Characterization of Microbial Shifts during the Production and Ripening of Raw Ewe Milk-Derived Idiazabal Cheese by High-Throughput Sequencing

Citation: Santamarina-García, G.; Hernández, I.; Amores, G.; Virto, M. Characterization of Microbial Shifts during the Production and Ripening of Raw Ewe Milk-Derived Idiazabal Cheese by High-Throughput Sequencing. Biology 2022,11, 769. https://doi.org/10.3390/ biology11050769 Academic Editor: Huizhong Chen Received: 13 April 2022 Accepted: 11 May 2022 Published: 18 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biology Article Characterization of Microbial Shifts during the Production and Ripening of Raw Ewe Milk-Derived Idiazabal Cheese by High-Throughput Sequencing Gorka Santamarina-García * , Igor Hernández , Gustavo Amores and Mailo Virto * Lactiker Research Group, Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; igor[email protected] (I.H.); gustavo.amor[email protected] (G.A.) *Correspondence: [email protected] (G.S.-G.); [email protected] (M.V.); Tel.: +34-945013799 (G.S.-G.); +34-945013099 (M.V.) Simple Summary: Idiazabal is a traditional cheese produced from raw ewe milk in the Basque Country (Southwestern Europe). The sensory properties of raw milk cheeses have been attributed, among other factors, to microbial shifts that occur during the production and ripening processes. In this study, we used high-throughput sequencing technologies to investigate the microbiota of Latxa ewe raw milk and the dynamics during cheese production and ripening processes. The microbiota of raw milk was composed of lactic acid bacteria (LAB), environmental bacteria and non-desirable bacteria. Throughout the cheese making and ripening processes, the growth of LAB was promoted, whereas that of non-desirable and environmental bacteria was inhibited. Moreover, some genera not reported previously in raw ewe milk were detected and clear differences were observed in the bacterial composition of raw milk and cheese among producers, in relation to LAB and environmental or non-desirable bacteria, some of which could be attributed to the production of flavour related compounds. Abstract: In this study, we used high-throughput sequencing technologies (sequencing of V3–V4 hypervariable regions of 16S rRNA gene) to investigate for the first time the microbiota of Latxa ewe raw milk and the bacterial shifts that occur during the production and ripening of Idiazabal cheese. Results revealed several bacterial genera not reported previously in raw ewe milk and cheese, such as Buttiauxella and Obesumbacterium. Both the cheese making and ripening processes had a significant impact on bacterial communities. Overall, the growth of lactic acid bacteria (LAB) (Lactococcus, Lactobacillus,Leuconostoc,Enterococcus,Streptococcus and Carnobacterium) was promoted, whereas that of non-desirable and environmental bacteria was inhibited (such as Pseudomonas and Clostridium). However, considerable differences were observed among producers. It is noteworthy that the starter LAB (Lactococcus) predominated up to 30 or 60 days of ripening and then, the growth of non-starter LAB (Lactobacillus,Leuconostoc,Enterococcus and Streptococcus) was promoted. Moreover, in some cases, bacteria related to the production of volatile compounds (such as Hafnia,Brevibacterium and Psychrobacter) also showed notable abundance during the first few weeks of ripening. Overall, the results of this study enhance our understanding of microbial shifts that occur during the production and ripening of a raw ewe milk-derived cheese (Idiazabal), and could indicate that the practices adopted by producers have a great impact on the microbiota and final quality of this cheese. Keywords: cheese quality; ripening; microbiota; bacterial diversity; 16S rRNA sequencing; PCoA 1. Introduction Idiazabal cheese is a semi-hard or hard cheese made exclusively from the raw milk of Latxa and/or Carranzana sheep, with a minimum ripening time of 60 days. Its production is located in the Basque Country (Southwestern Europe) and has a Protected Designation Biology 2022,11, 769. https://doi.org/10.3390/biology11050769 https://www.mdpi.com/journal/biology Biology 2022,11, 769 2 of 21 of Origin (PDO) [ 1 ]. Most of the producers attached to the Idiazabal PDO are small family dairies that lead the whole process, from livestock management to cheese making and final sales. Although Idiazabal cheese production is a strictly regulated process, producers may use different practices that may affect the characteristics of the final product. The most considerable differences in production practices are noticed in the management and feeding of the herd, leading to differences in milk quality [ 2 ]; in the use of artisanal or commercial rennet, or in the parameters selected during cheese making and ripening, since the Idiazabal PDO specifications establish ranges [3]. Idiazabal cheese, as other cheeses prepared from raw milk, has a richer and more intense aromatic profile compared with those produced from pasteurized milk [ 4 , 5 ]. Such interesting sensory properties of raw milk cheeses have previously been attributed, among other factors, to the complex dynamics of microbial composition during cheese making and ripening [ 5 , 6 ]. The quality