Recovery and differentiation of long ripened cheese microflora through a new cheese-based cultural medium

A partial picture of the typical microflora of PDO Parmigiano Reggiano cheese was achieved by studying the cultivability of lactic acid bacteria associated with its manufacturing and ripening. A comprehensive sampling design allowed for the analysis of the cheese microflora during its production over 20 months of ripening. An innovative cheese agar medium (CAM) was prepared after testing 18 formulations all based on grated Parmigiano Reggiano ripened cheese. During cheese manufacturing and ripening, different samples were sampled and their microflora was recovered using CAM in comparison with other traditional media. Colonies which formed units from the different agar media tested were picked and isolated; the phylogenetic positions of 154 isolated strains were studied at level of species by 16S-rRNA gene sequencing. CAM seems to be able to recover the minority population coming from milk and whey starter, hardly estimable, during the first hours of production, on traditional media.     

Pyroglutamic acid in cheese: presence, origin, and correlation with ripening time of Grana Padano cheese

Pyroglutamic acid is present in many cheese varieties and particularly in high amounts (0.5 g/100 g of cheese) in extensively ripened Italian cheeses (Grana Padano and Parmigiano Reggiano) that are produced with thermophilic lactic acid bacteria as starters. The mechanism of pyroglutamic acid formation in cheese seems to be mostly enzymatic, as demonstrated by the presence of only L-pyroglutamic acid enantiomer. Thermophilic lactobacilli are involved in pyroglutamic acid production, as suggested by the low pyroglutamic acid content found in Bagos, a ripened Italian mountain cheese produced without addition of starter. Because milk pasteurization did not influence the pyroglutamic acid content in the ripened Grana Padano cheese, the formation of pyroglutamic acid mainly depends on the whey starter microflora rather than that of raw milk. Pyroglutamic acid concentration is linearly correlated (R2 = 0.94) with the age of Grana Padano cheese.     

Invited review: Microbial evolution in raw-milk,long-ripened cheeses produced using undefined natural whey starters

The robustness of the starter culture during cheese fermentation is enhanced by the presence of a rich consortium of microbes. Natural starters are consortia of microbes undoubtedly richer than selected starters. Among natural starters, natural whey starters (NWS) are the most common cultures currently used to produce different varieties of cheeses. Undefined NWS are typically used for Italian cooked, long-ripened, extra-hard, raw milk cheeses, such as Parmigiano Reggiano and Grana Padano. Together with raw milk microbiota, NWS are responsible for most cheese characteristics. The microbial ecology of these 2 cheese varieties is based on a complex interaction among starter lactic acid bacteria (SLAB) and nonstarter lactic acid bacteria (NSLAB), which are characterized by their different abilities to grow in a changing substrate. This review aims to summarize the latest findings on Parmigiano Reggiano and Grana Padano to better understand the dynamics of SLAB, which mainly arise from NWS, and NSLAB, which mainly arise from raw milk, and their possible role in determining the characteristics of these cheeses. The review is presented in 4 main sections. The first summarizes the main microbiological and chemical properties of the ripened cheese as determined by cheese-making process variables, as these variables may affect microbial growth. The second describes the microbiota of raw milk as affected by specific milk treatments, from milking to the filling of the cheese milk vat. The third describes the microbiota of NWS, and the fourth reviews the knowledge available on microbial dynamics from curd to ripened cheese. As the dynamics and functionality of complex undefined NWS is one of the most important areas of focus in current food microbiology research, this review may serve as a good starting point for implementing future studies on microbial diversity and functionality of undefined cheese starter cultures.     

Production of pyroglutamic acid by thermophilic lactic acid bacteria in hard-cooked mini-cheeses

Pyroglutamic acid is present in high amounts (0.5g/ 100g) in many cheese varieties-and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano. An in vivo model system for cooked mini-cheese production and ripening acceleration was set up to demonstrate the ability of thermophilic lactic acid bacteria, used as a starter, to produce pyroglutamic acid (pGlu). In mini-cheeses stored at 38 and 30 degrees C for up to 45 d, all starters tested produced different amounts of pGlu. In descending order of pGlu production, the bacteria analyzed were: Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. lactis. Evidence for the presence of glutamine to pGlu cyclase activity in lactic acid bacteria was provided. Cell lysates obtained from cultures of L. helveticus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and S. thermophilus showed the ability to cyclize glutamine to pGlu, resulting in processing yields from 1.4 to 30.3%, depending on the subspecies. Formation of pGlu from free glutamine appeared to be similar to that observed using a glutamine-glutamine dipeptide substrate. Under the experimental conditions applied, pGlu aminopeptidase activity was only detected in L. helveticus. Thus, pGlu formation in long-ripened cooked cheese may depend on the activity of thermophilic lactic acid bacteria.     

