Relationships among house, rind and core microbiotas during manufacture of traditional Italian cheeses at the same dairy plant

Food Microbiology 54 (2016) 115e126

Contents lists available at ScienceDirect

Food Microbiology journal homepage: www.elsevier.com/locate/fm

Relationships among house, rind and core microbiotas during manufacture of traditional Italian cheeses at the same dairy plant Maria Calasso a, Danilo Ercolini b, Leonardo Mancini a, Giuseppina Stellato b, Fabio Minervini a, Raffaella Di Cagno a, Maria De Angelis a, *, Marco Gobbetti a a b

Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy Department of Agricultural Sciences, Division of Microbiology, University of Naples Federico II, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2015 Received in revised form 5 October 2015 Accepted 12 October 2015 Available online 22 October 2015

This study was aimed at establishing the relationships between house, rind and core microbiotas of cheese varieties manufactured at the same industrial dairy plant. Caciotta and Caciocavallo Pugliese cheeses were chosen as model systems. Mesophilic lactobacilli, cocci and, especially, thermophilic cocci were the most abundant cultivable bacteria found on equipment, which were located in the production area. According to cell counts, catabolic profiles of microbial communities deriving from equipment, and cheese core and rind differed. As shown by 16S rRNA targeted metagenomics, Streptococcus thermophilus dominated the communities from knife surface, brine tank, curds and core cheeses as well as it was the main colonizing bacterium from drain table, rinds and ripening room of Caciocavallo Pugliese cheeses. Compared to S. thermophilus, the other starters used (Lactococcus lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus) showed low relative abundance in cheeses and/or colonization capability. A set of other genera/species, which varied depending on the equipment surfaces and cheese making, contributed to the formation of a rather heterogeneous house microbiota. Representatives from such communities had (e.g., Lactobacillus casei, Lactobacillus plantarum group) or not (e.g., Actinobacteria) the capacity to colonize cheeses, which depended on the variety (Caciocavallo Pugliese or Caciotta cheese) and layer (rind or core). Other genera/species were mainly associated to the rind and ripening room of Caciotta (Staphylococcus species and Brochothrix spp.) or Caciocavallo Pugliese (Chromohalobacter and Sphingomonas) cheeses. © 2015 Elsevier Ltd. All rights reserved.

Keywords: House microbiota Rind and core cheese microbiotas 16S rRNA targeted metagenomics Streptococcus thermophilus Caciocavallo Pugliese Caciotta

1. Introduction Cheeses and related dairy environments are colonized by complex microbial communities, the latter of which being in several cases insufficiently investigated (Almeida et al., 2014). Dairy environment comprises equipment, brine tanks and ripening rooms, which harbor and act as vectors of microbes deriving from raw milk, starters, water, air, salt and human skin (Montel et al., 2014). Such resident microbes, defined as the house microbiota, unavoidably interact with the microbial populations from raw milk, primary, secondary and/or adjunct starter cultures during cheese manufacture (Bokulich and Mills, 2013; Somers et al., 2001; Su
arez et al., 1992). Raw milk acts as a direct source of microbes (e.g. non-

* Corresponding author. Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, via Amendola 165/a, 70126 Bari, Italy. E-mail address: [email protected] (M. De Angelis). http://dx.doi.org/10.1016/j.fm.2015.10.008 0740-0020/© 2015 Elsevier Ltd. All rights reserved.

starter lactic acid bacteria) mainly in cheeses made without milk pasteurization and as an indirect source by enriching the microbiota of equipment for making cheeses with thermally treated milk (Montel et al., 2014; Somers et al., 2001). Usually, dairy environment is rich of bacteria mainly belonging to Firmicutes, Actinobacteria and Proteobacteria phyla, yeasts and molds (Bokulich and Mills, 2013; Montel et al., 2014; Stellato et al., 2015). Bacterial species contaminating equipment (e.g., vat milk and tank used for milk coagulation) could grow, survive and also dominate in cheese during ripening. The wooden surface of the vats used for the manufacture of several PDO cheeses represents an important source of living microorganisms, which may play a key role in flavour formation during cheese ripening (Licitra et al., 2007; Lortal et al., 2009). The composition of biofilms associated with wooden vats used for making French cheese varieties revealed the presence of several dairy lactic acid bacteria, including starters, such as Streptococcus thermophilus, Lactobacillus helveticus and Lactococcus

116

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

lactis, and non-starter lactic acid bacteria, such as Lactobacillus plantarum and Lactobacillus casei (Licitra et al., 2007; Didienne et al., 2012). The capacity of strains from the house microbiota or used as starters to transfer and colonize equipment or other cheeses manufactured in the same dairy plant depends on various factors, which include environmental conditions, microbial metabolic potential and interactions with other microorganisms (Montel et al., 2014; Stellato et al., 2015). Environmental and technological factors (e.g., chemical and microbiological composition of milk, pasteurization, type of coagulation, stretching, shaping, salting, temperature and time of ripening) could affect the structure of the house microbiota which, in turn, influences the composition of the cheese microbiota (Callon et al., 2011; Irlinger and Mounier, 2009). Within the same cheese variety, there is a complex and poorly understood network of interactions between biotic (e.g., microbial interactions) and abiotic (e.g., pH, aw, redox potential and chemical composition) factors, which determine continuous changes in the microbial balance during cheese ripening (Montel et al., 2014). Recently, high-throughput sequencing was used to investigate the house microbiota of artisanal cheeses (Bokulich and Mills, 2013) and the microbial composition of the rinds of an Austrian hard cheese (Schornsteiner et al., 2014). Compared to cheese core, the rind shows largely different environmental parameters, which affect the microbial communities and the biochemical features (Almena-Aliste and Mietton, 2014). The rind microbiota plays a key role on the sensory characteristics of cheese and protects against pathogens and spoilage microorganisms (Irlinger and Mounier, 2009). Studies that aim at establishing the relationships between house and cheese microbiotas may represent a novel approach to study the interactive microbial drivers, which are responsible for the nutritional, safety and sensory features of artisanal and industrial cheeses. In this study, two traditional Italian cheese varieties, Caciotta and Caciocavallo Pugliese, were made at the same dairy industry since long time ago and chosen as model systems. Caciotta cheese is mostly made from raw or pasteurized cow's milk or blends of ewe's and cow's milk, using thermophilic lactic acid bacteria starters (mainly S. thermophilus and Lactobacillus delbrueckii subsp. lactis) and dry or brine salting (Aquilanti et al., 2011; Di Cagno et al., 2011). Caciocavallo Pugliese is a “pasta-filata” cheese manufactured from raw cow's milk using natural whey or commercial thermophilic (mainly S. thermophilus and L. helveticus) and mesophilic (e.g., Lc. lactis) cultures (De Angelis and Gobbetti, 2011; Kindstedt et al., 2004). This study was aimed at establishing, through culturedependent and -independent approaches, the relationships between house, rind and core microbiotas of Caciotta and Caciocavallo Pugliese cheeses, which were manufactured at the same industrial dairy plant. 2. Materials and methods 2.1. Industrial dairy plant Sampling (house and cheese microbiotas) was carried out at the industrial dairy plant Ignalat srl (Noci, Bari), which is located in Southern Italy. Caciotta and Caciocavallo Pugliese were manufactured at this dairy plant by more than ten years and were chosen as model cheeses. According to the International legislation (Anonymous, 2004; EFSA, 2008; FDA, 2007), the dairy industry only uses stainless steel equipment. Three daily cheese making batches were considered for each variety of cheese. The same milk for each daily batch was used for the manufacture of both cheese varieties. Caciotta and Caciocavallo Pugliese cheeses were manufactured in the same curd tank after cleaning. Both cheese varieties were salted in the same brine tank. The plan of the dairy industry and