of raw milk, use of starters and their intrinsic characteristics, type of rennet used and ripening time are some factors that determine the cheese microbiota [5,7–9] . The microbiota of milk has a diverse and complex composition, but it is mainly composed of lactic acid bacteria (LAB) [ 5 , 10 ]. In Idiazabal cheese, the most common LAB are Lactococcus,Lactobacillus and Leuconostoc [ 11 , 12 ]. These bacteria metabolize the lactose present in milk, generating lactic acid and other compounds, such as acetic acid, ethanol and diacetyl. These compounds, along with others produced during ripening, determine the sensory properties of cheese [ 13 ]. Although LAB are predominant, other lowabundance microorganisms are also part of the microbial ecosystem of cheese [ 10 , 14 , 15 ], and consequently contribute to the quality of the final product [16,17]. The characteristics of LAB and other microorganisms present in Idiazabal cheese have been described and related to its sensory properties in several studies [ 11 , 12 , 18 , 19 ]. However, these studies were performed 20 years ago using culture-dependent methods. Nowadays, high-throughput sequencing (HTS) technologies are used to monitor microbial communities in different fermented products [ 20 – 22 ], including cheese [ 23 – 25 ]. The HTS techniques allow the detection of a large number of bacteria, including those present in relatively small numbers [ 26 – 29 ], those present in a viable but non-cultivable state (VBNC) [ 30 ] and those not detected by other culture-dependent or independent methods [30–34]. The vast majority of studies on cheese focus on cheese produced from cow milk [24,35,36] , and only a few studies have been carried out on cheese produced from the raw milk of ewe [37–39] . Moreover, little is known about the bacterial composition of raw ewe milk [ 40 – 42 ] and how it changes during cheese making and ripening processes [26,43,44]. Therefore, this study aimed to (1) characterize the bacterial communities of the raw milk of Latxa ewe; (2) analyse the effect of cheese making and ripening processes on bacterial populations; and (3) study the potential differences among producers producing the same type of cheese. To the best of our knowledge, no comprehensive metagenomic study has been conducted to date on raw ewe milk-derived cheeses. Moreover, although Idiazabal cheese has an internationally recognized PDO [ 1 ], no HTS studies have been performed to characterize its bacterial populations. 2. Materials and Methods 2.1. Milk and Cheese Sampling To analyse the microbiota of Latxa ewe raw milk and Idiazabal cheeses, samples were collected from four artisanal Idiazabal PDO cheese producers (identified as A, B, C and D), whose dairies were situated in different geographic locations throughout the Basque Country. Milk was kept in refrigeration tanks before cheese making. Cheeses were produced from the collected milk samples, according to specifications issued by the Idiazabal Designation of Origin Regulatory Board [ 3 ], using Choozit MM 100 LYO 50 DCU (mixture of Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar. diacetylactis) (DuPont NHIB Ibérica S.L., Barcelona, Spain) as the starter. Milk was coagulated using artisanal rennet prepared from the stomachs of Latxa lambs (extracted during the first month of lactation, cleaned, dried, salted and ground, as described Biology 2022,11, 769 3 of 21 previously [ 45 ]) or commercial rennet NATUREN ® 195 Premium (Chr. Hansen Holding A/S, Hørsholm, Denmark). Cheese ripening was carried out in chambers maintained at 8–14 ◦ C temperature and 80–95% relative humidity. Cheeses were collected in duplicate at six time points during ripening (1, 7, 14, 30, 60 and 120 days). Therefore, a total of 4 raw milk samples and 48 cheese samples were analysed. Samples were collected and transported to the laboratory under refrigerated conditions (3 ◦C) for analysis. 2.2. DNA Extraction DNA extraction was performed immediately after sample arrival, following the method described by Erkus et al. [ 46 ], with some modifications. To extract DNA from cheese samples, 10 g of each sample was suspended in 90 mL of 2% (w/v) sterile sodium citrate (pH 8.0), and homogenized in a stomacher (Masticator Basic 400; IUL Instruments, Königswinter, Germany) six times, each for 20 s ON and 10 s OFF. Then, 1.5 mL of the resulting suspension was centrifuged at 8000 × gfor 10 min at 4 ◦ C, and the fat-containing supernatant was discarded. The obtained pellet was resuspended in 600 µ L of sodium citrate, and centrifuged three times at 8000 × gfor 10 min at 4 ◦ C. DNA was extracted with DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. To extract DNA from milk samples, 10 mL of raw milk from each sample was processed as described above, however, without the need for homogenisation in the stomacher. 2.3. Library Preparation and Sequencing HTS analysis was performed in the Sequencing and Genotyping Unit of the Genomic Facility/SGIker (supported by UPV/EHU, MICINN, GV/EJ, FSE) of the University of the Basque Country. The 16S rRNA gene library was prepared using Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA), according to the 16S rRNA gene metagenomics workflow of Illumina. The V3–V4 regions of the 16S rRNA gene were amplified by PCR (forward primer: 5 0 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC TACGGGNGGCWGCAG-3 0 ; reverse primer: 5 0 -GTCTCGTGGGCTCGGAGATGTGTA TAAGAGACAGGACTACHVGGGTATCTAATCC-3 0 ) as described by Klindworth et al. [ 47 ]. Then, 16S rRNA gene sequencing was performed on the Illumina MiSeq platform using the MiSeq Reagent Kit v3 (2 ×300 bp) (Illumina Inc.). 