Search Results (44)

Consumer interest in vegetarian, ethical, and clean-label foods is reviving the use of plant-derived milk coagulants. Cardosins from Cynara cardunculus (“thistle”) are aspartic proteases with strong clotting activity, yet their technological impact in cheese remains under-explored. This study compared a commercial thistle extract (PC) with traditional bovine rennet rich in chymosin (AC) during manufacture and 60-day ripening of Caciotta cheese. Classical compositional assays (ripening index, texture profile, color, solubility) were integrated with scanning electron microscopy, three-dimensional surface reconstruction, and descriptive sensory analysis. AC cheeses displayed slower but sustained proteolysis, yielding a higher and more linear ripening index, softer body, greater solubility, and brighter, more yellow appearance. Imaging revealed a continuous protein matrix with uniformly distributed, larger pores, consistent with a dairy-like sensory profile dominated by milky and umami notes. Conversely, PC cheeses underwent rapid early proteolysis that plateaued, producing firmer, chewier curds with lower solubility and darker color. Micrographs showed a fragmented matrix with smaller, heterogeneous pores; sensory evaluation highlighted vegetal, bitter, and astringent attributes. The data demonstrate that thistle coagulant can successfully replace animal rennet but generates cheeses with distinct structural and sensory fingerprints. The optimization of process parameters is therefore required when targeting specific product styles. Full article

Background: Zoonotic infection with Mycobacterium bovis continues to occur, particularly in regions lacking bovine tuberculosis surveillance and where the consumption of unpasteurized dairy products, including artisanal cheeses, is common. We describe the clinical and microbiological characteristics, diagnostic procedures, and treatment outcomes of individuals with HIV with M. bovis infection. Methods: We conducted a retrospective study analyzing sociodemographic, clinical, microbiological, and computed tomography (CT) data, as well as treatment outcomes, in 12 patients with HIV with confirmed M. bovis infection. These findings were compared with those of 14 individuals with HIV diagnosed with Mycobacterium tuberculosis infection during the same period. Results: Consumption of unpasteurized dairy products was significantly associated with M. bovis. Patients with M. bovis infection had higher CD4+ T-cell counts compared to those with M. tuberculosis infection (p = 0.01, r = 0.45). All M. bovis cases presented with extrapulmonary disease. CT imaging in M. bovis infection more frequently demonstrated retroperitoneal lymphadenopathy, hepatosplenomegaly, and splenic abscesses compared to M. tuberculosis infection. Microbiological identification was exclusively from extrapulmonary sites in all M. bovis cases. Surgical interventions, including abscess drainage or splenectomy, were significantly more common among M. bovis patients. Conclusions: M. bovis infection in individuals with HIV is characterized by consistent extrapulmonary, often abdominal, involvement. Surgical procedures are frequently required for both diagnosis and management. Targeted efforts to identify M. bovis are warranted, particularly in high-burden regions where unpasteurized dairy consumption remains prevalent. Full article

Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. Full article

Sweet whey (SW), a by-product of cheese production, has potential immunomodulatory properties that could be beneficial in preventing inflammation-related diseases. This study investigated the effects of SW derived from bovine, caprine, ovine, or an ovine/caprine mixture of milk on inflammation-related gene expression in THP-1-derived macrophages, both with and without LPS stimulation. Cells were treated with SW-D-P3 (a fraction smaller than 3 kDa produced by in vitro digestion), and the expression of inflammation-related genes was assessed using quantitative PCR. Results showed that the expression of TLR2 and ICAM1 was attenuated in non-LPS-stimulated macrophages treated with SW-D-P3, regardless of animal origin. Moreover, the expression of TLR4, IL1B, and IL6 was decreased and the expression of an NF-κB subunit RELA and CXCL8 was elevated in a subset of samples treated with SW-D-P3, depending on the milk source. In LPS-challenged cells, the expression of CXCL8 was upregulated and the expression of IRF5 and TNFRSF1A was downregulated in SW-D-P3-treated cells, regardless of animal origin. On the other hand, a number of inflammation-related genes were differentially expressed depending on the animal origin of the samples. Moreover, the higher IL10 expression observed in cells treated with ovine/caprine SW-D-P3 compared to those treated with SW-D-P3 of bovine, caprine, or ovine origin suggests an anti-inflammatory response, in which alternatively activated macrophages (M2 polarization phenotype) may participate. Overall, these findings suggest that incorporating SW into the food industry, either as a standalone ingredient or supplement, may help to prevent inflammation-related diseases. Full article