information on surfaces sampled are shown in Fig. S1. 2.2. Cheese manufacture Cow's milk used had the following characteristics: lactose 4.9%, protein 3.3%, fat 3.6%, and pH 6.6. For making Caciotta cheese, milk was pasteurized (71
C for 15 s), cooled at 37
C and inoculated with commercial (Sacco, Cadorago, Como, Italy) S. thermophilus and L. delbrueckii subsp. lactis starters (initial cell density for each species of approximately 7.5 ± 0.11 and 7.3 ± 0.14 log CFU/mL, respectively). The initial cell density of each starter was determined by plate count using M17 agar (S. thermophilus) or MRS agar (Oxoid, Ltd., Basingstoke, UK) (L. delbrueckii subsp. lactis) under conditions of anaerobiosis at 42
C for 48 h. After incubation for 30 min at 37
C, liquid calf rennet (35 mL/100 L) was added and coagulation took place within 30 min. After whey drainage and molding, curds were stored for approximately 4 h at room temperature. Salting was carried out by immerging (5 h) cheeses in brine (37% NaCl, wt/ vol). After salting, cheeses were further stored at room temperature for 12 h. Ripening was at 9
C (relative humidity ca. 73%) for 50 days. The weight of the cheeses was approximately 1.5 kg. For making Caciocavallo Pugliese, raw cow's milk was heated at 37
C and inoculated with commercial (Sacco) Lc. lactis, S. thermophilus, and L. helveticus starters (initial cell density of ca. 7.5 ± 0.25, 7.4 ± 0.13 and 5.1 ± 0.09 log CFU/mL, respectively). The initial cell density of each starter was determined by plate count using M17 agar (Lc. lactis, S. thermophilus) or MRS agar (Oxoid) (L. helveticus) under conditions of anaerobiosis at 25
C (Lc. lactis) and 42
C (S. thermophilus and L. helveticus), respectively, for 48 h. After incubation for 30 min at 37
C, liquid calf rennet (35 mL/100 L) was added and coagulation took place within 30 min. First, the coagulum was coarsely (by hand) cut, then it was held under whey at 37
C for 2 h, and finally mechanically cut to get particles of 1.5e2.0 cm3. After the curd had reached the value of pH of 5.25 (ca. 5 h), it was stretched in hot water (80
C) and molded. Cheeses were salted (10 h) in brine, following the same procedure as that used for Caciotta cheese, and ripened (9
C, relative humidity of ca. 75%) for 60 days. The weight of the cheeses was approximately 1.5 kg. Both varieties of cheese were washed with water and salt (3e4 times) during ripening. Except for Lc. lactis, the dairy industry used the same starter formulas by more than five years. Lc. lactis was used for making Caciocavallo Pugliese cheese only starting from September 2014. 2.3. Sampling Swab samples were collected in the production area just before daily cheese making, and after disinfection and washing procedures from the previous working day. The wet swabbing technique was used on the surfaces of the following equipment/tools/facilities: tube for pasteurized milk, stainless steel tank used for the coagulation of milk, knife used for curd cutting, draining table and brining tank (Fig. S1). Plastic molds used for Caciotta cheese were also analyzed. Stretching and molding machines and cooling tank for Caciocavallo Pugliese cheese were also surface swabbed. In detail, sterile rayon tip swabs (Nuova Aptaca Srl, Canelli, Asti, Italy) were moistened with sterile saline (NaCl, 9 g/L) solution using aseptic technique to prevent cross-contamination and streaked across a 100 cm2 square area of the target surface (Lahou and Uyttendaele, 2014), making enough vertical S-strokes to cover the entire sampling area. The sampling area of 100 cm2 was delimited by a sterile paper square. Swabs were inserted into tubes containing 3.5 mL of Amies gel transport medium without charcoal, in order to protect microorganisms from adverse conditions until culture-

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

dependent analysis (within 2 h since sampled) (MacFaddin, 1985). Adjacent 100 cm2-wide areas were sampled with sterile rayon tip swabs moistened with RNAlater®. Swabs were inserted into tubes containing 3.0 mL of RNAlater®, transferred to laboratory in dry ice and stored at 80
C for extraction of total bacterial genomic DNA. For Caciotta cheese, raw and pasteurized milk, curd immediately after coagulation, and cheeses after 1 (post-brining) (C1), 10 (C10), 20 (C20), 30 (C30), 40 (C40) and 50 (C50) days of ripening were collected from each of the three batches. For Caciocavallo Pugliese cheese, raw milk, curd immediately after coagulation and stretching, and cheeses after 1 (CC1), 10 (CC10), 20 (CC20), 30 (CC30), 40 (CC40) and 50 (CC50) days of ripening were collected from each of the three batches. Wall (Wall), floor (Floor), racks for Caciotta (C Racks) and sticks for Caciocavallo Pugliese ripening (CC Sticks) were surface swabbed, as described above. All samples were transported to the laboratory under refrigerated condition (ca. 4
C). For each cheese sample, two swabs with total 200 cm2-wide rinds and 100 g of core cheese were sampled and analyzed immediately (microbiological analysis) or frozen (80
C) (extraction of total bacterial genomic DNA). All samples were collected from three batches of each variety of cheese in three different days, and analyzed in triplicate (total nine analyses for each variety of cheese). All the samples analyzed were arranged into three groups, named Production Area, Caciotta cheese and Caciocavallo Pugliese cheese group, respectively, as reported in the Fig. S1. 2.4. Microbiological analyses Microbial counts of presumptive mesophilic and thermophilic lactobacilli, mesophilic and termophilic cocci, micrococci and staphylococci, enterococci, total coliforms, yeasts, and molds were determined as described previously (Gobbetti et al., 1997). Presumptive mesophilic and thermophilic lactobacilli were enumerated in MRS agar, under conditions of anaerobiosis at 30
C and 42
C, respectively, for 48 h. Presumptive mesophilic and thermophilic cocci were enumerated in M17 agar under conditions of anaerobiosis at 30
C and 42
C, respectively, for 48 h. Micrococci and staphylococci and enterococci were counted in Baird Parker agar plus egg yolk tellurite and Slanetz-Bartley agar (Oxoid), respectively, at 37
C for 48 h. Total coliforms were counted using Violet red bile lactose (Oxoid) at 37
C for 24 h. Yeasts were enumerated in Sabouraud Dextrose agar (SDA) (Oxoid) supplemented with chloramphenicol (0.1 g/L), incubating the plates at 30
C for 48 h. Molds were counted in Wort agar (Oxoid), incubating the plates at 25
C for 120 h. Except for Slanetz-Bartley agar, the media for enumeration of bacteria were supplemented with cycloheximide at 0.17 g/L. Enumeration of microbial groups contaminating the surface of equipment/tools/facilities and rinds of the cheeses was carried out shaking the swabs in 10 mL of sterile saline solution. One or 0.1 mL of the suspension containing microbial cells was, directly or after serial dilution, pour- or spreadplated, respectively. Prior to microbiological analyses of milk, curd, core of cheese or brine, 20 g of sample were homogenized with 180 mL of a sterile sodium citrate (2% wt/vol) solution (De Pasquale et al., 2014a). 2.5. Community-level catabolic profiles (CLCP) Carbon source utilization patterns of the microbiota of the surface of equipment/tools/facilities, raw milk, curds, rind or core of the cheeses were assessed using Biolog 96-well Eco micro-plates (Biolog, Inc., Hayward, CA) (Crecchio et al., 2004). Micro-plates contained 31 different carbon sources (carbohydrates, carboxylic acids, polymers, amino acids, amines, and miscellaneous

117

substrates) in triplicate. The initial first dilution of cellular suspension, used for microbiological analyses, was diluted into sterile sodium chloride solution to reach 65% transmittance, as recommended by the manufacturer and, subsequently, dispensed (150 mL) into each of the 96 wells of the micro-plates. The microplates were incubated at 30
C in the dark, and the color development was measured at 590 nm every 24 h by a micro-plate reader (Biolog Microstation). Results were used for calculating three indices (Shannon, 1948a, 1948b; Zak et al., 1994). Shannon's diversity (H0 ), indicating the substrate utilization pattern, was calculated as follows: H0 ¼ S pi ln (pi), where pi is the ratio of the activity of a particular substrate to the sums of activities of all substrates at 120 h. Substrate richness (S), expressing the number of different substrates used, was calculated as the number of wells with a corrected absorbance greater than 0.25. Substrate evenness (E), defined as the equitability of activities across all utilized substrates, was calculated as follows: E ¼ H0 /log S. 2.6. Extraction of total bacterial DNA Ninety milliliters of sterile saline solution were added to 10 g of milk, curd or cheese (previously stored in RNAlater® at 80
C) and homogenized for 5 min. Homogenates were centrifuged (1000 g, 5 min, 4
C) and the supernatants were recovered and centrifuged (5000 g, 15 min, 4
C). The pellet was suspended in 0.5 mL of sterile saline solution and this suspension was used for extraction of total DNA. Cell suspensions in RNAlater® (3.0 mL) collected through surface swabs of equipment or rind of cheeses were centrifuged (16,000 g, 2 min, 4
C). Pellet was used for extraction of total DNA. Extraction of DNA was carried out by using the FastDNA™ e Spin Kit for Soil (MP Biomedicals, Solon, OH, USA), according to the manufacturer's instructions. The concentration and purity of extracted DNA were assessed by spectrophotometric determination (Nanodrop ND-1000, Thermo Fisher Scientific Inc.). 2.7. 16S rRNA gene amplicon library preparation and sequencing Microbial diversity was studied by pyrosequencing of the amplified V1eV3 region of the 16S rRNA gene. For each samples (milk, curd, cheese, brine or swab), DNA extracted from three batches was pooled and used for pyrosequencing analysis. A fragment of 520 bp was amplified by using primers and PCR conditions described previously (Ercolini et al., 2012). 454 adaptors were included in the forward primer, followed by a 10ebp samplespecific multiplex identifier (MID). After agarose gel electrophoresis was performed, PCR products were purified twice by using an Agencourt AMPure kit (Beckman Coulter, Milan, Italy) and quantified by using the QuantiFluor system (Promega, Milan, Italy). The amplicon was used for pyrosequencing on a GS Junior platform (454 Life Sciences, Roche, Italy) according to the manufacturer's instructions and using titanium chemistry. 2.8. Bioinformatics and data analysis Raw reads were first filtered according to the 454 processing pipeline. Sequences were then analyzed by using QIIME 1.7.0 software following a pipeline previously described (De Filippis et al., 2014). Sequences that passed the quality filter were denoised (Reeder and Knight, 2010), and singletons were excluded. Operational taxonomic units (OTU), defined by 97% similarity, were picked by using the uclust method (Edgar, 2010), and the representative sequences, chosen as the most abundant in each cluster, were submitted to the RDPII classifier (Wang et al., 2007) to obtain the taxonomy assignment and the relative abundance of each OTU by using the Greengenes 16 S rRNA gene database (McDonald et al.,

