Canestrato Pugliese Cheese Research Articles

Native and non native sheep breed differences in canestrato pugliese cheese quality: a resource for a sustainable pastoral system

Canestrato Pugliese is an Italian uncooked hard cheese made by the Protected Designation of Origin (PDO) status. In the past, it was manufactured with milk from local sheep breeds (Altamurana and Leccese) while in recent years it has almost entirely been made with milk from non-native sheep breeds (Sarda and Comisana). The aim of the study was to investigate the breed effect on the quality of Canestrato Pugliese cheese by comparing two native (Altamurana and Leccese) and two non-native (Sarda and Comisana) sheep breeds. The experiment was carried out at the experimental farm of CREA-ZOE (Apulia region, Southern Italy) using a flock set-up of four sheep breeds: Altamurana, Leccese, Sarda, and Comisana. All sheep fed pasture supplemented with 200 g/sheep/day concentrate at each milking. For each breed, three cheese-makings of Canestrato Pugliese were carried out for three consecutive days following the PDO technology. At two and four months of ripening, cheese was analysed for gross composition, fatty acid profile, nutritional indexes, and volatile organic compounds. Significant differences were found between breeds in the fatty acid profile and nutritional indexes (P ≤ 0.05). Canestrato Pugliese from Comisana, Leccese, and Sarda had a higher dry matter and fat content than that from Altamurana breed (P ≤ 0.05). Cheeses from Altamurana and Comisana showed a higher content of unsaturated and omega-3 fatty acids and a better omega-6/omega-3 ratio than the others (P ≤ 0.05). The best Health Promoting Index was detected in Altamurana, Comisana, and Leccese cheeses (P ≤ 0.05). Additionally, sheep breed affected the content of volatile organic compounds (P ≤ 0.05). The highest value of volatile organic compounds was observed in cheeses from Leccese breed (P ≤ 0.05). The discriminant analysis performed on cheese data shows a separation between native and non-native sheep breeds. The present study reveals that the breed has an evident effect on the fatty acid and volatile organic compound profile of Canestrato Pugliese.

Causal relationship between microbial ecology dynamics and proteolysis during manufacture and ripening of protected designation of origin (PDO) cheese Canestrato Pugliese.

Pyrosequencing of the 16S rRNA gene, community-level physiological profiles determined by the use of Biolog EcoPlates, and proteolysis analyses were used to characterize Canestrato Pugliese Protected Designation of Origin (PDO) cheese. The number of presumptive mesophilic lactococci in raw ewes' milk was higher than that of presumptive mesophilic lactobacilli. The numbers of these microbial groups increased during ripening, showing temporal and numerical differences. Urea-PAGE showed limited primary proteolysis, whereas the analysis of the pH 4.6-soluble fraction of the cheese revealed that secondary proteolysis increased mainly from 45 to 75 days of ripening. This agreed with the concentration of free amino acids. Raw ewes' milk was contaminated by several bacterial phyla: Proteobacteria (68%; mainly Pseudomonas), Firmicutes (30%; mainly Carnobacterium and Lactococcus), Bacteroidetes (0.05%), and Actinobacteria (0.02%). Almost the same microbial composition persisted in the curd after molding. From day 1 of ripening onwards, the phylum Firmicutes dominated. Lactococcus dominated throughout ripening, and most of the Lactobacillus species appeared only at 7 or 15 days. At 90 days, Lactococcus (87.2%), Lactobacillus (4.8%; mainly Lactobacillus plantarum and Lactobacillus sakei), and Leuconostoc (3.9%) dominated. The relative utilization of carbon sources by the bacterial community reflected the succession. This study identified strategic phases that characterized the manufacture and ripening of Canestrato Pugliese cheese and established a causal relationship between mesophilic lactobacilli and proteolysis.

Effects of different surface treatments on ripening of Canestrato Pugliese cheese

The effect of different surface treatments (with olive oil, liquid smoke or pimaricin) on a number of physico-chemical characteristics (namely, moisture, NaCl content, pH, total and soluble nitrogen fractions, lipolytic processes and mould production) of the core and the rind zones of Canestrato Pugliese cheese were studied during the whole ripening period (100 days). Treated cheese data were statistically compared with data obtained for untreated cheese. While for the core zone proteolytic and lipolytic processes, together with all the other physico-chemical properties studied, remained unaffected by the surface treatment, in the layer close to the rind these were influenced significantly by the surface treatment in a manner correlated with the fungal growth.