This study critically examines the limitations of the official Italian methodology used for detecting bovine adulteration milk in Protected Designation of Origin (PDO) Mozzarella di Bufala Campana (MdBC). This method focuses on the whey fraction of cheese samples, which comprises about 1% of total MdBC proteins, and is based on a high-performance liquid chromatography (HPLC) quantification of the bovine β-lactoglobulin A (β-Lg A) as a marker. Here, we have demonstrated that this official methodology suffers from measurement inconsistencies due to its reliance on raw bovine whey standards, which fail to account for β-Lg genetic polymorphisms in real MdBC samples and protein thermal modifications during cheesemaking. To overcome these limitations, we propose a dual proteomics-based approach using matrix-assisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS) and nano-HPLC-electrospray (ESI)−tandem mass spectrometry (MS/MS) analysis of MdBC extracted whey. MALDI-TOF-MS focused on identifying proteotypic peptides specific to bovine and buffalo β-Lg and α-lactalbumin (α-La), enabling high specificity for distinguishing the two animal species at adulteration levels as low as 1%. Complementing this, nano-HPLC-ESI-MS/MS provided a comprehensive profile by identifying over 100 bovine-specific peptide markers from β-Lg, α-La, albumin, lactoferrin, and osteopontin. Both methods ensured precise detection and quantification of bovine milk adulteration in complex matrices like pasta filata cheeses, achieving high sensitivity even at minimal adulteration levels. Accordingly, the proposed dual proteomics-based approach overcomes challenges associated with whey protein polymorphism, heat treatment, and processing variability, and complements casein-based methodologies already validated under European standards. This integrated framework of analyses focused on whey and casein fraction enhances the reliability of adulteration detection and safeguards the authenticity of PDO buffalo mozzarella, upholding its unique quality and integrity. Full article

Cow’s milk contains A1- and A2-β-caseins. The breakdown of A1-β-casein produces β-casomorphin-7 (BCM-7), a peptide with opioid-like properties that is associated with health aspects. In addition, A1- and A2-β-casein have different technological properties. The aim of the present study was to investigate whether cheese produced from the milk of homozygous A1A1 and A2A2 cows varies in terms of its physicochemical parameters and BCM-7 concentration. These parameters were analyzed during initial cheese processing, six weeks of ripening and 84 days of storage, including additional microbiological analyses during the storage period. The pH values of the A1A1 cheeses were higher than those of the A2A2 cheeses from the beginning of production until the starter culture bacteria were added. The yellowness values of the A1A1 cheeses were lower until the salt bath treatment. Water activity, lightness, hardness, fat, protein, NaCl and dry matter content, as well as color and microbiological parameters, were not affected by the β-casein genotype. BCM-7 concentrations were higher in the A1A1 cheeses after pressing and during ripening. We found mainly comparable quality characteristics and slightly different BCM-7 levels in the A1A1 and A2A2 cheeses. From this point of view, both varieties are equally suitable for cheese production. Full article

Clostridium species are known for their impact on animal and human health, but also for the spoilage of foodstuffs. Their spores contaminate milk and result in germination and gas production, the latter being particularly evident in the cheeses that suffer severe depreciation. To address this issue, the Primary Production Department of the IZSLER institute in Brescia, Italy conducts the Most Probable Number (MPN) method on bovine milk samples collected from Northern Italian dairies between 2004 and 2023. This approach leverages two semi-quantitative protocols, S2 and S3, to detect Clostridium species spore forms upon customer request. Here, we would like to present an a-posteriori analysis on the results of the S2 and S3 protocols. The goal of this study is to highlight the differences between these two methods and provide evidence of the actual decrease in Clostridium species in raw cow milk over a 20-year period. Our analysis shows that client demand for S2 has progressively decreased, while S3’s has remained constant, and both protocols reveal a significant reduction in positives; furthermore, S3’s greater sensitivity made it more responsive to environmental changes. This highlights the necessity of choosing the appropriate testing protocol that accounts for both regulatory standards and environmental factors. Overall, our findings underscore the importance of continued monitoring to manage Clostridium species contamination and ensure milk quality. Full article

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan^®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk. Full article