118

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

2012). Alpha (rarefaction, Good's coverage, Chao1 richness and Shannon diversity indices) and beta diversity measures were calculated and plotted using QIIME (De Filippis et al., 2014). 2.9. Statistical analyses All analyses were carried out in three replicates for three batches (total 9 analyses for each variety of cheese). Data were subjected to one-way ANOVA and pair-comparison of treatment means was achieved by Tukey's procedure at P < 0.05, using the statistical software Statistica for Windows (Statistica 6.0, 1998). 3. Results 3.1. Enumeration of cultivable bacteria, yeasts and molds Relatively selective media were used to enumerate cultivable microbes (Table 1). Cell densities from surfaces were correlated with the sample areas and expressed as CFU/cm2. Data from milk, curds, and cheese rind and core were reported as CFU/g. Before cheese making, the surfaces of equipment located in the production area were already and variously contaminated by presumptive mesophilic lactobacilli (<1 to 3.36 log CFU/cm2), thermophilic lactobacilli (ca. <1 to 2.57 log CFU/cm2), mesophilic cocci (<1 to 3.96 log CFU/cm2), thermophilic cocci (ca. 1.22e3.56 log CFU/ cm2), enterococci (<1 to 3.19 log CFU/cm2), micrococci and staphylococci (<1 to 1.81 log CFU/cm2), coliforms (0.55e1.18 log CFU/ cm2), yeasts (<1 to 2.69 log CFU/cm2) and molds (<1 to 2.04 log CFU/cm2). The level of presumptive mesophilic lactobacilli in raw milk was 4.61 ± 0.09 log CFU/g. A consistent decrease was found into pasteurized milk (1.04 ± 0.18 log CFU/g; P < 0.05) (Table 1). As expected, such a decrease was a common feature, which was found also for the other microbial groups. The highest value of mesophilic lactobacilli (6.60 ± 0.09 log CFU/g; P < 0.05) was observed in Caciocavallo Pugliese cheese curd, after the pH attained the value of 5.25. As expected, a decrease occurred after curd stretching. During ripening, mesophilic lactobacilli increased by ca. 1 and 2 log cycles in the core of Caciotta and Caciocavallo Pugliese cheeses, respectively. At rind level, the highest cell density (4.38 ± 0.05 log CFU/ cm2; P < 0.05) was observed after 30 days of Caciotta cheese ripening. The cell density of mesophilic lactobacilli in the rind of Caciocavallo Pugliese cheese decreased by almost 2 log cycles throughout ripening. Mesophilic lactobacilli contaminated ripening rooms, mainly the racks used for making Caciotta cheese. At day 1, the highest number (9.00 ± 0.12 log CFU/g; P < 0.05) of presumptive thermophilic lactobacilli was found in the core of Caciotta cheese. During ripening, thermophilic lactobacilli progressively decreased by ca. 2 log cycles. An opposite trend was observed in the core of Caciocavallo Pugliese cheese. The disappearance of thermophilic lactobacilli from the rind was observed after 0 and 40 days of Caciotta and Caciocavallo Pugliese cheese ripening, respectively. Presumptive mesophilic cocci were already present in raw milk (4.65 ± 0.18 log CFU/g) (Table 1). At day 1, the cell density was 4.97 ± 0.21 log CFU/g. A progressive decrease (ca. 2 log cycles) was observed during ripening. Compared to core, the rind of Caciotta cheese harbored a lower (P < 0.05) numbers of mesophilic cocci. Caciocavallo Pugliese cheese had the highest numbers of mesophilic cocci, which decreased (ca. 2 log cycles) both at core and rind levels during ripening. Low levels of mesophilic cocci were found in the ripening rooms of both the cheese varieties. Presumptive thermophilic cocci were present at 4.23 ± 0.11 log CFU/g in raw milk (Table 1). Curds contained the highest levels (>8.5 log CFU/g). At rind level, the highest cell density

(>5 log CFU/cm2) of thermophilic cocci was found before ripening (day 1). A decrease was observed throughout ripening, especially for Caciocavallo Pugliese cheese. Thermophilic cocci were the microbial group detected at the highest cell density (>3 log CFU/cm2) in ripening rooms of both cheese varieties. Presumptive enterococci were detected in raw (2.40 ± 0.08 log CFU/g) but not in pasteurized milk (Table 1). After 10 days of ripening, the value of 5.82 ± 0.17 log CFU/g was found in the core of Caciotta cheese. Subsequently, a decrease of ca. 1 log cycle was observed. The values for Caciocavallo Pugliese cheese were lower. Apart from the varieties, rind cheeses contained low cell density (<2.0 log CFU/cm2) of enterococci, which disappeared at the end of ripening. No enterococci were detectable in the ripening rooms. Presumptive micrococci and staphylococci were found in raw (2.30 ± 0.15 log CFU/g) but not in pasteurized milk (Table 1). At core level, they were detectable almost exclusively in Caciotta cheese. At 30 days, the value of 5.31 ± 0.23 log CFU/g was reached. Then, they decreased by ca. 2 log cycles. At rind level (mainly at 30 days), the numbers of micrococci and staphylococci of Caciotta cheese were higher than those found for Caciocavallo Pugliese cheese. These bacteria were also present in the ripening rooms of both cheese varieties. Presumptive coliforms were detected in raw (3.10 ± 0.16 log CFU/g) but not in pasteurized milk (Table 1). Such bacteria progressively disappeared during ripening. Ripening rooms of both cheese varieties were contaminated by coliforms. Yeasts and molds were detectable in raw milk but disappeared in the core and rind of Caciocavallo Pugliese cheese during ripening. Ripening rooms and, especially, racks for Caciotta cheese were contaminated by yeasts and molds. 3.2. Community-level catabolic profiles The microbial communities deriving from milk, dairy environment (production area and ripening rooms) and equipment, curds, and core and rind of Caciotta and Caciocavallo Pugliese cheeses showed different capacities to utilize various carbon sources (amino acids, carbohydrates, carboxylic acids, amines, and polymers) (Fig. 1). The substrate utilization pattern (H0 index) and substrate richness (S index) values were calculated (Table 2). Overall, polymers (tween 40 and tween 80), carbohydrates (Dcellobiose, alpha-D-lactose, beta-methyl-D-glucoside, D-xylose, Dmannitol, N-acetyl-D-glucosamine, glucose-1-phosphate), amino acids (e.g., L-serine, L-asparagine, L-phenylalanine) and carboxylic acids (D-glucosaminic acid, D-galactonic acid-g-lactone, D-galacturonic acid, 2-hydroxy benzoic acid, 4-hydroxy benzoic acid, ghydroxybutyric acid, a-ketobutyric acid, glycyl-L-glutamic acid, and D-malic acid) were the carbon sources most used. The microbial communities from curd tank and cheese rinds, especially at 20e30 days, showed the highest number of metabolized substrates. Compared to Caciotta cheese, the microbial community from Caciocavallo Pugliese was characterized by higher H0 and S indices. The microbial community from ripening room of Caciocavallo Pugliese cheese also showed wide metabolic capacity. Glycyl-Lglutamic acid, phenyl ethylamine and putrescine were mainly metabolized by the microbial communities of rind samples and ripening room of Caciocavallo Pugliese cheese. 3.3. Richness and diversity of house and cheese microbiomes based on 16S rRNA gene sequencing data analysis To more in depth investigate the relationships between house and cheese microbiota, and to highlight the role of the former in directing the cheese microbial communities, the pyrosequencing of

Table 1 Cell numbers (log CFU/g or log CFU/cm2) of microbial groups in the dairy environment (production area and ripening rooms) and equipment, raw and pasteurized cows' milk (R milk; P milk), curds, core (c) and rind (r) microbiota of Caciotta and Caciocavallo Pugliese cheeses. Both cheese varieties were analyzed after one (T1), ten (T10), twenty (T20), thirty (T30), forty (T40) and fifty (T50) days of ripening. Samples

Mesophilic lactobacilli

Thermophilic lactobacilli

Mesophilic cocci

Thermophilic cocci

Enterococci

Micrococci and staphylococci

Coliforms

Yeasts

Molds

<1 MN 2.57 ± 0.06 GH <1 MN. <1 MN <1 MN <1 MN 1.40 ± 0.03L <1 MN 1.71 ± 0.04I

<1O 3.96 ± 0.08F 2.78 ± 0.06H <1O 1.62 ± 0.03M 2.01 ± 0.05L 2.44 ± 0.05HI 3.06 ± 0.06 GH 2.35 ± 0.05I

1.31 ± 0.03N 3.56 ± 0.07H 2.55 ± 0.05L 1.76 ± 0.03 MN 2.05 ± 0.04M 1.23 ± 0.01N 3.03 ± 0.06I 1.22 ± 0.03N 1.95 ± 0.04M

<1L 3.19 ± 0.07E <1L <1L 1.06 ± 0.02I <1L 1.07 ± 0.02I <1L 1.41 ± 0.03G

<1M 1.60 ± 0.03H <1M <1M <1M <1M 1.81 ± 0.04 GH <1M 1.69 ± 0.04H

<1G <1G 0.55 ± 0.15G 1.05 ± 0.14F <1G <1G <1G <1G 1.18 ± 0.09F

<1L 2.69 ± 0.06E <1L <1L 2.22 ± 0.05F <1L 1.61 ± 0.03H <1L 1.40 ± 0.03I

<1H 1.88 ± 0.04E <1H <1H 1.40 ± 0.03F <1H 2.04 ± 0.05E 1.22 ± 0.02FG 1.95 ± 0.04E

3.90 ± 0.09F 1.10 ± 0.02M 8.20 ± 0.15AB 9.00 ± 0.12A 7.74 ± 0.24B 7.68 ± 0.18B 7.62 ± 0.13B 7.54 ± 0.15B 6.66 ± 0.07C 3.87 ± 0.12F 2.30 ± 0.06H <1 MN <1 MN <1 MN <1 MN <1 MN 2.92 ± 0.08G