2.4. Bioinformatic Analysis Quality filtering and trimming of raw reads were performed using the MiSeq Reporter software (Illumina), and taxonomic classification was performed using the MG-RAST web data analysis tool [ 48 ], based on the Silva SSU database [ 49 ]. Since the sequencing of most variable regions of the 16S rRNA gene is effective up to the genus level, and seldom discriminates among species adequately [ 50 ], the taxonomic classification was performed up to the genus rank. Rarefaction curves were also generated using MG-RAST. 2.5. Statistical Analysis Relative bacterial abundance (%) was calculated based on the identified sequences, and three significant figures were used to express the results. The IBM SPSS statistical package version 26.0 (IBM SPSS Inc., Chicago, IL, USA, 2019) was used for data preparation and analysis. Mann–Whitney Utest and Kruskal-Wallis analysis of variance with Bonferroni correction were performed using the SPSS package. The objective was to estimate differences in reads and operational taxonomic units (OTUs) between milk and cheese samples, and to analyse the influence of producer, cheese making and ripening time factors on bacterial phyla and genera abundance. To determine the direction and strength of correlations among the main bacterial genera, Spearman’s rank correlation coefficients were calculated using SPSS, and displayed as a heat map in RStudio version 1.3.959 and R version 3.6.3 [ 51 ] using the “gplots” package [ 52 ]. To analyse the effect of producer and ripening time factors on the abundance of the main bacterial genera, Permutational Multivariate Biology 2022,11, 769 4 of 21 Analysis of Variance (PERMANOVA) was computed in R using the “vegan” package [ 53 ]. Principal Component Analysis (PCA) of the main bacterial genera was performed using their log-transformed, when necessary, and Unit Variance scaled abundance data, and plotted using the SIMCA software (version 15.0.0.4783; Umetrics AB, Umeå, Sweden). The number of principal components (PCs) was determined by eigenvalues (greater than 1.5) and cross validation. The aim was to study microbial dynamics in cheeses according to producer and ripening time factors. An Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was performed in SIMCA to confirm whether microbial communities of samples differed according to the producer. Alpha and beta diversity indices were calculated by taking into account the sequence abundance of all bacterial genera present in milk and cheese samples. Alpha diversity was assessed in R using different packages, depending on the objective: “tidyverse” package for data cleaning and preparation for analysis [ 54 ]; “BiodiversityR” package for calculating Shannon, Simpson, Inverse Simpson, Berger and Shannon evenness (Jevenness and Eevenness) diversity indices [ 55 ]; and “vegan” package for calculating Chao1 and ACE diversity indices. Significant differences among producers for each diversity index were analysed in SPSS using Kruskal-Wallis test. Beta diversity indices (Bray–Curtis and Jaccard dissimilarities) were calculated using the “vegan” package of R, and plotted into a Principal Coordinate Analysis (PCoA) model using the “APE” package of R [56]. 3. Results and Discussion 3.1. Characteristics of 16S rRNA Gene Sequencing Data A total of 10,798,992 16S rRNA gene sequences were obtained from Latxa ewe raw milk and Idiazabal cheese samples (n= 52), with an average sequence length of 348 ±101 bp , mean GC content of 53 ± 5% and 10,388 OTUs. Altogether, 24 bacterial phyla, 209 families and 645 genera were identified. Further details of the reads, OTUs and number of identified phyla, families and/or genera are summarised in Table 1. The number of sequences obtained from cheese samples was significantly greater than those obtained from milk samples (p ≤ 0.001), although no significant differences were observed in the number of identified OTUs between the two sample types (p > 0.05). Moreover, both milk and cheese samples obtained from different producers showed significant dissimilarities in the number of reads (p ≤ 0.01) and identified OTUs (p ≤ 0.001), with producer A being clearly distinct from the other three producers. In general, the rarefaction curves showed a clear and strong stabilizing tendency (Figure S1), indicating sufficient sampling of microbial communities. Overall, this study reports a greater number of sequence reads, OTUs and taxonomic identifications in raw ewe milk and cheese than previous studies [14,38,39,44]. 