Animal Science Publications Animal Science
2-2018
Autochthonous facility-specific microbiota dominates washed- rind Austrian hard cheese surfaces and its production environment
Narciso M. Quijada University of Veterinary Medicine Vienna
Evelyne Mann University of Veterinary Medicine Vienna
Martin Wagner University of Veterinary Medicine Vienna
David Rodríguez-Lázaro Universidad de Burgos
Marta Hernández Instituto Tecnológico Agrario de Castilla y León
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Abstract Cheese ripening involves the succession of complex microbial communities that are responsible for the organoleptic properties of the final products. The food processing environment can act as a source of natural microbial inoculation, especially in traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an artisanal washed-rind hard cheese produced in the western part of Austria without the addition of external ripening cultures. Here, the composition of the bacterial communities present on VB rinds and on different processing surfaces from two ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. VB production conditions (long ripening time, high salt concentration and low temperatures) favor the growth of psychro- and halotolerant bacteria. Several bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas dominated VB and were also found on most environmental surfaces. Analysis of OTUs shared between different surfaces suggests that VB rind bacteria are inoculated naturally during the ripening from the processing environment and that cheese surfaces exert selective pressure on these communities, as only those bacteria better adapted flourished on VB rinds. This study analyzed VB processing environment microbiota and its relationship with VB rinds for the first time, elucidating that the processing environment and the cheese microbiota should be considered as microbiologically linked ecosystems with the goal of better defining the events that take place during cheese maturation.
Keywords Cheese rind, bacteria, 16S rRNA gene cloning, Vorarlberger Bergkäse
Disciplines Bacteriology | Environmental Microbiology and Microbial Ecology | Food Processing | Genetics and Genomics
Comments This is a manuscript of an article published as Quijada, Narciso M., Evelyne Mann, Martin Wagner, David Rodríguez-Lázaro, Marta Hernández, and Stephan Schmitz-Esser. "Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment." International journal of food microbiology 267 (2018): 54-61. Posted with permission.
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Authors Narciso M. Quijada, Evelyne Mann, Martin Wagner, David Rodríguez-Lázaro, Marta Hernández, and Stephan Schmitz-Esser
This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/ans_pubs/525 *Manuscript with Line Numbers Click here to view linked References
1 Autochthonous facility-specific microbiota dominates washed-rind Austrian hard
2 cheese surfaces and its production environment
3
4
5 Narciso M. Quijada1,2, Evelyne Mann1, Martin Wagner1, David Rodríguez-Lázaro3,
6 Marta Hernández2, Stephan Schmitz-Esser1,4*
7
8 1 Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Vienna, Austria
9 2 Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de
10 Castilla y León, Valladolid, Spain
11 3 Division of Microbiology, Department of Biotechnology and Food Science,
12 Universidad de Burgos, Burgos, Spain
13 4 Current address: Department of Animal Science, Iowa State University, Ames, IA, USA
14
15 Keywords: Cheese rind, bacteria, 16S rRNA gene cloning, Vorarlberger Bergkäse
16
17 Corresponding author:
18 Stephan Schmitz-Esser, Department of Animal Science, Iowa State University, 3222
19 NSRIC, 1029 North University Boulevard, Ames, IA, 50011, USA, +1-515-294-2739,
1
21 Abstract
22 Cheese ripening involves the succession of complex microbial communities that are
23 responsible for the organoleptic properties of the final products. The food processing
24 environment can act as a source of natural microbial inoculation, especially in
25 traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an
26 artisanal washed-rind hard cheese produced in the western part of Austria without the
27 addition of external ripening cultures. Here, the composition of the bacterial
28 communities present on VB rinds and on different processing surfaces from two
29 ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning
30 and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study.
31 VB production conditions (long ripening time, high salt concentration and low
32 temperatures) favor the growth of psychro- and halotolerant bacteria. Several
33 bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas
34 dominated VB and were also found on most environmental surfaces. Analysis of OTUs
35 shared between different surfaces suggests that VB rind bacteria are inoculated
36 naturally during the ripening from the processing environment and that cheese
37 surfaces exert selective pressure on these communities, as only those bacteria better
38 adapted flourished on VB rinds. This study analyzed VB processing environment
39 microbiota and its relationship with VB rinds for the first time, elucidating that the
40 processing environment and the cheese microbiota should be considered as
41 microbiologically linked ecosystems with the goal of better defining the events that
42 take place during cheese maturation.
