Functional fresh cheeses can serve as effective carriers of bioactive compounds and probiotic cultures. This study developed a Casu Axedu cheese fortified with olive leaf phenolics and a probiotic-candidate Lactiplantibacillus plantarum (LP) strain. Three formulations were produced from thermised sheep milk: a control (CA-I), a cheese with LP (CA-IL) and a cheese combining olive leaf extract (OLE; 20 mg GAE 100 g−1 milk) and LP (CA-IOL). In all experimental cheeses, inulin was included as a dietary fibre, and lactose hydrolysis was performed to improve product accessibility for lactose-intolerant consumers. Cheeses were stored at 3 ± 1 °C for 30 days and analysed for polyphenol profile, antioxidant capacity, physicochemical and nutritional characteristics, microbiological quality, sensory properties and consumer acceptance. OLE fortification approximately doubled the total polyphenols and produced a more than threefold increase in FRAP antioxidant capacity in CA-IOL, with a high percentage of the main olive leaf phenolics retained after 30 days. Composition, fatty acid profile, vitamin A/E and cholesterol contents were unaffected by OLE and LP addition; all formulations met “high in protein” and lactose-free label criteria (according to the guidelines issued by the Italian Ministry of Health DGISAN Communication No. 27673 of 7 July 2015), and the inulin-based “source of fibre” claim was supported by formulation and mass-balance calculation. Presumptive lactobacilli remained at high counts throughout storage in the LP-containing cheeses, and OLE neither impaired the lactic microflora nor promoted coliform growth. OLE addition increased perceived bitterness, astringency and olive-like notes in CA-IOL, moderately reducing liking scores relative to those of the control among the tested consumer panel (5.25 ± 1.7 at 30 d); mean liking nonetheless remained above the neutral point of the 9-point hedonic scale. Overall, combining inulin, olive leaf extract and a probiotic-candidate L. plantarum strain enabled the production of a Casu Axedu cheese with enhanced antioxidant properties and improved nutritional features while retaining acceptable sensory quality. This approach represents a promising strategy for the valorisation of olive by-products and the development of innovative sheep milk dairy products consistent with circular economy principles.

Several plant-based milk coagulants, such as those extracted from Cynara cardunculus, are available as alternatives to animal-derived rennet for cheese production. The increasing use of plant-derived rennet substitutes requires an assessment of their performance on different types of cheese, information that is often lacking. The aim of this study was to evaluate the impact of a commercial rennet derived from C. cardunculus, compared to calf rennet, used as a control, on the quality of a long-ripened, hard, cooked sheep’s milk cheese. The control and experimental cheeses, produced in three cheesemaking trials, were sampled 1 day after production and at 12 and 18 months of ripening and assessed for their physico-chemical, textural, and sensory characteristics. No significant differences were observed in the features of the produced cheeses, in relation to the coagulant used. Notably, despite the long ripening period, in this type of cheesemaking technology, C. cardunculus rennet did not result in higher levels of proteolysis, softer texture or increased bitterness, features often associated with this type of plant-based coagulant. The outcomes of the present study may be of interest to the dairy industry for the selection of alternative milk-clotting agents in the production of specific sheep’s milk cheese categories.

In this study, yoghurt from sheep milk was formulated with spent myrtle berry (SMB) powder to enhance its polyphenol content and antioxidant activity. The incorporation of SMB powder into sheep milk yoghurt had a significant effect on its technological properties. Despite the increase in total polyphenols (8.46 mg/100 g FW vs. 1.35 mg/100 g FW in the control) and consequently in the antioxidant activity (1.76 vs. 0.41 in the control), SMB powder caused a substantial alteration of the yoghurt structure, resulting in a reduction in firmness and stickiness of 52.4% and 61% and a threefold increase in syneresis compared to the control, consequently leading to a reduction in gel strength. The analysis of the microstructure revealed a disruption of the protein network that weakened the gel microstructure. Consequently, subsequent studies will be dedicated to the optimisation of the process through the incorporation of thickening agents to enhance consistency and increase the water-holding capacity. Conversely, the incorporation of SMB into yoghurt did not result in a detrimental impact on sensory quality. The present study is the first to demonstrate the potential of SMB powder as a sustainable functional ingredient in dairy applications.

