Valorization of chicken feather through dekeratinization by keratinolytic Bacillus species to amino acid

The poultry meat processing sector generates chicken feathers as by-products, and they are 90percent keratin in composition. Keratin is an insoluble and structural protein that shows recalcitrance to hydrolysis by classical proteolytic enzymes, including trypsin, pepsin, and papain. Keratinases are a group of proteolytic enzymes endowed with keratin degradation into peptides and amino acids. They are recently gaining traction for their multifaceted potential application in the green industrial space. Hence, keratinolytic bacteria previously isolated from dumpsite were identified using 16S rDNA sequencing. The optimal fermentation conditions were determined for enhanced extracellular keratinase production and chicken feather degradation. Also, the amino acid analysis of the chicken feather hydrolysates was carried out. The biochemical properties of the keratinases were also determined. Based on 16S rDNA sequencing and phylogenetic analysis, the isolates coded as SSN-02 and HSN-03 showed a high percentage of sequence homology with Bacillus spp.; hence, they were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM, respectively. Bacillus sp. NFH5 showed optimal keratinase production of 1149.99 ± 80.99 U/mL after 96 h of incubation time, in optimized fermentation conditions that included pH (4.0), chicken feather (1.5percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). Similarly, Bacillus sp. FHNM demonstrated the maximum keratinase production of 480 ± 41.14 U/mL 144 h post cultivation, in optimized fermentation conditions with pH (7.0), chicken feather (2.0percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). For Bacillus sp. NFH5 chicken feather hydrolysate, the amino acids in relatively higher concentration (>1.0g/100g sample) include arginine (1.8), serine (1.16), aspartic acid (1.95), glutamic acid (2.47), proline (1.16) and glycine (1.45). Bacillus sp. FHNM feather hydrolysates, contained (g/100g of sample): arginine (1.9), serine (1.4), aspartic acid (2.5), glutamic acid (2.51), glycine (1.51), proline (1.13), leucine (1.030, histidine (1.25), and lysine (1.06) (g/100g of sample) in high concentration. The keratinases were optimally active at pH 8.0. Bacillus sp. FHNM showed an optimal temperature of 100 oC; while Bacillus sp. NFH5 keratinase displayed optimal activity at 90 oC. EDTA and 1,10-phenanthroline inhibited the keratinases, and the inhibition pattern indicated that they belong to metalloprotease. Keratinase from Bacillus sp. FHNM showed considerable residual activity in the presence of Co²⁺ (93percent), Fe³⁺ (99percent), and K⁺ (94percent). Bacillus sp. NFH5 keratinase retained 92percent, 92percent, 93percent of the original activity against Ba²⁺, Na⁺ and Fe³⁺ treatment. Bacillus sp. FHNM keratinase was remarkably stable after 60 min of detergents treatment with residual activity of 89percent, 96percent, 81percent, 73percent, 96percent, 88percent, 88percent and 98percent for Omo, Surf, Ariel, Sunlight, Prowash, Freshwave, Sky, and Evaklin, respectively. Maq impacted the enzyme stability negatively, with residual activity of 48percent after 60 min of incubation. Additionally, keratinase Bacillus sp. NFH5 retained 68percent, 78percent, 80percent, 84percent, 57percent, 80percent, 98percent, 106percent and 106percent of the original activity against Omo, Surf, Ariel, Sunlight, Maq, Prowash, Freshwave, Sky and Evaklin, respectively. Therefore, these results suggest that Bacillus spp. could be ideal candidates for sustainable production of active keratinases and valorization of the abundantly generated keratinous biomass. The stability displayed by keratinases from Bacillus sp. FHNM and Bacillus sp. NFH5 suggests their promising candidacy for detergent formulation.Thesis (MSc) -- Faculty of Science and Agriculture, 202

Valorization of chicken feather through dekeratinization by keratinolytic Bacillus species to amino acid