43
44
2
45 1. Introduction
46
47 Cheese is one of the oldest fermented foods. There is proven evidence for cheese
48 making in the sixth millennium B.C. and its manufacture is strongly related with
49 regionally different technical, social and economic conditions (Irlinger et al., 2015;
50 Salque et al., 2013;). Particularly in long-ripened raw milk cheeses, cheese
51 fermentation involves a complex and dynamic microbiome that establishes through
52 the ripening process and strongly influences the characteristics of the final products
53 including also food safety, as cheese microbiota can act as a natural barrier for
54 pathogens and spoilage microorganisms (reviewed by Boldyreva et al., 2016).
55 Cheese rinds microbiota differs largely from core cheese microbiota and can develop
56 either by direct inoculation of external ripening cultures and/or by natural colonization
57 of microbes from the processing environment, which is in continuous contact with
58 cheese rinds (Bokulich and Mills, 2012; Irlinger et al., 2015; Monnet et al., 2015). The
59 processing environment encompasses many surfaces such as equipment, brine tanks,
60 ripening rooms, vats, benches, clothes and human skin that can act as potential
61 sources of microorganisms (Kousta et al., 2009; Montel et al., 2014). The successful
62 colonization of these autochthonous microbial communities, summed up in the term
63 “house microbiota”, relies on their ability to cope with environmental conditions such
64 as low temperature, high salt content, humidity, pH, moisture control (brining,
65 pressing), cleaning procedures and competition against other microbes (Bokulich and
66 Mills, 2013; Mounier et al., 2006). Deacidification of the cheese surface by yeasts and
67 molds which consume lactate and produce ammonia as well as extensive washing of
68 cheeses with salt favors the establishment of acid-sensitive and salt- and psychro-
3
69 tolerant bacteria, such as coagulase-negative cocci (CNC, mainly Staphylococcus
70 equorum), Gram-positive coryneforms (Brevibacterium, Corynebacterium) and Gram-
71 negative bacteria such as Halomonas (Coton et al., 2012; Delbes et al., 2007; Gori et
72 al., 2013; Irlinger and Mounier, 2009; Mounier et al., 2005; Rea et al., 2007). Microbial
73 populations involved in cheese ripening are often also found in the processing
74 environment and it has been demonstrated that facility-specific “house” microbiota is
75 implicated in the development of brand-specific characteristics of many traditionally
76 manufactured cheeses (Bokulich and Mills, 2013; Dolci et al., 2009; Mounier et al.,
77 2006; Van Hoorde et al., 2010). Moreover, exploring the relationships between
78 ripening and production facility environment can be very useful to understand the
79 impact of the house microbiota on the hygienic safety of the processed cheese
80 products (Bokulich and Mills, 2013; Calasso et al., 2016; Mounier et al., 2006; Stellato
81 et al., 2015). In contrast to our current study, prior studies have mainly focused on
82 short-ripened soft or semi-soft cheeses. Washed-rind technology is used to
83 homogenously spread microorganisms on the cheese surface to favor implantation of
84 desirable bacteria, but is also sensitive to cross-contamination events due to the
85 magnitude of manipulative steps applied.
86 Austrian Vorarlberger Bergkäse (VB) is an artisanal hard cheese produced in the
87 western part of Austria (Vorarlberg) and has a protected designation of origin (PDO).
88 Highly similar types of cheeses are produced in the adjacent regions of Austria and
89 Germany. Its manufacture consists of traditional techniques including the use of raw
90 cow milk exclusively from alpine pastures with the addition of starter cultures. VB is
91 brined either in a brine bath or by dry salting surface treatment and no other
4
92 treatment, such as the addition of external ripening cultures, is applied during
93 ripening, which last from three to up to 18 months.
94 In a previous study, the bacterial and fungal composition of the rind microbiota of VB
95 was assessed by using 16S and 18S rRNA cloning and Sanger sequencing, revealing a
96 high diversity on VB cheese rinds with Gram-negative bacteria being particularly
97 abundant (Schornsteiner et al., 2014). The question remained vital how the surface of
98 the brined cheese is influenced by yet uncharacterized microbial communities
99 originating from food-contact and non-food-contact surfaces in the ripening cellar.
100 We aimed to characterize the microbial communities present in VB rinds and in
101 environmental surfaces including air filters, floors, racks, shelves and walls from two
102 different ripening cellars from the same cheese production facility to identify shared
103 bacteria between the different environments in this dairy plant.