Autochthonous whey-based (scotta-innesto) starters are increasingly recognized as a valuable resource to preserve microbial biodiversity and sensory identity in hard sheep milk cheeses produced under PDO-type specifications. In this study, two indigenous scotta-innesto cultures collected in Sardinia (Italy) in the 1960s were compared with a widely used commercial starter in pilot-scale hard sheep milk cheese manufacture produced according to Pecorino Romano PDO specifications. To this end, an integrated multidisciplinary approach was employed by combining starter metagenomics, culture-dependent microbiology, LC-HRMS-based untargeted metabolomics, targeted aroma volatile analysis, and descriptive sensory analysis. The autochthonous consortia were dominated by Lactobacillus delbrueckii and Streptococcus thermophilus and showed a higher abundance of protease and peptidase genes as well as pathways linked to acetyl-CoA metabolism and alcohol formation. Conversely, the commercial starter comprised higher proportions of Lactobacillus helveticus and Lactococcus spp., together with the enrichment of the acetoin and diacetyl pathways. Moreover, by focusing on ripening, cheeses produced with the autochthonous starters showed a lower accumulation of purine catabolites, such as hypoxanthine, and higher levels of 1-methyladenosine, methionine and dimethylglycine, suggesting a potential enhancement of purine salvage and biosynthetic activity. Also, the starter culture influenced the synthesis and the accumulation of selected key aroma compounds, with higher 2-hexanol and 1-butanol in cheeses inoculated with autochthonous starters, while acetoin and ketones were found as key aroma markers in commercial-starter cheeses. Finally, saltiness and pungency, hardness, and crystal perception emerged as the most discriminant sensory attributes, with autochthonous-starter cheeses showing higher intensity scores.

This study investigated element distribution during the sheep milk process by analyzing raw sheep milk and six derived fractions. Because natural concentrations of toxic elements were below detection limits, milk was also spiked with As, Cd, Hg, Pb, and Sn to track their distribution. Na, K, Rb, Cs, and As accumulated in aqueous fractions (whey and scotta). Ca, Zn, Cd, Sn, Pb, Fe, and Cu were largely retained in pecorino cheese. Mg and Mo partitioned almost equally between cheese and scotta whey. Se was concentrated in scotta and ricotta, and Hg was distributed across cheese, ricotta, and scotta. Nutritionally, pecorino cheese is confirmed to be rich in Ca, and a source of Zn, Se, and Mo; ricotta is a source of Se; and scotta whey retains soluble elements with potential applications in functional foods.

The production of Fiore Sardo cheese is regulated by the specification of the Protected Designation of Origin (PDO), which aims to guarantee the specific area of production, the know-how of local producers, and the specific use of raw milk from Sarda sheep. The thermization of milk is a sub-pasteurization process that is commonly used in cheese-making to lower the bacterial load and increase the shelf life of the product; it is therefore a cause of non-compliance with the PDO specification of Fiore Sardo cheese, allowing producers to gain practical and economic advantages. In this work, NIR spectroscopy coupled with multivariate discriminant analysis was used to identify the thermal treatment of milk in Fiore Sardo cheese samples. Cheeses were produced using raw milk (38 °C), low-thermized milk (57 °C for 30 s), and high-thermized milk (68 °C for 30 s). The NIR spectra of the cheeses were used to build discriminant models for individuating the thermal treatment of the processed milk. The obtained discriminant models were able to correctly classify about 90% of the Fiore Sardo cheese samples. This method could be suitable as a screening technique to authenticate Fiore Sardo PDO cheese.