4.65 ± 0.18 EF 1.13 ± 0.05 MN 3.24 ± 0.10G 4.97 ± 0.21E 4.34 ± 0.13 EF 3.84 ± 0.11F 3.28 ± 0.10G 3.17 ± 0.09G 3.01 ± 0.09 GH 3.88 ± 0.12F 2.79 ± 0.08H 2.17 ± 0.05IL 1.30 ± 0.02L <1O <1O <1O <1O

4.23 ± 0.11FG 1.00 ± 0.03O 9.01 ± 0.22B 9.10 ± 0.23AB 9.18 ± 0.16AB 9.72 ± 0.14A 9.69 ± 0.09A 9.22 ± 0.18AB 8.98 ± 0.17B 5.02 ± 0.15E 4.87 ± 0.11 EF 4.69 ± 0.13F 4.78 ± 0.04 EF 4.99 ± 0.14E 4.02 ± 0.07G 3.04 ± 0.08I 3.26 ± 0.09I

2.40 ± 0.08F <1L 2.60 ± 0.08F 3.57 ± 0.09DE 5.82 ± 0.17A 5.69 ± 16AB 5.29 ± 0.12B 5.10 ± 0.14B 4.30 ± 0.13C 1.75 ± 0.06FG 1.45 ± 0.04G 0.94 ± 0.02 GH 1.08 ± 0.01H <1L <1L <1L <1L

2.30 ± 0.15FG <1M <1M <1M 3.50 ± 0.09D 3.63 ± 0.11D 5.31 ± 0.23A 3.58 ± 0.06D 3.53 ± 0.04D 1.71 ± 0.05H 2.25 ± 0.07FG 4.13 ± 0.08BC 4.46 ± 0.10AB 4.30 ± 0.12B 3.56 ± 0.06D 1.40 ± 0.02HI 3.94 ± 0.07C

3.10 ± 0.16C <1G <1G 3.75 ± 0.14BC 3.01 ± 0.01C 2.48 ± 0.03CD 2.86 ± 0.12C 1.36 ± 0.11E <1G 3.64 ± 0.36BC 2.52 ± 0.18CD 1.44 ± 0.11E <1G <1G <1G 2.17 ± 0.26CD 2.32 ± 0.06CD

3.50 ± 0.08C <1L 1.22 ± 0.04L 1.56 ± 0.03M 3.01 ± 0.07D 2.70 ± 0.08E 2.00 ± 0.05G 1.91 ± 0.03G 1.45 ± 0.01I 2.55 ± 0.07 EF 3.40 ± 0.10C 2.83 ± 0.08DE 2.25 ± 0.06F 1.45 ± 0.04I 1.25 ± 0.04L 1.99 ± 0.05G 1.95 ± 0.01G

2.26 ± 0.05D 1.78 ± 0.04 EF 1.45 ± 0.04F 2.10 ± 0.06D 2.60 ± 0.08C 3.41 ± 0.11AB 4.00 ± 0.12A 3.11 ± 0.08B 2.90 ± 0.09BC 3.13 ± 0.09B 3.96 ± 0.12A 2.88 ± 0.06BC 2.60 ± 0.07C 2.50 ± 0.07C 2.44 ± 0.03CD 1.48 ± 0.02F 3.78 ± 0.10A

3.90 ± 0.09F 5.30 ± 0.03DE 5.53 ± 0.18D 5.16 ± 0.12E 5.32 ± 0.18DE 5.42 ± 0.09D 5.62 ± 0.19D 6.18 ± 0.20CD 6.59 ± 0.22C 6.79 ± 0.23C 2.10 ± 0.08HI 1.95 ± 0.07I 1.77 ± 0.04I 1.00 ± 0.01M <1 MN <1 MN <1 MN 1.42L

4.65 ± 0.18 EF 8.54 ± 0.29A 7.69 ± 0.26AB 7.47 ± 0.22AB 7.63 ± 0.11AB 7.03 ± 0.14B 6.80 ± 0.13BC 6.83 ± 0.23BC 6.40 ± 0.24C 5.30 ± 0.16D 3.23 ± 0.11G 2.63 ± 0.10HI 2.30 ± 0.11I 1.95 ± 0.05L 1.69 ± 0.02LM 1.59 ± 0.03M 1.03 ± 0.07N 1.87 ± 0.18L

4.23 ± 0.11FG 8.69 ± 0.27BC 8.89 ± 0.30B 8.98 ± 0.31B 8.97 ± 0.18B 8.93 ± 0.13B 9.20 ± 0.05AB 8.80 ± 0.33B 8.55 ± 0.29BC 8.20 ± 0.22C 5.51 ± 0.18DE 3.22 ± 0.11I 3.15 ± 0.10I 3.05 ± 0.08I 2.81 ± 0.09IL 2.68 ± 0.04L 3.15 ± 0.10I 3.87 ± 0.12 GH

2.40 ± 0.08F 4.34 ± 0.15C 3.70 ± 0.13D 4.00 ± 0.14CD 4.05 ± 0.08CD 4.30 ± 0.04C 4.33 ± 0.09C 4.45 ± 0.15C 4.00 ± 0.13CD 3.51 ± 0.11DE 0.84 ± 0.02H <1L <1L <1L <1L <1L <1L <1L

2.30 ± 0.15FG 1.40 ± 0.05HI 1.30 ± 0.01I 0.55 ± 0.04LM 0.50 ± 0.01LM <1M <1M <1M <1M <1M 1.80 ± 0.05 GH 0.84 ± 0.02L 0.82 ± 0.03L 0.32 ± 0.01M 0.25 ± 0.01M 0.26 ± 0.02M 0.30 ± 0.01M 1.25 ± 0.04I

3.10 ± 0.16C 4.21 ± 0.18B 5.10 ± 0.26A 3.51 ± 0.11BC 2.94 ± 0.24C 2.61 ± 0.08CD 2.52 ± 0.26CD 2.33 ± 0.17CD 1.29 ± 0.28E <1G 2.08 ± 0.04D 1.41 ± 0.11E 1.12 ± 0.07F 0.83 ± 0.01G <1G <1G 1.33 ± 0.15E 1.48 ± 0.08E

3.50 ± 0.08C 4.52 ± 0.15B 5.44 ± 0.18A 3.62 ± 0.12C 3.60 ± 0.07C 2.15 ± 0.04F 1.48 ± 0.01I <1L <1L <1L 2.71 ± 0.06E 2.18 ± 0.04F 1.94 ± 0.03G <1L <1L <1L 1.25 ± 0.04L 1.79 ± 0.06 GH

2.26 ± 0.05D 2.11 ± 0.07D 2.04 ± 0.05E <1H <1H <1H <1H <1H <1H <1H <1H <1H 1.15 ± 0.02G <1H <1H <1H 1.93 ± 0.06E 2.57 ± 0.08C

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

Production area Tube <1N Curd tank 3.36 ± 0.07 GH Knife <1N C Drain table <1N CC Drain table 1.99 ± 0.04L CC Stretch machine <1N CC Mold machine 1.67 ± 0.03LM CC Cooling tank <1N Brine tank 1.44 ± 0.03M Caciotta cheese R Milk 4.61 ± 0.09F P Milk 1.04 ± 0.18 MN Curd 6.25 ± 0.07C T1-c 6.78 ± 0.21B T10-c 6.87 ± 0.22B T20-c 6.93 ± 0.30B T30-c 7.33 ± 0.17AB T40-c 7.30 ± 0.11AB T50-c 7.10 ± 0.08AB T1-r 3.48 ± 0.09 GH T10-r 3.32 ± 0.07 GH T20-r 3.89 ± 0.11G T30-r 4.38 ± 0.05F T40-r 4.13 ± 0.12FG T50-r 3.48 ± 0.06 GH Wall <1N Racks 4.45 ± 0.11F Caciocavallo Pugliese cheese R Milk 4.61 ± 0.09F Curd 6.17 ± 0.04C Acid curd 6.60 ± 0.09BC Stretched curd 5.84 ± 0.14D T1-c 5.10 ± 0.17E T10-c 5.82 ± 0.19D T20-c 6.48 ± 0.22BC T30-c 6.84 ± 0.23B T40-c 7.63 ± 0.25A T50-c 7.85 ± 0.26A T1-r 2.96 ± 0.10HI T10-r 2.66 ± 0.04I T20-r 2.05 ± 0.07L T30-r 2.04 ± 0.06L T40-r 2.01 ± 0.05L T50-r 1.30 ± 0.03M Sticks <1N Floor 1.40 ± 0.01M

AeO Values within a column followed by different letters are significantly different (P < 0.05). R Milk, Raw milk; P Milk, Pasteurized Milk; Tube, tube from milk tank to curd tank; Curd tank, Tank for the coagulation of milk; Knife, knife used to break the curd; C Drain table, table to drain the curd of Caciotta cheese; CC Drain table, table to drain the curd of Caciocavallo cheese; CC Stretch machine, machine to stretch the curd of Caciocavallo cheese; CC Mold machine, machine to mold the curd of Caciocavallo cheese; CC Cooling tank, tank to cool the forming Caciocavallo cheese; Brine Tank, tank with brine; Wall, wall of ripening room of Caciotta cheese; Racks, racks of ripening room of Caciotta cheese; Acid curd, curd of Caciocavallo cheese when the pH reached the value of 5.25; Stretch curd, curd of Caciocavallo cheese after the stretching; Sticks, sticks for ripening of Caciocavallo cheese; Floor, floor of ripening room of Caciocavallo cheese.

