A busca por tecnologias que visem minimizar o impacto ambiental da indústria coureira vem aumentando constantemente. A utilização de enzimas e de produtos oxidantes na etapa de depilação da pele é uma alternativa que permitem a redução do potencial poluidor da indústria do couro, além de possibilitar a redução do tempo de processo. Neste trabalho buscou-se reunir os benefícios do uso de enzimas, produzidas pelo cultivo de duas linhagens de Bacillus subtilis (BLBc 11 e BLBc 17), e peróxido de hidrogênio, através da associação destes produtos em um processo de depilação enzimático-oxidativo, alternativo ao processo convencional (cal e sulfeto de sódio). Foram realizados testes iniciais a fim de avaliar as melhores condições de depilação, verificando-se que as melhores condições seriam: realizar a primeira etapa somente com o extrato enzimático, alcalinizar o meio com 1% de hidróxido de sódio e realizar, então, a etapa oxidativa com peróxido de hidrogênio, com o tempo total de processo em torno de 4 h. Os testes de depilação enzimática-oxidativa foram realizados nestas condições, aplicando as enzimas nas concentrações de 100 U g-1 de pele e 300 U g-1 de pele, e o peróxido de hidrogênio nas concentrações de 4 % e 8 %. As peles foram visualmente avaliadas, observando-se que em todos os ensaios os pelos não foram totalmente removidos, conseguindo-se sua remoção com posterior raspagem mecânica. Os pelos foram removidos intactos, com a raiz. Foram realizados testes comparativos entre o método de depilação proposto, a depilação convencional e a depilação puramente enzimática. O banhos residuais foram avaliados através de análises de pH, nitrogênio total Kjeldhal, sólidos dissolvidos totais, fixos e voláteis, glicosaminoglicanos, proteoglicanos e hidroxiprolina. Os resultados foram satisfatórios, mostrando que o método de depilação com o uso de enzimas e peróxido de hidrogênio pode ser considerado uma alternativa viável ao uso de cal e sulfeto de sódio. / The search for technologies aiming at minimizing the environmental impact of leather industry is constantly increasing. The use of enzymes and oxidants in the unhairing step is an alternative which allow reducing the pollution of the leather industry, in addition to enabling the reduction of process time. In this study we sought to bring together the benefits of using enzymes, produced by two strains of Bacillus subtilis (BLBc 11 e BLBc 17), and hydrogen peroxide through the combination of these products in an enzymatic-oxidative unhairing, alternative to the conventional process (lime and sodium sulfide). Initially, tests were carried out to assess the best conditions for hair removal, verifying that the best conditions are: perform the first step only with the enzymatic extract liquid, alkalinization with 1% sodium hydroxide and, then, the oxidation step with hydrogen peroxide, with the total process time around 4 h. Tests for enzyme-oxidative unhairing were performed under these conditions, applying enzymes at concentrations of 100 U g-1 of skin and 300 U g-1 of skin, and hydrogen peroxide at concentrations of 4% and 8%. The skins were visually evaluated, observing that in all the tests the hairs were not completely removed, achieving its removal with subsequent mechanical scraping. The hairs were removed intact, with the roots. Tests were conducted comparing the proposed unhairing method, the conventional unhairing and purely enzymatic unhairing performed with enzymatic extract produced by strains BLBc 11 and BLBc 17. The residual baths were evaluated through analysis of pH, total Kjeldhal nitrogen, total dissolved solids, fixed and volatile, glycosaminoglycans, proteoglycans and hydroxyproline. The results were satisfactory, showing that the enzymatic-oxidative unhairing using enzymes and hydrogen peroxide proposed here can be considered a viable alternative to the use of lime and sodium sulfide.