The poultry meat processing sector generates chicken feathers as by-products, and they are 90percent keratin in composition. Keratin is an insoluble and structural protein that shows recalcitrance to hydrolysis by classical proteolytic enzymes, including trypsin, pepsin, and papain. Keratinases are a group of proteolytic enzymes endowed with keratin degradation into peptides and amino acids. They are recently gaining traction for their multifaceted potential application in the green industrial space. Hence, keratinolytic bacteria previously isolated from dumpsite were identified using 16S rDNA sequencing. The optimal fermentation conditions were determined for enhanced extracellular keratinase production and chicken feather degradation. Also, the amino acid analysis of the chicken feather hydrolysates was carried out. The biochemical properties of the keratinases were also determined. Based on 16S rDNA sequencing and phylogenetic analysis, the isolates coded as SSN-02 and HSN-03 showed a high percentage of sequence homology with Bacillus spp.; hence, they were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM, respectively. Bacillus sp. NFH5 showed optimal keratinase production of 1149.99 ± 80.99 U/mL after 96 h of incubation time, in optimized fermentation conditions that included pH (4.0), chicken feather (1.5percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). Similarly, Bacillus sp. FHNM demonstrated the maximum keratinase production of 480 ± 41.14 U/mL 144 h post cultivation, in optimized fermentation conditions with pH (7.0), chicken feather (2.0percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). For Bacillus sp. NFH5 chicken feather hydrolysate, the amino acids in relatively higher concentration (>1.0g/100g sample) include arginine (1.8), serine (1.16), aspartic acid (1.95), glutamic acid (2.47), proline (1.16) and glycine (1.45). Bacillus sp. FHNM feather hydrolysates, contained (g/100g of sample): arginine (1.9), serine (1.4), aspartic acid (2.5), glutamic acid (2.51), glycine (1.51), proline (1.13), leucine (1.030, histidine (1.25), and lysine (1.06) (g/100g of sample) in high concentration. The keratinases were optimally active at pH 8.0. Bacillus sp. FHNM showed an optimal temperature of 100 oC; while Bacillus sp. NFH5 keratinase displayed optimal activity at 90 oC. EDTA and 1,10-phenanthroline inhibited the keratinases, and the inhibition pattern indicated that they belong to metalloprotease. Keratinase from Bacillus sp. FHNM showed considerable residual activity in the presence of Co²⁺ (93percent), Fe³⁺ (99percent), and K⁺ (94percent). Bacillus sp. NFH5 keratinase retained 92percent, 92percent, 93percent of the original activity against Ba²⁺, Na⁺ and Fe³⁺ treatment. Bacillus sp. FHNM keratinase was remarkably stable after 60 min of detergents treatment with residual activity of 89percent, 96percent, 81percent, 73percent, 96percent, 88percent, 88percent and 98percent for Omo, Surf, Ariel, Sunlight, Prowash, Freshwave, Sky, and Evaklin, respectively. Maq impacted the enzyme stability negatively, with residual activity of 48percent after 60 min of incubation. Additionally, keratinase Bacillus sp. NFH5 retained 68percent, 78percent, 80percent, 84percent, 57percent, 80percent, 98percent, 106percent and 106percent of the original activity against Omo, Surf, Ariel, Sunlight, Maq, Prowash, Freshwave, Sky and Evaklin, respectively. Therefore, these results suggest that Bacillus spp. could be ideal candidates for sustainable production of active keratinases and valorization of the abundantly generated keratinous biomass. The stability displayed by keratinases from Bacillus sp. FHNM and Bacillus sp. NFH5 suggests their promising candidacy for detergent formulation.Thesis (MSc) -- Faculty of Science and Agriculture, 202

Leaf tensile strength, "in vitro" digestibility, and fiber component relationships in tall fescue

Em um programa de melhoramento de forrageiras, a identificação de plantas com elevado valor nutritivo, é um dos fatores importantes para melhorar o desempenho animal. Interrelações significativas entre características físicas e químicas talvez possam ser utilizadas como um critério para a seleção de novas cultivares de festuca-alta de maior valor nutritivo. Os objetivos deste experimento foram: a. avaliar a tensão foliar (TF) e a digestibilidade in vitro da matéria seca (DIVMS) e dos constituintes da parede celular; b. analisar as associações entre essas características com a TF e c. determinar a eficiência da TF como uma técnica para a seleção de novos germoplasmas de festuca-alta, com forragem de melhor qualidade. Setenta e um genótipos de festuca alta foram avaliados neste experimento. Foi encontrada uma variabilidade genética significativa para TF nesta população de festuca-alta. Geralmente, as correlações de TF com DIVMS e componentes da parede celular não foram significativas, enquanto que as correlações de DIVMS com os componentes da parede celular foram altamente significativas e negativas. Conforme esperado, houve baixa repetibilidade de correlações de caracteres nos dois anos. A "análise de coeficientes de trilha" demonstrou que a celulose e a lignina foram os caracteres com os maiores efeitos diretos na TF e na DIVMS.In a forage breeding program it is necessary to identify germplasms with high forage quality, able to positively influence animal performance. The interrelationships between leaf tensile strength (LTS) and chemical composition parameters may provide a useful criterion for screening tall fescue (Festuca arundinacea Schreb.) for herbage quality in a breeding program. Seventy-one genetically diverse genotypes of tall fescue were randomly selected from a broad-based population to evaluate LTS (leaf tensile strength), in vitro dry matter digestibility (IVDMD), acid detergent fiber (ADF), neutral detergent fiber (NDF), cellulose (CELL), lignin (ADL), hemicellulose (HEMI), ADL/ADF and ASH, to determine the associations among these traits with LTS, and the importance of LTS as a technique for screening tall fescue for herbage quality. Significant genetic variation was found in this gene pool for LTS. The majority of the correlations of LTS with IVDMD and with fiber components were not significantly different from zero while correlations of IVDMD with fiber components were highly significant and negative. As expected, character correlation repeatabilities between years were usually low. Path coefficient analysis showed that CELL and ADL were two important components with large direct effects on LTS and on IVDMD