104
105 2. Material and methods
106
107 2.1. Dairy plant, cheese production and sampling
108 For this study, two ripening cellars (A2 and A3) in an Austrian dairy plant have been
109 sampled in November 2012. Cellar A2 contains VB from 0 to 4 months ripening time
110 (short-ripening cellar), cellar A3 contains VB aged from 4 to 18 months (long-ripening
111 cellar). Temperature in short- and long-ripening cellars was 13.5 ºC and 10 ºC,
112 respectively. VB undergo different brining treatments in each cellar: daily treatment
113 with 20% NaCl solution for VB in the short ripening cellar and weekly treatment with
114 10% NaCl solution in the long ripening cellar. For more details regarding the ripening
115 cellars and the dairy plant, see Schornsteiner et al. (2014). Surface swab samples were
5
116 taken during ripening with sterile dry sponge sticks (3 M) from the wooden shelves,
117 racks, walls, floor and air filters, sampling an area of approximately one m2 for each
118 surface. With the exception of the air filters, within each ripening cellar, three different
119 areas (i.e. shelves, racks, walls and floor) were sampled at the same time. For the
120 cheese rinds, the surface of 25 to 30 cheese wheels was sampled with sterile scalpels
121 and pooled for DNA extraction.
122
123 2.2. DNA extraction, 16S rRNA gene amplification, cloning and sequencing
124 100 ml sterile 1 × PBS buffer was added to the sponges in a sterile bag and
125 homogenized for seven min in a lab blender (Stomacher 3500 Seward, UK).
126 Subsequently, the cells were pelleted at 11,000 × g for 20 min at 4 °C. The three
127 corresponding samples (i.e. from shelves, racks or walls) were then pooled and DNA
128 isolation was performed from 250 mg of the remaining pellet using the MoBio
129 PowerSoil Kit according to the manufacturer’s instructions. To investigate the bacterial
130 microbiota in the different environmental samples, 16S rRNA gene PCR was performed
131 using the primers 616F (Juretschko et al., 1998) and 1492R (Lane, 1991). Each PCR
132 reaction was performed in a final volume of 50 μL, containing 0.2 pM of each primer,
133 0.8 mM dNTP-mix (TaKaRa, Saint-Germain-en-Laye, France), 1×Ex Taq Buffer (TaKaRa),
134 0.025 U TaKaRa Ex Taq HS (TaKaRa), 5 μL DNA template and DEPC-treated water
135 (Thermo Scientific, Vienna, Austria). 16S rRNA gene PCR was performed under the
136 following conditions: initial denaturation at 95 °C for 5 min, 25 cycles at: 94 °C for 40 s,
137 at 52 °C for 40 s, and at 72 °C for 1 min and a final elongation at 72 °C for 7 min.
6
138 PCR amplicons were ligated into the pSC-A-amp/kan PCR cloning vector using the
139 StrataClone PCR Cloning Kit (Agilent Technologies, Vienna, Austria) and transformed
140 into competent cells following the manufacturer’s instructions.
141 One gene library was created for each environment (air filter, floor, shelves, racks and
142 wall; 96 clones per environment) and for cheese rinds (48 clones per rind) from both
143 cellars. In total, 1056 clones were sequenced using the vector-specific primers M13F
144 and M13R, yielding approximately 1500 bp high-quality 16S rRNA gene sequences per
145 clone. Sequencing was performed at LGC Genomics (Berlin, Germany). Fifty-four and
146 58 clone sequences from cheese rinds from short- and long-ripening cellars,
147 respectively, were taken from our previous study (Schornsteiner et al., 2014) and
148 included in the sequence analysis.
149
150 2.3. Sequence analysis
151 All sequences were analyzed using mothur (Schloss et al., 2009) with the following
152 parameters: minimum sequence length of 900 bp and the maximal number of
153 ambiguities set to five. Chimeric sequences were excluded with “chimera.uchime” and
154 “chimera.bellerophon”. After quality control and removal of chimeric sequences, 1059
155 sequences remained and were aligned to the SILVA SSURef 119 reference database
156 (Pruesse et al., 2007). Sequences were clustered into operational taxonomic units
157 (OTUs) using a distance limit of 0.01 (=99% similarity). This resulting OTU classification
158 was used for all further analyses on all taxonomic levels. Representative sequences of
159 OTUs with an abundance higher than 0.1% were double checked for correct taxonomic
160 assignment against type strains by using the RDP seqmatch tool (Cole et al., 2014).
161 Plotting was carried out in R environment (https://www.r-project.org/) by using the
7
162 enveomics.R, factoextra and vegan packages. Heatmaps were created using JColorGrid
163 (Joachimiak et al., 2006). The OTU table was formatted to a BIOM file using mothur
164 (“make.biom”) and used to create a bipartite graph (“make_otu_network.py”) by using
165 QIIME 1.9.1 software (Caporaso et al., 2010). OTU networks were visualized using
166 Cytoscape 3.3.0. (Shannon et al., 2003). Nodes represent either samples or bacterial
167 OTUs. Connections were drawn between OTUs and the samples they belong, with
168 edge weights defined as the number of sequences from each OTU that occurred in
169 each sample. The BIOM file was also used to calculate Weighted and Unweighted
170 Unifrac with QIIME 1.9.1 software and imported to R environment to perform Principal
171 Coordinates Analysis (PCoA). Venn diagrams were obtained by using Bioinformatics
172 and Evolutionary Genomics software (Shade and Handelsman, 2012).
