Prediction of indicators of cow diet composition and authentication of feeding specifications of Protected Designation of Origin cheese using mid-infrared spectroscopy on milk.

The ability of mid-infrared spectroscopy (MIR) to predict indicators (1) of diet composition in dairy herds and (2) for the authentication of the cow feeding restrictions included in the specification of 2 Protected Designation of Origin (PDO) cheeses (Cantal and Laguiole) was tested on 7,607 bulk milk spectra from 1,355 farms located in the Massif Central area of France. For each milk sample, the corresponding cow diet composition data were obtained through on-farm surveys. The cow diet compositions varied largely (i.e., from full grazing for extensive farming systems to corn silage-based diets, which are typical of more intensive farming systems). Partial least square regression and discriminant analysis were used to predict the proportion of different feedstuffs in the cows' diets and to authenticate the cow feeding restrictions for the PDO cheese specifications, respectively. The groups for the discriminant analysis were created by dividing the data set according to the threshold of a specific feedstuff. They were issued based on the specifications of the restriction of the PDO cheese. The pasture proportion in the cows' diets was predicted by MIR with an coefficient of determination in external validation (R2V) = 0.81 and a standard error of prediction of 11.7% dry matter. Pasture + hay, corn silage, conserved herbage, fermented forage, and total herbage proportion in the cows' diets were predicted with a R2V >0.61 and a standard error of prediction <14.8. The discrimination models for pasture presence, pasture ≥50%, and pasture ≥57% in the cows' diets achieved an accuracy and specificity ≥90%. A sensitivity and precision ≥85% were also observed for the pasture proportion discrimination models, but both of these indexes decreased at increasing thresholds from 0 to 50, and 57% pasture in the cows' diets. An accuracy ≥80% was also observed for pasture + hay ≥72%, herbage ≥50%, pasture + hay ≥25%, absence of fermented herbage, absence of corn silage, and corn silage ≤30% in the cows' diets, but for several models, either the sensitivity or precision was lower than the accuracy. Models built on the simultaneous respect of all the criteria of the feeding restrictions of PDO cheese specifications achieved an accuracy, specificity, sensitivity, and precision >90%. Both the regression and discriminant MIR models for bulk milk can provide useful indicators of cow diet composition and PDO cheese specifications to producers and consumers (farmers, dairy plants).

Selected Cardoon (Cynara cardunculus L.) Genotypes Suitable for PDO Cheeses in Mediterranean Regions.

Cardoon flower extract is a traditional and exclusive rennet used for some PDO cheeses in several Mediterranean regions, due to its extremely high concentration in cardosins. In this preliminary study, six individual cardoon genotypes (1M - 6M) were selected because they revealed a wide and consistent diversity of total and specific cardosin concentrations in flowers. During three growing seasons, the stability of 12 biochemical characteristics of flower extracts and 26 plant morphological descriptors was confirmed. Surprisingly, the cardosin profiles of each genotype, based on four main groups A0, A1, A and B, were stable during the annual flower harvesting period and over all three years using ion-exchange chromatography and native-PAGE electrophoresis. This knowledge will allow an improvement in the quality and standardization of cardosin profiles from cardoon flowers used for cheese production and other innovative applications. The results obtained are promising for the development of a plant breeding program based on biochemical and morphological characteristics in order to obtain the most adapted plant architecture for combined purposes related to specific cardosins composition, flower and plant biomass production, and ease of harvesting.

1H-NMR Approach for the Discrimination of PDO Grana Padano Cheese from Non-PDO Cheeses.

Protected Designation of Origin cheeses are products with high-quality standards that can claim higher prices on the market. For this reason, non-PDO cheeses with lower quality can be mislabeled as PDO or mixed with it for economic gain especially when the product is in a shredded form. Luckily, the production of PDO cheese is subjected to strict procedural specification rules that result in a product with a defined profile of its metabolites, which can be used for authentication purposes. In this study, an NMR metabolomic approach combined with multivariate analysis was implemented to build a classification model able to discriminate PDO Grana Padano cheese from a large dataset of competitors. The great advantage of the proposed approach is a simple sample preparation, obtaining a holistic overview of the analyzed samples. The untargeted approach highlighted a "typical profile" of Grana Padano samples, which could be used for protection purposes. In parallel, the targeted results allowed us to identify potential chemicals, such as lactate, some amino acids and lipids. These initial results could open the road to a potential new additional tool to check the authenticity of PDO cheeses in the future.