In this study, the lipidomic alterations in cooked-curd ovine cheese were comprehensively assessed by comparing samples produced using either vegetal (Cynara cardunculus L.) or animal (calf) rennet. Lipid extracts were analyzed using high-resolution liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and the resulting data were processed through multivariate statistical analyses to explore compositional changes across 18 months of ripening. The use of cardoon rennet, led to distinct modifications in the lipid profile compared to conventional calf rennet. A broad range of lipid classes, including phosphatidylserines, triacylglycerols, free fatty acids, phosphatidylcholines, ceramides, sphingomyelins, phosphatidylethanolamines, and monoacylglycerols, were significantly affected by the type of rennet employed. In contrast, the ripening process mainly influenced the abundance and composition of free fatty acids, reflecting ongoing lipolysis and lipid remodeling. To enhance classification and biomarker discovery, a forward stepwise interval partial least squares discriminant analysis (PLS-DA) was applied, enabling the identification of two optimal feature subsets for discriminating samples based on rennet type and ripening stage. The resulting models achieved high classification accuracies of 88 % for rennet type (cardoon vs. calf) and 91 % for ripening time (24 h, 12 months, 18 months), underscoring the strong lipidomic signature linked to both technological variables. These results allowed to better understand how the type of rennet and the ripening process can change the composition of lipids in cheese. This knowledge can be useful for improving product quality, ensuring authenticity, and developing new types of traditional cheeses with specific features.

A freeze-dried natural starter culture (NSC) was developed and assessed for its suitability in producing a semi-cooked, 60-day-ripened cheese resembling the protected designation of origin (PDO) Pecorino Sardo. The culture, derived from raw ewe’s milk from a dairy farm involved in the study, without thermal decontamination to preserve indigenous microbiota, was enriched with two strain-level-characterised, biodiverse mixtures of Streptococcus thermophilus (Str-mix) and Lactobacillus delbrueckii (Lb-mix). This study evaluated the technological robustness and adaptability of NSC enriched with biodiverse Str-mix and Lb-mix across three different artisanal dairy-processing environments with varying milk compositions and equipment levels at plants located in different geographic areas. During cheesemaking, technological, physico-chemical, and compositional parameters were monitored, along with microbial characterisation of milks and 1-day cheeses. After 60 days of ripening, cheeses were characterised from the microbiological, physico-chemical, and compositional perspectives. Furthermore, nutritional labelling was established, and consumer acceptance was determined. Results showed that the starter (NSC + Str-mix + Lb-mix) demonstrated strong and reproducible technological performance in all dairies, regardless of the milk’s chemical and microbial composition variability. Sensory quality was preserved in cheeses ripened for up to 180 days. These preliminary findings seem to support the use of freeze-dried, raw-milk-derived natural cultures in artisanal cheesemaking as a way to preserve microbial diversity and to reconnect with traditional practices that enhance both the tangible and intangible assets of modern society.

Although PDO Pecorino Sardo is one of the oldest traditional cheeses of Sardinia, Italy, data on its nutritional properties and food safety are lacking. In particular, significant amounts of lactose and galactose may be a health concern for consumers. The primary objective of this study is to quantify, using a validated GC-FID method, the residual lactose and galactose content in “maturo” (i.e., ripened for at least two months) Protected Denomination of Origin (PDO) Pecorino Sardo cheese. A statistically representative sampling from seven dairies distributed throughout Sardinia has been selected for this aim. In addition to lactose and galactose, two of their metabolites (i.e., glucose and tagatose, respectively) and a bioactive polyol like myo-inositol were quantified. The concentration of lactose (mean 26 mg kg−1, range 4–90 mg kg−1) was below the strictest limit set in the European Union (i.e., 100 mg kg−1), while the galactose content was found to be in an amount (mean: 76 mg kg−1, range: 10–200 mg kg−1) that even patients afflicted with severe galactosemia, albeit with some circumspection, could consume this cheese. Ripening (two to four months) had no significant effect on the amount of all analytes, while a slight decrease in galactose levels was observed during the manufacturing season. Finally, the amounts of glucose, tagatose, and myo-inositol are constant in the range of a few tens of mg kg−1.