119

120

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

Fig. 1. Permutation analysis of the community-level catabolic profiles, which were found in the dairy production area (equipment), raw cow's milk, curd, core and rind of cheese and related ripening room of Caciotta (C) and Caciocavallo Pugliese (CC) cheeses. Both cheese varieties were analyzed after one, ten, twenty, thirty, forty and fifty days of ripening. c, core; r, rind. R Milk, raw milk; Tube, tube from milk tank to curd tank; Curd Tank, tank for the coagulation of milk; Knife, knife used to break the curd; C Drain Table, table to drain the curd of Caciotta cheese; CC Drain table, table to drain the curd of Caciocavallo cheese; CC Stretch Machine, machine to stretch the curd of Caciocavallo cheese; CC Mould Machine, machine to mold the curd of Caciocavallo cheese; CC Cooling tank, tank to cool the forming Caciocavallo cheese; Brine Tank, tank with brine; C curd, curd of Caciotta cheese; C1-c to C50-c, samples of Caciotta cheese after 1e50 days of ripening; C1-r to C50-r, swab from surface of Caciotta cheese after 1e50 days of ripening; C Wall, wall of ripening room of Caciotta cheese; C Racks, racks of ripening room of Caciotta cheese; CC curd, curd of Caciocavallo cheese; CC acid curd, curd of Caciocavallo cheese when the pH reached the value of 5.25; CC stretch curd, curd of Caciocavallo cheese after the stretching; CC1-c to CC50-c, samples of Caciocavallo cheese after 1e50 days of ripening; CC1-r to CC50-r, swab from surface of Caciocavallo cheese after 1e50 days of ripening; CC Sticks, sticks for ripening of Caciocavallo cheese; CC Floor, floor of ripening room of Caciocavallo cheese.

16S rRNA gene was carried out. A total of 237,304 raw sequence reads were obtained (average number of sequences per sample: 4013; average sequence length: 495 bp). The values of alpha diversity parameters are reported in Table S1. The highest values of Chao1 richness and Shannon diversity indices were found for curd tank (666.56 and 5.07, respectively). Compared to cheese core, the rind had a higher (P < 0.05) Chao1 richness index. The sticks used to

ripen Caciocavallo Pugliese cheese had higher values (P < 0.05) of Chao1 richness and Shannon diversity compared to racks used for Caciotta ripening (502.08 vs. 56.11 and 4.23 vs. 2.99, respectively). All samples showed a Good's estimated sample coverage (ESC) above 97.8%, indicating a satisfactory description of the microbial diversity. The community structure was also analyzed using three

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

phylogeny-based beta-diversity measures (Fig. S2). With few exceptions, samples from the production area were not differentiated based on bacterial-lineage-specific principal-coordinate analysis with a weighted UniFrac distance matrix. 3.4. House and cheese bacterial microbiomes Overall, six phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Deinococcus-Thermus) and one candidate division (TM7) were identified (Fig. 2). Firmicutes and Proteobacteria dominated the production area. The only exceptions were found for the tube of pasteurized milk and the molding machine where Actinobacteria prevailed. Proteobacteria and Bacteroidetes dominated the raw milk. As expected, Firmicutes markedly

121

increased in curd and cheeses at day 1. Compared to core cheese, the rind showed a higher abundance of Proteobacteria and other phyla, especially after 20 days of ripening. The relative abundance of OTU at genus and species levels is shown in Fig. 3. Within Actinobacteria, Microbacterium lacticum and Dermacoccus dominated the tube of pasteurized milk and mold machine for Caciocavallo cheese, respectively (Fig. 3A). Arthrobacter and Brevibacterium were mainly identified in the rind of Caciotta cheese, while Propionibacterium in Caciocavallo Pugliese. Chryseobacterium, belonging to the phylum Bacteroidetes, was mainly associated to raw milk and Caciocavallo Pugliese cheese. Within Firmicutes, OTU belonging to starter (Lc. lactis and S. thermophilus) and non-starter lactic acid bacteria (L. casei group, L. plantarum group, Lactobacillus alimentarius, Enterococcus and Lactococcus

Table 2 Community-Level Catabolic Profiles (CLCPs) of dairy environment (production area and ripening rooms) and equipment, raw cows' milk (R Milk), curds, core (c) and rind (r) microbiota of Caciotta (C) and Caciocavallo Pugliese (CC) cheeses. Both cheese varieties were analyzed after one (C1, CC1), ten (C10, CC10), twenty (C20, CC20), thirty (C30, CC30), forty (C40, CC40) and fifty (C50, CC50) days of ripening.

Production area

Caciotta cheese

Caciocavallo Pugliese cheese

Sample

H0

Tube Curd tank Knife C Drain table CC Drain table CC Stretch machine CC Mold machine CC Cooling tank Brine tank C Curd C1-c C10-c C20-c C30-c C40-c C50-c C1-r C10-r C20-r C30-r C40-r C50-r C Wall C Racks R Milk CC Curd CC Acid curd CC Stretched curd CC1-c CC10-c CC20-c CC30-c CC40-c CC50-c CC1-r CC10-r CC20-r CC30-r CC40-r CC50-r CC Sticks CC Floor

0.71 5.16 2.81 2.93 3.00 3.98 2.84 3.22 5.06 3.91 3.17 3.25 3.16 3.18 3.35 3.54 3.52 3.59 5.12 5.08 3.12 3.05 3.56 3.51 2.82 3.00 3.12 2.99 3.02 2.84 3.00 3.04 3.11 3.10 3.17 3.41 5.13 5.09 5.14 4.92 5.01 5.42

E ¼ H0 /logS

S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.22 0.07 0.13 0.12 0.10 0.11 0.07 0.22 0.10 0.08 0.16 0.12 0.09 0.11 0.15 0.16 0.08 0.22 0.21 0.15 0.13 0.02 0.18 0.12 0.11 0.02 0.10 0.11 0.01 0.12 0.09 0.01 0.15 0.12 0.17 0.23 0.24 0.26 0.22 0.58 0.13

1.25 21.02 14.53 15.32 15.48 18.39 13.04 17.18 20.52 18.24 15.40 17.12 16.80 17.00 17.34 17.78 17.73 18.30 20.05 20.60 15.61 15.54 18.21 17.78 13.10 14.62 16.92 15.79 15.60 13.71 15.80 15.53 15.89 15.53 15.75 18.55 20.52 20.65 20.94 16.88 18.19 20.35

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.59 0.41 0.43 0.43 0.32 0.37 0.48 0.57 0.51 0.03 0.18 0.41 0.48 0.29 0.50 0.50 0.51 0.56 0.28 0.44 0.14 0.51 0.50 0.37 0.41 0.27 0.17 0.44 0.38 0.01 0.44 0.25 0.14 0.33 0.52 0.57 0.58 0.59 0.47 0.51 0.57

0.57 0.25 0.19 0.19 0.19 0.22 0.22 0.19 0.25 0.21 0.21 0.19 0.19 0.19 0.19 0.20 0.20 0.20 0.26 0.25 0.20 0.20 0.20 0.20 0.22 0.21 0.18 0.19 0.19 0.21 0.20 0.20 0.20 0.20 0.20 0.18 0.25 0.25 0.25 0.29 0.83 0.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.02 0.01 0.01 0.01 0.00 0.01 0.01 0.0 0.01 0.01 0.01 0.01 0.01 0.01 0.0 0.0 0.01 0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.0 0.01 0.0 0.01 0.04 0.01

Each value was expressed as the mean ± standard deviations (n ¼ 3) analyzed in duplicate. H0 , Shannon's diversity; S, substrate richness; E, substrate evenness. Tube, tube from milk tank to curd tank; Curd tank, tank for the coagulation of milk; Knife, knife used to break the curd; C Drain table, table to drain the curd of Caciotta cheese; CC Drain table, table to drain the curd of Caciocavallo cheese; Brine tank, tank with brine; CC Stretch machine, machine to stretch the curd of Caciocavallo cheese; CC Mold machine, machine to mold the curd of Caciocavallo cheese; CC Cooling tank, tank to cool the forming Caciocavallo cheese; R Milk, Raw milk; P Milk, Pasteurized milk; C Curd, Caciotta curd immediately after coagulation; C1-c to C50-c, cores of Caciotta cheese after 1 (post-brining) to 50 days of ripening; C1-r to C50-r, rinds of Caciotta cheese after 1 (post-brining) to 50 days of ripening; CC Curd, Caciocavallo cheese curd immediately after coagulation; CC Acid curd, Caciocavallo cheese curd when the pH reached the value of 5.25; CC Stretched curd, Caciocavallo cheese curd after stretching; CC1-c to CC50-c, cores of Caciocavallo cheese after 1 (post-brining) to 50 days of ripening; CC1-r to CC50-r, rinds of Caciocavallo cheese after 1 (post-brining) to 50 days of ripening; C Wall, wall of Caciotta cheese ripening room; C Racks, racks for Caciotta cheese ripening; CC sticks, sticks for Caciocavallo Pugliese cheese ripening; CC Floor, floor of Caciocavallo cheese ripening room.