Comparative analysis of microbial succession and proteolysis focusing on amino acid pathways in Asiago-PDO cheese from two dairies.

In this study, a comprehensive and comparative analysis was conducted on Italian Asiago-PDO cheese obtained from two different dairies named Dairy I and Dairy II using industrial and natural fermented milk, respectively. The analysis encompassed the evaluation of chemical composition, the succession of the microbiota during manufacture and ripening, and proteolysis mainly focusing on free individual amino acid (FAA) profiles. A metagenomic approach was used to investigate the cheese microbiome functionality. Differences in gross chemical composition were more evident during ripening, with Dairy II showing higher variability within batches. The microbiota varied significantly between the two dairies and ripening stages. The choice of starter culture shaped the microbiota during production and affected the microbial diversity of non-starter lactic acid bacteria (NSLAB) originated from the raw milk during ripening. Peptide chromatographic profiles and FAA concentrations increased as ripening progressed, with Dairy I showing higher production of FAA. Functional analysis of the metagenomes linked species to specific amino acid metabolism/catabolism pathways. The amino acid metabolism pathways, particularly those related to aromatic amino acids, lysine, and branched-chain amino acids, were affected by the presence of specific NSLAB species, which differed between the two dairies. The results obtained in this study reveal the impact of starter culture on peculiar cheese microbiota assemblies, which selectively targets amino acid pathways, providing insights into the potential flavor and aroma characteristics of Asiago-PDO cheese.

Effectiveness of modified atmosphere and vacuum packaging in preserving the volatilome of Stelvio PDO cheese over time.

We aimed to assay the effectiveness of vacuum or modified atmosphere packaging in preserving the organoleptic characteristics of already ripened slices of Stelvio Protected Designation of Origin cheese during 3 months of storage. A multi-omics panel, including metagenomic and metabolomic analyses, was implemented together with physicochemical and sensory analyses. Among the 177 volatiles identified, 30 out of the 50 potent odorants were found to be prevalent, regardless of packaging. Isovaleric acid showed the highest relative intensity in all samples. Caproic and caprylic acids always increased during storage, while metabolites such as dodecane and 2,3-butanediol always decreased. Slow proteolysis occurred during storage, but did not differentiate cheese samples. The type of packaging differentiated the microbiota and volatile profile, with modified atmosphere packaging keeping the volatilome more stable. Out of the 50 potent odorants, 9 were relevant to sample discrimination, with 8-nonen-2-one, 2-nonanone, and caproic acid being more abundant in stored samples.

Tracing the identity of Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" cheese using NMR spectroscopy and multivariate data analysis.

BACKGROUND: Nuclear magnetic resonance (NMR) spectroscopy is one of the well-established tools for food metabolomic analysis, as it proved to be very effective in authenticity and quality control of dairy products, as well as to follow product evolution during processing and storage. The analytical assessment of the EU mountain denomination label, specifically for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" (Mountain-CQ) cheese, has received limited attention. Although it was established in 2012 the EU mountain denomination label has not been much studied from an analytical point of view. Nonetheless, tracing a specific profile for the mountain products is essential to support the value chain of this specialty. RESULTS: The aim of the study was to produce an identity profile for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" (Mountain-CQ) cheese, and to differentiate it from Parmigiano Reggiano PDO samples (conventional-PDO) using 1H NMR spectroscopy coupled with multivariate data analysis. Three different approaches were applied and compared. First, the spectra-as-such were analysed after proper preprocessing. For the other two approaches, Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) was used for signals resolution and features extraction, either individually on manually-defined spectral intervals or by reapplying MCR-ALS on the whole spectra with selectivity constraints using the reconstructed "pure profiles" as initial estimates and targets. All approaches provided comparable information regarding the samples' distribution, as in all three cases the separation between the two product categories conventional-PDO and Mountain-CQ could be highlighted. Moreover, a novel MATLAB toolbox for features extraction via MCR-ALS was developed and used in synergy with the Chenomx library, allowing for a putative identification of the selected features. SIGNIFICANCE: A first identity profile for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" obtained by interpreting the metabolites signals in NMR spectroscopy was obtained. Our workflow and toolbox for generating the features dataset allows a more straightforward interpretation of the results, to overcome the limitations due to dimensionality and to peaks overlapping, but also to include the signals assignment and matching since the early stages of the data processing and analysis.