The aim of this work was develop a technological process for the manufacturing of an ice cream from sheep milk, enriched with both functional ingredients and probiotic bacteria. The studied process involved the use of an enriched milk (EM) obtained by mixing predetermined amounts of sheep skimmed milk concentrated by ultrafiltration (retentate), cream from sheep’s milk and whey, microparticulated whey proteins (MWP), obtained by ultrafiltration of sweet sheep whey as a source of whey proteins, marine algal oil from Schizochytrium spp. as a source of the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), inulin as a prebiotic fiber, and locust bean gum as a stabilizer. The resulting EM was inoculated with starter and aroma cultures together with the probiotic culture of Bifidobacterium animalis subsp. lactis (BB-12®) in order to obtain a fermented functional product (FFP) with a physico-chemical composition similar to that of EM. FFP was the main ingredient (~80%, w/w) in the ice cream mixture. Two sucrose-alternative sweeteners (trehalose and erythritol), together with dextrose, were subsequently added to obtain the final ice cream formulation. The resulting ice cream met three nutritional claims: “Source of protein”, “Source of fiber” and “High in omega-3 fatty acids” listed in Regulations (EC) No 1924/2006 and (EU) No 116/2010. Furthermore, the ice cream satisfied the requirement of “probiotic food” according to the Italian Ministry of Health’s guidelines for probiotics. The nutritional characteristics of the ice cream, including the concentration of the probiotic culture, remained stable up to 120 days of storage at −20 ± 2 °C.

The effect of different sub-pasteurization heat treatments and different ripening times was investigated in this work. The metabolite profiles of 95 cheese samples were analyzed using GC-MS in order to determine the effects of thermal treatment (raw milk, 57 degrees C and 68 degrees C milk thermization) and ripening time (105 and 180 days). ANOVA test on GC-MS peaks complemented with false discovery rate correction was employed to identify the compounds whose levels significantly varied over different ripening times and thermal treatments. The univariate t-test classifier and Partial Least Square Discriminant Analysis (PLS-DA) provided acceptable classification results, with an overall accuracy in cross-validation of 76% for the univariate model and 72% from the PLSDA. The metabolites that mostly changed with ripening time were amino acids and one endocannabinoid (i.e., arachidonoyl amide), while compounds belonging to the classes of biogenic amines and saccharides resulted in being strongly affected by the thermization process.

The great majority of infant formula (FM) for neonate’s nutrition are produced using ingredients from cow milk. Recently, some countries, such as China and New Zealand, are turning their attention to the use of ovine milk ingredients for FM production. In this study, a pilot plant process has been set up to produce infant formula ingredients from Sarda sheep milk. To meet the nutritional needs of neonates (0–6 and 6–12 months of age) two different liquid milk-derived formulations (IF1 and IF2, respectively) obtained mixing whole milk, skimmed milk, and whey milk ultrafiltration concentrate (retentate) were produced. Compositional analysis of milk, retentate, and the final IFs showed that the two formulations contain elements of nutritional interest, such as well-balanced content of high biological value proteins (casein:whey proteins ratio of 30:70 and 60:40 for IF1 and IF2, respectively), vitamin A, E and B5, cholesterol, minerals, nucleotides, free amino acids and essential fatty acids (n–6:n–3 ~1), compatible with the growth and development needs of neonates. Therefore, the obtained IF1 and IF2 can be proposed as valuable ovine dairy ingredients for FM manufacturing. Further studies will be necessary to verify the adaptability of the developed process from laboratory to industrial scale application.

The growing scientific interest in the role of food in promoting human health and wellbeing has profoundly influenced consumers’ perceptions and attitudes towards nutrition, leading to the advent of a new class of foods, called functional foods, which are currently one of the fastest growing food-producing sectors, particularly in the dairy industry. The cluster project “Diversification in sheep & goat Sardinian dairy production” was built and carried out, based on requests from ten Sardinian dairy companies, to plan and implement experimental protocols directed to develop new production processes, according to the latest health and nutritional guidelines. Consequently, the following different interconnected research lines were developed: lactose-free dairy products; low-fat dairy products; dairy products enriched with added functional ingredients. The studied processes were based on the modification of cheese milk or whey, through the elimination of or reduction in one or more components with negative health effects or by adding functional ingredients. Therefore, a total of six different dairy products were developed: two from sheep milk and whey and four from goat milk. The technological processes adopted were typically those of Ricotta, fresh and soft cheeses. Contextually, their adaptability to the industrial equipment available in the cluster dairy companies was verified, and most of them were successfully transferred. These novel dairy products meet the current market demand, which shows a greater interest in fresh and short-ripened dairy products, with a low energy intake and high nutritional value. Moreover, can represent an example of the diversification in the sheep and goat dairy sector.