173
174 2.4. Accession numbers
175 The 16S rRNA gene clone sequences have been submitted to GenBank and are
176 available under accession numbers MF091711 to MF092719.
177
178 3. Results
179
180 3.1. Analysis of 16S rRNA gene clone libraries
181 After quality control, 1059 sequences with an average length of 1435 bp remained.
182 Clustering the sequences at 99% identity revealed 236 OTUs. Rarefaction curves
183 (Figure S1) and Good’s coverage estimator (Table S1) suggest that the number of
184 clones sequenced were sufficient to cover the microbial diversity of the environments
185 studied. Shannon and Chao1 diversity indices showed higher diversity in
8
186 environmental surfaces from the short-ripening cellar (A2) than from the long-ripening
187 cellar (A3) overall, while long-ripened VB rinds were more diverse than short-ripened
188 VB rinds (Table S1).
189
190 3.2. Analysis of the microbial community composition of the cellars and cheese rinds
191 Overall, four different phyla (Actinobacteria, Bacteroidetes, Firmicutes and
192 Proteobacteria) were identified. Proteobacteria was the most abundant overall and
193 dominated most cellar surface environments (Figure 1A). Actinobacteria was the most
194 abundant phylum on cheese rinds from both cellars.
195 The relative abundance of the 12 most abundant genera is shown in Figure 1B. The
196 most abundant genera were Halomonas, Brevibacterium and Staphylococcus (29.1%,
197 10.1% and 7.8% of all clones, respectively). Halomonas was the dominant genus in
198 most samples and was present in all environmental and VB rinds samples except for
199 the shelves of the long ripening cellar. Other Proteobacteria such as
200 Pseudoalteromonas, Pseudomonas and Psychrobacter were present on environmental
201 surfaces but not in VB rinds. Brevibacterium was the most abundant actinobacterial
202 genus, and dominated VB rinds from both cellars (37.2% and 23.8% in the short- and
203 long-ripening cellar, respectively) and shelves from the short-ripening cellar (22.2%).
204 Brevibacterium was present in all the other environments except for the floor from the
205 short-ripening cellar. Corynebacterium and Leucobacter were present in cheese rinds
206 from both cellars and also in other environments. Firmicutes was mainly represented
207 by Staphylococcus, which was present in all samples except shelves and racks from the
208 long-ripening cellar. Psychroflexus was the most abundant representative of
209 Bacteroidetes and was found in some environmental surfaces but not in VB rinds.
9
210 Figure 1B evidenced that, despite the different rind washing conditions that are
211 applied to VB in each cellar, the microbial composition of VB rinds is similar.
212 Taxonomic assignments of the 50 most abundant OTUs are shown in Table S2 and the
213 relative abundance of the 25 most abundant OTUs within each environment is
214 represented in Figure 2. OTU1 was the most abundant OTU and most similar to
215 Halomonas meridiana (97.5% similarity) and was present in environmental surfaces
216 from both cellars but not in VB rinds. Interestingly, Halomonas boliviensis (OTU10,
217 98.2% similarity) was the most abundant Halomonas representative in VB rinds and
218 especially abundant in the short-ripened ones, where it was the second most abundant
219 OTU (24.4% relative abundance). Apart from VB rinds, it was only found in low
220 abundance in the racks and the air filter from the short-ripening cellar. Staphylococcus
221 equorum (OTU2) and Brevibacterium linens (OTU3) were present in VB rinds and
222 environmental surfaces from both cellars. Almost all of the most abundant OTUs were
223 present in both cellars. Some of those OTUs were present in both cheese rinds and
224 were also shared with other surfaces of their cellar: e.g. Staphylococcus equorum
225 (OTU2), Brevibacterium linens (OTU3) and Halomonas boliviensis (OTU10). Other OTUs,
226 such as Halomonas meridiana (OTU1), Pseudoalteromonas haloplanktis (OTU4),
227 Halomonas alkaliantarctica (OTU6), Halomonas variabilis (OTU7) and Psychrobacter
228 faecalis (OTU12) were present only on environmental surfaces.
229
230 3.3. Analysis of OTUs shared between environments reveals differences between
231 “cheese” and “house” microbiota
232 Comparison of the OTUs shared between samples revealed that the highest overlap
233 was found when comparing VB rinds from both cellars (Figure S2). 14 OTUs,
10
234 representing 84.2% of all cheese rind clones, were shared between VB rinds from both
235 cellars. Environmental surfaces of short- and long-ripening cellars shared 33 OTUs
236 (62.2% of all environmental surface clones). These values were higher compared to the
237 number of OTUs shared between VB rinds and surfaces from the same cellar.