Impact of LAB from Serpa PDO Cheese in Cheese Models: Towards the Development of an Autochthonous Starter Culture.

Serpa is a protected designation of origin (PDO) cheese produced with raw ewes' milk and coagulated with Cynara cardunculus. Legislation does not allow for milk pasteurization and starter culture inoculation. Although natural Serpa's rich microbiota allows for the development of a unique organoleptic profile, it also suggests high heterogeneity. This raises issues in the final sensory and safety properties, leading to several losses in the sector. A possible solution to overcoming these issues is the development of an autochthonous starter culture. In the present work, some Serpa cheese Lactic acid bacteria (LAB)-isolated microorganisms, previously selected based on their safety, technological and protective performance, were tested in laboratory-scale cheeses. Their acidification, proteolysis (protein and peptide profile, nitrogen fractions, free amino acids (FAA)), and volatiles generation (volatile fatty acids (VFA) and esters) potential was investigated. Significant differences were found in all parameters analyzed, showing a considerable strain effect. Successive statistical analyses were performed to compare cheese models and Serpa PDO cheese. The strains L. plantarum PL1 and PL2 and the PL1 and L. paracasei PC mix were selected as the most promising, resulting in a closer lipolytic and proteolytic profile of Serpa PDO cheese. In future work, these inocula will be produced at a pilot scale and tested at the cheese level to validate their application.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

Consumer Preferences for Cheese Products with Quality Labels: The Case of Parmigiano Reggiano and Comté.

The paper examined the potential demand for a food specialty dairy product, cheese, with alternative multiple labels. A random-parameter logit model was applied to interpret the results of online discrete choice experiments (DCE) for the elicitation of the preference of the cheese consumers surveyed in two European countries, France (n = 400) and Italy (n = 408). We analyzed consumers' choices of quality-labeled cheeses, i.e., protected-designation-of-origin (PDO)-labeled Parmigiano Reggiano and Comté. Other features were tested, such as organic (Comté) and Mountain Product (Parmigiano Reggiano) labels, companies' brands and price. The paper contributes to the literature on credence attributes by examining consumers' willingness to pay (WTP) for differentiated cheese products in two EU countries, and by identifying the effects of personal characteristics, in terms of socio-demographics and level of product involvement, on the differences in preferences. The results show that price was the most important attribute in both countries, followed by the PDO quality label, particularly when paired with the second quality feature. Two cheese consumer segments were identified via latent class models in each country, helping producers to improve their marketing of agri-food products with a high gastronomic value and differentiation potential.

Organoleptic Chemical Markers of Serpa PDO Cheese Specificity.

Serpa is a protected designation of origin cheese produced with a vegetable coagulant (Cynara cardunculus L.) and raw ovine milk. Despite the unique sensory profile of raw milk cheeses, numerous parameters influence their sensory properties and safety. To protect the Serpa cheese quality and contribute to unifying their distinctive features, some rheologic and physicochemical parameters of cheeses from four PDO producers, in distinct seasons and with different sensory scores, were monitored. The results suggested a high chemical diversity and variation according to the dairy, month and season, which corroborates the significant heterogeneity. However, a higher incidence of some compounds was found: a group of free amino acids (Glu, Ala, Leu, Val and Phe), lactic and acetic acids, some volatile fatty acids (e.g., iC4, iC5, C6 and C12) and esters (e.g., ethyl butanoate, decanoate and dodecanoate). Through the successive statistical analysis, 13 variables were selected as chemical markers of Serpa cheese specificity: C3, C4, iC5, C12, Tyr, Trp, Ile, 2-undecanone, ethyl isovalerate, moisture content on a fat-free basis, the nitrogen-fractions (maturation index and non-protein and total nitrogen ratio) and G' 1 Hz. These sensory markers' identification will be essential to guide the selection and development of an autochthonous starter culture to improve cheese quality and safety issues and maintain some of the Serpa authenticity.