A busca por tecnologias que visem minimizar o impacto ambiental da indústria coureira vem aumentando constantemente. A utilização de enzimas e de produtos oxidantes na etapa de depilação da pele é uma alternativa que permitem a redução do potencial poluidor da indústria do couro, além de possibilitar a redução do tempo de processo. Neste trabalho buscou-se reunir os benefícios do uso de enzimas, produzidas pelo cultivo de duas linhagens de Bacillus subtilis (BLBc 11 e BLBc 17), e peróxido de hidrogênio, através da associação destes produtos em um processo de depilação enzimático-oxidativo, alternativo ao processo convencional (cal e sulfeto de sódio). Foram realizados testes iniciais a fim de avaliar as melhores condições de depilação, verificando-se que as melhores condições seriam: realizar a primeira etapa somente com o extrato enzimático, alcalinizar o meio com 1% de hidróxido de sódio e realizar, então, a etapa oxidativa com peróxido de hidrogênio, com o tempo total de processo em torno de 4 h. Os testes de depilação enzimática-oxidativa foram realizados nestas condições, aplicando as enzimas nas concentrações de 100 U g-1 de pele e 300 U g-1 de pele, e o peróxido de hidrogênio nas concentrações de 4 % e 8 %. As peles foram visualmente avaliadas, observando-se que em todos os ensaios os pelos não foram totalmente removidos, conseguindo-se sua remoção com posterior raspagem mecânica. Os pelos foram removidos intactos, com a raiz. Foram realizados testes comparativos entre o método de depilação proposto, a depilação convencional e a depilação puramente enzimática. O banhos residuais foram avaliados através de análises de pH, nitrogênio total Kjeldhal, sólidos dissolvidos totais, fixos e voláteis, glicosaminoglicanos, proteoglicanos e hidroxiprolina. Os resultados foram satisfatórios, mostrando que o método de depilação com o uso de enzimas e peróxido de hidrogênio pode ser considerado uma alternativa viável ao uso de cal e sulfeto de sódio. / The search for technologies aiming at minimizing the environmental impact of leather industry is constantly increasing. The use of enzymes and oxidants in the unhairing step is an alternative which allow reducing the pollution of the leather industry, in addition to enabling the reduction of process time. In this study we sought to bring together the benefits of using enzymes, produced by two strains of Bacillus subtilis (BLBc 11 e BLBc 17), and hydrogen peroxide through the combination of these products in an enzymatic-oxidative unhairing, alternative to the conventional process (lime and sodium sulfide). Initially, tests were carried out to assess the best conditions for hair removal, verifying that the best conditions are: perform the first step only with the enzymatic extract liquid, alkalinization with 1% sodium hydroxide and, then, the oxidation step with hydrogen peroxide, with the total process time around 4 h. Tests for enzyme-oxidative unhairing were performed under these conditions, applying enzymes at concentrations of 100 U g-1 of skin and 300 U g-1 of skin, and hydrogen peroxide at concentrations of 4% and 8%. The skins were visually evaluated, observing that in all the tests the hairs were not completely removed, achieving its removal with subsequent mechanical scraping. The hairs were removed intact, with the roots. Tests were conducted comparing the proposed unhairing method, the conventional unhairing and purely enzymatic unhairing performed with enzymatic extract produced by strains BLBc 11 and BLBc 17. The residual baths were evaluated through analysis of pH, total Kjeldhal nitrogen, total dissolved solids, fixed and volatile, glycosaminoglycans, proteoglycans and hydroxyproline. The results were satisfactory, showing that the enzymatic-oxidative unhairing using enzymes and hydrogen peroxide proposed here can be considered a viable alternative to the use of lime and sodium sulfide.