238 Weighted and Unweighted distances were calculated to compare samples based on
239 the relative abundance or presence/absence of OTUs, respectively, and represented as
240 principal coordinate analysis (PCoA, Figure 3). Differences between cheese rind and
241 environmental surface microbiota can be seen, as cheese rinds clustered together and
242 separated from the environmental surfaces except of the shelves from the short-
243 ripening cellar.
244 Differences are clearly visible between cheese rinds and surface-related samples as an
245 OTU-network in Figure 4. S. equorum (OTU2), the OTU that was the most represented
246 between environments, appears in the middle of the network. B. linens (OTU3), was
247 shared by many environments but was mostly abundant in cheese rinds and so it
248 appears closer to these samples, whereas H. meridiana appears closer to
249 environmental surfaces and separated from cheese rind samples.
250
251 4. Discussion
252
253 The cheese producing environment is inhabited by autochthonous microbiota that play
254 a key role in cheese ripening and several studies have been performed to elucidate the
255 relationships between cheese and “house” microbiota. Here, we applied near full-
256 length 16S rRNA gene cloning and sequencing to provide much higher taxonomic
257 resolution than in studies using short-read next generation sequencing technologies or
11
258 other typing techniques such as DGGE, TGGE or RFLP. In addition, we applied stringent
259 OTU clustering based on 99% 16S rRNA gene similarity (most studies use 97%),
260 allowing a more specific (species-level) tracking of shared OTUs between samples.
261 Low temperature of the ripening cellars and washing of VB wheels with saline
262 generates high salt concentrations that favor the growth of psychro- and halotolerant
263 bacteria such as Halomonas, Oceanisphaera, Pseudoalteromonas and Psychrobacter
264 that are usually found in marine environments (Irlinger et al., 2015; Monnet et al.,
265 2015). Bokulich and Mills (2013) found these bacterial taxa in milk-handling surfaces
266 and suggested that they were part of the style-specific microbiome enriched by the
267 washed-rind processing technology. Halomonas has been previously detected in
268 washed-rind cheeses, especially in short-ripened cheeses, where it seems to develop
269 important functions during ripening (Coton et al., 2012; Dugat-Bony, 2016; Mounier et
270 al., 2005; Mounier et al., 2009; Quigley et al., 2012; Schornsteiner et al., 2014; Wolfe et
271 al., 2014;). Accordingly, we found Halomonas in higher abundance in short-ripened VB
272 rinds than long-ripened ones. Halomonas was the most abundant genus in our study
273 and dominated most environmental surfaces suggesting that it is well adapted to VB
274 ripening conditions. Nevertheless, 99% similarity-OTU level analysis indicated that not
275 all Halomonas OTUs seem to colonize both VB rind and environmental surfaces.
276 Interestingly, the most abundant OTU overall, H. meridiana (OTU1), was found in most
277 environmental samples and not in VB rinds, where H. boliviensis (OTU10, 98.2%
278 identity) was the most abundant Halomonas OTU. The H. boliviensis-like OTU10
279 showed 99.7% 16S rRNA gene-sequence similarity to the most abundant OTU found on
280 VB rinds in our previous study, also identified as H. boliviensis. These two OTUs thus
12
281 most likely represent the same Halomonas boliviensis strains that might be particularly
282 well adapted to VB rinds.
283 In our study, Oceanisphaera, Pseudoalteromonas, Pseudomonas and Psychrobacter
284 were found mainly in environmental samples. These genera have been described
285 previously in cheese-processing environments and European washed-rind cheeses,
286 where they might play a role during ripening, particularly Psychrobacter and
287 Pseudoalteromonas (Bokulich and Mills, 2013; Calasso et al., 2016; Irlinger et al., 2015;
288 Quigley et al., 2012; Stellato et al., 2015;Wolfe et al., 2014).
289 Coryneforms such as Brevibacterium, Brachybacterium, and Corynebacterium
290 dominated VB rinds from both cellars. Coryneforms are important in cheese ripening
291 due to their proteolytic activity and the production of volatile compounds and
292 ammonia (Eliskases-Lechner and Ginzinger, 1995; Rattray and Fox, 1999; Schröder,
293 2011). They have been found to dominate cheese rinds (Delcenserie et al., 2014;
294 Dugat-Bony, 2016; Gori et al., 2013; Mounier et al., 2005; Rea et al., 2007; Quigley et
295 al., 2012) and washed-rind cheese ripening room surfaces (Bokulich and Mills, 2013).