Long-Aged Parmigiano Reggiano PDO: Trace Element Determination Targeted to Health.

The concentrations of four health-related trace elements were measured using Atomic Absorption Spectroscopy in long-ripened (24- and 40-months) Parmigiano Reggiano (PR) PDO cheese, obtained from both summer and winter milk. To date, there are limited data on PR trace element concentrations, and no data about long-ripened cheese, especially when ripened for 40 months. Thus, the aim of this investigation is to determine chromium, manganese, selenium, and zinc concentrations, improving the available data on these trace elements and increasing knowledge of the biological properties of PR linked to their content in this cheese. The results show that 40-month ripened PR is a source of selenium and chromium, according to definitions under the European Regulation 1924/2006, as a 30 g cheese portion contains 11 ± 2 µg (summer milk) and 10 ± 1 µg (winter milk) of selenium and 8 ± 1 µg (summer and winter milk) of chromium, providing in excess of 8.25 and 6 µg per portion, respectively. This represents 15% of nutrient reference intake values for adults. These findings allow for the claim to be made that PR possesses the health properties ascribed to food sources of selenium and chromium according to European Regulation 432/2012.

The Impact of Different Inoculation Schemes on the Microbiota, Physicochemical and Sensory Characteristics of Greek Kopanisti Cheese throughout Production and Ripening.

Kopanisti is a Greek PDO cheese, which is traditionally produced by the addition of an amount of over-mature Kopanisti, called Mana Kopanisti, to initiate cheese ripening. The aim of this study was the production of four types of Kopanisti cheese (A-D) using pasteurized cow milk, and a combination of the following starters/adjuncts in order to test their ability to be used in Kopanisti cheese production: A: Lactococcus lactis subsp. lactis and Lacticaseibacillus paracasei, B: L. lactis and Lc. paracasei/Mana Kopanisti, C: L. lactis and Lc. paracasei/Ligilactobacillus acidipiscis and Loigolactobacillus rennini, D: Lig. acidipiscis and Loig. rennini. Throughout production and ripening, classical microbiological, metataxonomics and physicochemical analyses were employed, while the final products (Day 35) were subjected to sensory analysis as well. Most interestingly, beta-diversity analysis of the metataxonomics data revealed the clusters constructed among the Kopanisti types based on the different inoculation schemes. On day 35, Kopanisti A-C types clustered together due to their similar 16S microbiota, while Kopanisti D was highly differentiated. On the contrary, ITS data clustered Kopanisti B and C together, while Kopanisti A and D were grouped seperately. Finally, based on the sensory evaluation, Kopanisti C appeared to have the most suitable bacteria cocktail for the Kopanisti cheese production. Therefore, not only were the conventional starters used, but also the Lig. acidipiscis and Loig. rennini strains could be used in a standardized Kopanisti cheese production that could lead to final products of high quality and safety.

Evolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertime.

Biochemical, volatile and textural profiles during manufacture and ripening were determined in samples of Castelmagno PDO cheese obtained from three different batches in the main artisan cheese plant of Castelmagno PDO production area. At the end of manufacture, samples were characterised by a pH of 6.57% and 52.4% moisture content. The HPLC analysis of organic acids and sugars showed the exhaustion of lactose content, while Urea-PAGE indicated extensive primary proteolysis of both ß-casein and αs1-casein. During ripening, cheeses were characterised by high degradation of ß-casein and αs1-casein, due to bacterial action. RP-HPLC profiles showed a high production of peptides eluted between 20 and 30min. In total, 92 volatile compounds were identified in cheese headspace. Texture profiles showed an increase in hardness, gumminess, chewiness and adhesiveness values, as well as a decrease in cohesiveness during ripening.

Evaluating the Authenticity of the Raw-Milk Cheese Fontina (PDO) with Respect to Similar Cheeses.

