Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation / Decomposição fúngica de resíduos de pena de frango em fermentação submersa e de estado sólido

Poultry industry is expanding rapidly and producing million tons of feather waste annually. Massive production of keratinaceous byproducts in the form of industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Keratinase released by a variety of microbes (bacteria and fungi) can be used for the effective treatment of keratin waste. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This study involves the isolation, characterization, and potential utility of fungal species for the degradation of chicken-feather waste through submerged and solid-state fermentation. The isolated fungus was identified and characterized as Aspergillus (A.) flavus. In a trial of 30 days, it was appeared that 74 and 8% feather weight was reduced through sub-merged and solid-state fermentation, respectively by A. flavus. The pH of the growth media in submerged fermentation was changed from 4.8 to 8.35. The exploited application of keratinolytic microbes is, therefore, recommended for the treatment of keratinaceous wastes to achieve dual benefits of remediation.

A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.

Safety Assessment of Keratin and Keratin-Derived Ingredients as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 8 keratin-derived ingredients, which function mainly as skin and hair conditioning agents in personal care products. The Panel reviewed relevant data provided in this safety assessment and concluded that the 8 keratin-derived ingredients are safe in the present practices of use and concentration described in this safety assessment.

Development of a novel keratin dressing which accelerates full-thickness skin wound healing in diabetic mice: In vitro and in vivo studies.

Impaired wound healing is a major medical problem in diabetes. The objective of this study was to determine the possible application of an insoluble fraction of fur-derived keratin biomaterial as a wound dressing in a full thickness surgical skin wound model in mice ( n = 20) with iatrogenically induced diabetes. The obtained keratin dressing was examined in vitro and in vivo. In vitro study showed the keratin dressing is tissue biocompatible and non-toxic for murine fibroblasts. Antimicrobial examination revealed the keratin dressing inhibited the growth of S. aureus and E. coli. In vivo studies showed the obtained dressing significantly ( p < 0.05) accelerated healing during the first week after surgery compared to control wounds. Keratin dressings were incorporated naturally into granulation and regenerating tissue without any visible signs of inflammatory response, which was confirmed by clinical and histopathological analysis. It is one of the first studies to show application of insoluble keratin proteins and its properties as a wound dressing. The obtained keratin dressing accelerated wound healing in mice with iatrogenically induced diabetes. Therefore, it can be considered as a safe and efficient wound dressing. Although future studies are needed to explain the molecular mechanism behind fur-derived keratin effect during the multilayer wound healing process, our findings may open the way for a new class of insoluble fur keratin dressings in chronic difficult to heal wounds treatment.

Chitosan scaffolds containing chicken feather keratin nanoparticles for bone tissue engineering.

Chicken feathers are considered as major waste from poultry industry. They are mostly constituted by a protein called keratin. In this study, keratin was prepared from chicken feathers and from where keratin nanoparticles (nKer) were synthesized. Since chitosan has excellent properties like controlled biodegradation and biocompatibility, we used keratin nanoparticles along with chitosan matrix as scaffolds (CS/nKer) and they were characterized by SEM, FT-IR and XRD analyses. There was a porous architecture in the scaffolds in the range to support cell infiltration and tissue ingrowth. The keratin nanoparticles had interaction with chitosan matrix and did not alter the semi crystalline nature of chitosan scaffolds. The biodegradation and protein adsorption of the scaffolds were significantly increased upon addition of keratin nanoparticles. The scaffolds were also found to be non-cytotoxic to human osteoblastic cells. Thus, CS/nKer scaffolds could serve as a potential biomimetic substrate for bone tissue engineering applications.

Biocompatibility and osseointegration of reconstituted keratin in an ovine model.

Reconstituted keratin has potential as a raw material for orthopaedic applications. The aim of this study was to investigate the in vivo biocompatibility and osseointegration of keratin materials in an ovine model. Six different modifications of the keratin polymer, based on porous or dense constructs, regenerated by either neutral or acidic treatment, with or without hydroxyapatite, were made as small rods and inserted into drilled round defects in the femur and tibia of sheep. Histology was carried out on samples taken at different time points up to 24 weeks postsurgery. All keratin implants showed similar histological profiles, which included granulation tissue surrounding and infiltrating the implants, followed by new bone formation radiating from the existing bone. By 8 weeks, new bone had grown to within a short distance of the implant surface, and in some places was in direct apposition to the keratin implant. In the 12 to 24-week period, there was peripheral resorption and infiltration of bony trabeculae with regard to the porous constructs only. The tissue reaction appeared to model that of a fairly inert material. Further work on improving the extent of osseointegration and acceleration of the biodegradation rate of reconstituted keratin is underway.

