Enzymatic Unhairing Research Articles

Resource Utilization of Bovine Hair Recycling from Enzymatic Unhairing during Leather Manufacturing

With the development of enzymatic unhairing technology in the leather industry, enhancing the recycling of bovine hair is a new issue emerging in leather-making. In this work, the effects of alkali concentration, time, and temperature in the pretreatment, as well as enzyme dosage, time, temperature, and pH in the following treatment on the dissolution performance and keratin yield of bovine hair were investigated. Hence, the extraction process of keratin from bovine hair by the alkali-enzyme method was optimized. The results sho that the optimal conditions for the keratin extraction are as follows: for the pretreatment with alkali, the alkali concentration is 0.4 M, the pretreatment time is 1.0 h, the pretreatment temperature is 65°C, and for the following enzymatic treatment, the enzyme dosage is 3324 U/g bovine hair, the action time is 4.0 h, action temperature is 45°C, and the pH is controlled between 8.5-11.5. Compared with the alkali method, the alkali-enzyme method increases the keratin yield from 24.4% to 58.1%, and the alkali dosage is reduced by 50%. The prepared keratin had high in vitro antioxidant activity, which provided a new idea for the resource utilization of bovine hair waste.

Key mechanism of enzymatic dehairing technology for leather-making: permeation behaviors of protease into animal hide and the mechanism of charge regulation

Enzymes have been widely used as alternatives to conventional chemicals in cleaner leather processes due to their advantages of meeting increasing environmental demands. Especially, enzymatic unhairing based on protease has been applied to leather-making for a long time, however, it still has the key problem of slow permeation in the animal hide, resulting in slow unhairing rate, poor hair removal effect, excessive proteolysis of hide collagen, and decreased leather quality. Aiming at the key problem of bio-unhairing technology, fluorescent labeling technique and confocal laser scanning microscopy were used to investigate the protease permeation behaviors into the animal hide based on well-prepared labeled proteases, as well as the quantitative analysis of the protease amount in different hide layers. The results show that the protease mainly permeates the bovine hide through transfollicular routes from the hair side, and although the intercellular-interfibrillar pathway also exists, it does not play an important role. Additionally, the protease permeation behaviors into the hide are greatly impacted by the charge states of the hide and protease proteins, depending on the isoelectric points (pI) of the proteins and solution pH values. When the solution pH is not between the pI values of the two proteins, the similarly charged protease can quickly and deeply penetrate the hide because of the electrostatic repulsion. The established mechanism provides a theoretical basis for developing an efficient enzymatic unhairing technology for leather-making, and this can also be applied to other processes involving the enzyme permeation into the hide or leather.Graphical

Recovery of Industrially Useful Hair and Fat from Enzymatic Unhairing of Goatskins during Leather Processing

Leather processing not only serves social needs by putting into use the meat industry’s by-products (hides and skins) but also makes a significant contribution to global economic growth through trade and job creation. In the wake of globalization, however, leather manufacturers are facing new challenges in meeting environmental imperatives and improving the utilization of wastes generated during leather processing. This study describes the recovery of hair and fatfrom fleshings obtained after enzymatic unhairing of goatskins using a protease from an isolate of Bacillus cereus Strain 1-p. The recovered hair and fats were further characterized to facilitate recommendations for different industrial applications. The following hair properties were visually examined and evaluated by hand; straight length, density and uniformity, hair strength and overall quality. The fats were analyzed by characterizing the fatty acid composition using theGas Chromatography-Mass Spectrometry (GC-MS analysis). The recovered hair was intact and rated to be of average to good quality. The fat characterization indicated that methyl 9Z-octadecenoate (9Z-heptadecenoic acid; oleic acid) was the most abundant fatty acid with an abundance of 31.65%. The sulfide-free fats and intact hair, therefore, were recommended for use in various industrial applications such as manufacturing of poultry feedstuff, organic fertilizers, biodiesel and biofuels, fatliquoring agents, soaps and cosmetics after further purification where necessary. The hair and fats recovered from this study are particularly advantageous over those recovered from sulfide unhairing systems as they are free from any sulfides or lime contamination thus easier to purify and use. The study concluded that the use of the enzyme extract from Bacillus cereus Strain 1-p to unhair goatskins facilitated the recovery of valuable hairand fats that can be used for other industrial applications.