Technological characterization of the natural lactic acid bacteria of artisanal Turkish White Pickled cheese

The aim of this study was to characterize the lactic acid bacteria (LAB) isolated from White Pickled cheeses produced with traditional methods; and to improve the quality of cheesemaking with a selection of bacterial cultures from artisanal White cheeses. LAB were isolated and identified from 30 White Pickled cheese samples collected from various cities in Turkey. Also, the numbers of several microbial groups (total aerobic mesophilic bacteria, LAB, enterococci, coliforms, moulds and yeasts) of cheese samples were enumerated. Lactobacilli, lactococci and enterococci were the most abundant microbial groups. The numbers of Enterococcus and Lactobacillus isolates were higher than those of the other LAB. Enterococcus faecalis (24.43%), Enterococcus faecium (17.61%) and Lactobacillus fermentum (19.88%) isolates were the most frequently isolated species. Lactococcus strains showed the highest acidifying activity, followed by Enterococcus and Lactobacillus strains. Proteolytic activity of Enterococcus faecalis strains was higher than that of the other enterococci species, except Enterococcus avium strains. Within lactobacilli strains, the highest mean proteolytic activity was that of Lactobacillus bifermentans, Lactobacillus brevis and Lactobacillus casei strains.     

新疆伊犁牧区传统手工奶酪中微生物多样性及其功能分析

为探究新疆伊犁牧区传统手工奶酪中的可培养乳酸菌和细菌多样性,首先采用Illumina MiSeq高通量测序技术对细菌16S rRNA基因的V1～V3可变区进行测序分析,结果显示：14 份奶酪中共存在493 种细菌属和780 种细菌种,其中奶酪中的优势细菌属和优势乳酸菌属为雷尔氏菌属和乳杆菌属,优势乳酸菌种为瑞士乳杆菌,且奶酪中还存在致病菌。在此基础上,利用传统培养法从MRS培养基中筛选出68 株可培养疑似乳酸菌,经生理生化实验及16S rDNA序列同源性分析技术对其进行属种鉴定。结果表明：68 株疑似乳酸菌分别为短乳杆菌、融合魏斯氏菌、屎肠球菌、粪肠球菌、耐久肠球菌、鸟肠球菌、棉籽糖肠球菌、植物乳杆菌、瑞士乳杆菌,其中融合魏斯氏菌为奶酪中的优势乳酸菌种。在乳酸菌的属种鉴定上,传统培养法能够更准确地将乳酸菌鉴定到种水平。奶酪微生物基因COG功能预测结果显示：奶酪中的微生物富含与氨基酸转运和代谢有关的基因。将各个样品的微生物群落组成与功能组成进行比较,发现各个样品的细菌群落组成虽存在明显差异,但其功能组成却具有高度相似性。     

Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology

The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.     

Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Headspace analysis of Italian and New Zealand parmesan cheeses

New Zealand is a leader in the global dairy industry. Milk powder is the principal export product, but there is also a prominent cheese manufacturing industry, catering more for the domestic market. The Selected Ion Flow Tube-Mass Spectrometric (SIFT-MS) technique was used to compare 4 New Zealand cheeses marketed as "parmesan" with 4 Italian Parmigiano Reggiano and Grana Padano cheeses. The cheese headspace was analyzed in real time without any sample preconcentration. Total of 38 volatile compounds in the cheese headspace were monitored with headspace concentrations varying between single digit parts per billion (ppb) to tens of parts per million (ppm). When the results were subjected to multivariate statistical analysis, a clear discrimination was found between the New Zealand "parmesan" and Italian cheeses based solely on the measured concentrations of these volatile compounds. If the volatile compounds used in the analyses were restricted to known odor-active compounds in Parmigiano Reggiano cheese, the ability to discriminate between the cheeses was maintained. The analyses also showed that it was possible to clearly differentiate between the different processing plants in individual countries. Important discriminatory volatiles in the samples tested were butanoic acid and phenylacetaldehyde for discriminating between Italian cheeses and ethyl butyrate, acetaldehyde and methylbutanals between New Zealand cheeses. We conclude that the New Zealand "parmesans" do not provide a good representation of the aroma of Italian "parmesans." PRACTICAL APPLICATION: SIFT-MS has been shown to clearly differentiate both country of origin and the manufacturer of "parmesan" cheeses made in Italy and New Zealand based on differences in volatile organic compounds. Thus this method will have benefit for use in the quality control of "parmesan" and other cheese varieties.     

Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria

The aim of this research was to study the bacterial populations involved in the production of artisanal Raschera PDO cheese (Italian Maritime Alps, northwest Italy) in order to collect preliminary knowledge on indigenous lactic acid bacteria (LAB). A total of 21 samples of Raschera PDO cheese, collected from six dairy farms located in the production area, were submitted to microbiological analysis. LAB were randomly isolated from M17 agar, MRS agar and KAA plates and identified by combining PCR 16S-23S rRNA gene spacer analysis, species-specific primers and 16S rRNA gene sequencing. Biodiversity of Lactococcus lactis subsp. lactis isolates was investigated by RAPD-PCR. LAB microflora showed the highest count values among all microbial groups targeted. They reached counts of 10(9) colony forming unit (cfu)/g in cheese samples after 3 days of salting and 15 days of ripening. Yeast population also showed considerable count values, while enterococci and coagulase-negative cocci (CNC) did not overcome 10(7)cfu/g. L. lactis subsp. lactis was the species most frequently isolated from Raschera PDO samples at all different production stages while in aged cheeses Lactobacillus paracasei was frequently isolated. RAPD-PCR highlighted that isolates of L. lactis subsp. lactis isolated from Raschera PDO were highly homogeneous.     

Improving the quality of European hard-cheeses by controlling of interactions between lactic acid bacteria and propionibacteria

This work aimed to improve the knowledge and the control of interactions between lactic acid bacteria (LAB) and propionibacteria (PAB) in order to control the propionic acid fermentation in European hard cheeses, and therefore their quality. Among more than 50 couples of strains LAB/PAB used in cheese making, some pairs of strains were chosen in function of quality results on cheeses (sensorial analysis). A whey model system and an alternative conductimetric method, with good reproducibility were used to study interactions. The lipolytic activity of PAB strains varied by a factor 4 to 5. The aroma of ripened Emmental cheese was linked to the level and the type of lipolysis in the cheese, correlated with lipolytic activity of PAB. The main pathways of the utilisation of lactate by PAB, studied by ¹³C RMN, were useful to understand the balance between final products: acetic, propionic acid, CO₂.     

Using PacBio sequencing to investigate the bacterial microbiota of traditional Buryatian cottage cheese and comparison with Italian and Kazakhstan artisanal cheeses

Traditional fermented dairy foods including cottage cheese have been major components of the Buryatia diet for centuries. Buryatian cheeses have maintained not only their unique taste and flavor but also their rich natural lactic acid bacteria (LAB) content. However, relatively few studies have described their microbial communities or explored their potential to serve as LAB resources. In this study, the bacterial microbiota community of 7 traditional artisan cheeses produced by local Buryatian families was investigated using single-molecule, real-time sequencing. In addition, we compared the bacterial microbiota of the Buryatian cheese samples with data sets of cheeses from Kazakhstan and Italy. Furthermore, we isolated and preserved several LAB samples from Buryatian cheese. A total of 62 LAB strains (belonging to 6 genera and 14 species or subspecies) were isolated from 7 samples of Buryatian cheese. Full-length 16S rRNA sequencing of the microbiota revealed 145 species of 82 bacterial genera, belonging to 7 phyla. The most dominant species was Lactococcus lactis (43.89%). Data sets of cheeses from Italy and Kazakhstan were retrieved from public databases. Principal component analysis and multivariate ANOVA showed marked differences in the structure of the microbiota communities in the cheese data sets from the 3 regions. Linear discriminant analyses of the effect size identified 48 discriminant bacterial clades among the 3 groups, which might have contributed to the observed structural differences. Our results indicate that the bacterial communities of traditional artisan cheeses vary depending on geographic origin. In addition, we isolated novel and valuable LAB resources for the improvement of cottage cheese production.     

Discrimination of commercial Caciocavallo cheeses on the basis of the diversity of lactic microflora and primary proteolysis

A wide variety of semi-hard pasta-filata cheese fall under the common name “Caciocavallo”, including Caciocavallo Silano, a Protected Denomination of Origin (PDO) cheese produced in Southern Italy. Multivariate analysis techniques were used to investigate the specificity of Caciocavallo Silano PDO cheeses within the area of production and to compare them with cheeses of similar denomination or cheeses produced with similar technology. The composition of lactic acid bacteria (LAB) communities at the species level and the primary proteolysis of commercial cheeses were assessed. A high variability was observed in the composition of LAB communities, and no clear relationship was found with either the region of origin or the cheese denomination. However, the microflora of PDO cheeses was less heterogeneous than that of non-PDO cheeses and composed mainly of non-starter lactic acid bacteria (NSLAB) such as Lactobacillus paracasei, L. fermentum, and L. plantarum. A high diversity in the proteolytic pattern of the cheeses was also found. This diversity was associated with the trends observed in the community of LAB populating each of the cheeses. The main problems concerning the protection of the denomination are discussed.     