This study presents an extensive investigation on the effect of pasteurization on raw whole ewe milk. Milk samples have been analyzed, throughout lactation (from February to July), by time-domain nuclear magnetic resonance (TD-NMR), collecting the characteristic TD-NMR relaxation parameters, proton longitudinal and transverse relaxation times (1H T1 and T2). Collected data aim at integrating previous NMR works, mainly focusing on dairy model systems (casein and whey proteins solutions and gels, reconstituted skim milk) and cheese, with specific reference to the effect of heat treatments. Whole ewe milk, from a single flock (Sarda sheep breed), was daily analyzed both as untreated (raw) and heat treated with a laboratory-scaled high-temperature, short-time treatment (72°C for 15 and 20 s). Moreover, molecular dynamics in milk were investigated by TD-NMR in different periods of lactation for the first time. As a consequence of high-temperature short-time treatment, 1H T1 and T2 consistently shifted to lower values with respect to raw counterparts. Statistical analysis indicated a significant decrease of T2 in treated samples, to an extent dependent on the heat treatment duration. A subset of dedicated experiments demonstrated that the observed T2 shift is largely ascribable to protein molecular rearrangements and, to a lesser extent, to the interaction of fat globules with proteins or other nonfat components (or both). In light of the crucial importance of detecting the application of a heat treatment to milk, the results reported here suggest TD-NMR relaxation parameters were able to describe heat-induced changes in molecular dynamics and interactions of milk components in a water-rich environment. The use of TD-NMR can be considered a potential suitable technique for quality control and assurance practices in the dairy industry. Upon statistical validation of methods, the application of TD-NMR in the dairy industry would take advantage of its low cost, reliability, and robustness.

This study was conducted to assess, for the first time, the survival of the pathogenic bacteria Listeria monocytogenes, Salmonella spp., Escherichia coli O157:H7, and Staphylococcus aureus during the ripening of protected designation of origin (PDO) Pecorino Romano cheese. A total of twenty-four cheese-making trials (twelve from raw milk and twelve from thermized milk) were performed under the protocol specified by PDO requirements. Sheep cheese milk was first inoculated before processing with approximately 106 colony-forming unit (CFU) mL−1 of each considered pathogen and the experiment was repeated six times for each selected pathogen. Cheese composition and pathogens count were then evaluated in inoculated raw milk, thermized milk, and cheese after 1, 90, and 150 days of ripening. pH, moisture, water activity, and salt content of cheese were within the range of the commercial PDO Pecorino Romano cheese. All the cheeses made from raw and thermized milk were microbiologically safe after 90 days and 1 day from their production, respectively. In conclusion, when Pecorino Romano cheese is produced under PDO specifications, from raw or thermized milk, a combination of factors including the speed and extent of curd acidification in the first phase of the production, together with an intense salting and a long ripening time, preclude the possibility of growth and survival of L. monocytogenes, Salmonella spp., and E. coli O157:H7. Only S. aureus can be still detectable at such low levels that it does not pose a risk to consumers.

Thermization is a sub-pasteurization heat treatment of cheese milk (at 57-68°C for 15-30 s) aimed to reduce the number of undesirable microbial contaminants with reduced heat damage to the indigenous milk enzymes. In this work, the effects of milk thermization on the compositional parameters, proteolysis indices, free fatty acid levels, and low molecular weight metabolite profiles of ovine cheese were studied. Cheese samples at different ripening stages and produced in 2 different periods of the year were analyzed. While the effects of milk thermization on cheese macro-compositional parameters and free fatty acid levels were not evident due to the predominant effects of milk seasonality and cheese ripening stage, the gas chromatography-mass spectrometry based metabolomics approach of ovine cheese produced from raw and thermized milk highlighted strong differences at the metabolite level. Discriminant analysis applied to gas chromatography-mass spectrometry data provided an excellent classification model where cheese samples were correctly classified as produced from raw or thermized milk. The metabolites that mostly changed due to the thermization process belonged to the classes of free amino acids and saccharides. Gas chromatography-mass spectrometry-based metabolomics has proven to be a valid tool to study the effect of mild heat treatments on the polar metabolite profile in ovine cheese.