Heterologous Expression of Alkaline Metalloproteinases in Bacillus Subtilis SCK6 for Eco-Friendly Enzymatic Unhairing of Goatskins

In this study, alkaline metalloprotease gene 1067 was cloned from Planococcus halotolerans SCU63T and heterologously expressed in Bacillus subtilis SCK6. Using Luria Bertani (LB) broth medium as the initial medium, the optimal medium was obtained through a series of fermentation and culture optimization (g/L): yeast extract (10), soybean powder (15), urea (20), potassium chloride (6.7), calcium chloride (13.3), NaCl (10). On the basis of the optimal medium, the highest enzymatic activity of 1259.21 U/mL could be obtained by culturing at 30°C for 40 h. with pH 8, inoculation amount of 4% and filling amount of 50 mL. EDTA inhibits protease activity and PMSF promotes it, indicating that it was a metalloprotease rather than a serine protease. The optimum reaction temperature of the protease is 70°C, and the optimum pH is 9. The metal ions Zn 2+, Co2+ and surfactant β-ME, Tween 80 can improve the activity of the protease. The results of unhairing, SEM and staining show that this metalloprotease can completely dehair goatskin. Compared with the conventional chemical method, the goatskin after enzymatic unhairing has softer texture and smoother surface, and there is no obvious damage to the goatskin.

A substrate protection approach to applying the calcium ion for improving the proteolysis resistance of the collagen.

Enzymatic dehairing, as a crucial part of cleaner leather processing, has reached processive advancement with potentially replacing the traditional hair removal due to increasing pressure from environmental demand. However, this cleaner technology based on proteases has a problem that the hide grain (collagen-rich structure) is susceptible to be hydrolyzed, decreasing the quality of finished leather. From the perspective of improving the stability of collagen fibers and their resistance to proteolysis, a method for protecting the hide grain during the enzymatic dehairing process was developed. The results showed that calcium ions had a swelling effect on collagen fibers under near-neutral conditions (pH 6.0-10.0), decreasing the thermal stability of collagen and the proteolysis resistance of collagen significantly. The alkaline environment (pH 10.0-12.0) will promote the dissociation of carboxyl groups in hide collagen, promoting the combination of calcium ions and carboxyl groups. This strategy can change the surface charge of collagen fibers and strengthen the connection between collagen fibers, thus improving protease resistance and the thermal stability of collagen. However, collagen fibers could swell violently once the alkalinity of the solution environment was extreme. Despite the above situation, calcium ion was still conducive to maintain the structural stability of collagen fibers. At pH 10.0-12.0, pretreating animal hide with a solution containing calcium ions can improve the protease resistance of hide grain, making the hide grain well-protected. This method provided an effective way to establish a safer enzymatic unhairing technology based on substrate protection. KEY POINTS: • A collagen protection method for hair removal of animal hide was developed. • This method applied calcium ions to collagen at alkaline conditions (pH 10.0-12.0). • Pretreatment results of calcium ions at different pH values on animal hide were compared.

Evaluation of the Bacillus cereus Strain 1-p Protease for the Unhairing of Goatskins during Leather Production

The unhairing stage of leather processing is associated with the production of significant amounts of solid and liquid wastes. The use of enzymes to replace the polluting sulphides currently used for unhairing is a viable alternative. Various proteases from different Bacillus cereus strains as well as many other bacterial strains have been used successfully for the unhairing of skins. However, no previous work has assessed the use of the crude alkaline protease extract from Bacillus cereus strain 1-p, a novel Bacillus cereus strain obtained from the shores of Lake Bogoria - a soda lake in Kenya – in the unhairing of goatskins. This study, therefore, evaluates the potential of the protease extract from the Bacillus cereus strain 1-p to unhair goatskins. Optimum variables for unhairing using the protease were investigated. Complete unhairing was achieved within 12 hours at 27°C and pH 12 using the crude enzyme. The period and temperature required for complete unhairing were significantly lower than that of other enzymatic unhairing techniques. Compared to the leather unhaired with sulphide, the leather unhaired with the enzyme did not only show superior organoleptic properties but also recorded comparable or superior physical properties, namely tensile strength (26.94 N/mm2), percentage elongation (76.29%), tear strength (43.59 N/mm), and distension at grain crack and burst (7.9 mm and 8.2 mm respectively). The wastewater from the enzymatic unhairing process recorded a significant reduction in biochemical oxygen demand (78%), chemical oxygen demand (83%), and the wastewater volume (50%) compared to the process that uses sulphide. It was concluded that the use of the crude protease extract from the Bacillus cereus strain 1-p in unhairing goatskins is feasible.