Sweet whey (SW) and yogurt acid whey (YAW) are dairy by-products of the cheese-making process and Greek-style yogurt production, respectively. Both of them are considered pollutants with huge volumes of SW and YAW produced due to the growing demand for dairy products worldwide. Moreover, whey-derived peptides, resulting from fermentation as well as from further hydrolysis during digestion, have been associated with various biological activities. In the present study, the angiotensin-converting enzyme (ACE)-inhibitory activity of 48 SW samples and 33 YAW samples from bovine, ovine, caprine, and ovine/caprine milk obtained were evaluated. Additionally, the SW and YAW digestates and two of their fractions (smaller than 10 kDa, SW-D-P10 and YAW-D-P10, and smaller than 3 kDa, SW-D-P3 and YAW-D-P3), which were obtained after in vitro digestion and subsequent ultrafiltration, were also subjected to evaluation. Our data indicated that the D-P10 and D-P3 fractions exhibited higher ACE-inhibitory activity compared to the corresponding values before digestion. The ACE-inhibitory capacity after in vitro digestion was higher for the ovine SW samples compared to their bovine and caprine counterparts. The effect of the D-P3 fraction on the inhibition of nitric oxide (NO) production and the expression of a selected panel of immune-response-related genes in LPS-stimulated RAW 264.7 macrophages was also evaluated. Fractions from both dairy by-products inhibited NO production in LPS-stimulated RAW 264.7 cells. Especially, ovine SW-D-P3 showed a strong NO inhibitory activity and suppressed inducible nitric oxide synthase (Nos2) mRNA levels. However, YAW-D-P3 could not trigger neither the gene expression of inflammatory macrophage mediators Nos2 and cyclooxygenase-2 (Ptgs2) nor tumor necrosis factor-α (Tnf) and interleukin 6 (Il6) in LPS-stimulated murine macrophages regardless of animal origin. These findings suggest that in vitro digestion could enhance the production of ACE-inhibitory peptides in both dairy by-products, while SW from ovine origin displays higher potential as an anti-inflammatory agent, effectively preventing excessive NO production. Full article

Brazilian artisanal cheeses have recently gained significant commercial prominence and consumer favor, primarily due to their distinctive sensory attributes and cultural and historical appeal. Many of these cheeses are made with raw milk and undergo a relatively short ripening period, sometimes ranging from 4 to 8 days, though it is usually shorter than the period stated by law. Moreover, there is insufficient evidence regarding the efficacy of a short ripening period in reducing certain zoonotic foodborne pathogens, such as Brucella spp., Coxiella burnetiid, and Mycobacterium bovis (as part of the Mycobacterium tuberculosis complex). Additionally, a literature analysis revealed that the usual ripening conditions of Brazilian artisanal cheeses made with raw milk may be inefficient in reducing the levels of some hazardous bacterial, including Brucella spp., Listeria monocytogenes, coagulase-positive Staphylococcus, Salmonella, and Coxiella burnetti, to the acceptable limits established by law, thus failing to ensure product safety for all cheese types. Moreover, the assessment of the microbiological safety for this type of cheese should be broader and should also consider zoonotic pathogens commonly found in bovine herds. Finally, a standardized protocol for evaluating the effectiveness of cheese ripening must be established by considering its peculiarities. Full article

The Podolica cattle breed is widespread in southern Italy, and its productivity is characterized by low yields and an extraordinary quality of milk and meats. Most of the milk produced is transformed into “Caciocavallo Podolico” cheese, which is made with 100% Podolica milk. Fourier Transform Infrared Spectroscopy (FTIR) is the technique that, in this research work, was applied together with machine learning to discriminate 100% Podolica milk from contamination of other Calabrian cattle breeds. The analysis on the test set produced a misclassification percentage of 6.7%. Among the 15 non-Podolica samples in the test set, 2 were misclassified and recognized as Podolica milk even though the milk was from other species. The correct classification rate improved to 100% when the same method was applied to the recognition of Podolica and Pezzata Rossa milk produced by the same farm. Furthermore, this technique was tested for the recognition of Podolica milk mixed with milk from other bovine species. The multivariate model and the respective confusion matrices obtained showed that all the 14 Podolica samples (test set) mixed with 40% non-Podolica milk were correctly classified. In addition, Pezzata Rossa milk produced by the same farm was detected as a contaminant in Podolica milk from the same farm down to concentrations as little as 5% with a 100% correct classification rate in the test set. The method described yielded higher accuracy values when applied to the discrimination of milks from different breeds belonging to the same farm. One of the reasons for this phenomenon could be linked to the elimination of the environmental variable. However, the results obtained in this work demonstrate the possibility of using FTIR to discriminate between milks from different breeds. Full article