Polyphasic characterization of indigenous lactobacilli and lactococci from PDO Canestrato Pugliese cheese

A polyphasic approach, involving both genotypic and phenotypic analyses was used to characterize 33 isolates of lactic acid bacteria from the raw milk Protected Designation of Origin (PDO) Canestrato Pugliese cheese, in order to select candidate strains that can be used as autochthonous starter cultures in the dairy industry. Randomly amplified polymorphic DNA (RAPD) analysis and clustering by the unweighted pair-group method with arithmetic averages (UPGMA) were used to evaluate the genotypic diversity, while phenotypic characterization was performed through miniaturized assays and traditional biochemical tests. Technological properties of major interest for cheese-making (acidification, tendency to lysis, proteolytic and peptidase activity) were also evaluated, and selected subset of data was statistically examined by the one-way ANOVA and the Tukey–Kramer HSD test, to highlight strain-specific and species-specific differences among the isolates. A high degree of diversity appeared in the phenotypic and technological traits in opposition to a relatively low genotypic diversity. Although none of the isolates showed the best performances in all the activities, an appropriate mixture of strains could be selected for providing an efficacious autochthonous starter culture.

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese were studied. Cheeses were manufactured from the bulk milk obtained from ewes subjected to four different experimental treatments: (1) low dietary crude protein (CP) of 13% in dry matter (DM) at a low ventilation rate (23.5 m3/h per ewe) (LPLV); (2) low dietary CP at a moderate ventilation rate (47 m3/h per ewe) (LPMV), (3) moderate dietary CP of 16% DM at a low ventilation rate (MPLV); and (4) moderate dietary CP dietary at a moderate ventilation rate (MPMV). Bulk milk and cheeses (at 1, 15, 45 and 90 d of ripening) were analysed for chemical composition, N fractions and plasmin-plasminogen activities. The pH 4.6-soluble and insoluble N fractions were analysed by urea-PAGE. Bulk milk from ewes receiving the low CP diet displayed higher casein and lower urea contents. Ewes subjected to the low ventilation rate displayed a higher plasminogen activity in milk, whereas no differences emerged among treatments in the conversion of zymogen to plasmin. During ripening the plasmin-plasminogen system in cheese did not display significant changes across treatments. At 90 d of ripening, the cheese produced with milk from ewes receiving the low CP diet and exposed to the low ventilation rate displayed more markedly stained bands in the area of gamma-caseins in pH 4.6-insoluble N fraction. As from 15 d of ripening, the pH 4.6-soluble fraction showed a greater number of bands in the LPMV and MPMV than in the LPLV and MPLV cheeses. The results showed that Canestrato Pugliese cheese manufactured with milk from ewes fed the low CP diet and exposed to the moderate ventilation rate was characterized by higher protein and casein content in the fresh cheese and by a greater proteolysis after 90 d of ripening.

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk protected designation of origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies. Thirty phenotypic assays and three molecular techniques (restriction fragment length polymorphism, partial sequencing of the 16S rRNA gene and recA multiplex PCR assay) were applied to the identification of 304 isolates from raw milk Canestrato Pugliese cheese. As a result, 168 of 207 isolates identified were ascribed to genus Enterococcus, 25 to Lactobacillus, 13 to Lactococcus and one to Leuconostoc. More in details among the lactobacilli, the species Lactobacillus brevis and Lactobacillus plantarum were predominant, including 13 and 10 isolates respectively, whereas among the lactococci, Lactococcus lactis subsp.cremoris [corrected] was the species more frequently detected (seven isolates). Except for the enterococci, phenotypic tests were not reliable enough for the identification of the isolates, if not combined to the genotype-based molecular techniques. The polyphasic approach utilized allowed 10 different LAB species to be detected; thus suggesting the appreciable LAB diversity of the autochthonous microbial population of the Canestrato Pugliese cheese. A comprehensive study of the resident raw milk Canestrato Pugliese cheese microbial population has been undertaken.

Effect of different ventilation regimens on ewes' milk and Canestrato Pugliese cheese quality in summer

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4.6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.

Comparison of mycoflora associated to Canestrato Pugliese cheese produced according three protocols

The associated mycoflora of the Canestrato Pugliese cheese produced from raw milk, pasteurized milk or by heating the curd in hot whey were compared. In order to evaluate the ability to colonize Canestrato Pugliese cheese, the effects of pH, salt content, temperature and nutritional source on growth of some strains were investigated. The cheese produced according to a traditional protocol harboured a more heterogeneous mycoflora than to modern protocols. However, the species most frequently isolated from our samples (Fusarium moniliforme, Aspergillus niger and Aspergillus flavus) were infrequently associated with cheese and indicated a strong contamination of the ripening rooms used for cheese ripening only for a short while. Moreover, since during mould growth a risk of mycotoxin production in the cheese exists, the isolation of mycotoxigenic moulds could represent a health concern. The results obtained emphasize the need to control relative humidity and hygienic conditions of the ripening houses and provide an improved understanding of fungi of Canestrato Pugliese cheese.