122

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

Fig. 2. Relative abundance (percent) of total bacteria, which were found at the phylum level in the dairy production area (equipment) (A), raw cow's milk, curd, core and rind of cheese and related ripening room of Caciotta (B) and Caciocavallo Pugliese (C) cheeses. Both cheese varieties were analyzed after one (T1), ten (T10), twenty (T20), thirty (T30), forty (T40) and fifty (T50) days of ripening. c, core; r, rind.

garvieae) were variously detected from the equipment (Fig. 3B). The OTU attribution to starter strains was presumptive since typing was omitted but the deliberated inoculum (initial cell density for each species of approximately 5.0e7.5 log CFU/mL) makes the matching almost certain. Although used as starter, Lc. lactis was detectable at very low level (<2%) in Caciocavallo Pugliese curd and cheeses under ripening. That from S. thermophilus (starter for both the cheeses) was the only OTU detected in almost all samples. The only exceptions were the tube of pasteurized milk and the racks to ripen Caciotta cheese. S. thermophilus was the dominant Firmicutes in knife, Caciocavallo Pugliese drain table, brine tank, raw milk, curds, core cheeses and ripening rooms. At rind level, S. thermophilus decreased throughout ripening and dominated only in Caciocavallo cheese. The equipment harbored very low level (<0.5% of relative abundance) of L. delbrueckii subsp. lactis, which was used as starter for making Caciotta cheese. This species co-dominated with S. thermophilus the Caciotta curd, but decreased throughout ripening, especially at rind level. Although not used as starter, L. delbrueckii subsp. lactis contaminated the curd of Caciocavallo Pugliese cheese and persisted during ripening. L. helveticus, the other starter used for making Caciocavallo Pugliese cheese, was detected at low level (<2%) in all cheeses. Regarding non-starter lactic acid bacteria, L. plantarum group dominated the curd tank and it was also detected in the molding machine and rind of Caciocavallo Pugliese cheese. L. alimentarius, Enterococcus and Lc. garvieae were subdominant species variously found in the equipment and cheese rind and core of both the varieties. Lactobacillus brevis was only found on curd tank. L. casei group and other Lactobacillus species were mainly associated to the

cheese core. Low levels (<2%) of Leuconostoc spp. were diffusely found, especially in cheese during ripening. Staphylococcus species (e.g., Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus hominis) and Brochothrix spp. were mainly detected in the rind and ripening room of Caciotta cheese. Staphylococcus aureus was only present in the ripening room of Caciocavallo Pugliese cheese. Except for tube of pasteurized milk and knife, Acinetobacter and Moraxellaceae were the Proteobacteria most largely present in the equipment from the production area, raw milk and Caciocavallo Pugliese cheese (Fig. 3C). Marinomonas spp., Vibrio spp., other Vibrionaceae, Psychrobacter, Cobetia and Pseudoalteromonas were mainly found in the cheese rind during ripening, especially in the case of Caciotta cheese. The presence of Chromohalobacter, Enterobacteriaceae and Sphingomonas was mainly observed in the rind and ripening room of Caciocavallo Pugliese cheese. As shown by PCoA (Fig. S3) and the 3-D plot of PCA (Fig. 4) based on the relative abundance of OTU at genus/species level, the samples from the production area were clearly differentiated from cheeses. No systematic differences were found between the relative abundance of genera/species, which were associated with the cheese core of Caciotta and Caciocavallo Pugliese. On the contrary, the cheese rinds of Caciotta or Caciocavallo Pugliese were clearly clustered in two different zones of the 3-D plot. 3.5. Several correlations between community level catabolic profiles and house and cheese bacterial microbiomes Several positive correlations (r > 0.7; P < 0.05) were found between the community level catabolic profiles and house and cheese

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

123

Fig. 3. Relative abundance (percent) of total Actinobacteria (A), Firmicutes (B) and Proteobacteria (C), which were found at the genus/species level in the dairy production area (equipment), raw cow's milk (milk), curd, core and rind of cheese and related ripening room of Caciotta (C) and Caciocavallo Pugliese (CC) cheeses. Both cheese varieties were analyzed after one (C1; CC1), ten (C10; CC10), twenty (C20; CC20), thirty (C30; CC30), forty (C40; CC40) and fifty (C50; CC50) days of ripening. c, core; r, rind.

124

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

Fig. 4. Score plot of the three principal components (PC) after principal component analysis (PCA) of the total bacteria, which were found at the genus/species level in the dairy production area (equipment), raw cow's milk, curd, core and rind of cheese and related ripening room of Caciotta (C) and Caciocavallo Pugliese (CC) cheeses. Both cheese varieties were analyzed after one (C1; CC1), ten (C10; CC10), twenty (C20; CC20), thirty (C30; CC30), forty (C40; CC40) and fifty (C50; CC50) days of ripening. c, core; r, rind; green, equipment and raw cow's milk; blue, curds and core of Caciocavallo Pugliese and Caciotta cheeses; orange, rind of Caciotta cheese and related ripening room (C Racks, C Wall); red, rind of Caciocavallo Pugliese cheese and related ripening room (CC Floor, CC Sticks).

bacterial microbiomes: M. lacticum was correlated with tween 80, D-xylose, D-mannitol, N-acetyl-D-glucosamine, and D-malic acid; Propionibacterium spp. with glycyl-L-glutamine; Dermanococcus, Propionibacterium linens, Lc. lactis and Lc. garvieae with L-threonine; Arthrobacter, Propionibacterium, Staph. equorum, Staph. hominis, Staph. sciuri and Vibrio with serine; and Acinetobacter and Arthrobacter with malic acid. 4. Discussion Dairy plants and equipment, harboring the house microbiota, and biotechnological parameters, including the use of starters, determine the composition of the cheese microbiota, which, in turn, affects the biochemical events occurring in cheese during ripening and contributes to hygienic, nutritional and sensorial quality of cheeses (Fox et al., 2000; Montel et al., 2014). Aiming at establishing the role of the house microbiota in directing the cheese microbial communities, contaminants from dairy plant and equipment, and bacteria from raw milk and cheese rind and core were investigated through culture-dependent and -independent approaches. Two different varieties of cheese (Caciotta and Caciocavallo Pugliese), manufactured at the same dairy industry and using different commercial starters, were chosen as model systems. Mesophilic lactobacilli, cocci and, especially, thermophilic cocci were the most abundant cultivable bacteria found on equipment, which were located in the production area (milk-handling surfaces). Sampling was carried out before daily cheese making, and after disinfection and washing procedures from the previous working day. Therefore, their persistent presence might be hypothesized. In general, bacteria in milk may adhere and aggregate

on stainless steel surfaces, resulting in biofilm formation on milk storage tanks and process lines (Marchand et al., 2012). The formation of biofilm is promoted through accumulation of microscopic particles of organic matter onto metal surfaces, and biofilm structure development is usually fast (8e12 h) (Bremer et al., 2009; Scott et al., 2007). Presumptive enterococci, micrococci, staphylococci, yeasts and molds were also found on equipment. Pathogenic bacteria, such as Staph. aureus, Listeria monocytogenes and Salmonella spp., were not cultivable within both house and cheese microbiotas. According to starter composition, thermophilic cocci (both cheese varieties) and lactobacilli (L. delbrueckii subsp. lactis was used for making Caciotta cheese) dominated curds and cheeses at 1 day. According to previous findings (De Pasquale et al., 2014b; Di Cagno et al., 2012), mesophilic lactobacilli increased in the core of both cheese varieties during ripening. Compared to Caciocavallo Pugliese, Caciotta cheese showed a higher contamination by presumptive staphylococci and micrococci, mainly at core level. Similar results were found for Caciotta (Lanciotti et al., 2007) and Caciocavallo cheese (Vernile et al., 2006), probably due to the environmental conditions of cheese making and ripening and the microbial interactions. Cultivable bacteria from cheese rinds of both varieties decreased during ripening. This agreed with a previous report on non-smearripened cow's milk cheeses (Monfredini et al., 2012). Except for presumptive enterococci, ripening rooms harbored cultivable bacterial microbiotas, which almost reflected those found in Caciotta or Caciocavallo Pugliese cheeses. According to cultivable microbiotas, the catabolic profiles of microbial communities deriving from equipment, and core and rind cheeses were different. This further confirmed the existence of partly diverse microbial ecosystems (Irlinger et al., 2015; Rea et al., 2007; Viljoen et al., 2003). As shown by 16S rRNA targeted metagenomics, the only OTU detectable in almost all house and cheese samples was S. thermophilus. Notwithstanding its contamination from raw milk, S. thermophilus was the only lactic acid bacterium commonly present in the starter formulas that were used to acidify the curd of both cheese varieties. A set of other microbial species flanked S. thermophilus in the structure of the house microbiota, with variations depending on the surface equipment. Artisanal cheese making facilities manufacturing fresh, bloomy-rind and smearripened cheeses, also showed distinct communities contaminating the surface equipment (Bokulich and Mills, 2013). Some of the representative species from the house microbiota did not have the capacity to consistently contaminate cheeses. It was the case of some Actinobacteria. M. lacticum, which dominated the community from the tube of pasteurized milk and was variously detected on cooling tank, and stretching and molding machines for making Caciocavallo Pugliese, did not contaminate cheeses. Almost the same was found for Dermacoccus. Other Actinobacteria (Arthrobacter, Brevibacterium and Propionibacterium) were at a nondetectable level on equipment surfaces, but were associated to cheese rinds of both the varieties (Irlinger et al., 2015). The capacity of starters to persist in cheeses and to contaminate equipment surfaces differed. Lc. lactis, which was used for making Caciocavallo Pugliese cheese, dominated the community of drain curd tables, stretching machine and brine tank but showed low relative abundance in the cheese core and rind. Under the conditions of the dairy industry of this study, the capacity of environmental contamination by Lc. lactis was markedly lower than that highlighted for S. thermophilus. In agreement, Lc. lactis was previously found to dominate raw milk without a detectable presence in Caciocavallo Pugliese cheese (De Pasquale et al., 2014b). The other starters, L. delbrueckii subsp. lactis and, especially, L. helveticus, also showed low relative abundance in cheeses and/or colonization capability. Nevertheless, contaminating levels of L. delbrueckii

