Antônio S. Egito

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazils semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Canola Cake as a Potential Substrate for Proteolytic Enzymes Production by a Selected Strain of Aspergillus oryzae: Selection of Process Conditions and Product Characterization

Oil cakes have excellent nutritional value and offer considerable potential for use in biotechnological processes that employ solid-state fermentation (SSF) for the production of high value products. This work evaluates the feasibility of using canola cake as a substrate for protease production by a selected strain of Aspergillus oryzae cultivated under SSF. The influences of the following process parameters were considered: initial substrate moisture content, incubation temperature, inoculum size, and pH of the buffer used for protease extraction and activity analysis. Maximum protease activity was obtained after cultivating Aspergillus oryzae CCBP 001 at 20°C, using an inoculum size of 107 spores/g in canola cake medium moistened with 40 mL of water to 100 g of cake. Cultivation and extraction under selected conditions increased protease activity 5.8-fold, compared to the initial conditions. Zymogram analysis of the enzymatic extract showed that the protease molecular weights varied between 31 and 200 kDa. The concentrated protease extract induced clotting of casein in 5 min. The results demonstrate the potential application of canola cake for protease production under SSF and contribute to the technological advances needed to increase the efficiency of processes designed to add value to agroindustrial wastes.

Peptide profile of Coalho cheese: A contribution for Protected Designation of Origin (PDO).

Coalho cheese of Ceará and the Jaguaribe region of Brazil has been studied to determine its peptide profile. Peptides generated by the action of peptidases upon cheese proteins were separated by reverse-phase HPLC to give 28 fractions. Peptide sequencing after MS/MS fragmentation enabled the identification of 116 different peptides; 74 of them originated from β-casein, 4 from βA2-casein, 4 from βA3-casein, 25 from αS1-casein, 5 from αS2-casein, and 4 from κ-casein. Phosphorylated peptides were identified, one from αS1-casein and 17 from β-casein. Other reports on the bioactivity of casein-derived peptides have shown that the β-casein peptide (193-209) exhibits immunomodulatory, antimicrobial and antihypertensive activity. The peptides β-casein (58-72), β-casein (193-202), αs1-casein (85-91), αs1-casein (1-9), as well as αs2-casein (189-197) have antihypertensive activity. The fragment αS1-casein (1-23) is an immunomodulatory and antimicrobial peptide. These results can be a marker to determine the authenticity of this Brazilian cheese.

Caracterização eletroforética de proteínas e estabilidade do leite em vacas submetidas à restrição alimentar

The objective of this work was to evaluate the feeding restriction effects on milk yield and on its electrophoretic protein profile, and to relate them to milk stability to the alcohol test. Two experiments were carried out with Jersey cows: in the first one, the effect of 40% feed restriction was evaluated on semiconfined cows; in the second one, the effect of 30% reduction in total digestive nutrients was evaluated on confined cows. Protein fractions were determined by electrophoresis and their quantification was done by image analysis. Milk samples were classified according to their stability in the 72°GL‑alcohol test. The 40% feed restriction for semiconfined cows decreased milk yield, but it did not change milk protein contents and stability; in this case, unstable milk showed larger contents of β‑casein and total proteins, but lower κ‑casein in comparison to stable milk. The 30% restriction of energy supply for confined cows did not reduce milk yield, but decreased the percentage of serum bovine albumin and milk stability; in this case, unstable and stable milk did not differ as for protein fractions.

Functional Properties of Lactobacillus mucosae Strains Isolated from Brazilian Goat Milk

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.

Insights into milk-clotting activity of latex peptidases from Calotropis procera and Cryptostegia grandiflora

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 10min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16kDa, 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.

Degradabilidade in situ do bagaço de cana-de-açúcar para pequenos ruminantes de raças naturalizadas do Nordeste brasileiro

Com o crescimento da industria sucroalcooleira, buscam-se alternativas de uso dos residuos gerados por ela. Os pequenos ruminantes de racas naturalizadas do Nordeste brasileiro mostram-se como grupos potenciais para que seja utilizado o bagaco de cana-de-acucar (BCA) nas dietas. Nesse contexto, objetivou-se determinar a degradabilidade ruminal in situ da materia seca (MS) e da fibra em detergente neutro (FDN) do BCA e os parâmetros ruminais em caprinos Moxoto e ovinos Morada Nova, fistulados no rumen. Contidos em sacos de nailon, 3g de BCA foram incubados no rumen nos tempos seis, 24 e 96 horas, determinando-se o conteudo de MS e FDN nos residuos obtidos. Nos tempos zero, seis e 12 horas apos a primeira refeicao, mediram-se no liquido ruminal pH e nitrogenio amoniacal (N-NH3) ruminal. O CMS nao diferiu entre caprinos e ovinos. O potencial de maxima degradacao da MS foi semelhante entre especies, e da FDN foi superior em caprinos. Ovinos apresentaram maiores tempo de colonizacao, taxa de degradacao e degradabilidade efetiva da MS e FDN. O pH nao diferiu entre as especies. Observou-se maior concentracao de N-NH3 ruminal em caprinos, no tempo zero. Diante da maior velocidade de degradacao da MS do BCA pelos ovinos, essa especie se mostra detentora de uma microbiota ruminal com crescimento mais eficiente sobre o BCA.