3.2. In-Depth Analysis of Microbial Shifts 3.2.1. Bacterial Composition of the Raw Milk of Ewe Milk is an important source of microorganisms in cheese [ 5 , 57 ]. A total of 21 bacterial phyla, 165 families and 455 genera were identified in raw milk samples. At the phylum level (Figure 1A, Table S1), Firmicutes (10.5–54.1%) and Proteobacteria (16.9–40.7%) were the most dominant, followed by Bacteroidetes (5.44–19.6%). Other phyla, with abundances higher than 1%, were detected only in milk samples obtained from some producers: Actinobacteria and Verrucomicrobia in samples obtained from producer C (3.75% and 1.33%, respectively) and D (2.97% and 2.75%, respectively), and Planctomycetes in samples from producer D (1.28%). In general, the predominance of Firmicutes and Proteobacteria in raw ewe milk is consistent with previous studies [ 26 , 40 , 42 ], although differences in the abundance of each phylum have been reported among milk samples collected from different breeds [ 26 , 40 – 42 ]. However, raw milk samples of Latxa ewe were characterized by the high-level abundance of Bacteroidetes and a notable presence of Verrucomicrobia and Planctomycetes in comparison with milk collected from other breeds [ 26 , 40 , 42 ]. This indicates a differential characteristic of Latxa ewe raw milk used for Idiazabal cheese production. Biology 2022,11, 769 5 of 21 Table 1. Metataxonomic data of Latxa ewe raw milk and Idiazabal cheese samples at 6 ripening times (1, 7, 14, 30, 60 and 120 days) from 4 producers (A, B, C and D) (n= 52). Producer Milk/Cheese Ripening Time (Day) Sample ID Bacterial Diversity Sequences OTUs Mbp Count Phyla Families Genera A Milk MA 66,989 8450 2.70 8 103 221 1 1 321,148 21,102 6.84 10 102 244 2 331,816 17,948 5.96 10 107 242 7 9 238,804 12,412 4.21 10 82 182 10 251,225 13,472 4.55 10 92 203 14 17 263,231 13,305 4.51 10 81 176 18 334,305 16,504 5.95 11 90 209 30 25 269,207 14,266 5.18 8 89 195 26 280,285 13,701 4.65 8 84 191 60 33 330,729 15,810 5.50 10 79 179 34 341,419 16,324 5.92 10 79 183 120 41 370,181 22,557 7.61 14 83 187 42 332,471 16,882 6.15 12 78 170 B Milk MB 6092 2504 0.769 10 66 136 13 257,407 12,967 4.39 9 90 178 4 184,670 10,745 3.66 7 80 158 711 160,436 9174 3.15 7 70 124 12 148,700 8429 3.09 9 67 117 14 19 193,164 8672 2.94 9 57 107 20 134,695 7270 2.48 7 55 107 30 27 140,484 2919 1.31 7 40 60 28 129,491 6422 2.33 6 43 88 60 35 295,377 13,046 4.38 10 64 125 36 157,505 7103 2.46 6 47 91 120 43 294,909 12,781 4.61 10 54 107 44 175,689 8629 3.13 10 53 95 C Milk MC 10,632 4889 1.41 10 66 135 15 237,563 11,167 3.76 10 83 165 6 200,211 9565 3.24 10 73 156 713 172,573 9232 3.16 9 72 136 14 105,377 2947 1.32 5 39 65 14 21 281,503 11,879 4.03 10 76 152 22 162,633 7438 2.54 8 57 112 30 29 374,652 14,534 5.20 8 75 164 30 178,935 7641 2.65 9 66 125 60 37 390,610 14,658 5.06 12 83 170 38 218,602 8265 2.84 8 57 105 120 45 116,160 5909 2.05 5 47 76 46 188,779 8633 2.99 7 51 83 D Milk MD 52,040 11,547 3.80 21 151 378 17 210,147 11,128 3.71 10 97 217 8 175,444 8825 3.03 11 91 197 715 136,796 6512 2.38 7 65 120 16 126,129 6062 2.11 8 56 113 14 23 194,767 8284 2.99 11 66 131 24 201,259 9138 3.09 14 73 139 30 31 161,519 7050 2.39 13 68 119 32 187,889 8164 2.78 10 66 136 60 39 86,466 4525 1.68 8 45 84 40 202,656 8933 3.28 7 65 127 120 47 205,505 9667 3.36 11 67 119 48 209,716 10,185 3.51 15 63 123 Biology 2022,11, 769 6 of 21 Biology 2022, 11, x FOR PEER REVIEW 6 of 22 3.2. In-Depth Analysis of Microbial Shifts 3.2.1. Bacterial Composition of the Raw Milk of Ewe Milk is an important source of microorganisms in cheese [5,57]. A total of 21 bacterial phyla, 165 families and 455 genera were identified in raw milk samples. At the phylum level (Figure 1A, Table S1), Firmicutes (10.5–54.1%) and Proteobacteria (16.9–40.7%) were the most dominant, followed by Bacteroidetes (5.44–19.6%). Other phyla, with abundances higher than 1%, were detected only in milk samples obtained from some producers: Actinobacteria and Verrucomicrobia in samples obtained from producer C (3.75% and 1.33%, respectively) and D (2.97% and 2.75%, respectively), and Planctomycetes in samples from producer D (1.28%). In general, the predominance of Firmicutes and Proteobacteria in raw ewe milk is consistent with previous studies [26,40,42], although differences in the abundance of each phylum have been reported among milk samples collected from different breeds [26,40–42]. However, raw milk samples of Latxa ewe were characterized by the high-level abundance of Bacteroidetes and a notable presence of Verrucomicrobia and Planctomycetes in comparison with milk collected from other breeds [26,40,42]. This indicates a differential characteristic of Latxa ewe raw milk used for Idiazabal cheese production. Figure 1. Relative abundance (%) of bacterial phyla and genera of Latxa ewe raw milk ((A,B), respectively) and 1-day-old ripened Idiazabal cheese samples ((C,D), respectively) produced by four producers (A, B, C, D). Figure 1. Relative abundance (%) of bacterial phyla and genera of Latxa ewe raw milk ( (A,B), respectively ) and 1-day-old ripened Idiazabal cheese samples (( C , D ), respectively) produced by four producers (A, B, C, D). A total of 24 genera with abundance greater than 1% were identified, and 10 of these genera showed abundance higher than 5% (Figure 1B, Table S2). Lactococcus ( 1.64–14.5% ), Eubacterium (0.0766–9.18%), Clostridium (0.183–6.09%), Leuconostoc (0–6.10%) and Staphylococcus (0.291–5.48%) were the most abundant genera within Firmicutes. Similarly, Pseudomonas (7.36–18.5%), Buttiauxella (0–14.1%), Serratia (0.0245–12.6%) and Raoultella (0–6.86%) showed the