The implementation of quality assurance schemes for the assessment of PDO food authenticity is an issue involving manufacturers, traders, retailers and consumers. In this respect, reliable analytical methods are needed to integrate paper-trailing information. The feasibility of distinguishing the Italian Fontina PDO cheese from the generic Fontal cheese was preliminarily evaluated on a set of commercial samples by measuring selected parameters (pH, alkaline phosphatase activity, content of copper, volatiles, extent of proteolysis) related to the different manufacturing processes. The relative profile of free amino acids proved to be a promising tool. A new set of 41 samples of Fontina PDO cheese was collected at representative dairies within the recognized production area and analyzed for free amino acids. A chemometric model of Fontina PDO cheese was built based on the mean content and standard deviation of 15 free amino acids. On this basis, all of the PDO samples were correctly identified, whereas all of the Fontal cheeses were recognized as different cheeses.

Influence of Cardoon Flower (Cynara cardunculus L.) and Flock Lactation Stage in PDO Serra da Estrela Cheese.

Serra da Estrela (SE) cheese is one of the most appreciated Portuguese cheeses, being produced only from raw ewe's milk, cardoon flower and salt. Cardoon takes part in two important processes in cheese production-coagulation and proteolysis-contributing to its unique features. Furthermore, milk chemical characteristics change during the milking season, being another factor that account for the high variability of cheese attributes. Therefore, the purpose of this work is to study the influence of cardoon flower (commercial, 6 M and 3 M) and flock lactation stage (November 2018, February and April 2019) in the final characteristics of SE cheese. The parameters analysed were moisture, protein, fat and salt contents, texture and colour. Results showed that flock lactation stage has the highest influence in all the studied characteristics, corresponding the early stages of lactation to the most protein-rich and low-fat cheeses. Cardoon flower affects mainly fat and rind colour. This study allows us to conclude that seasonal changes in ewe's milk have a considerable impact in cheese attributes, and that although cardoon type had a more restrained effect, when used with expertise it may help adjust cheese sensory characteristics in order to obtain a final product that matches consumer acceptability requirements.

Assessing the authenticity of animal rennet using δ15N analysis of chymosin.

Chymosin is a protease that curdles the milk casein. Animal rennet was the first discovered source of chymosin and its use is mandatory for the production of PDO cheeses such as Parmigiano Reggiano and Grana Padano. Of the alternatives, fermentation-produced chymosin is the most competitive because it functions in a similar way, but is much cheaper. Analytical tools are necessary in order to distinguish the 2 types of chymosin and verify the compulsory use of animal rennet in the production of PDO cheeses. In this work, a method to analyse 15N/14N in chymosin after extraction was developed. The δ15N values of animal rennet range from 5.7‰ to 8‰, whereas the δ15N values of fermentation-produced chymosin are significantly lower, ranging from -5.3‰ to 2.2‰. A threshold value of 5.7‰ was defined for authentic animal rennet. Addition of fermentation-produced chymosin to animal rennet, or its complete substitution, can be therefore detected.

Cheese: Food Perception and Food Choice.

In light of the increasing interest in the economic and socio-political impact of the 'traditional food' trend, it is essential to understand the determinant factors that lead to traditional consumer choices. The standardization of sensory quality evaluation methods marks the pressing need for food product certification, particularly foods with specific sensory characteristics, such as those with a Protected Designation of Origin (PDO). Consumer perception of particular foods, especially for foods that are culturally and socially contingent, such as cheese, must be understood as both a psychophysical reflex and a learned social practice. Consumers create their own perceptions based on the overall intrinsic or extrinsic cheese characteristics, mainly sensory characteristics that reflect others' attributes. These characteristics are normally linked to the specific cheese manufacture process. Some patents propose the use of adapted cheesemaking equipment (EP1982582A2), suitable for the manufacture of small-scale cheeses, such as some PDO cheese. Thus, sensory evaluation of any kind of cheese is based, in the initial phase, on knowledge of the sensory methods for cheese evaluation and, in a second phase, on the familiarity of the cheese characteristics and verbalization of desirable and undesirable attributes. This paper presents a case study based on the traditional food product, Évora cheese, assembled with PDO cheeses, whose sensory and physicochemical quality attributes are essential in order to obtain this designation and ensure the genuine properties that characterize them, as well as ascertaining exactly how they are perceived and further accepted by the consumer.

