Search Results (26)

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

The process of camel milk’s transformation into cheese is a delicate operation due to various difficulties in achieving coagulation. This study investigates the processing challenges of camel milk in the production of camel milk cheese using chicken gizzard inner lining extract (CGLE) as a coagulant. The crude extract presents an extraction yield of 55.05 ± 1.8% and a pH = 4.40 ± 0.05. The optimal coagulation conditions were pH 5 and temperature 45 °C. A fresh camel milk cheese was produced using CGLE and characterized as CME. The cheese yield of the CME was 26.88 ± 0.42%, which was higher than that obtained with chymosin (CC) at 12.66 ± 0.12%. The pH and acidity were 5.29 ± 0.09 and 56.25 ± 1.25°D. The gross composition of camel cheese (CME) was determined in comparison to (CC) fat (13.50 ± 2.82%), proteins (11.61 ± 0.19%), and dry matter (38.85 ± 1.22%). The sensory analysis demonstrated significant differences (p < 0.05) between the CME and CC in terms of white color, acidic taste, and consistency. Therefore, CME presents an overall acceptability in comparison to the control. The chicken gizzard inner lining extract could be used as an efficient coagulant for the production of fresh camel cheese. Full article

Low-moisture Mozzarella cheese (LMMC) was manufactured in a dairy factory by stretching fresh curd in hot water, with the addition of 0–30% commercial curd (stored curd) purchased as a semi-finished product. Two commercial fermentation-produced camel chymosins, CC-M and CC-S, were employed as coagulants. The chemical, rheological and microstructural properties of LMMC were assessed during storage. The results demonstrated that cheese composition was not significantly influenced by curd addition. The use of CC-S promoted a slight increase of fat matter with respect to the CC-M samples because of the higher proteolytic specificity and clotting activity of the CC-S enzyme. A higher extent of proteolysis was found in LMMC manufactured with CC-M. The textural properties evaluated during storage revealed an increase in meltability, adhesiveness and springiness over time. The amount of added curd had only a minor effect. The melting behaviour was significantly influenced by proteolysis during the 35-day storage period. Overall, the proteolysis during aging was the most impactful factor affecting the properties of LMMC. Full article

Milk coagulants play a crucial role in defining curd characteristics. The objective of this study was to compare the coagulation dynamics of two commonly used coagulants in cheesemaking: cardoon flower extract (Cynara cardunculus L.) and commercial chymosin, using sheep milk from four different origins in the Baixo Alentejo region of Portugal, as the substrate. Milk composition was determined using the MilkoScan 133B, while the milk-clotting time (MCT) was measured following ISO 23058/IDF 199:2006 guidelines with slight modifications and coagulation kinetics, and technological properties were evaluated using the Optigraph apparatus. The results demonstrate that the type of coagulant impacts the coagulation properties of sheep milk. Pearson’s correlation analysis indicated that milk samples with higher protein content exhibited longer coagulation times but resulted in firmer curds. On the other hand, the use of cardoon flower extract introduced greater variability compared to chymosin, with a delayed onset of coagulation, reduced curd firmness, and increased variability in enzymatic kinetics. These results suggest that cardoon extract, while traditional, introduces greater heterogeneity in curd formation compared to the more consistent action of chymosin. Full article