Segmentation of Parmigiano Reggiano dairies according to cheese-making technology and relationships with the aspect of the cheese curd surface at the moment of its extraction from the cheese vat

Parmigiano Reggiano cheese dairies develop specific cheese-making strategies to adapt the variable characteristics of raw, not standardized milk to the final goal of obtaining cheese consistent with the standard. Analyzing 1,175 cheese-making reports from 30 out of 383 dairies associated with the Parmigiano Reggiano Consortium in 2010 and 2011, 4 groups of Parmigiano Reggiano dairies using specific cheese-making technologies were discriminated by means of multiple linear discriminant analysis. Cheese makers manage cheese-making practices to obtain curd with different roughness properties, classified according to jargon words such as “rigata” and “giusta” or synonyms, because they believe that the roughness of the cheese curd surface immediately after the extraction from the vat is associated with different whey-draining properties and to the final outcome of the cheese. The aspect of the surfaces of the curds produced by the 4 groups of dairies was different according to the technology applied by each group. Cutting of the coagulum when it is still soft for a longer time and faster cooking of the cheese curd grains were associated with a less rough appearance of the surface of the curd, whereas under the opposite conditions, cutting the coagulum when it is firm for a shorter time, led to a curd with a rougher surface. These findings partially support the traditional feeling of Parmigiano Reggiano cheese makers, who consider the curd surface aspect one of the main drivers for their technological choices; to date, however, no data are provided about correlation between the aspect of the curd and the quality of the ripened cheese. If a sufficiently strong correlation could be demonstrated by the future development of the research, the operational effectiveness of Parmigiano Reggiano dairies will be able to largely benefit from the availability of sound and early process markers.     

Diversity of lactic acid bacteria in two Flemish artisan raw milk Gouda-type cheeses

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to study the diversity of lactic acid bacteria (LAB) in two Flemish artisan raw milk Gouda-type cheeses. In parallel, conventional culturing was performed. Isolates were identified using (GTG)(5)-PCR and sequence analysis of 16S rRNA and pheS genes. Discriminant analysis revealed some differences in overall LAB diversity between the two batches and between the two cheeses. Within each batch, the diversity of 8- and 12-week-old cheeses was relatively similar. Conventional isolation mainly revealed the presence of Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rhamnosus and Pediococcus pentosaceus. PCR-DGGE revealed the presence of three species of which no isolates were recovered, i.e. Enterococcus faecalis, Lactobacillus parabuchneri and Lactobacillus gallinarum. Conversely, not all isolated bacteria were detected by PCR-DGGE. We recommend the integrated use of culture-dependent and -independent approaches to maximally encompass the taxonomic spectrum of LAB occurring in Gouda-type and other cheeses.     

Lactic acid bacteria profiles and tyramine and tryptamine contents of Turkish tulum cheeses

Tulum cheese, commonly produced in Turkey, is a traditional Turkish dairy product made from raw milk. In this research, 20 samples of tulum cheese were purchased from different markets in Isparta and Ankara (Turkey) and the populations of lactic acid bacteria (LAB) and contents of biogenic amines of Turkish tulum cheeses were investigated. The mean counts of LAB (as log10 cfu/g) were 7.22±0.67 on MRS agar, 6.93±1.77 on M17 agar, 7.28±0.72 on APT agar and 5.98±2.20 on ADA agar. Isolates of LAB obtained from 20 tulum cheeses were characterised as lactobacilli (48.5%), enterococci (32.7%), lactococci (15.8%) and streptococci (3.0%). Lactobacillus paracasei ssp. paracasei, Lactobacillus bifermentans and Enterococcus faecium predominated among the LAB in the samples of tulum cheeses; these organisms represented 30.2% of the isolates. As amino acids, tyrosine and tryptophan were determined in tulum cheeses. Tyramine and tryptamine, biogenic amines, were found at 0.00–329.00 and 0.32–40.44 mg/kg, respectively.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.     

Evaluation of genetic polymorphism among Lactobacillus rhamnosus non-starter Parmigiano Reggiano cheese strains

Parmigiano Reggiano (PR) is an Italian cooked, long-ripened cheese made with unheated cow's milk and natural whey starter. The microflora is involved in the manufacturing of this cheese, arising from the natural whey starter, the raw milk and the environment. Molecular studies have shown that mesophilic non-starter lactic acid bacteria (NSLAB) are the dominant microflora present during the ripening of PR. In this study, a characterisation of Lactobacillus rhamnosus isolated from a single PR manufacturing and ripening process is reported, using a combination of genotypic fingerprinting techniques (RAPD-PCR and REP-PCR). The intraspecies heterogeneity evidenced for 66 strains is correlated to their abilities to adapt to specific environmental and technological conditions. The detection of biotypes that correlate with specific moments in cheese ripening or differential development throughout this process suggests that these strains may have specific roles closely linked to their peculiar technological properties.     

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes’ milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes’ milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.