296 Brevibacterium was the most abundant coryneform genus found in this study and its
297 most abundant OTU was assigned to B. linens (OTU3). B. linens is important in flavor
298 and carotenoid production, the latter being responsible for the red pigmentation of
299 red-smear cheeses (Bockelmann, 2010; Eliskases-Lechner and Ginzinger, 1995; Rattray
300 and Fox, 1999). B. linens was more abundant in short-ripened VB rinds than in long-
301 ripened ones. The reduction in abundance of B. linens associated with longer ripening
302 periods has been previously reported and explained by the lack of growth nutrients
303 produced by yeasts in the beginning of ripening and insufficient competitiveness
13
304 against other surface colonizers (Feurer et al., 2004; Goerges et al., 2008; Gori et al.,
305 2013; Mounier et al., 2005).
306 Firmicutes was mainly represented by Staphylococcus equorum (OTU2), which was
307 present in both VB rinds but was most abundant in long-ripened ones, confirming
308 previous studies that associated S. equorum with long-ripened cheeses (Gori et al;
309 2013; Irlinger et al., 1997). S. equorum produces extracellular proteolytic and lipolytic
310 enzymes that are responsible for color and aromatic characteristics of cheeses and
311 antimicrobial substances that can help its competition against other microorganisms
312 (Carnio et al., 2000; Kastman et al., 2016). It is one of the most frequently identified
313 species in washed, natural and mold rind cheeses (Delcenserie et al., 2014; Irlinger et
314 al., 2015; Quigley et al., 2012; Wolfe et al., 2014) and also part of the “house”
315 microbiota from washed-rind cheese making plants (Bokulich and Mills; 2013; Mounier
316 et al., 2006). A recent study showed that S. equorum is stimulated by fungi of the
317 genus Scopulariopsis (Kastman et al., 2016). Interestingly, we found Scopulariposis to
318 be the most abundant fungal phylotype on VB cheese rinds in our previous study
319 (Schornsteiner et al., 2014), which might explain the high abundance of S. equorum
320 particularly on VB rinds.
321 Sphingobacterium and Psychroflexus were the main representatives of the phylum
322 Bacteroidetes. Sphingobacterium was found mainly on long-ripened VB rinds and has
323 been associated with raw milk microbiota and cheese rinds, although its functions in
324 cheese ripening are still unknown (Delcenserie et al., 2014; Dugat-Bony et al., 2016;
325 Hantsis-Zacharov and Halpern, 2007; Quigley et al., 2013; Schmidt et al., 2012; Wolfe
326 et al., 2014;). Psychroflexus was found in environmental surfaces in this study and on
14
327 cheese rinds in other recent studies (Delcenserie et al., 2014, Dugat-Bony et al., 2016;
328 Seiler et al., 2012).
329 No external surface ripening cultures are added during VB production, supporting the
330 formation of an indigenous, highly stable, “house” microbiota on VB rinds. Cheese
331 production conditions including ripening time, salt concentration and temperature
332 strongly influence the microbial communities and select for those microbiota that are
333 better adapted to growth under its specific conditions. The environmental conditions
334 of VB production seem to favor the growth of certain bacterial genera, such as
335 Brevibacterium and Halomonas.
336 The origin of the “house” microbiota is still unclear. Milk can act as a source of
337 inoculation as several cheese-related microorganisms such as Corynebacterium and
338 Staphylococcus can be part of the raw milk microbiota but also of the human skin
339 microbiota, which can act as another source of inoculation (Kable et al., 2016; Mounier
340 et al., 2006; Quigley et al., 2013).
341 Microbial composition analysis evidenced complex microbiome relationships in the VB
342 ripening environment. VB rinds represent a selective environment, as only certain
343 bacterial groups such as Brevibacterium, S. equorum, H. boliviensis, Corynebacterium,
344 Leucobacter and Sphingobacterium seem to be able to flourish, although their
345 abundance in short- and long-ripened VB varied. Nevertheless, the changes in VB rind
346 microbiota are less pronounced compared with the environmental surfaces.
347 It should be noted that also population densities in the different environments may
348 have a big impact on the transfer of microorganisms between different environments.
349 It is conceivable that e.g. the population density of the shelf microbiota is higher and
350 therefore its contribution to cheese microbiota may be more significant that the
15
351 contribution of microorganisms from the walls or floors. As we performed a cultivation
352 independent analyses, no CFUs were determined that could provide more insights into
353 the population densities in different ripening cellar environments. Similarities between
354 shelves and short-ripened VB rinds microbiota suggest transfer of microorganisms
355 between both surfaces in these production conditions. This transfer does not seem to
356 occur in the long-ripening cellar, as long-ripened VB rinds microbiota appeared
357 dispersed in the environmental surfaces of the cellar. Longer ripening times might
358 favor the establishment of certain communities adapted to survive under the
359 conditions of the different niches, which can act (or have acted) as source of indirect
360 inoculation.
