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Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Caseins (αs1-, αs2, β-, and κ-caseins) form the major protein fraction of milk, irrespective of their origins. They are able to form well-ordered colloidal structures in association with colloidal calcium phosphate, named casein micelles. Chymosin-mediated milk coagulation takes place through loss of casein micelles’ stability by hydrolyzing κ-caseins. This process is critical for the quality of cheese and other milk derivatives. Therefore, many microscopy techniques have been used to understand the structural aspects underlying the integrity of casein micelles during chymosin action. However, these technologies can be costly and laborious. In this study, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to study milk coagulation by chymosin. Following 15 min of chymosin action, the AFM images showed the start of the formation of casein micelle aggregates. After 30–45 min, the micelles continued aggregating, forming structures such as bunches of grapes. Finally, after 45–60 min, these structures formed large clusters of casein micelles. After 60 min, the zeta potential did not drop to zero when the milk clotted, but still had a negative value, suggesting that the κ-caseins on the micelle surface were not totally hydrolyzed. The results corroborate those previously described and suggest this tool as an alternative to study the milk-clotting process at the ultrastructural level induced by proteases.

Sapucaia or Lecythis pisonis Cambess. is an ornamental tree that produces edible, tasty, and nutritious nuts, and can be used for timber production. Sapucaia has potential as a functional food, owing to optimal nutritional and particularly Se levels. The present study sought to characterize the seeds of 21 sapucaia matrices from forest remnants of a neotropical hotspot (Atlantic Forest) and farms in two Brazilian states. Biometrics, germination, vigor, lignin content in the teguments, minerals content, and phenotypic and molecular diversity were analyzed. The seeds of matrices 16 and 21 were the most vigorous. Nuts from matrices 21 and 17 contained the highest amounts of Se. Matrices 5, 8, and 21 were the most phenotypically distant; whereas matrix 21 was the most genetically distant. Importantly, we validated a new non-destructive and efficient X-ray-based methodology for internal and densitometric analysis of sapucaia seeds, and demonstrated a high genetic divergence among matrices.

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in β-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow’s milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow’s milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow’s milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy., Instituto de Estudios Inmunológicos y Fisiopatológicos

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl2. However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process., Centro de Investigación de Proteínas Vegetales