Milk is the most consumed liquid food in the world due to its high nutritional value and relatively low cost, characteristics that make it vulnerable to adulteration. One of the most common types of milk adulteration involves the undeclared addition of cow’s milk to milk from other mammalian species, such as goats, sheep, buffalo or donkeys. The incidence of such adulteration not only causes a crisis in terms of commercial market and consumer uncertainty but also poses a risk to public health, as allergies can be triggered by proteins in undeclared cow’s milk. In this study, a specific qualitative touchdown (TD) PCR method was developed to detect the undeclared addition of cow’s milk in goat and sheep milk based on the discrimination of the peak areas of the melting curves after the modification of bovine-specific primers. The developed methodology has high specificity for the DNA templates of other species, such as buffalos and donkeys, and is able to identify the presence of cow’s milk down to 1%. Repeatability was tested at low bovine concentrations of 5% and 1% and resulted in %RSD values of 1.53–2.04 for the goat–cow assay and 2.49–7.16 for the sheep–cow assay, respectively. The application of this method to commercial goat milk samples indicated a high percentage of noncompliance in terms of labeling (50%), while a comparison of the results to rapid immunochromatographic and ELISA kits validated the excellent sensitivity and applicability of the proposed PCR methodology that was able to trace more adulterated samples. The developed assays offer the advantage of multiple detection in a single run, resulting in a cost- and time-efficient method. Future studies will focus on the applicability of these assays in dairy products such as cheese and yogurt. Full article

Bovine whey cheese (WC) is a product from southern European countries that presents some challenges: its production process involves high energy inputs; the yield is low; and WC has a short shelf life. The application of ultrafiltration (UF) to bovine whey before manufacture of WC and the employment of protective cultures can reduce these disadvantages. The objective of this research was the production of whey cheeses using ultrafiltrated bovine cheese whey with added probiotics or probiotics plus protective cultures. Three types of WC were produced: control CW without any addition (C); CW with the addition of the probiotic Lactobacillus acidophilus (LA5); and CW with the addition of Lactobacillus acidophilus plus a protective culture containing Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus (LA5FQ4). The WCs were stored under refrigerated conditions for 28 days. The products with added cultures presented lower pH values and higher titratable acidities when compared to the control. Sample LA5 presented the lowest pH and the highest titratable acidity, while LA5FQ4 presented intermediate values. Slight differences were observed between products regarding color parameters, chiefly resulting from storage time. The samples with added cultures were firmer when compared to the control, with LA5 cheeses showing the highest values at the end of the storage. Lactic acid bacteria (LAB) counts were on the order of log 8–9 CFU/g for the products with added cultures. Lower levels of yeasts and molds were detected on the sample with the protective culture (LA5FQ4), so that by the end of storage they presented counts one log cycle lower than C and LA5. Hence, the beneficial impact of the protective culture on the shelf life of the product is evident. Regarding sensory evaluation, LA5FQ4 cheeses obtained the highest scores for all parameters evaluated. It can be concluded that the use of UF associated with the addition of protective cultures can be very useful to reduce the energy consumption of the manufacturing process, to prolong the shelf life of WC and to improve its sensory properties. Full article

Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains Lactococcus lactis subsp. lactis ZGBP5-51, Enterococcus faecium ZGBP5-52 and Enterococcus faecalis ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (⁷⁵QFLPYPYYAKPA⁸⁶, ⁴⁰VAPFPEVFGK⁴⁹, ¹¹⁷ARHPHPHLSF¹²⁶), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates. Full article

The objective of this study was to develop fresh and matured cheeses with different bovine colostrum levels, aiming to promote the consumption of dairy products with the addition of colostrum. Four different cheese formulations were produced with a mixture of 0:100, 15:85, 20:80, and 25:75, bovine colostrum:milk (v:v), and aged for 0, 10, 20, and 40 days. Milk, colostrum, and fresh and matured cheeses were submitted to physicochemical characterization. Moreover, microbiological quality, yield, texture profile, color, and sensory acceptance of cheese samples were evaluated. Colostrum supplementation favored low acidity, high moisture, a pH range of 5.0–6.2, and water activity of 0.94–99. Sensory attributes and overall evaluation of all cheese formulations achieved an Acceptability Index above 70, indicating good acceptability. Since cheese with colostrum presented the potential to be used as human food, assessing the presence of colostrum bioactive components in those dairy products is a promising goal for further research. Full article