Cheese production is an ancient practice to preserve a perishable food, such as milk, for a long time. The first step of cheese processing involves the addition of rennet, which contains the enzymes necessary for the hydrolysis and coagulation of the caseins present in milk. Typically, animal-derived rennet, such as calf rennet containing chymosin, are used as source of enzymes for cheese processing. Alternatively, microbial chymosin or recombinant chymosin is used. However, recently, plant-derived rennet such as the ones derived from thistle and bitter orange flowers and from artichoke (Cynara cardunculus var. scolymus) have also been demonstrated to be valid sources of enzymes for cheese processing. Therefore, herein, different plant and fruit extracts were tested and compared for their ability to coagulate milk caseins. In particular, beyond artichoke and cardoon (Cynara cardunculus) extracts, those from pineapple (Ananas comosus (L.) Merr.), papaya (Carica papaya L.), common fig (Ficus carica L.) milky sap, and oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm.) were investigated for their proteolytic, esterase, and milk-clotting activities. The extracts were then exploited as vegetable and fruit rennet for the experimental production of cheeses, which were examined, after 30 days of maturation, for their moisture, fat, protein, and free fatty acid (FFA) content. Interestingly, the artichoke, cardoon, and thistle mushroom extracts showed high proteolytic activity compared to calf rennet, while the level of esterase activity appeared to be similar for all the extracts. The papaya extract showed the lowest proteolytic and esterase activity. Although the pH, moisture, fat, and protein contents were very similar to those of cheese made with calf rennet, the medium- and long-chain FFAs broadly differed among produced cheeses, with variations in the lipid quality indices. Full article

Inflammation is a natural protective reaction of the body against endogenous and exogenous damage, such as tissue injuries, trauma, and infections. Thus, when the response is adequate, inflammation becomes a defense mechanism to repair damaged tissue, whereas when the response is inadequate and persistent, the increase in inflammatory cells, cytosines, and chymosins impair tissue regeneration and promote a response harmful to the organism. One example is chronic tissue inflammation, in which a simple lesion can progress to ulcers and even necrosis. In this situation, the anti-inflammatory medications available in therapy are not always effective. For this reason, the search for new treatments, developed from medicinal plants, has increased. In this direction, the plants Agave sisalana (sisal) and Punica granatum (pomegranate) are rich in saponins, which are secondary metabolites known for their therapeutic properties, including anti-inflammatory effects. Although Brazil is the world’s leading sisal producer, approximately 95% of the leaves are discarded after fiber extraction. Similarly, pomegranate peel waste is abundant in Brazil. To address the need for safe and effective anti-inflammatory treatments, this study aimed to create a topical mucoadhesive gel containing a combination of sisal (RS) and pomegranate residue (PR) extracts. In vitro experiments examined isolated and combined extracts, as well as the resulting formulation, focusing on (1) a phytochemical analysis (total saponin content); (2) cytotoxicity (MTT assay); and (3) a pharmacological assessment of anti-inflammatory activity (phagocytosis, macrophage spreading, and membrane stability). The results revealed saponin concentrations in grams per 100 g of dry extract as follows: SR—29.91 ± 0.33, PR—15.83 ± 0.93, association (A)—22.99 ± 0.01, base gel (G1)—0.00 ± 0.00, and association gel (G2)—0.52 ± 0.05. In MTT tests for isolated extracts, cytotoxicity values (µg/mL) were 3757.00 for SR and 2064.91 for PR. Conversely, A and G2 exhibited no cytotoxicity, with increased cell viability over time. All three anti-inflammatory tests confirmed the presence of this activity in SR, PR, and A. Notably, G2 demonstrated an anti-inflammatory effect comparable to dexamethasone. In conclusion, the gel containing SR and PR (i.e., A) holds promise as a novel herbal anti-inflammatory treatment. Its development could yield economic, social, and environmental benefits by utilizing discarded materials in Brazil. Full article

Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A₂₈₀ units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The K_m, k_cat and k_cat/K_m values of moose chymosin were 4.7 μM, 98.7 s⁻¹, and 21.1 μM⁻¹ s⁻¹, respectively. The pattern of change in the coagulation activity as a function of pH and Ca²⁺ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg²⁺, Zn²⁺, Co²⁺, Ba²⁺, Fe²⁺, Mn²⁺, Ca²⁺, and Cu²⁺ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers. Full article