A busca por tecnologias que visem minimizar o impacto ambiental da indústria coureira vem aumentando constantemente. A utilização de enzimas e de produtos oxidantes na etapa de depilação da pele é uma alternativa que permitem a redução do potencial poluidor da indústria do couro, além de possibilitar a redução do tempo de processo. Neste trabalho buscou-se reunir os benefícios do uso de enzimas, produzidas pelo cultivo de duas linhagens de Bacillus subtilis (BLBc 11 e BLBc 17), e peróxido de hidrogênio, através da associação destes produtos em um processo de depilação enzimático-oxidativo, alternativo ao processo convencional (cal e sulfeto de sódio). Foram realizados testes iniciais a fim de avaliar as melhores condições de depilação, verificando-se que as melhores condições seriam: realizar a primeira etapa somente com o extrato enzimático, alcalinizar o meio com 1% de hidróxido de sódio e realizar, então, a etapa oxidativa com peróxido de hidrogênio, com o tempo total de processo em torno de 4 h. Os testes de depilação enzimática-oxidativa foram realizados nestas condições, aplicando as enzimas nas concentrações de 100 U g-1 de pele e 300 U g-1 de pele, e o peróxido de hidrogênio nas concentrações de 4 % e 8 %. As peles foram visualmente avaliadas, observando-se que em todos os ensaios os pelos não foram totalmente removidos, conseguindo-se sua remoção com posterior raspagem mecânica. Os pelos foram removidos intactos, com a raiz. Foram realizados testes comparativos entre o método de depilação proposto, a depilação convencional e a depilação puramente enzimática. O banhos residuais foram avaliados através de análises de pH, nitrogênio total Kjeldhal, sólidos dissolvidos totais, fixos e voláteis, glicosaminoglicanos, proteoglicanos e hidroxiprolina. Os resultados foram satisfatórios, mostrando que o método de depilação com o uso de enzimas e peróxido de hidrogênio pode ser considerado uma alternativa viável ao uso de cal e sulfeto de sódio. / The search for technologies aiming at minimizing the environmental impact of leather industry is constantly increasing. The use of enzymes and oxidants in the unhairing step is an alternative which allow reducing the pollution of the leather industry, in addition to enabling the reduction of process time. In this study we sought to bring together the benefits of using enzymes, produced by two strains of Bacillus subtilis (BLBc 11 e BLBc 17), and hydrogen peroxide through the combination of these products in an enzymatic-oxidative unhairing, alternative to the conventional process (lime and sodium sulfide). Initially, tests were carried out to assess the best conditions for hair removal, verifying that the best conditions are: perform the first step only with the enzymatic extract liquid, alkalinization with 1% sodium hydroxide and, then, the oxidation step with hydrogen peroxide, with the total process time around 4 h. Tests for enzyme-oxidative unhairing were performed under these conditions, applying enzymes at concentrations of 100 U g-1 of skin and 300 U g-1 of skin, and hydrogen peroxide at concentrations of 4% and 8%. The skins were visually evaluated, observing that in all the tests the hairs were not completely removed, achieving its removal with subsequent mechanical scraping. The hairs were removed intact, with the roots. Tests were conducted comparing the proposed unhairing method, the conventional unhairing and purely enzymatic unhairing performed with enzymatic extract produced by strains BLBc 11 and BLBc 17. The residual baths were evaluated through analysis of pH, total Kjeldhal nitrogen, total dissolved solids, fixed and volatile, glycosaminoglycans, proteoglycans and hydroxyproline. The results were satisfactory, showing that the enzymatic-oxidative unhairing using enzymes and hydrogen peroxide proposed here can be considered a viable alternative to the use of lime and sodium sulfide.

Content: The toxicity and unpleasant smell of hydrogen sulphide (H2S) gas is an issue for the leather industry that has been contained rather than eliminated in tannery practice. Completely eliminating H2S from tanneries while maintaining practical and economically feasible processing is still a big challenge to be addressed. Significant progress has, though, been made by introducing robust and reliable low sulphide unhairing systems based on selective soaking and specific enzymatic liming auxiliaries. These systems allow the reduction of sodium sulphide offers from the typical 2.5% to 1% of pelt weight. These lower levels reduce the amount of hydrogen sulphide gas released into the environment from the liming float, as well as the amount of sulphide that is carried over in the hide to subsequent processing steps. Overall, the H2S problem is not eliminated, but significantly reduced with this technology. In a further evolution of the technology, organic thio compounds can be used to fully or partially replace the already low levels of sulphide required, and thus allow to operate with offers well below 1%, or even completely without inorganic sulphide. Due to their reductive power, the organic thio compounds react with keratin in a similar way as inorganic sulphides, but they are oxidized much faster, which is advantageous for the effluent load. Alternatively to, or in combination with organic thio compounds, H2S scavengers can be used to reduce or eliminate hyrogen sulphide released from liming floats. Different types of scavengers are available, but the selection is limited for technical and economic reasons. The paper on hand shows how the release of hydrogen sulphide from beamhouse operations can be effectively controlled by a combination of technologies involving more effective unhairing with lower amounts of reductive agents, replacement of inorganic sulphide by organic thio compound and the use of H2S scavengers. Take-Away: - The release of toxic H2S from Beamhouse operations can be effectively controlled using modern unhairing technologies allowing to use no or low sulphide. - These modern unhairing systems are based on a combination of selective soaking, unhairing assisted by enzymes with specific activity and the use of organic thio compounds.