Lactose, galactose and glucose determination in naturally "lactose free" hard cheese: HPAEC-PAD method validation.

A chromatographic method by HPAEC-PAD was developed and in-house validated for the quantification of low sugar levels in hard cheese, specifically Grana Padano PDO cheese. Particular attention was paid to the extraction procedure, due to residual microbial and enzymatic activities. Specificity in detection and linearity were verified. Recoveries ranged from 93% for lactose to 98% for glucose and galactose. The obtained LOD and LOQ values were, respectively, 0.25 and 0.41mg/100g for lactose, 0.14 and 0.27mg/100g for galactose, and 0.16 and 0.26mg/100g for glucose. The method was applied to 59 samples of Grana Padano PDO cheese: galactose showed the highest concentration and variability among the samples (1.36±0.89), compared to both lactose (0.45±0.12) and glucose (0.46±0.13). Considering the very low levels of sugars detected, authentic PDO Grana Padano could be safely included in the diet of people suffering from lactose intolerance.

Development of a Quantitative GC-MS Method for the Detection of Cyclopropane Fatty Acids in Cheese as New Molecular Markers for Parmigiano Reggiano Authentication.

Cyclopropane fatty acids (CPFA), as lactobacillic acid and dihydrosterculic acid, are components of bacterial membranes and have been recently detected in milk and in dairy products from cows fed with corn silage. In this paper, a specific quantitative gas chromatography-mass spectrometry (GC-MS) method for the detection of CPFA in cheeses was developed, and the quality parameters of the method (limit of detection, limit of quantitation, and intralaboratory precision) were assessed. Limit of detection and quantitation of CPFA were, respectively, 60 and 200 mg/kg of cheese fat, and the intralaboratory precision, determined on three concentration levels, satisfied the Horwitz equation. This method was applied to 304 samples of PDO cheeses of certified origin, including Parmigiano Reggiano (Italy), Grana Padano (Italy), Fontina (Italy), Comté (France), and Gruyère (Switzerland). Results showed that CPFA were absent in all of the cheeses whose Production Specification Rules expressly forbid the use of silages (Parmigiano Reggiano, Fontina, Comté, and Gruyère). CPFA were instead present in variable concentrations (300-830 mg/kg of fat) in all of the samples of Grana Padano cheese (silages admitted). A mix of grated Parmigiano Reggiano and Grana Padano was also prepared, showing that the method is able to detect the counterfeiting of Parmigiano Reggiano with other cheeses up to 10-20% Grana Padano content. These results support the hypothesis that CPFA can be used as a marker of silage feedings for cheeses, and the data reported can be considered a first attempt to create a database for CPFA presence in PDO cheeses.

rRNA-based monitoring of the microbiota involved in Fontina PDO cheese production in relation to different stages of cow lactation.

Fontina Protected Denomination of Origin (PDO) cheese is a full-fat semi-cooked cheese traditionally made in Northwest Italy (Aosta Valley) and manufactured from raw cow's milk. The management of cattle farms in Aosta Valley calls for seasonal migration to high pastures during the summer and the concentration of calving during the autumn and the beginning of the winter. Based on cattle physiology and given to calving seasonality, three cow lactation phases i.e. post-partum, oestrus and early gestation, can be identified and an effect could be hypothesized on average milk composition and on cheese quality. The aim of the present paper was to investigate the bacterial dynamics during Fontina PDO cheese manufacturing and ripening, in relation to the different lactation stages, in order to evaluate a possible correlation between microbiota and phase of lactation. For this purpose, microbial RNA analysis was carried out by RT-PCR coupled with DGGE and high-throughput sequencing. A good performance of the starter cultures was highlighted throughout Fontina PDO manufacturing and ripening; in fact, the starter prevailed against the autochthonous microbiota. Thus, the microbial activity, which was supposed to affect the final quality of Fontina PDO cheese, appeared to be strictly associated to the presence of the starter, which did not show any difference in its performance according to the different stages of cow lactation. Therefore, the results of this research highlighted a negligible correlation between the microbiota of raw milk and the organoleptic quality and typicity of Fontina cheese in relation to lactation seasonality.