A method for the design and synthesis of a metallopolymer composite (CoNP) based on cobalt nanoparticles using the hyperbranched polyol process was developed. It was shown that hyperbranched polyester polyols in a melted state can be both a reducing agent and a stabilizer of metal nanoparticles at the same time. The mechanism of oxidation of hyperbranched polyol was studied using diffuse reflectance IR spectroscopy. The process of oxidation of OH groups in G4-OH started from 90 °C and finished with the oxidation of aldehyde groups. The composition and properties of nanomaterials were determined with FT-IR and UV-Vis spectroscopy, Nanoparticle Tracking Analysis (NTA), thermogravimetric analysis (TG), powder X-ray diffraction (XRD), NMR relaxation, and in vitro biological tests. The cobalt-containing nanocomposite (CoNP) had a high colloidal stability and contained spheroid polymer aggregates with a diameter of 35–50 nm with immobilized cobalt nanoparticles of 5–7 nm. The values of R₂ and R₁ according to the NMR relaxation method for CoNPs were 6.77 mM·ms⁻¹ × 10⁻⁵ and 4.14 mM·ms⁻¹ × 10⁻⁵ for, respectively. The ratio R₂/R₁ = 0.61 defines the cobalt-containing nanocomposite as a T₁ contrast agent. The synthesized CoNPs were nonhemotoxic (HC₅₀ > 8 g/mL) multifunctional reagents and exhibited the properties of synthetic modulators of the enzymatic activity of chymosin aspartic proteinase and exhibited antimycotic activity against Aspergillus fumigatus. The results of the study show the unique prospects of the developed two-component method of the hyperbranched polyol process for the creation of colloidal multifunctional metal–polymer nanocomposites for theranostics. Full article

In this study, the enzyme-induced and acid coagulation of raw milk samples were investigated, and these two processes were monitored by determining the changes in dielectric and rheological behavior to see if any correlations could be found between these two properties. For the enzyme-induced experiments, 250 cm³ of raw milk sample and industrial chymosin were used, and during the acid coagulation, industrial thermophile yogurt culture was added to the same amount of milk. The apparent viscosity was monitored with a vibro viscometer, and the dielectric constant was measured in a frequency range of 200–2400 MHz with an open-ended dielectric assessment kit (DAK) connected to a vector network analyzer (VNA). Our experimental findings revealed that during both enzyme-induced and acid coagulation, two distinct phases could be found in terms of dielectric and rheological properties, and regardless of the molecular mechanisms behind the aggregation of casein micelles, a strong correlation (with R²-values ranging between 0.967 and 0.992) can be found between the extent of the apparent viscosity and dielectric constant, suggesting that dielectric measurements might be adequately used in these dairy processing technologies. These results verified the applicability of the non-destructive dielectric measurements in dairy technologies. Full article

Optical sensor arrays are widely used in obtaining fingerprints of samples, allowing for solutions of recognition and identification problems. An approach to extending the functionality of the sensor arrays is using a kinetic factor by conducting indicator reactions that proceed at measurable rates. In this study, we propose a method for the discrimination of proteins based on their oxidation by sodium hypochlorite with the formation of the products, which, in turn, feature oxidation properties. As reducing agents to visualize these products, carbocyanine dyes IR-783 and Cy5.5-COOH are added to the reaction mixture at pH 5.3, and different spectral characteristics are registered every several minutes (absorbance in the visible region and fluorescence under excitation by UV (254 and 365 nm) and red light). The intensities of the photographic images of the 96-well plate are processed by principal component analysis (PCA) and linear discriminant analysis (LDA). Six model proteins (bovine and human serum albumins, γ-globulin, lysozyme, pepsin, and proteinase K) and 10 rennet samples (mixtures of chymosin and pepsin from different manufacturers) are recognized by the proposed method. The method is rapid and simple and uses only commercially available reagents. Full article

In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow’s, goat’s, ewes’, mare’s and camel’s milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast (Pichia pastoris GS115/pGAPZαA/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow’s, goat’s, ewes’, camel’s and mare’s milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures. Full article