Content: Unhairing process is usually considered as the most polluted process in leather production. The conventional method of unhairing which using lime and sodium sulfide produces a large amount of sludge and waste water. In order to reduce pollution, we developed a novel unhairing enzyme and named as WB600-KerT, which possesses low collagen-degrading ability and high keratin-degrading ability in previous study. The objective of this study is to study the properties and effect of WB600-Kert to replace traditional chemicals in unhairing process. It found that the protease of WB600-Kert exhibited optimum keratin activity at 40°C. Compared with commercial unhairing enzyme and conventionally sodium sulfide methods, WB600 exhibited better unhairing effect and higher efficiency. The results indicated that goat skin unhaired with WB600 achieve enough softness, shrinkage temperature and tear strength as well as conventionally sodium sulfide methods Furthermore, if adding a small amount of sodium sulfide, the unhairing process could be accelerated while the unhairing effect was further improved. Generally speaking, this enzyme showed good application potential in unhairing process and was effective for reducing pollution which may promote the development of leather industry. Take-Away: 1. The protease of WB600-KerT was firstly applied to hair removal. 2. The addition of little sodium sulfide in WB600-KerT could enhance unhairing efficiency.

Content: The removal of hair from a hide or skin by dissolving it with a mixture of lime and sulfide is a fundamentally understood feature of leather technology. Or is it? For a long time, it has been accepted within the leather literature that, in water, sulfide may be present as either hydrogen sulfide (H2S), hydrosulfide (HS-) or sulfide (S2-), depending on the pH. pH < 6 6 < pH < 12 pH > 12 H2S(aq) ⇌ HS-(aq) ⇌ S2-(aq) The generally accepted mechanism of hair burning is sulfide attack at the cystine disulfide linkages in keratin. Also, it is believed that the unhairing reaction only proceeds at an appreciable rate in the presence of the dianionic S2- species, because that fits with the technological observation that unhairing reactions only proceed at pH greater than 12. However, recent publications have provided substantive proof that the S2- species does not exist in aqueous media at any pH: researchers were unable to observe any evidence of the S2- species in a solution of Na2S dissolved in hyper-concentrated NaOH and CsOH using Raman spectroscopy. The assigned second pKa for removal of the second proton has now been estimated to be 19, making the concentration of S2-(see below) vanishingly small. HS- ⇋ S2- + H+ There is a clear contradiction between the currently accepted mechanism for sulfide unhairing with the evidenced speciation of sulfide species in aqueous environment. Here the implications for this important process are discussed and possible alternative mechanisms postulated that fit with the new knowledge. Take-Away: It is a truism that we must understand the mechanistic principles of a process in order to control it. Here, we have a big change in thinking for ‘sulfide unhairing’, so it is vital that we understand the implications for leather science and leather technology of that change.

Content: Nowadays, tannery pollution is of great concern worldwide. The unhairing and fiber bundle-opening processes contribute the majority of the pollution by the use of sodium sulfide and calcium hydroxide, which were proposed to be replaced by neutral protease combined with (potassium chloride) KCl, (lithium bromide)LiBr/1-allyl-3-methylimidazolium chloride([AMIm]Cl) in the present work. Proper amount of KCl can speed up the unhairing with the grain not destroyed by the neutral protease. Four methods for unhairing and fiber bundle-opening were used as follows: 1. Two steps in different floats as KCl/neutral protease unhairing, followed by LiBr/[AMIm]Cl for fiber bundle-opening; 2. Two steps in different floats as neutral protease unhairing, followed by LiBr/[AMIm]Cl for fiber bundle-opening; 3. One step in the same float as neutral protease for unhairing firstly and then LiBr/[AMIm]Cl for fiber bundle-opening. 4. One step in the same float as neutral protease/KCl for unhairing firstly and then LiBr/[AMIm]Cl for fiber bundle-opening. It was found that using neutral protease/KCl solution for unhairing and LiBr/[AMIm]Cl solution for fiber bundle-opening is the best in fiber bundle-opening at the liming process. Besides, all the methods used here are better than the traditional liming processes (C) from viewpoints of unhairing and fiber bundleopening. Take-Away: 1. The addition of potassium chloride can improve the stability of neutral protease solution. 2. The catalytic efficiency of the enzyme was improved because of adding potassium chloride can enhance the contact rate of neutral protease with substrate. 3. Ionic liquids have less hydrolysis of collagen and larger dissolution of polysaccharides.

