Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber.

BACKGROUND: Hair is composed mainly of keratin protein and a small amount of lipid. Protein hydrolysates, in particular those with low molecular weight distribution have been known to protect hair against chemical and environmental damage. Many types of protein hydrolysates from plants and animals have been used in hair and personal care such as keratin hydrolysates obtained from nails, horns and wool. Most of these hydrolysates are obtained by chemical hydrolysis and hydrothermal methods, but recently hydrolyzed hair keratin, feather keratin peptides, and feather meal peptides have been obtained by enzymatic hydrolysis using Bacillus spp in submerged fermentation. RESULTS: Keratin peptides were obtained by enzymatic hydrolysis of keratinases using Bacillus subtilis AMR. The microorganism was grown on a feather medium, pH 8.0 (1% feathers) and supplemented with 0.01% of yeast extract, for 5 days, at 28°C with agitation. The supernatant containing the hydrolysates was colleted by centrifugation and ultra filtered in an AMICON system using nano-membranes (Millipore - YC05). The Proteins and peptides were analyzed using HPTLC and MALDI-TOF-MS. Commercial preparations of keratin hydrolysates were used as a comparative standard. After five days the feather had been degraded (90-95%) by the peptidases and keratinases of the microorganism. MALDI-TOF mass spectrometry showed multiple peaks that correspond to peptides in the range of 800 to 1079 Daltons and the commercial hydrolysate was in the range of 900 to 1400 Da. HPTLC showed lower molecular mass peptides and amino acids in the enzymatic hydrolysate when compared with the commercial hydrolysate . A mild shampoo and a rinse off conditioner were formulated with the enzymatic hydrolysate and applied to hair fibers to evaluate the hydration, with and without heat, using a Corneometer® CM 825. The hydration was more efficient with heat, suggesting a more complete incorporation of hydrolysates into the fibers. Scanning Electron Microscopy showed deposits of organic matter in the junction of the cuticles that probably collaborates to the sealing of the cuticles, increasing the brightness and softness. CONCLUSIONS: These results show that the enzymatic method to produce keratin peptides for hair care products is an attractive and eco- friendly method with a great potential in the cosmetic industry.

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi.

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman's complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97kDa protein band in cellular extract and an 85kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.

Peptidase profiles from non-albicans Candida spp. isolated from the blood of a patient with chronic myeloid leukemia and another with sickle cell disease.

Candida lipolytica and Candida rugosa were isolated from blood samples from a patient with chronic myeloid leukemia (31 years old) and a patient with sickle cell disease (1-year-old), respectively. Isolates were grown for 48 h at 37 degrees C in either Sabouraud or tryptone soy broth (TSB). Peptidases were tested for using substrate sodium dodecyl sulfate-polyacrylamide gels with gelatin, casein, bovine serum albumin (BSA) or hemoglobin. Enzymography analyses were made on the following substrates: human albumin, IgG and human fibrinogen, which had been incubated with the concentrated supernatants. For C. lipolytica, a approximately 60-kDa gelatin-degrading serine proteolytic activity was found in the TSB supernantant as well as a metallopeptidase activity capable of hydrolysing human albumin, IgG and human fibrinogen. With C. rugosa, albumin, IgG and human fibrinogen substrates were degraded by an aspartyl-like peptidase activity. Supernatants from C. rugosa also showed three serine proteolytic activities towards gelatin (approximately 50 kDa, TSB), casein ( approximately 94 kDa, TSB) and BSA ( approximately 120-kDa, Sabouraud), in addition to a metallopeptidase capable of degrading casein ( approximately 110 kDa, Sabouraud). Little is known about peptidases of C. rugosa and C. lipolytica and this preliminary data may prove useful for future work on host-parasite relationship and antifungal agents.

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine.

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.

Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis.

The FML antigen of Leishmania donovani, in combination with either Riedel de Haën (R), QuilA, QS21 saponins, IL12 or BCG, was used in vaccination of an outbred murine model against visceral leishmaniasis (VL). Significant and specific increases in anti-FML IgG and IgM responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a and IgG2b and delayed type of hypersensitivity to L. donovani lysate (DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups achieved the highest IgG2a response. QuilA-FML developed the strongest DTH and QS21-FML animals showed the highest serum IFN-gamma concentrations. The reduction of parasitic load in the liver in response to each FML-vaccine formulation was: 52% (P<0.025) for BCG-FML, 73% (P<0.005) for R-FML, 93% (P<0.005) for QuilA-FML and 79.2% (P<0.025) for QS21-FML treated animals, respectively. Protection was specific for R-FML and QS21-FML while the QuilA saponin treatment itself induced 69% of LDU reduction. The FML-saponin vaccines promote significant, specific and strong protective effects against murine visceral leishmaniasis. BCG-FML induced minor and non-specific protection while IL12-FML, although enhancing the specific antibody and IDR response, failed to reduce the parasitic load of infected animals.