highest abundance within Proteobacteria, and Chryseobacterium ( 0–11.7% ) within Bacteroidetes. Differences were observed among milk samples obtained from different producers (Table S2). While Pseudomonas and Lactococcus were identified as the main genera common to all analysed raw milk samples, the remaining genera were characteristic of each producer. The abundance of the rest of genera classified as “others” and unclassified sequences was remarkable (6.25–22.4% and 15.6–50.8%, respectively). Differences observed in the microbial composition of milk samples at the phylum and genus levels among producers (Tables S1 and S2) could be caused by various factors such as differences in lactation stage, flock management and feeding, or sources of microorganisms, for instance, mammary gland diseases or microorganisms contaminating the teat surface, practices and materials employed during milking or dairy environment [ 5 , 10 , 15 , 41 ]. Moreover, these factors could explain the differences observed in bacterial communities between the raw milk of Latxa ewe and that of other ewe breeds [40–42]. Biology 2022,11, 769 7 of 21 The identified bacterial genera were divided into three groups: LAB, comprising genera previously classified as LAB [ 57 ]; environmental bacteria, including bacteria derived from the natural environment [ 58 ]; and non-desirable bacteria, containing genera exhibiting a pathogenic potential [ 59 ] or related to spoilage [ 60 ]. The LAB identified in this study included the genera Lactococcus,Leuconostoc,Enterococcus,Lactobacillus,Carnobacterium and Streptococcus. These gram-positive bacteria have frequently been identified in dairy products [ 57 , 61 ], and their presence in the raw milk of ewe breeds, other than Latxa, has been confirmed by HTS, albeit at different abundances [42–44]. The environmental bacterial genera identified in this study included Obesumbacterium, Roseburia and Prosthecobacter. These genera have been isolated from different natural sources, such as soil, fresh and salt water as well as animal and human gut [ 62 – 64 ]; however, to the best of our knowledge, no study has reported their presence in raw ewe milk. The non-desirable bacterial genera identified in this study included Pseudomonas, Clostridium,Staphylococcus and Bacillus, which are widely known pathogens [ 59 , 65 ]. For instance, Pseudomonas is the most important psychrotrophic bacteria in raw milk, which may even predominate in refrigerated milk [ 10 ]. It comes from natural environment [ 66 ] and has been related to hygiene conditions [ 67 , 68 ]. Some species belonging to the genera Buttiauxella, Serratia,Chryseobacterium,Eubacterium,Raoultella,Ruminococcus,Pantoea,Stenotrophomonas, Bacteroides,Flavobacterium and Acinetobacter have also been described as opportunistic or as emerging pathogens [ 69 – 79 ]. Moreover, some of these genera, such as Serratia and Clostridium, are also related to milk spoilage, resulting in off-flavours [ 80 ] and to the cheese blowing defect because of CO 2 production [ 81 ]. The presence of these bacteria in raw ewe milk has been reported only in a few studies [ 42 , 59 , 82 , 83 ]. To the best of our knowledge, the genera Buttiauxella,Serratia,Eubacterium,Raoultella,Ruminococcus and Bacteroides have not been identified in raw ewe milk so far. 3.2.2. Bacterial Shifts during the Cheese Making Process Next, the effect of the cheese making process, which encompasses all the production stages from milk to 1-day-old ripened cheeses, on microbiota was analysed. In this way, the bacterial composition of Latxa ewe raw milk and 1-day-old ripened Idiazabal cheese was compared. In 1-day-old ripened cheese samples, bacteria belonging to 19 phyla, 160 families and 450 genera were detected; thus, the number of identified bacterial families and genera were similar between 1-day-old ripened cheese and raw milk samples, but the number of bacterial phyla identified in cheese was less than that identified in raw milk. However, the cheese making process had a great impact on the abundance of bacterial communities (Figure 1C,D, Tables S1 and S2). At the phylum rank (Figure 1C, Table S1), the relative abundance of Firmicutes increased remarkably in 1-day-old ripened Idiazabal cheese samples (63.7–94.7%), while that of Proteobacteria decreased (2.61–22.4%), although remaining as the second most important phyla. In general, the abundances of the rest of phyla decreased, although the effect of the cheese making process was not statistically significant in all cases. The abundance of sequences classified as “others” was considerably reduced (<0.01%), and unidentified sequences accounted for lower, yet remarkable, abundance (1.21–18.6%). To date, very few HTS studies have analysed the effect of the cheese making process on bacterial communities in raw ewe milk cheeses [43,44] , and even fewer at the phylum rank [ 26 ]. In comparison to raw ewe milk-derived cheeses, more HTS studies have been conducted on cow milk-derived cheeses [ 29 , 84 ]. De Pasquale et al. [ 26 ] have reported an increase in Firmicutes abundance and a decrease in Proteobacteria abundance in Canestrato Pugliese raw ewe milk-derived cheese, but the changes were more drastic than those observed in this study. No information