Screening of additives to reduce grain damage risk on unhairing by proteinase K

Enzymatic unhairing is a cleaner strategy for leather-making. It is a potential alternative to the traditional hair-burning process. However, several shortcomings, such as uncontrolled enzymatic reaction, and risk of grain looseness and damage have restricted the broad application of enzymatic unhairing. In this work, metal ions and organic additives were screened for lessening the hydrolytic activity of proteinase K to collagen fiber. Then, the selected additives were applied to the enzymatic unhairing process for bovine hide. The results showed that a suitable concentration of metal ions (Cu (II), Fe (III) and Al (III)) and organic additives (salicylate, laurate, adipate, gallate and epicatechin (ECG)) could diminish approximately 35% of the hydrolytic activity of proteinase K to collagen fibers. Then, the additives were applied for the bovine hide enzymatic unhairing process. Hydroxyproline determination in the unhairing float shows that applying additives could reduce collagen hydrolysis. The morphology results showed that the grain damage could be significantly reduced with the addition of the screened additives in the proteinase K enzymatic unhairing system, whereas the addition of ECG and gallate significantly slowed down the unhairing speed. This outcome provides new potential to reduce the risk of grain damage in enzymatic unhairing process.Graphical abstract

Enzyme-induced changes in skin and its implications

Surface charge of the skin matrix is an extremely important parameter in leather making process because effectivity of the chemicals largely depends on the surface charge of the skin matrix. In the present research work, isoelectric point (Ip) and point of zero charge (pzc) are used to predict the distribution of electric potential and charge at the skin tissue–liquid interface after chemical and enzymatic unhairing of the skin. In this study, the determination of zeta potential and isoelectric point for relimed tissues processed by chemical and enzymatic methods was performed using a zeta potential analyzer and a titrimetric method. These results indicate that enzyme-treated tissues had lower isoelectric point than the chemical-treated tissues, which has been explained by studying the amino acid composition of the tissues. The tissue samples were also characterized by FTIR–ATR and solid-state 13C NMR spectroscopy to understand the chemical/structural changes occurred due to enzymatic treatment. The lower Ip/pzc of the tissue obtained by enzymatic process ensured maximum uptake of chemicals, making it an eco-benign process.

A novel unhairing enzyme produced by heterologous expression of keratinase gene (kerT) in Bacillus subtilis

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8h to 24h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.

Towards environmentally friendly skin unhairing process: A comparison between enzymatic and oxidative methods and analysis of the protein fraction of the related wastewaters

A huge amount of by-products, wastes and chemicals are generated during leather processing, originating environmental problems due to the great amount of pollutants released, in particular for the unhairing step. Due to this reason, several strategies are studied today to reduce its impact. In this work two different methods, developed at a pilot scale level in semi-industrial reactors, were compared: an unhairing method based on proteolytic enzymes (Alcalase from Bacillus licheniformis) and an oxidative one based on chemical agents (sodium hydroxide and hydrogen peroxide). The efficiency of the unhairing was evaluated in both cases. The impact of both treatments on the nitrogen fraction in wastewaters was determined in order to assess the suitability of these by-products for a possible recovery in food/feed industry: the protein amount, the total and free amino acids, peptides and the percentage of amino acid racemization were determined on broths, washing residues and skins. The results indicated that both methods have the potential to be more sustainable alternatives to the current ones. The oxidative method seemed to be the most efficient and less expensive unhairing methodology, and does not require a further step after the end of the treatment as in the case of enzymatic unhairing process. On the other side, the wastewaters of enzymatic treatments, quite rich in good quality proteins, could be recovered for feed/food formulations, whereas the wastewaters coming from the oxidative treatments, due to the oxidative damage, only for lesser value applications.