Toxicity evaluation of chicken calamus keratin conduit as a tissue-engineering scaffold biomaterial.

OBJECTIVE: To evaluate the toxicity of chicken calamus keratin (CCK) conduit as a tissue-engineered scaffold material. METHODS: The chemical composition of the leaching solution of CCK was determined by means of ultraviolet spectrometry, and the toxic effects of the solution was evaluated by skin sensitization test in rats, intracutaneous stimulation test in rabbits, acute systemic toxicity test in mice, and cytotoxicity test in L929 cells. RESULTS: The leaching solution of CCK consisted mainly of middle-molecular-weight peptides with a small quantity of macromolecular proteins. Skin sensitization test in rats showed that application of the CCK leaching solution caused no obvious skin reddening, regional edema, or skin necrosis. Intracutaneous injection of the leaching solution in rabbits did not induce obvious skin stimulation manifested by intradermal erythema or edema. In acute systemic toxic test, administration of the leaching solution in mice caused no death, organ dysfunction, cyanosis, tremor, severe peritoneal irritation, ptosis, or dyspnoea. In vitro cytotoxicity test indicated that the cell toxicity of the CCK leaching solution was approximately at 0 level. CONCLUSION: CCK contained in the treated chicken calamus easily undergoes hydrolysis to release mainly some peptides which do not induce obvious toxic effects, suggesting the safe potential applications of CCK conduit as a tissue-engineering biomaterial.

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense.

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.

Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum.

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human beta-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human alpha-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 x 10(5) PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10-50 micro M), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.

Demonstration of the pathogenic effect of point mutated keratin 9 in vivo.

A wild type keratin 9 (K9) cDNA and a point mutated keratin 9 cDNA were injected subcutaneously into mouse skin. The hemagglutinin tag staining of the wild type K9 cDNA injected specimens mainly showed a homogeneous pattern, whereas the point mutated K9 cDNA injected specimens mainly showed a granular pattern in the suprabasal cells. Double staining of K9 and the endogenous keratin revealed the incorporation of de novo synthesized K9 into the keratin network. These results demonstrate that (1) a naked DNA transfection into mouse skin can detect the pathogenic changes of point mutated keratin in vivo and (2) the keratin 9 mutation disrupts the keratin network formation in the suprabasal cells in vivo.

The physicochemical and biopharmaceutical properties of fragmented keratin as a new drug carrier.

Two types of fragmented keratin were prepared from buffalo horn and hoof using savinase and Na2S, and their physicochemical and biopharmaceutical properties were examined in mice. The number-average molecular weight of enzymatically fragmented keratin (E-FK), chemically fragmented keratin (C-FK), and fragmented gelatin (FG) were 8000, 33,000, and 6600, respectively. The systematic acute toxicity of FKs was significantly low. Moreover, the immunogenicity of FKs was significantly lower than that of superoxide dismutase. FKs and FG were partially hydrolyzed by trypsin. FKs were digested easily by alpha-chymotrypsin, but FG underwent less hydrolysis under the same conditions. FKs were bound to plasma proteins, including albumin, and also to some proteins in liver and kidney homogenates. In plasma, E-FK was hydrolyzed slowly, but in liver and kidney homogenates it showed slightly faster hydrolysis. In contrast, FG was not hydrolyzed in any of the media used here. After intravenous administration of FKs and FG to mice, these molecules were rapidly eliminated from the plasma. E-FK and C-FK were taken up into the kidneys (CLuptake, kidney; 10,400, 11,600 microliters/h/g), and then gradually excreted in urine. FG was excreted rapidly into urine (CLurine; 6360 microliters/h). Interestingly, C-FK was also taken up into the liver (CLliver; 4820 microliters/h). These results indicated that fragmented keratins are biodegradable materials and might be used as new types of liver- and kidney-specific targeting carriers.