361 Despite of the higher taxonomic resolution provided by near full-length 16S rRNA
362 genes, it is not possible to prove that the same OTUs represent the same strains. In
363 general, molecular biology based approaches such as those used in our study have
364 inherent well-known possible biases such as DNA extraction, primer binding biases,
365 and cloning bias which can influence the results of such studies. The absence of certain
366 phylotypes in some surfaces in our study might alternatively be explained by chance
367 only and these phylotypes might have been recovered if greater sequencing depth was
368 employed or these phylotypes may have been present initially and were outnumbered
369 by other bacteria. It should also be mentioned that additional sampling e.g. at more
370 time points to include more cheese batches would be needed to obtain more reliable
371 insights into the community composition on VB rinds and the ripening cellar
372 environment.
373 Further investigations using shotgun sequencing might be performed in order to better
374 understand the complex mechanisms involved in cheese rind and “house” microbiota
16
375 and its potential use as facility-specific surface ripening cultures. The relative
376 abundance and the metabolic activity of certain ripening cultures in Reblochon-style
377 cheese using metranstriptomics has been described recently, demonstrating the
378 strength of shotgun sequencing methods (Monnet et al., 2016)
379 The processing environment and cheese rind microbiota may be considered as a whole
380 with the goal of better defining the events that take place during cheese production.
381 Further investigations on the facility-specific house microbiota may allow selecting
382 facility-specific surface ripening cultures, well adapted to the specific production
383 conditions and responsible for the specific (regional) properties of artisanal cheeses.
384 This study shows for the first time the relationship between cheese rinds and facility-
385 specific microbiota from Austrian Vorarlberger Bergkäse and the importance of non-
386 inoculated autochthonous microbiota dominating VB rinds. Future studies such as
387 additional and deeper cheese rind and production environment community
388 composition surveys and genome sequencing of cheese rind bacteria or ripening
389 experiments will be needed to better understand the functional contributions of
390 specific cheese rind bacteria to cheese ripening and to complement community
391 composition surveys.
392
393 ACKNOWLEDGEMENTS
394 We would like to acknowledge Monika Dzieciol, Stefanie Wetzels, and Elisa
395 Schornsteiner for their help during sampling and processing of the samples.
396
397 Funding
17
398 N.M.Q. holds a Ph.D. studentship from the Spanish National Institute for Agriculture
399 and Food Research and Technology (INIA) co-financed by the European Social Fund
400 (fellowship FPI2014-020) and his work at the University of Veterinary Medicine Vienna
401 was financed by COST action STSM-FA1202.
402
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FIGURES
Figure 1. Relative abundance of the phyla (A) and the 12 most abundant genera (B) of bacteria found in cheese rind and cheese production environment.
Figure 2. Heatmap showing the relative abundance of the 25 most abundant OTUs within each cellar and the different surfaces sampled in each cellar.
Figure 3: Principal coordinate analysis (PCoA) of 16S rRNA gene OTUs based on
Weighted (A) and Unweighted (B) UniFrac distances. Dots are colored regarding the ripening cellar they belong to (black: short-ripening cellar, A2; white: long-ripening cellar, A3).
Figure 4. OTU sharing network. Light blue nodes represent the different OTU and the size of each node is proportional to their relative abundance. The diamond shaped nodes represent the samples and are colored regarding the cellar they belong: yellow for cellar the short-ripening cellar (A2), red for the long-ripening cellar (A3). OTUs and the samples they belong to are connected by colored lines corresponding to the type of sample. The 10 most abundant OTUs are also highlighted.
27
Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Supplementary material
Table S1. Species richness and diversity estimates for 16S rRNA gene libraries. All values were calculated with mothur at the 1% distance level.