Leaf tensile strength, "in vitro" digestibility, and fiber component relationships in tall fescue

Em um programa de melhoramento de forrageiras, a identificação de plantas com elevado valor nutritivo, é um dos fatores importantes para melhorar o desempenho animal. Interrelações significativas entre características físicas e químicas talvez possam ser utilizadas como um critério para a seleção de novas cultivares de festuca-alta de maior valor nutritivo. Os objetivos deste experimento foram: a. avaliar a tensão foliar (TF) e a digestibilidade in vitro da matéria seca (DIVMS) e dos constituintes da parede celular; b. analisar as associações entre essas características com a TF e c. determinar a eficiência da TF como uma técnica para a seleção de novos germoplasmas de festuca-alta, com forragem de melhor qualidade. Setenta e um genótipos de festuca alta foram avaliados neste experimento. Foi encontrada uma variabilidade genética significativa para TF nesta população de festuca-alta. Geralmente, as correlações de TF com DIVMS e componentes da parede celular não foram significativas, enquanto que as correlações de DIVMS com os componentes da parede celular foram altamente significativas e negativas. Conforme esperado, houve baixa repetibilidade de correlações de caracteres nos dois anos. A "análise de coeficientes de trilha" demonstrou que a celulose e a lignina foram os caracteres com os maiores efeitos diretos na TF e na DIVMS.In a forage breeding program it is necessary to identify germplasms with high forage quality, able to positively influence animal performance. The interrelationships between leaf tensile strength (LTS) and chemical composition parameters may provide a useful criterion for screening tall fescue (Festuca arundinacea Schreb.) for herbage quality in a breeding program. Seventy-one genetically diverse genotypes of tall fescue were randomly selected from a broad-based population to evaluate LTS (leaf tensile strength), in vitro dry matter digestibility (IVDMD), acid detergent fiber (ADF), neutral detergent fiber (NDF), cellulose (CELL), lignin (ADL), hemicellulose (HEMI), ADL/ADF and ASH, to determine the associations among these traits with LTS, and the importance of LTS as a technique for screening tall fescue for herbage quality. Significant genetic variation was found in this gene pool for LTS. The majority of the correlations of LTS with IVDMD and with fiber components were not significantly different from zero while correlations of IVDMD with fiber components were highly significant and negative. As expected, character correlation repeatabilities between years were usually low. Path coefficient analysis showed that CELL and ADL were two important components with large direct effects on LTS and on IVDMD

Genetic characterization of type A enterotoxigenic Clostridium perfringens strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains. © 2009 Deguchi et al

Association of insularity and body condition to cloacal bacteria prevalence in a small shorebird

Do islands harbour less diverse disease communities than mainland? The island biogeography theory predicts more diverse communities on mainland than on islands due to more niches, more diverse habitats and availability of greater range of hosts. We compared bacteria prevalences ofCampylobacter,ChlamydiaandSalmonellain cloacal samples of a small shorebird, the Kentish plover (Charadrius alexandrinus) between two island populations of Macaronesia and two mainland locations in the Iberian Peninsula. Bacteria were found in all populations but, contrary to the expectations, prevalences did not differ between islands and mainland. Females had higher prevalences than males forSalmonellaand when three bacteria genera were pooled together. Bacteria infection was unrelated to bird's body condition but females from mainland were heavier than males and birds from mainland were heavier than those from islands. Abiotic variables consistent throughout breeding sites, like high salinity that is known to inhibit bacteria growth, could explain the lack of differences in the bacteria prevalence between areas. We argue about the possible drivers and implications of sex differences in bacteria prevalence in Kentish plovers