The aim of this work was to measure the physico-chemical and the colorimetric parameters of ovaries from Mugil cephalus caught in the Tortoll lagoon (South-East coast of Sardinia) along the steps of the manufacturing process of Bottarga, together with the rheological parameters of the final product. A lowering of all CIELab coordinates (lightness, redness and yellowness) was observed during the manufacture process. All CIELab parameters were used to build a Linear Discriminant Analysis (LDA) predictive model able to determine in real time if the roes had been subdued to a freezing process, with a success in prediction of 100%. This model could be used to identify the origin of the roes, since only the imported ones are frozen. The major changes of all the studied parameters (p < 0.05) were noted in the drying step rather than in the salting step. After processing, Bottarga was characterized by a pH value of 5.46 (CV = 2.8) and a moisture content of 25% (CV = 8), whereas the typical per cent amounts of proteins, fat and NaCl, calculated as a percentage on the dried weight, were 56 (CV = 2), 34 (CV = 3) and 3.6 (CV = 17), respectively. The physical chemical changes of the roes during the manufacturing process were consistent for moisture, which decreased by 28%, whereas the protein and the fat contents on the dried weight got respectively lower of 3% and 2%. NaCl content increased by 3.1%. Principal Component Analyses (PCA) were also performed on all data to establish trends and relationships among all parameters. Hardness and consistency of Bottarga were negatively correlated with the moisture content (r = -0.87 and r = -0.88, respectively), while its adhesiveness was negatively correlated with the fat content (r = -0.68).

The aim of this work was to evaluate the effect of sheep milk protein-to-fat ratio (PFR) standardization, on the technological, physico-chemical and rheological properties of PDO Pecorino Romano cheese in the summer period (June and July). Two levels of PFR were investigated: i) similar to 0.90 standardised PFR (SPFR) and ii) similar to 0.76 natural PFR (NPFR). SPFR cheese had a lower fat content (34.2 +/- 0.4 vs 36.8 +/- 0.4) and a higher protein content (27.1 +/- 0.3 vs 25.6 +/- 0.8) than NPFR cheese. Consequently it was more compact, cohesive and hard compared to the latter. These aspects may contribute to reduce the time of ripening required to achieve a cheese suitable for grating use. In PDO Pecorino Romano cheese, this feature is monitored by the moisture in nonfat substance (MNFS), which at 8 months of ripening must be <= 47.0%. The standardization of milk contributed to achieve the suitable value earlier (46.0 +/- 0.3 vs 47.2 +/- 0.5). The standardization of the protein/fat ratio in sheep milk did not induce any significant effect on the lipolysis or proteolysis progress in the cheese during ripening. Increasing the PFR significantly increased the levels of protein but, significantly, reduced the levels of moisture in nonfat substance, fat-in-dry matter and salt-in-moisture. The percentage of milk fat recovered in the SPFR cheese was significantly higher than in the NPFR cheese. Increasing the PFR led to a significant decrease in the actual cheese yield, but a significant increase occurred in the normalized cheese yield. The final results prove that a standardization of PFR in cheese milk has a marked effect on cheese composition, fat recovery, and cheese yield.

A few studies have been published on the distribution of oxytetracyline (OTC) residues present in milk among cheese, whey, and milk protein fractions, throughout cheese-making, most of them, focused on the effect of pasteurization and Ultra High Temperature (UHT) treatments, and carried out on cow milk. This study aimed to investigate the impact of a thermisation treatment of ovine milk spiked with oxytetracycline at MRL (maximum residue limit) and half MRL, on the fate of the molecule, and the effect of OTC residues on starter culture development and cheese composition. The antibiotic recovery and partition from milk into whey and cheese were assessed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Starter and non-starter microflora development was monitored by viable plate counts. Milk thermisation did not affect OTC recovery, partition and cheese chemical composition. On a dry matter basis, an OTC reduction between 15 and 19% was calculated in 60-day cheese, at MRL and half MRL, respectively. OTC caused a dose-dependent difference in the time required to reach pH 5.60, which was significantly higher (P < 0.05) at MRL level compared to half MRL and control, allowing coliform bacteria to reach 6 log CFU g(-1) in 1-day MRL OTC cheeses.