0.11 to 8.81 ± 0.15 log cfu/g after day 2 and then remained stable during the next 5 days of fermentation. The MSA counts increased from 5.38 ± 0.20 log cfu/g after inoculation to their maximum of 7.46 ± 0.22 log cfu/g after 2 days of fermentation. Afterwards, they declined to 6.59 ± 0.13 log cfu/g. During the strong acidification setup, the pH dropped from 5.80 ± 0.08 to 4.67 ± 0.12 after 4 days of 3
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Fig. 1. Counts of Lactobacillus sakei CTC 494 (grey squares), Staphylococcus carnosus 833 (white diamonds), and S. xylosus 3PA6 (black diamonds), as well as the pH profiles (grey circles) of fermentations in liquid meat simulation medium (MSM) with a mild (a, b) or strong (c, d) acidification profile. Percentages of counts of S. carnosus 833 (white bars) and S. xylosus 3PA6 (black bars) were determined based on colony morphology.
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fermentation and remained constant till day 7. The LAB counts increased from 5.89 ± 0.10 to 9.14 ± 0.13 log cfu/g after 3 days of fermentation and then stabilized around this value. The initial level of CNS at 5.92 ± 0.23 log cfu/g increased to 7.24 ± 0.11 log cfu/g after 1 day of fermentation and then slowly decreased to 6.29 ± 0.09 log cfu/g after 7 days. With respect to the species diversity of CNS in the meat fermentation models, only S. carnosus and S. xylosus were isolated, outperforming other CNS species from the background microbiota. As compared to the experiments in MSM, S. xylosus generally displayed an enhanced competitiveness (Fig. 2b,d). At the mild acidification profiles (Fig. 2b), S. xylosus even was the most prevalent CNS species till day 3, followed by a co-prevalence with S. carnosus after day 7. In contrast, when the pH dropped below 5.00 (Fig. 2d), the relative abundance of the S. xylosus isolates declined from 70% (day 2) to 25% (day 3). By the end of the experiment, S. carnosus was the only CNS species isolated.
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Fermented sausages were produced to investigate how S. carnosus behaved at different acidification levels and how its fitness was affected by the presence or absence of S. xylosus in conventional starter cultures for sausage fermentation (Fig. 3). The aw values were similar for all investigated batches, decreasing from an initial value of 0.965 to 0.936 after acidification and 0.897 at the end of maturation. To evaluate the competitiveness of S. carnosus at its most unfavourable conditions of high pH, a trial in slightly acidified sausages with no carbohydrates added was carried out during a conventional starter culture set-up with (Fig. 3a,b) or without (Fig. 3c,d) the addition of S. xylosus. In general, the pH showed a similar decrease for both experimental variants, decreasing from pH 5.74 to pH 5.51 after 2 days of fermentation, followed by an increase to pH 5.81–5.92 after 28 days. Also, the patterns of the MRS and MSA counts as well as the pH evolution were comparable for both variants. In all cases, whether or not S. xylosus was added as a co-starter culture, the conditions were
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Fig. 2. Counts of lactic acid bacteria (grey squares) and coagulase-negative staphylococci (black triangles), as well as the pH profiles (grey circles) and the species diversity of the staphylococcal communities of mince-based meat fermentation models inoculated with a starter culture of S. carnosus 833, S. xylosus 3PA6, and L. sakei CTC 494, with a mild (a, b) or strong (c, d) acidification profile. Species were identified as S. carnosus (white bars) and S. xylosus (black bars) by gene-sequencing.
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Fig. 3. Counts of lactic acid bacteria (grey squares) and coagulase-negative staphylococci (black triangles), as well as the pH profiles (grey circles) and the species diversity of the staphylococcal communities of fermented sausages produced on pilot-scale with (a, b) no carbohydrates added and inoculated with S. carnosus 833, S. xylosus 2S7–2, and L. sakei CTC 494; (c, d) no carbohydrates added and inoculated with S. carnosus 833 and L. sakei CTC 494; (e, f) glucose added (final concentration of 0.25%, m/m) and inoculated with S. carnosus 833 and L. sakei CTC 494; and (g, h) glucose added (final concentration of 0.75%, m/m) and inoculated with S. carnosus 833 and L. sakei CTC 494. Species were identified as S. carnosus (white bars), S. xylosus (black bars), S. equorum (light grey bars), and S. saprophyticus (dark grey bars) by gene-sequencing.
