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The use of crude aqueous extracts of Cynara cardunculus flowers as coagulants in the production of high-quality sheep and goat cheeses—as are the cases of several Portuguese and Spanish cheese varieties with Protected Designation of Origin status—has been maintained since ancient times. The unique rheological attributes and sensory properties characteristic of these cheeses have always suggested that this plant coagulant (and, therefore, its isolated milk-clotting proteases) could be used as alternative rennet in the dairy industry, particularly suited for the production of sheep and goat cheeses. However, the lack of standardization of C. cardunculus crude flower extracts, whose quality and performance depends on numerous factors, has always hampered the application of this plant rennet in industrial production scales. To overcome these limitations, and to aim at developing more effective solutions with potential for scalability of production and commercial application, several strategies have been undertaken in more recent years to establish new cardoon-based rennets. This review provides an overview on these developments and on the currently available solutions, which range from producing standardized formulations of native cardoon enzymes, to the optimization of the heterologous production of cardosins and cyprosins to generate synthetic versions of these milk-clotting enzymes. Challenges and emerging opportunities are also discussed.

We evaluated the effects of fermentation time and acid casein content on the microbial rennet obtained by solid-state fermentation using wheat bran as the carbon source. The experiments used two fermentation times (72 and 96 h), while acid casein content was 1.5, 2.0, 2.5, and 3.0 g. Rennet strength from eight enzymatic extracts was measured using pasteurized whole milk. Rennet strength of samples from 72 h of fermentation showed an increase when acid casein content increased. The rennet strength increased at 96 h of fermentation with increasing amount of casein (up to 2.5 g), and then decreased with the largest addition (3.0 g) of casein. Coagulation time for the sample with highest rennet strength was 420 s.

The reduced availability and the increasing prices of calf rennet, coupled to the growing global demand of cheese has led, worldwide, to explore alternative clotting enzymes, capable to replace traditional rennet, during the cheesemaking. In addition, religious factors and others related to the vegetarianism of some consumers, have led to alternative rennet substitutes. Nowadays, several plant-derived milk-clotting enzymes are available for cheesemaking technology. Many efforts have also been made to compare their effects on rheological and sensory properties of cheese to those arising from animal rennet. However, vegetable clotting enzymes are still partially suitable for cheesemaking, due to excessive proteolytic activity, which contribute to bitter flavor development. This review provides a literature overview of the most used vegetable clotting enzymes in cheese technology, classified according to their protease class. Finally, clotting and proteolytic activities are discussed in relation to their application on the different cheesemaking products.

The aim of this study was to evaluate the combined effect of coagulant type (animal rennet, vegetal rennet, microbial rennet, or recombinant chymosin) and microbial transglutaminase on sensory properties. Results show that cheese coagulated with vegetal rennet exuded the highest amount of whey. Cheeses obtained using transglutaminase were less grainy, more soluble, creamier and showed a greater milk intensity and fresh cheese aroma. The effect of transglutaminase on moisture and persistency was different depending on the type of coagulant used. It can be concluded that microbial transglutaminase is a useful tool to modify sensory characteristics of cheese traditionally coagulated with animal rennet.

Since ancient times, thistles have been used as clotting agents in the production of traditional cheeses, particularly in the Mediterranean area. In recent years, their use in cheesemaking has increased to satisfy the growing requests from vegetarian consumers. In this research paper, four different cheeses, typical of the Mediterranean area, were evaluated from a nutritional point of view: Caciofiore (from Italy) and Torta del Casar (from Spain), both typically produced using vegetable rennet, and Queso de Murcia al vino (from Spain) and Feta (from Greece), traditionally produced using animal rennet. All the cheeses were manufactured according to their traditional cheesemaking procedures and used as controls. Experimental cheeses were produced using aqueous extracts obtained from flowers of either spontaneous or cultivated thistles indigenous to the Mediterranean area (respectively Onopordum tauricum for Caciofiore, and Cynara humilis for Torta del Casar, Queso de Murcia al vino, and Feta). All cheeses were characterized for fat-soluble and mineral compounds to assess their nutritional adequacy according to the recommended daily intake of each evaluated nutrient. All the cheeses were found to be a good source of vitamin A, calcium and phosphorus, with an optimal Ca/P molar ratio, except for Feta. By consuming the recommended serving (50 g) of the studied cheeses, the salt and cholesterol intake is, on average, 16.4% and 15.9%, respectively of recommended intake. The use of aqueous thistle extracts in cheesemaking appears to have no effect on the nutritional quality of the studied cheeses.