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

subsp. lactis were found in the curd of Caciocavallo Pugliese cheese, even though it was not used as starter. Although detectable in almost all house and cheese samples, the relative abundance of S. thermophilus varied. It dominated the communities from knife surface, brine tank, curds and core cheeses, as well as it was the main colonizing bacterium from drain table, rinds and ripening room of Caciocavallo Pugliese cheeses. Probably, the consistent colonization of other equipment by S. thermophilus was prevented due to bacterial interactions within communities having variable composition. As previously shown, S. thermophilus strains dominated during the manufacture (without starters) of traditional Caciocavallo type cheese and persisted in wooden vats (Settanni et al., 2012). All non-starter lactic acid bacteria genera/species (e.g., L. casei group and L. plantarum group), which were detected in cheese core and rind of both the varieties, were also detectable within the house microbiota. Probably due to the technology used (e.g., stretching, cheese hanging), the rinds of Caciocavallo Pugliese and Caciotta cheese were colonized by different microbial communities. Brochothrix, a Firmicutes spoilage bacterium mainly found in meat and fish products (De Filippis et al., 2013; Parlapani et al., 2015), dominated the communities of Caciotta cheese rind and racks in the ripening room. Coagulase-negative, halotolerant Staphylococcus species were always present in cheese rind, especially in Caciotta cheese. Staphylococcus sp. were the most frequently identified species in washed, natural and mold rind cheeses (Irlinger et al., 2015). Probably due to a previous contamination, non-cultivable Staph. aureus was identified in the ripening room floor for Caciocavallo Pugliese, but the cheese was free from staphylococcal contamination. Similar to a previous study (Irlinger et al., 2015), a-Proteobacteria were not detectable. The only exception was Sphingomonas, which mainly associated to Caciocavallo Pugliese cheese (this study) and soft American mold rind cheeses (Irlinger et al., 2015). bProteobacteria (mainly Novospirillum itersonii) were identified in raw milk without further colonization. No cheese colonization was found for some g-Proteobacteria (e.g., Acinetobacter species and other Moraxellaceae), which were mainly associated to raw milk and milk-handling surfaces. On the contrary, marine halotolerant g-Proteobacteria (Marinomonas spp., Vibrio spp., Psychrobacter, Cobetia, Pseudoalteromonas, Chromohalobacter) were identified in the equipment and dominated the rind of Caciotta and/or Caciocavallo Pugliese cheese. As previously found in some European washed rind cheeses (Irlinger et al., 2015), g-Proteobacteria may play a role during ripening of several varieties. A set of genera/species constitute bacterial communities, which varied depending on the equipment surfaces and cheese making, and all together formed a rather heterogeneous house microbiota. Representatives from such communities had (e.g., non-starter lactic acid bacteria and g-Proteobacteria) or not (e.g., Actinobacteria) the capacity to colonize cheeses, depending on the variety (Caciocavallo Pugliese or Caciotta cheese) and layer (rind or core). Lactic acid bacteria starters showed diverse capacities to colonize cheeses and especially equipment of dairy plant (e.g., Lc. lactis vs. S. thermophilus), thus hypothesizing the house microbiota as an indirect source of starter inoculation (e.g., L. delbrueckii subsp. lactis). Acknowledgments We thank the industrial plant Ignalat, located in Noci, Bari (Apulia region), Italy, for the supply of cow's milk, cheese manufacture, and technical support. This work was funded by Ministero  e della Ricerca, Ministero dello Svidell'Istruzione, dell'Universita luppo Economico and Fondo Europeo di Sviluppo Regionale

125

(PON02_00186_3417037, project PROINNO_BIT). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fm.2015.10.008. References
bert, A., Abraham, A.L., Rasmussen, S., Monnet, C., Pons, N., Almeida, M., He s, C., Loux, V., Batto, J.M., Leonard, P., Kennedy, S., Ehrlich, S.D., Pop, M., Delbe Montel, M.C., Irlinger, F., Renault, P., 2014. Construction of a dairy microbial genome catalog opens new perspectives for the metagenomic analysis of dairy fermented products. BMC Genom. 15, 1101. http://dx.doi.org/10.1186/14712164-15-1101. Almena-Aliste, M., Mietton, B., 2014. Cheese classification, characterization, and categorization: a global perspective. Microbiol. Spectr. 2 http://dx.doi.org/ 10.1128/microbiolspec.CM-0003-2012. Anonymous, 2004. Regulations on materials intended to come into contact with food EC1935/2004. Off. J. Eur. Union. L338, 1e17. Aquilanti, L., Babini, V., Santarelli, S., Osimani, A., Petruzzelli, A., Clementi, F., 2011. Bacterial dynamics in a raw cow's milk Caciotta cheese manufactured with aqueous extract of Cynara cardunculus dried flowers. Lett. Appl. Microbiol. 52, 651e659. http://dx.doi.org/10.1111/j.1472-765X.2011.03053.x. Bokulich, N., Mills, D., 2013. Facility-specific “house” microbiome drives microbial landscapes of artisan cheesemaking plants. Appl. Environ. Microbiol. 79, 5214e5223. http://dx.doi.org/10.1128/AEM.00934-13. Bremer, P.J., Seale, B., Flint, S., Palmer, J., 2009. Biofilms in dairy processing. In: Fratamico, P.M., Annous, B.A., Gunther, N.W. (Eds.), Biofilms in the Food and Beverage Industries. Woodhead Publishing in Food Science, Technology and Nutrition, Cambridge, UK, pp. 396e431. Callon, C., Picque, D., Corrieu, G., Montel, M.C., 2011. Ripening conditions: a tool for the control of Listeria monocytogenes in uncooked pressed type cheese. Food Control 22, 1911e1919. http://dx.doi.org/10.1016/j.foodcont.2011.05.003. Crecchio, C., Gelsomino, A., Ambrosoli, R., Minati, J.L., Ruggiero, P., 2004. Functional and molecular responses of soil microbial communities under differing soil management practices. Soil Biol. Biochem. 36, 1873e1883. http://dx.doi.org/ 10.1016/j.soilbio.2004.05.008. De Angelis, M., Gobbetti, M., 2011. Cheese: pasta-filata cheeses: traditional pastafilata cheese. In: Fuquay, J.W., Fox, P.F., McSweeney, P.L.H. (Eds.), Encyclopedia of Dairy Sciences, second ed., vol. 1. Elsevier Academic Press, San Diego, CA, pp. 745e752. De Filippis, F., La Storia, A., Stellato, G., Gatti, M., Ercolini, D., 2014. A selected core microbiome drives the early stages of three popular italian cheese manufactures. PLoS One 9, e89680. http://dx.doi.org/10.1371/journal.pone.0089680. De Filippis, F., La Storia, A., Villani, F., Ercolini, D., 2013. Exploring the sources of bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing. PLoS One 8, e70222. http://dx.doi.org/10.1371/ journal.pone.0070222. De Pasquale, I., Calasso, M., Mancini, L., Ercolini, D., La Storia, A., De Angelis, M., Di Cagno, R., Gobbetti, M., 2014a. Causal relationship between microbial ecology dynamics and proteolysis during manufacture and ripening of Canestrato Pugliese PDO cheese. Appl. Environ. Microbiol. 80 (14), 4085. http://dx.doi.org/ 10.1128/AEM.00757-14. De Pasquale, I., Di Cagno, R., Buchin, S., De Angelis, M., Gobbetti, M., 2014b. Microbial ecology dynamics reveal a succession in the core microbiota involved in the ripening of pasta filata Caciocavallo Pugliese cheese. Appl. Environ. Microbiol. 80, 6243e6255. http://dx.doi.org/10.1128/AEM.02097-14. Di Cagno, R., De Pasquale, I., De Angelis, M., Buchin, S., Calasso, M., Fox, P.F., Gobbetti, M., 2011. Manufacture of Italian Caciotta-type cheeses with adjuncts and attenuated adjuncts of selected non-starter lactobacilli. Int. Dairy J. 21, 254e260. http://dx.doi.org/10.1016/j.idairyj.2010.12.007. Di Cagno, R., De Pasquale, I., De Angelis, M., Gobbetti, M., 2012. Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli. J. Dairy Sci. 95, 4784e4795. http://dx.doi.org/10.3168/jds.20115283. Didienne, R., Defargues, C., Callon, C., Meylheuc, T., Hulin, S., Montel, M.C., 2012. Characteristics of microbial biofilm on wooden vats (“gerles”) in PDO Salers cheese. Int. J. Food Microbiol. 156, 91e101. http://dx.doi.org/10.1016/ j.ijfoodmicro.2012.03.007. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460e2461. http://dx.doi.org/10.1093/bioinformatics/ btq461. Ercolini, D., De Filippis, F., La Storia, A., Iacono, M., 2012. “Remake” by highthroughput sequencing of the microbiota involved in the production of water buffalo mozzarella cheese. Appl. Environ. Microbiol. 78, 8142e8145. http:// dx.doi.org/10.1128/AEM.02218-12. EFSA, 2008. Guidance Document on the Submission of a Dossier on a Substance to Be Used in Food Contact Materials for Evaluation by EFSA by the Pannel on Additives, Flavourings, Processing Aids and Materials in in Contact with Food (AFC). Eur. Food Saf. Authority, pp. 1e125. FDA, 2007. Determining the Regulatory Status of Components of a Food Contact