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Time (days) decreasing experimental control but increasing realism were adopted to investigate the competitive behaviour of S. carnosus in the presence or absence of S. xylosus and to verify how this was affected by acidification. A general finding was that acidification favoured S. carnosus over S. xylosus, although the latter CNS species could take over when the pH drop was small. These results agreed with previous findings that have demonstrated the prevalence of S. xylosus in mildly acidified fermented sausages (Blaiotta et al., 2004; Cocolin et al., 2001; Iacumin et al., 2006). The results of the three experimental designs differed with respect to the fitness of S. xylosus, which seemed more at ease in the meatbased fermentation model systems. The latter suggested that, in contrast to the liquid simulation approach, the effect of a solid meat matrix gave a selective advantage to S. xylosus, which is known to be welladapted to meat and animal skin environments (Leroy et al., 2017; Vanderhaeghen et al., 2015; Vermassen et al., 2014, 2016). This CNS species possesses the metabolic flexibility to adapt to various substrates and environmental niches (Dordet-Frisoni et al., 2007; Nagase et al., 2002; Shale et al., 2005) and may enhance its persistence by the formation of biofilms (Planchon et al., 2006). Staphylococcus carnosus is of major technological interest for meat fermentation, being safe to use and generally having good nitrate reductase activity for proper colour development (Sánchez Mainar et al., 2017). Yet, this CNS species is only rarely recovered from spontaneously fermented meats, which seems at odds with its common use as the main staphylococcal component within conventional meat starter
unfavourable for S. carnosus that was outcompeted quickly. In contrast, S. xylosus became the most prevalent CNS species by the end of the fermentation (day 2), whether or not added as a starter culture. In the latter case, Staphylococcus equorum was also present but in low numbers and only at day 28. In the acidified experimental variants, S. xylosus lost its competitive benefit, giving rise to prevalence by S. carnosus (Fig. 3e-h). When sausages were prepared with a low concentration of carbohydrates added (0.25%, m/m), the pH dropped to 5.25 ± 0.11 at day 4, whereas at the high level of carbohydrates added (0.75%, m/m), the pH dropped to pH 4.90 ± 0.05. During both experimental variants, the initial decrease in pH values was followed by a rise. The initial counts of presumable CNS started at around 6.41 ± 0.26 log cfu/g for both experimental variants, amounting to about 7.01 ± 0.31 log cfu/g after 4 days of fermentation. The LAB counts started at 6.44 ± 0.12 log cfu/ g, reaching their highest value of 7.92 ± 0.22 log cfu/g after 4 days of fermentation. At all time points investigated, S. carnosus dominated the fermentations, especially at the low pH scenario. Isolates belonging to S. xylosus and Staphylococcus saprophyticus species were found too, but only at the intermediate acidification profiles and in numbers lower than 25% (Fig. 3f).
4. Discussion During the present study, three different experimental designs of 5
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Acknowledgements
cultures, whether or not in combination with S. xylosus (Leroy et al., 2006). This apparently contradicts both the suggestion that the genome of S. carnosus provides excellent adaptability to the fermented meat matrix (Rosenstein et al., 2009) and its more pronounced durability in acidified environments compared to other CNS species, including S. xylosus (Ravyts et al., 2010). Acid tolerance may be ascribed to the finding that strains of this species usually possess several acid stresscoping strategies, such as arginine deiminase (Sánchez Mainar et al., 2014) and amino acid decarboxylation activities (Stavropoulou et al., 2015). The results obtained during the present study underlined that the performance of S. carnosus was enhanced in environments where acidification was pronounced and when other CNS species were outcompeted. In Northern-European fermented sausages, which are generally of a more acidic nature than Southern-European ones, S. carnosus starter cultures are therefore generally found to be robust, steering the meat fermentation process relatively easily (Janssens et al., 2012; Ravyts et al., 2010; Stahnke et al., 2002). The reason for the apparently paradoxal absence of S. carnosus in spontaneously fermented sausages remains unclear. It may be that S. carnosus is only occasionally encountered on raw meat (Janssens et al., 2012). As such, a very low initial presence would be insufficient to generate a meaningful population of S. carnosus during fermentation, despite the competitive features that characterize this CNS species. The reason for its scarceness in fresh meat may relate to a poor adaptation to animal skin compared to other CNS species, resulting in low contamination levels after slaughter (Vanderhaeghen et al., 2015). Alternatively, some of the specific ecological determinants within unfermented meat may not be supportive for its growth, including the relatively high pH (usually around 5.6–5.8). From the present study, it became clear that dominance of the CNS communities by S. carnosus is indeed not to be taken for granted in low-acidic environments, so that other CNS species may take over the lead even when S. carnosus is added as the sole CNS starter culture. In fermentations without carbohydrates added, the technological fitness of S. carnosus was so low that it was outperformed by strains of CNS species that were originating from the background of the meat, mostly belonging to S. xylosus and to a lesser degree S. equorum. In fermented sausages with a low concentration of carbohydrates added, S. saprophyticus emerged from the meat background besides S. xylosus, but only in low proportions. The latter three species are indeed among the CNS species that are most commonly found in spontaneously fermented sausages with a low to mild acidification profile (Drosinos et al., 2005; Fonseca et al., 2013; García-Varona et al., 2000; Greppi et al., 2015; Papamanoli et al., 2002; Pisacane et al., 2015). The findings of the present study also met the finding that S. xylosus is the dominant CNS species isolated from slightly fermented sausages, whereas S. carnosus has been found only in more acidic sausages (Aymerich et al., 2003). In conclusion, it seemed that the inclusion of both S. carnosus and S. xylosus in conventional meat starter cultures offers more technological flexibility than when only one of these CNS species would be used. This is particularly the case since different types of fermented sausage may rely on different degrees of acidification. The results obtained during the present study indeed suggested that conventional starter cultures may behave differently when applied in dissimilar technological set-ups or using different recipes, with possible repercussions on product quality. Further studies should therefore explore how conventional starter cultures behave during meat fermentation if they are supplemented with other species of CNS, as to evaluate the impact of starter culture innovation on CNS ecology and its technological implications. Although most of the differences in competitiveness of CNS in meat seem to be situated at species level, further in-depth exploration of strain variability will have to indicate to which degree the latter can also have some influence on the overall community patterns.
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