The objective of this study is to characterize purebred Arab mares' milk through monitoring its physicochemical and microbiological composition during different lactation stages and to test its ability to coagulate. Sixteen purebred Arabian mares were selected among 45 mares with approximately the same foaling dates. Milk samples were collected once a week for each mare during 4 months of lactation and analysed in three replicates for physicochemical composition, nitrogen fractions, somatic cell count and total bacterial count. Coagulation assays were carried out in triplicates on fresh mare's milk using increasing doses of calf rennet, starter (Streptococcus thermophilus) and CaCl2. Arab mare's milk is characterized by an alkaline pH, low-fat and protein contents and high lactose content. The somatic cell and bacterial counts are very low, indicating good health status of mares. Fat and protein contents decreased as the stage of lactation progressed. However, as lactation number increased, fat level tended to increase while protein level tended to decrease. Clotting assays proved that it is possible to coagulate Arabian mare’s milk using appropriate doses of calf rennet and starter, while respecting the coagulation procedures and parameters.

Calcium can be added to cheese milk to influence the coagulation process and to increase cheese yield. Calcium compounds used in the dairy industry show substantial differences in their practical application. Therefore, this study aimed to evaluate the potential use of 0, 5, 10, 15, and 20 mg Ca 100 g−1 of milk in the form of calcium gluconate, lactate, and carbonate as alternatives to calcium chloride in manufacturing fresh acid rennet cheese from high-pasteurized (90 °C, 15 s) goat’s milk. The pH value of the cheese was reduced most strongly by the addition of increasing doses of calcium lactate (r = −0.9521). Each cheese sample showed increased fat content with the addition of calcium. Only calcium chloride did not reduce protein retention from goat’s milk to cheese. The addition of 20 mg Ca 100 g−1 of milk in the form of gluconate increased cheese yield by 4.04%, and lactate reduced cheese yield by 2.3%. Adding each calcium compound to goat’s milk significantly increased Ca and P levels in the cheese (p ≤ 0.05). The highest Ca levels were found in cheese with the addition of 20 mg Ca 100 g−1 of milk in the form of lactate. In all groups, similar contents of Mn, Mo, and Se were found. Calcium addition significantly affected cheese hardness, while higher calcium concentrations increased hardness. Carbonate caused the greatest increase in the cohesiveness of cheese. The addition of calcium compounds increased the adhesiveness and springiness of cheese compared to controls. The cheese with calcium chloride had the highest overall acceptability compared to the other cheese samples. The addition of calcium carbonate resulted in a lower score for appearance and consistency, and influenced a slightly perceptible graininess, sandiness, and stickiness in its consistency, as well as provided a slightly perceptible chalky taste.

Casein as the major protein of milk is a promising protein source for biopolymer fibers. Current casein-based fibers are fabricated by dissolving caseins in alkaline media and wet spinning in a coagulation bath containing harsh chemicals. In milk, casein is present in so-called casein micelles (CMs). Based on the rennet-induced aggregation, we developed a process that can be applied for the spinning of micellar casein fibers in a sustainable way without the use of harsh chemicals. Fabricated fibers show a surface with a characteristic microstructure, which can also be detected embedded in a network structure inside the fiber. The fibers are stable under acidic and neutral conditions and decompose in alkaline media down to aggregates with sizes comparable to the characteristic microstructure. The so far reached tensile properties of the micellar fiber are between low and mid double-digit percentage range compared to casein azlons.

Milk coagulation is a process used for the formulation of different dairy products such as cheese. In this process, milk undergoes changes in its chemical stability thanks to acidification or enzymatic reactions. Traditionally, milk coagulation has been carried out with rennet of animal origin, but recently, the research of new types of rennet such as microbial rennet and vegetable rennet has increased. This study aims to present an organized review of the most relevant information on lactic coagulation, its relationship with vegetable rennets, and the importance of the botanical genus Cynara in the extraction of vegetable rennets, focusing on the coagulant potential of artichoke (Cynara scolymus). We conducted this literature review and found that lactic coagulation and vegetable rennets are linked through the enzymatic activity of the latter. The results of the main studies demonstrated a strong relationship between vegetable rennets and protease enzymes as well as the presence of these enzymes in extracts of cardoon (Cynara scolymus) and artichoke (Cynara scolymus). In addition, studies highlight the presence of thistle extracts in artisanal cheese preparations in the Iberian Peninsula. Based on the results of the studies, a comparison between cheeses made with vegetable rennet and those made with traditional rennet was also carried out. Although the results show that the use of vegetable rennet in the manufacture of cheese can confer undesirable characteristics, the use of extracts from Cynara plants demonstrates that vegetable rennets have an industrial potential, especially the one obtained from artichoke (Cynara scolymus) due to its high availability. Nevertheless, specific studies are required for a better understanding and application of this rennet.

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4°C. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl₂ resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55°C, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.