Sample No. of No. of OTUs Chao1 Shannon Good´s coverage clones Rinds A2 78 23 32.43 2.44 0.85 Shelves A2 90 35 50.00 3.31 0.83 Racks A2 92 27 61.00 2.51 0.82 Wall A2 95 39 102.00 2.91 0.71 Air Filter A2 94 33 120.75 2.45 0.71 Floor A2 86 37 102.00 2.95 0.70 Rinds A3 80 26 41.60 2.87 0.84 Shelves A3 92 17 108.00 1.47 0.85 Racks A3 89 31 101.00 2.76 0.76 Wall A3 82 23 34.00 2.36 0.85 Air Filter A3 91 19 37.33 1.89 0.88 Floor A3 90 42 89.25 3.17 0.69
Table S2: Taxonomic assignment of the 50 most abundant OTUs from VB cheese rinds and production environment
OTU No. of Relative Identity Acces sion Best RDP type strain match clones abundance to best number (%) RDP type strain (%) 1 90 8.50 97.5 AJ306891 Halomonas meridiana DSM 5425 2 80 7.55 99.9 AB009939 Staphylococcus equorum ATCC 43958T 3 56 5.29 96.9 X77451 Brevibacterium linens DSM 20425 4 55 5.19 98.9 X67024 Pseudoalteromonas haloplanktis ATCC 14393 5 52 4.91 97.5 AJ430344 Comamonas aquatica LMG 2370 6 50 4.72 98.0 AJ564880 Halomonas alkaliantarctica CRSS 7 37 3.49 97.9 AJ306893 Halomonas variabilis DSM 3051 8 35 3.31 97.4 KF591598 Oceanisphaera profunda SM1222 9 28 2.64 99.1 FR714910 Psychroflexus halocasei WCC 4520 10 27 2.55 98.2 AY245449 Halomonas boliviensis LC1 11 23 2.17 99.8 AF074384 Pseudomonas gessardii CIP 105469 12 20 1.89 99.5 AJ421528 Psychrobacter faecalis Iso -46 13 16 1.51 98.3 JX152779 Brevibacterium jeotgali SJ5 -8 14 16 1.51 97.5 EF144148 Halomonas subterranea ZG16 15 16 1.51 99.2 AY228479 Yaniella halotolerans YIM 70085 16 15 1.42 98.4 KC876052 Brachybacterium ginsengisoli DCY80 17 15 1.42 95.9 GQ246672 Leucobacter denitrificans M1T8B10 18 15 1.42 97.9 AY243343 Brevibacterium permense VKM Ac -2280 19 15 1.42 98.7 AJ222815 Corynebacterium variabile DSM 20132 20 12 1.13 95.9 FN908501 Sphingobacterium lactis DSM 22361 21 11 1.04 97.6 AF212204 Halomonas sulfidaeris ATCC BAA -803 22 10 0.94 96.7 JF432053 Pseudomonas formosensis CC -CY503 23 9 0.85 99.9 AM040495 Providencia vermicola OP1 24 8 0.76 97.8 FJ196022 Marinobacter antarcticus ZS2 -30 25 8 0.76 97.4 DQ645593 Halomonas olivaria C17 26 8 0.76 97.5 AJ704395 Marinobacter maritimus CK47 27 8 0.76 98.4 AJ306891 Halomonas meridiana DSM 5425 28 7 0.66 97.2 FN908483 Pseudomonas litoralis 2SM5 29 6 0.57 99.3 Z93440 Acinetobacter johnsonii ATCC 17909T 30 6 0.57 99.5 JX986974 Alcaligenes aquatilis LMG 22996 31 5 0.47 97.7 AJ306888 Halomonas aquamarina DSM 30161 32 5 0.47 98.7 AB354933.1 Halomonas neptunia ATCC BAA -805 33 5 0.47 98.4 X81665 Acinetobacter lwoffii DSM 2403 34 5 0.47 97.2 AJ417388 Halomonas halocynthiae KMM 1376 35 5 0.47 98.3 AY243344 Brevibacterium antiquum VKM Ac -2118 36 5 0.47 98.5 AF288370 Idiomarina loihiensis L2TR 37 5 0.47 98.5 AF288370 Idiomarina loihiensis L2TR 38 4 0.38 99.0 X97887 Nocardiopsis listeri DSM 40297T 39 4 0.38 99.5 X91031 Brachybacterium alimentarium CNRZ 925 40 4 0.38 98.2 X81665 Acinetobacter lwoffii DSM 2403 41 4 0.38 95.3 JX986974 Alcaligenes aquatilis LMG 22996 42 4 0.38 99.7 AM040490 Providencia heimbachae DSM 3591 43 4 0.38 96.0 X80745 Arthrobacter protophormiae DSM 20168 44 4 0.38 99.0 AF267152 Corynebacterium casei LMG S -19264 45 4 0.38 96.2 AY962237 Halomonas kenyensis AIR -2 46 4 0.38 94.4 AJ306894 Halomonas venusta DSM 4743 47 3 0.28 95.8 AF445248 Atopostipes suicloacalis PPC79 48 3 0.28 98.8 AY842259 Psychrobacter celer SW -238 49 3 0.28 90.0 EF421176 Halomonas xianhensis A-1 50 3 0.28 98.9 EF114311 Bacillus isronensis B3W22
Figure S1. Rarefaction curves for 16S rRNA gene libraries (0.01 distance) of the different environments in short- and long-ripening cellars (A2 and A3, respectively).
Figure S2. Venn diagram showing the number of shared OTUs between VB rinds and VB processing environment samples.