Comparison of the microbiological, compositional, biochemical, volatile profile and sensory characteristics of three Italian PDO ewes’ milk cheeses

Three batches of three Italian ewes’ milk cheeses (Canestrato Pugliese, Fiore Sardo and Pecorino Romano), which were manufactured under the Protected Denomination of Origin (PDO), were compared for microbiological, compositional, biochemical, volatile profile and sensory characteristics. After ca. 1 year of ripening, the three cheeses had a complex microbial population, composed mainly of non-starter lactic acid bacteria (NSLAB); several species of mesophilic lactobacilli and enterococci were identified. Pecorino Romano had a very high level of NaCl, 8.7±0.1% w/w. The level of pH 4.6-soluble nitrogen increased in the order: Canestrato Pugliese>Fiore Sardo>Pecorino Romano. Urea-PAGE electrophoresis showed that the hydrolysis of α s1 -casein was more consistent than β -casein. RP-HPLC profiles of the ethanol-soluble and ethanol-insoluble fractions of the pH 4.6-soluble nitrogen showed differences between the cheeses which agreed with the content of free amino acids. Glutamic acid, histidine, valine, isoleucine, leucine and phenylalanine were the free amino acids present at the highest levels in all the cheeses. Sixty-two volatile components were identified by Gas Chromatography-Mass Spectrometry analysis of steam distillates of the cheeses. The volatile profile of the three cheeses differed significantly. Esters were the main volatiles in the Canestrato Pugliese cheese but were the lowest in Fiore Sardo cheese. Ketones and alcohols were the principal class of volatile components in Fiore Sardo and Pecorino Romano cheeses, respectively. Lactones and products of the breakdown of the sulphur-containing amino acids were found at considerable levels in all cheeses, but aldehydes were present at low levels. Only 30 carboxylic acids were identified; Canestrato Pugliese had the highest content of total acids. Butanoic, hexanoic, octanoic and decanoic acids represented the 30–33% of the total carboxylic acids in the cheeses. The cheeses were subjected to descriptive sensory analysis; 19 discriminating and 2 descriptive attributes were analysed by Principal Component Analysis (PCA). The sensory characteristics of the three ewes’ milk cheeses were distinct.

Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot whey

Canestrato Pugliese cheeses were produced from raw ewes' milk (R and R II cheeses), pasteurized ewes' milk (P cheese) and by heating the curd in hot whey according to a traditional protocol (T cheese). R II differed from R cheese mainly by having been produced from raw milk with a higher number of somatic cells, 950.000 vs. 750.000 ml −1, respectively. Compared to P and T cheeses, R and R II cheeses had a higher concentration (one or two orders of magnitude) of cheese-related bacteria such as adventitious mesophilic lactobacilli, enterococci and staphylococci. At the end of ripening, all cheeses contained less than 1.0 log cfu g −1 of total and fecal coliforms, and Escherichia coli and Staphylococcus aureus were not detected. As shown by phenotypic identification and RAPD-PCR, R cheese contained the largest number of mesophilic lactobacilli species and the greatest diversity of strains within the Lactobacillus plantarum species. Primary proteolysis did not differ appreciably among the cheeses. On the contrary, both urea-PAGE and the RP-HPLC analyses of the water-soluble N fractions showed the more complex profiles in cheeses produced by raw milks. R and R II cheeses had the highest values of water-soluble N/total N (ca. 30%) and the highest concentration of total free amino acids (ca. 40 mg g −1) which approached or exceeded those reported for Italian cheeses with very high level of proteolysis during ripening. The main differences between R–R II and P–T cheeses were the concentrations of aspartic acid, proline, alanine, isoleucine, histidine and lysine. The water-soluble extracts of R and R II cheeses contained levels of amino-, imino- and di-peptidase activities, which were about twice those found in P and T cheeses. Cheeses differed slightly in the concentration of total free fatty acids that ranged between 1673 and 1651 mg kg −1 in R and R II cheeses, and 1397 and 1334 mg kg −1 in P and T cheeses. Butyric, caproic, capric, palmitic, oleic and linoleic acids were found at the highest concentrations.