could be found in the literature concerning the effect of the manufacturing process on the remaining phyla. The effect of the cheese making process on the main bacterial genera is shown in Figure 1D and Table S2. Within LAB, Lactococcus was the most abundant genus in 1-day-old ripened cheese samples collected from all producers (52.5–93.2%), although a notably lower abundance was observed for producer A. The effect of the cheese making process Biology 2022,11, 769 8 of 21 on the remaining genera in the LAB group varied with the producer. The abundance of Lactobacillus decreased in cheese samples collected from all producers, except producer A (<0.200% in all producers); abundances of Leuconostoc and Carnobacterium were higher in producer A samples (4.48% and 4.40%, respectively); abundances of Streptococcus and Enterococcus were slightly higher in the cheese samples of producer A (0.507% and 0.458%, respectively) and producer D (0.993% and 0.892%, respectively). The genus Lactococcus was predominant during the cheese making process because of its presence in the starter culture, confirming that bacteria comprising the starter culture grow and predominate, as has been previously observed for Pecorino Siciliano cheese [ 39 ]. The proliferation of non-starter LAB (NSLAB) has been reported previously, although there are clear differences according to the type of cheese. For instance, Lactococcus and Lactobacillus have been reported as predominant in Caciofiore della Sibilla cheese [ 43 ], whereas Lactococcus,Carnobacterium and Enterococcus predominate in Canestrato Pugliese cheese [26]. In general, the abundance of non-desirable bacteria was less than 1% after cheese making, although the abundance of some bacterial genera, namely Buttiauxella (0–5.79%), Serratia (0.00179–2.16%) and Raoultella (0.0151–1.43%), was maintained at a remarkable level or even increased in cheese obtained from some producers (Figure 1D, Table S2). The opportunistic bacteria Hafnia,Brevibacterium and Psychrobacter [ 85 – 87 ], which had low abundance in milk (<1%), also increased their abundances in the cheese of some producers (0.00282–9.62%, 0.0210–2.43% and 0.00168–2.28%, respectively). Notably, these bacterial genera exhibit lipase and/or protease activities [ 88 – 90 ], and produce interesting volatile compounds (such as 1-hexanol, 1-propanol, propyl butanoate or butyl butanoate), affecting cheese quality [ 91 – 93 ]. Overall, the abundance of environmental bacteria decreased during cheese making, although Obesumbacterium maintained a remarkable abundance in samples from producer A (1.89%). Moreover, the environmental genus Chromohalobacter [ 94 ], which showed low abundance in milk, exhibited higher abundance in cheese, especially that obtained from producer C (1.78%). The abundance of bacteria classified as “others” and of unidentified bacteria in cheese (1.31–2.74% and 1.26–18.6%, respectively) was lower than that in milk (Figure 1D, Table S2). Suppression of the growth of environmental and nondesirable bacteria, such as Pseudomonas or Staphylococcus, during cheese making has been reported previously [ 26 , 43 ]. Nonetheless, little has been reported about the prevalence of opportunistic or emerging pathogens and environmental bacteria after the cheese making process using raw ewe milk. De Pasquale et al. [26] have detected Raoultella in Canestrato Pugliese cheese but not in raw milk and Alegría et al. [ 32 ] have reported a prevalence of Chromohalobacter in fresh Oscypek cheese. Hafnia and Psychrobacter have been identified in other cheeses prepared from raw ewe milk [ 38 , 95 ], although the effect of the cheese making process on the abundance of these bacteria is unknown. The cheese making process adds other factors that can influence the bacterial communities [ 9 ], in addition to factors that determine the milk microbiota (Section 3.2.1). Briefly, the conversion of milk to cheese decreases the pH to 4.5–5.3, which interferes with the growth of most bacteria, except LAB [ 9 , 57 ]. The NaCl concentration of the brine and low salt tolerance of most bacteria only facilitate the growth of LAB [ 57 ] and halophiles, such as Psychrobacter [ 96 ] and Chromohalobacter [ 94 ]. The decrease in moisture content and water activity (a w ) also suppresses the proliferation of most bacteria, except LAB, because of their resistance to reduced a w values [ 9 , 57 ]. Moreover, variation that occurs in the redox potential during the conversion of milk to cheese only allows the growth of facultative or obligate anaerobic bacteria [ 57 ]. It is worth mentioning that artisanal rennet employed for the production of some raw ewe milk cheeses could be an important source of microorganisms, for example LAB [ 8 ]. The use of lamb rennet paste containing pregastric lipase results in higher lipolysis and the development of the characteristic flavour of Idiazabal cheese [ 97 ]. Although artisanal rennet contains high levels of a wide range of microorganisms, including aerobic mesophilic bacteria [ 98 ], no significant differences have been detected in microbial counts in Idiazabal cheeses prepared using artisanal or commercial rennet [ 99 ]; however, it would be interesting to elucidate this aspect using Biology 2022,11, 769 9 of 21 culture-independent methods, such as HTS. Finally, it has been observed that small differences in the environment of dairy facilities producing artisanal cheeses can lead to the development of site-specific “household” microbiota [ 100 ]. Therefore, these factors could explain the differences in bacterial composition observed among different raw ewe milk cheeses and among producers producing the same type of cheese. 