126

M. Calasso et al. / Food Microbiology 54 (2016) 115e126

Material. U.S. Food and Drug Administration. Fox, P.F., Guinee, T.P., Cogan, T.M., McSweeney, P.L.H., 2000. Fundamentals of Cheeses Sciences. Aspen Publishers, Gaithersburg, MD, USA, ISBN 978-0-83421260-2, pp. 251e277. Gobbetti, M., Burzigotti, R., Smacchi, E., Corsetti, A., De Angelis, M., 1997. Microbiology and biochemistry of Gorgonzola cheese during ripening. Int. Dairy J. 7, 519e529. http://dx.doi.org/10.1016/S0958-6946(97)00047-2.
linck, S., Dugat-Bony, E., 2015. Cheese rind microbial comIrlinger, F., Layec, S., He munities: diversity, composition and origin. FEMS Microbiol. Lett. 362 (2), 1e11. http://dx.doi.org/10.1093/femsle/fnu015. Irlinger, F., Mounier, J., 2009. Microbial interactions in cheese: implications for cheese quality and safety. Curr. Opin. Biotechnol. 20, 142e148. http://dx.doi.org/ 10.1016/j.copbio.2009.02.016. Kindstedt, P., Caric, M., Milanovic, S., 2004. Pasta-filata cheeses. In: Fox, P.F., McSweeney, P.L.H., Cogan, T.M., Guinee, T.P. (Eds.), Cheese: Chemistry, Physics and Microbiology, third ed., vol. 2. Elsevier Academic Press, London, United Kingdom, pp. 251e277. Lahou, E., Uyttendaele, M., 2014. Evaluation of three swabbing devices for detection of Listeria monocytogenes on different types of food contact surfaces. Int. J. Environ. Res. Public Health 11, 804e814. http://dx.doi.org/10.3390/ ijerph110100804. Lanciotti, R., Patrignani, F., Iucci, L., Guerzoni, M.E., Suzzi, G., Belletti, N., Gardini, F., 2007. Effects of milk high pressure homogenization on biogenic amine accumulation during ripening of ovine and bovine Italian cheeses. Food Chem. 104, 693e701. Licitra, G., Ogier, J.C., Parayre, S., Pediliggieri, C., Carnemolla, T.M., Falentin, H., Madec, M.N., Carpino, S., Lortal, S., 2007. Variability of bacterial biofilms of the “tina” wood vats used in the Ragusano cheese-making process. Appl. Environ. Microbiol. 73, 6980e6987. http://dx.doi.org/10.1128/AEM.00835-07. Lortal, S., Di Blasi, A., Madec, M.N., Pediliggieri, C., Tuminello, L., Tanguy, G., Fauquant, J., Lecuona, Y., Campo, P., Carpino, S., Licitra, G., 2009. Tina wooden vat biofilm: a safe and highly efficient lactic acid bacteria delivering system in PDO Ragusano cheese making. Int. J. Food Microbiol. 132, 1e8. http://dx.doi.org/ 10.1016/j.ijfoodmicro.2009.02.026. MacFaddin, J.F., 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams and Wilkins, Baltimore, MD. Marchand, S., De Block, J., De Jonghe, V., Coorevits, A., Heyndrickx, M., Herman, L., 2012. Biofilm formation in milk production and processing environments; influence on milk quality and safety. Compr. Rev. Food Sci. Food Saf. 11, 133e147. http://dx.doi.org/10.1111/j.1541-4337.2011.00183.x. McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen, G.L., Knight, R., Hugenholtz, P., 2012. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610e618. http://dx.doi.org/10.1038/ismej.2011.139. Monfredini, L., Settanni, L., Poznanski, E., Cavazza, A., Franciosi, E., 2012. The spatial distribution of bacteria in Grana-cheese during ripening. Syst. Appl. Microbiol. 35, 54e63. http://dx.doi.org/10.1016/j.syapm.2011.07.002. Montel, M.C., Buchin, S., Mallet, A., Delbes-Paus, C., Vuitton, D., Desmasures, N., Berthier, F., 2014. Traditional cheeses: rich and diverse microbiota with associated benefits. Int. J. Food Microbiol. 177, 136e154. http://dx.doi.org/10.1016/

j.ijfoodmicro.2014.02.019. Parlapani, F.F., Haroutounian, S.A., Nychas, G.J.E., Boziaris, I.S., 2015. Microbiological spoilage and volatiles production of gutted European sea bass stored under air and commercial modified atmosphere package at 2
C. Food Microbiol. 50, 44e53. http://dx.doi.org/10.1016/j.fm.2015.03.006. €rges, S., Gelsomino, R., Brennan, N.M., Mounier, J., Vancanneyt, M., Rea, M.C., Go Scherer, S., Swings, J., Cogan, T.M., 2007. Stability of the biodiversity of the surface consortia of Gubbeen, a red-smear cheese. J. Dairy Sci. 90, 2200e2210. http://dx.doi.org/10.3168/jds.2006-377. Reeder, J., Knight, R., 2010. Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat. Methods 7, 668e669. http:// dx.doi.org/10.1038/nmeth0910-668b. Schornsteiner, E., Mann, E., Bereuter, O., Wagner, M., Schmitz-Esser, S., 2014. Cultivation-independent analysis of microbial communities on Austrian raw milk hard cheese rinds. Int. J. Food Microbiol. 180, 88e97. http://dx.doi.org/ 10.1016/j.ijfoodmicro.2014.04.010. Scott, S.A., Brooks, J.D., Rakonjac, J., Walker, K.M.R., Flint, S.H., 2007. The formation of thermophilic spores during the manufacture of whole milk powder. Int. J. Dairy Technol. 60, 109e117. http://dx.doi.org/10.1111/j.1471-0307.2007.00309.x.
, G., Bellina, V., Francesca, N., Moschetti, G., Settanni, L., Di Grigoli, A., Tornambe Bonanno, 2012. A., Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a traditional Caciocavallo type cheese. Int. J. Food Microbiol. 155, 73e81. http://dx.doi.org/10.1016/ j.ijfoodmicro.2012.01.022. Shannon, C.E., 1948a. A mathematical theory of communication. Bell Syst. Tech. J. 27, 379e423. http://dx.doi.org/10.1002/j.1538-7305.1948.tb01338.x. Shannon, C.E., 1948b. A mathematical theory of communication. Bell Syst. Tech. J. 27, 623e656. http://dx.doi.org/10.1002/j.1538-7305.1948.tb00917.x. Somers, E.B., Johnson, M.E., Wong, A.C., 2001. Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment. J. Dairy Sci. 84, 1926e1936. http://dx.doi.org/10.3168/jds.S0022-0302(01)74634-6. Stellato, G., De Filippis, F., La Storia, A., Ercolini, D., 2015. Coexistence of lactic acid bacteria and potential spoilage microbiota in a dairy processing environment. Appl. Environ. Microbiol. 81, 7893e7904. http://dx.doi.org/10.1128/AEM.0229415.
s, C.M., Criado, M.T., 1992. Adherence of psychrotrophic bacteria Su
arez, B., Ferreiro to dairy equipment surfaces. J. Dairy Res. 59, 381e388. Vernile, A., Spano, G., Lecce, A., Tarantino, D., Beneduce, L., Massa, S., 2006. Microbiological and chemical characteristics of “monti dauni meridionali” caciocavallo cheese. Adv. Food Sci. 28, 2e6. Viljoen, B.C., Khoury, A.R., Hattingh, A., 2003. Seasonal diversity of yeasts associated with white-surface mould-ripened cheeses. Food Res. Int. 36, 275e283. http:// dx.doi.org/10.1016/S0963-9969(02)00169-2. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261e5267. http://dx.doi.org/10.1128/AEM.00062-07. Zak, J., Willig, M., Moorhead, D., Wildman, H., 1994. Functional diversity of microbial communities: a quantitative approach. Soil Biol. Biochem. 26, 1101e1108. http:// dx.doi.org/10.1016/0038-0717(94)90131-7.