Content: Preservation (or) curing is an important unit process for transportation and storage of raw hides/skins without any deterioration. Popular preservation process is mostly achieved by reducing the moisture content of hides/skins using common salt (NaCl). Usage of salt in preservation process leads to generation of large amount of contaminated salt, total dissolved solids (TDS) and consume huge amount of water for subsequent rehydration step. On the other hand, lime-sodium sulphide based reductive process is commonly employed for the removal of hair from hides/skins. This process leads to generation of lime sludge and possible evolution of toxic hydrogen sulphide gas thereby making the working atmosphere more unpleasant. Several alternative techniques for preservation as well as unhairing process have been developed individually to replace salt and sulfide, respectively. However, a single compound performing dual functions such as preservation and unhairing action in neutral pH conditions has not explored so far. In the present work, a novel formulation has been developed, which possess the both preservation and unhairing potential, and applied on the hides/skins for storage up to 6 months at ambient conditions without dehydration. Low level of sulphide was used during alkaline fiber opening for complete removal of hair. The strength and organoleptic properties are on par with salted skins/hides. The developed process completely eliminates the use of salt and 75% sulphide and also reduces the time and water required for soaking process. The developed system reduces 85% of pollution load discharged from soaking and unhairing processes. Take-Away: One pot system for salt free preservation and low sulfide dehairing for sustainable leather manufacture.

Content: Beamhouse area is an important step in leather technology, either in the final quality of the leather or in the contribution to the contamination of effluents. In unhairing process, the use of enzymes as sulphide assistants can reduce sulfhidric gas emissions to reach permitted levels for health. The characterization of enzymatic extracts allows controlling the proteolysis mechanism so that its action does not attack the reticular structure. A new fungal enzyme extracts were applied on submerged bovine skin in the soaking and unhairing steps. Fungal strains were isolated from alkaline soils of coast of Buenos Aires province and enzymatic extracts (EE) were obtained by submerged culture with bovine hair waste from hair-saving unhairing process as inducer in the production of keratinolytic enzymes. This activity was determined by quantitative test and the most productive strains were selected: Clonostachys rosea (CR), Emericellopsis minima (EM), Paecilomyces lilacinus (PL), Penicillium sp (Psp), Fusarium oxysporum (FO), Acremonium sp (A) and an unidentified filamentous fungus strain with sterile yellow mycelium (SYM). EE were tested at laboratory scale on submerged bovine skin with biocide 0,2% w/w wet skin, anionic 0,1% and non-ionic 0,5% tensioactives in soaking and unhairing respectively and incubated 48 h at 37°C in shaking thermostatic bath. Controls were tested in the same conditions without EE. Morphological changes in the skin were observed by scanning electron microscopy (SEM). Pieces of bovine skin were fixed, post-fixed in formaldehyde 4% and dehydrated in alcohol solutions, treated by critical point drying, metalized and analyzed in SEM. It was observed with FO, EM and A extracts empty hair follicles, absence of epidermis, papillary layer of dermis exposed, hairs enclosed by detached hair follicle sheaths and collagen fibers without characteristic patterns. With PL and SYM EE some hairs were detached while Psp and CR EE did not produce changes. In control samples epidermis and hair without modification were observed. In the quantitative test, keratinolytic activities increased in the following order: A<PL<Psp<CR<FO<SYM. Submerged culture with keratin as inductor produced keratinolytic enzymes useful for unhairing step. Fusarium oxysporum extract showed the greatest effect on the skin, thus the relationship between keratinolytic activity and depilatory effect was found. However, it is necessary to find the optimal conditions to avoid the damage of collagen and enable its application as a sustainable technology. Take-Away: A new fungal isolates applicated to bovine skin in beamhouse processes

Content: Unhairing process brought serious pollution, and enzyme application for replacing polluting chemicals in unhairing process attracted much attention in recent years. However, the unhairing enzymes haven’t been accepted widely in actual production due to low purity, complex composition and poor stability. To solve these problems, unhairing enzyme is suggested to be improved by genetic modification in this research. The High-keratinase-producing gene (KerT), which was extracted from B. amyloliquefaciens TCCC11319, was introduced into the B.subtilis WB600 by heterologous expression. Because Bacillus subtilis WB600 is deficient in six extracellular proteases, this process successfully reduced the collagenolytic protease content in crude broth as well as improved the keratinase content. Meantime, the recombinant KerT produced by B.subtilis WB600 had the obviously unhairing effect to remove hairs. The results showed that the collagen degradability of recombinant KerT was slightly and it did not cause any adverse effects on the hide quality. This research will contribute to the development of unhairing enzyme, and the novel unhairing enzyme might be applied as the key factor for the advanced cleaning biotechnology in leather production process. Take-Away: 1. The keratinase gene KerT was firstly reported and analyzed which was extracted from B. amyloliquefaciens TCCC11319. 2. The collagenolytic protease activity of unhairing enzyme was successfully inhibited by heterologous expression of kerT. 3. The unhairing effect of this novel unhairing enzyme was similar to current Sulphur-lime method without damaging hide structure.