3.2.3. Bacterial Shifts during the Cheese Ripening Process Finally, the effect of the ripening process on the bacterial composition of cheese was studied. A total of 23 phyla, 197 families and 583 genera were identified throughout the cheese ripening process; thus, the number of bacterial families and genera was higher during cheese ripening than in raw milk and in cheese after the cheese making process. At the phylum level (Table S1), the abundance of Firmicutes increased (from a mean of 79.4% at 1 day of ripening to 97.7% at 120 days of ripening), while that of Proteobacteria decreased sharply (from 8.56% to 0.116%). In general, abundance of the remaining phyla not predominant during the cheese making process and that of “others” and unidentified bacteria were reduced, except Actinobacteria in the cheese of some producers; nonetheless, the change in abundance levels was not significant for all phyla. Overall, the predominance of Firmicutes and reduction in the abundance of Proteobacteria and remaining phyla have previously been reported in other raw ewe milk cheeses such as Liqvan cheese [38,44]. The cheese ripening time had a considerable impact on bacterial abundance at the genus level, resulting in large differences among producers (Figure 2, Table S2). Within LAB, Lactococcus remained the most dominant genus in Idiazabal cheese during ripening at all times and for all producers (mean abundance: 74.9% at 1 day of ripening, and 74.5% at 120 days of ripening), except producer A, which showed notably lower proportions of Lactococcus. The effect of ripening time on bacterial abundance was significant only for Lactobacillus, with an increase in its abundance for all producers (from 0.0949% to 8.96%), while the evolution of the abundance of the remaining genera varied with the producer. In cheeses from producer A, the abundance of Leuconostoc, which increased after cheese making, was unquestionably promoted by ripening time (from 4.48% to 31.0%), whereas that of Carnobacterium decreased (from 4.40% to 0.330%). In cheeses obtained from producers A and D, the abundances of Streptococcus and Enterococcus, which increased during the cheese making process, also increased during ripening (from 0.750% to 4.52% and from 0.675% to 2.12%, respectively). Overall, taking into account LAB dynamics ( Table S2 ) and their correlations during ripening time (Figure 3), a clear pattern was observed. The abundance of Lactococcus decreased over 30 or 60 days of ripening, depending on the producer, when NSLAB (Leuconostoc,Lactobacillus,Streptococcus and Enterococcus) began to proliferate. In other words, from the first ripening month on these NSLAB begin to proliferate and become an important part of the final microbiota of the cheese. The predominance of bacteria added as part of the starter culture has also been previously reported during the ripening of other raw ewe milk cheeses, such as Pecorino Siciliano cheese [ 38 , 39 ]. However, lactose depletion, salt concentration, and low pH and temperature decrease the viability of starter LAB, and depending on lysis rates, the NSLAB gain importance [ 101 ]. The proliferation of Lactobacillus,Leuconostoc,Streptococcus or Enterococcus has also been observed in other raw ewe milk cheeses [ 26 , 38 , 39 , 95 ]; however, the NSLAB composition of other types of raw ewe milk cheeses is different from that of Idiazabal cheese [ 38 , 39 , 43 , 44 ]. These differences are important, since NSLAB affect, among others, the proteolysis and lipolysis of cheese, and consequently, its final properties, including flavour and texture [102–104]. Biology 2022,11, 769 16 of 21 Biology 2022, 11, x FOR PEER REVIEW 16 of 22 Figure 6. PCoA of bacterial beta diversity at genus rank based on Bray-Curtis (A) and Jaccard (B) dissimilarities. Taking together, alpha and beta diversity indices confirmed the results of the indepth analysis of microbial shifts, univariate analysis (Kruskal-Wallis test) and multivariate analyses (PERMANOVA, PCA and OPLS-DA). The cheese making and ripening processes had an undoubted impact on the bacterial communities. Overall, bacteria from the starter culture predominated at the beginning of ripening, but after 30 or 60 days of ripening, the bacteria from raw milk, especially NSLAB, began to proliferate and become noticeable. Nonetheless, clear differences in the microbial composition of raw ewe milk and cheese samples were observed among producers, which could indicate that differences in practices, such as flock management and milking, as well as parameters selected during cheese making and ripening processes would determine the final microbiota. 