Raw milk adulteration with cheese whey is a major problem that affects the dairy industry. The objective of this work was to evaluate the adulteration of raw milk with the cheese whey obtained from the coagulation process, with chymosin enzyme using casein glycomacropeptide (cGMP) as an HPLC marker. Milk proteins were precipitated with 24% TCA; with the supernatant obtained, a calibration curve was established by mixing raw milk and whey in different percentages, which were passed through a KW-802.5 Shodex molecular exclusion column. A reference signal, with a retention time of 10.8 min, was obtained for each of the different percentages of cheese whey; the higher the concentration, the higher the peak. Data analysis was adjusted to a linear regression model, with an R² of 0.9984 and equation to predict dependent variable (cheese whey percentage in milk) values. The chromatography sample was collected and analyzed by three tests: a cGMP standard HPLC analysis, MALDI-TOF spectrometry, and immunochromatography assay. The results of these three tests confirmed the presence of the cGMP monomer in adulterated samples with whey, which was obtained from chymosin enzymatic coagulation. As a contribution to food safety, the molecular exclusion chromatography technique presented is reliable, easy to implement in a laboratory, and inexpensive, compared with other methodologies, such as electrophoresis, immunochromatography, and HPLC-MS, thus allowing for the routine quality control of milk, an important product in human nutrition. Full article

In the present study, a kiwifruit aqueous extract was developed and used as a coagulant enzyme in cheesemaking. In detail, polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the presence of actinidin, the kiwifruit enzyme involved in κ-casein hydrolysis, in different tissues (pulp, peel, and whole fruit) of ripe and unripe kiwifruits. Data revealed the presence of the enzyme both in the peel and in the pulp of the fruit. Although the aqueous extract obtained from the kiwifruit peel was able to hydrolyze semi-skimmed milk, it did not break down κ-casein. The aqueous extract obtained from the pulp showed a hydrolytic activity toward both κ-casein and semi-skimmed milk. The values for milk-clotting and proteolytic activity of the kiwifruit pulp extract were evaluated at different temperatures and pH parameters in order to obtain a high value of the MCA/PA ratio; we found that a temperature of 40 °C in combination with a pH value of 5.5 allowed us to obtain the best performance. In addition, the data revealed a higher hydrolytic activity of the enzymatic preparation from ripe kiwifruits than that from unripe ones, suggesting the use of the extract from pulp of ripe kiwifruits in the laboratory-scale cheesemaking. The data showed that 3% (v/v) of the ripe kiwifruit pulp extract determined a curd yield of 20.27%, comparable to chymosin yield. In conclusion, the extraction procedure for kiwifruit aqueous extract proposed in the present study was shown to be a fast, cheap, chemical-free, and ecofriendly technology as a plant coagulant for cheese manufacturing. Full article

It is well known that cheese yield and quality are affected by animal genetics, milk quality (chemical, physical, and microbiological), production technology, and the type of rennet and dairy cultures used in production. Major differences in the same type of cheese (i.e., hard cheese) are caused by the rennet and dairy cultures, which affect the ripening process. This review aims to explore current technological advancements in animal genetics, methods for the isolation and production of rennet and dairy cultures, along with possible applications of microencapsulation in rennet and dairy culture production, as well as the challenge posed to current dairy technologies by the preservation of biodiversity. Based on the reviewed scientific literature, it can be concluded that innovative approaches and the described techniques can significantly improve cheese production. Full article

The use of casein glycomacropeptide (CGMP) as a protein substitute in phenylketonuria (PKU) has grown in popularity. CGMP is derived from κ casein and is a sialic-rich glycophosphopeptide, formed by the action of chymosin during the production of cheese. It comprises 20–25% of total protein in whey products and has key biomodulatory properties. In PKU, the amino acid sequence of CGMP has been adapted by adding the amino acids histidine, leucine, methionine, tyrosine and tryptophan naturally low in CGMP. The use of CGMP compared to mono amino acids (L-AAs) as a protein substitute in the treatment of PKU promises several potential clinical benefits, although any advantage is supported only by evidence from non-PKU conditions or PKU animal models. This review examines if there is sufficient evidence to support the bioactive properties of CGMP leading to physiological benefits when compared to L-AAs in PKU, with a focus on blood phenylalanine control and stability, body composition, growth, bone density, breath odour and palatability. Full article