4. Conclusions This is the first HTS study carried out with the objective of characterizing the microbiota of Latxa ewe raw milk and examining the bacterial shifts that occur during the production and ripening of Idiazabal cheese. This research confirms that HTS techniques allow a better understanding of the microbial communities, which could not be achieved previously using culture-dependent techniques. Several bacterial genera were detected for the first time in raw ewe milk and cheese. Both the cheese making process and ripening time had a remarkable impact on bacterial communities, although considerable differences were observed among producers. Thus, the use of raw milk and the practices and conditions employed by each producer for flock management, milking and cheese making and ripening could determine the microbiota. The growth of LAB was promoted throughout the cheese making and ripening processes, whereas that of non-desirable and environmental bacteria was inhibited. However, LAB composition differed among producers, and the growth of NSLAB was promoted after 30 or 60 days of ripening. In addition, in some cases, bacteria related to the production of volatile compounds (such as Hafnia, Brevibacterium and Psychrobacter) showed notable abundance during the first few weeks of ripening. Figure 6. PCoA of bacterial beta diversity at genus rank based on Bray-Curtis ( A ) and Jaccard (B) dissimilarities. Taking together, alpha and beta diversity indices confirmed the results of the in-depth analysis of microbial shifts, univariate analysis (Kruskal-Wallis test) and multivariate analyses (PERMANOVA, PCA and OPLS-DA). The cheese making and ripening processes had an undoubted impact on the bacterial communities. Overall, bacteria from the starter culture predominated at the beginning of ripening, but after 30 or 60 days of ripening, the bacteria from raw milk, especially NSLAB, began to proliferate and become noticeable. Nonetheless, clear differences in the microbial composition of raw ewe milk and cheese samples were observed among producers, which could indicate that differences in practices, such as flock management and milking, as well as parameters selected during cheese making and ripening processes would determine the final microbiota. 4. Conclusions This is the first HTS study carried out with the objective of characterizing the microbiota of Latxa ewe raw milk and examining the bacterial shifts that occur during the production and ripening of Idiazabal cheese. This research confirms that HTS techniques allow a better understanding of the microbial communities, which could not be achieved previously using culture-dependent techniques. Several bacterial genera were detected for the first time in raw ewe milk and cheese. Both the cheese making process and ripening time had a remarkable impact on bacterial communities, although considerable differences were observed among producers. Thus, the use of raw milk and the practices and conditions employed by each producer for flock management, milking and cheese making and ripening could determine the microbiota. The growth of LAB was promoted throughout the cheese making and ripening processes, whereas that of non-desirable and environmental bacteria was inhibited. However, LAB composition differed among producers, and the growth of NSLAB was promoted after 30 or 60 days of ripening. In addition, in some cases, bacteria related to the production of volatile compounds (such as Hafnia,Brevibacterium and Psychrobacter) showed notable abundance during the first few weeks of ripening. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biology11050769/s1, Figure S1: Rarefaction curves of microbial populations of the studied samples from each producer. Each graph represents a producer (A, B, C and D) and each line is coloured according to the Sample ID; Figure S2: Scores and loadings plots of Biology 2022,11, 769 17 of 21 PCA based on main bacterial genera of Idiazabal cheeses from 4 producers (A, B, C and D). Samples are coloured according to the producer and labels indicate samples identification; Table S1: Mean and standard deviation of bacterial phyla of Latxa ewe raw milk and Idiazabal cheese samples at 6 ripening times (1, 7, 14, 30, 60 and 120 days) from 4 producers (A, B, C and D) (n= 52).; Table S2: Mean and standard deviation of bacterial genera of Latxa ewe raw milk and Idiazabal cheese samples at 6 ripening times (1, 7, 14, 30, 60 and 120 days) from 4 producers (A, B, C and D) (n= 52): Table S3: α -diversity indices of Latxa ewe raw milk and Idiazabal cheese samples at 6 ripening times (1, 7, 14, 30, 60 and 120 days) from 4 producers (A, B, C and D) (n= 52). Author Contributions: Conceptualization, methodology, validation, investigation and resources, G.S.-G., I.H., G.A. and M.V; formal analysis and data curation, G.S.-G., I.H. and G.A. writing—original draft preparation, G.S.-G.; writing—review and editing, G.S.-G., I.H., G.A. and M.V.; visualization, G.S.-G.; supervision, project administration and funding acquisition, M.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Basque Government, grant to Research Groups number IT944-16. G. Santamarina-García received a predoctoral grant from the University of the Basque Country (UPV/EHU). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data are not publicly available yet as some data sets are being used for additional publications. 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