Cleaner processing: a sulphide-free approach for depilation of skins.

The conventional unhairing process in leather making utilises large amount of lime and sodium sulphide which is hazardous and poses serious waste disposal concerns. Under acidic conditions, sodium sulphide liberates significant quantities of hydrogen sulphide which causes frequent fatal accidents. Further, the conventional unhairing process involves destruction of the hair leading to increased levels of biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS) and total suspended solids (TSS) in the effluent. A safe approach is needed to overcome such environmental and health problems through an eco-benign process. The present study deals with a clean technology in which the keratinous body is detached from the dermis using enzymes produced from Bacillus crolab MTCC 5468 by solid state fermentation (SSF) as an alternative to noxious chemicals. Complete unhairing of skin could be achieved with an enzyme concentration of 1.2% (w/w). The bio-chemical parameters of the spent liquor of the enzymatic process were environmentally favourable when compared with conventional method. The study indicates that the enzymatic unhairing is a safe process which could be used effectively in leather processing to alleviate pollution and health problems.

A Novel Approach to Enzymatic Unhairing and Fiber Opening of Skin Using Enzymes Immobilized on Magnetite Nanoparticles

One of the prerequisites for making leather is to remove the inter-fibrillar proteins and noncollagenous materials, such as hair, flesh, etc. These proteinous and nonproteinous materials are removed in several steps, cumulatively known as the pretanning (or beam house) operations. A paradigm shift from chemical- to enzyme-based processes ensured that these noncollagenous materials were removed using enzymatic digestion rather than brutal osmotic forces employing chemicals like lime and sulfide. In order to make sure that a cocktail of enzymes (protease + amylase) have broad application and stability, their immobilization onto matrices that can enable overcoming such drawbacks is essential. This work, taking clues from the catalytic applications of nanoparticle-immobilized enzymes looks at metal oxide nanoparticle-immobilized enzymes for unhairing and fiber opening applications in a facile manner. Iron oxide (Fe3O4) nanoparticles have been selected for the present study since metal oxides are proven as an ...

Cohesive system for enzymatic unhairing and fibre opening: an architecture towards eco-benign pretanning operation

Abstract Pretanning operation is always a challenging task in leather processes due to high generation of solid waste. Cleaner leather processing is a new avenue to exterminate the existing pollution complications. Enzymes are the best alternate to conventional chemicals in the context of cleaner leather processing. In the current investigation, pretanning processes are carried out with enzymes. Protease is used for unhairing and amylase is used to remove inter-fibrillary materials by treating with skin/hide matrix. Protease and amylase are suitably formulated (fibrozyme) and applied as a single system to substitute conventional liming/reliming processes. Fibrozyme with a concentration of 3.5% is optimized by drum method. Unhairing efficiency, extent of fibre opening through pore size evaluation, pollution load, morphology features evaluated through staining and SEM (Scanning Electron Microscope), physical strength characteristics, organoleptic properties and visual assessment of crust leathers are assessed. Unhairing efficiency of fibrozyme has matched with conventional liming system in terms of hair removal and general appearances. Shrinkage temperature of about 112 °C was observed in enzyme treated wet blue leathers. The physical strength of the crust leathers has met the UNIDO norms. Scanning electron microscopy study substantiates no distortion of fibres. The fibres are compact and uniformly opened up as well as distributed in enzymatic process. In single step enzyme process, there is reduction in pollution load. The study implies the role of enzyme in tanning industry and gives a new bioresource avenue to the existing cleaner processing systems.

Environmentally friendly hide unhairing: Enzymatic-oxidative unhairing as an alternative to use of lime and sodium sulfide

Various studies have been conducted to develop technologies that minimize the environmental concerns associated with the leather industry. The use of enzymes and oxidizing products during the unhairing step reduces pollution by tanneries as well as process time. In this study, were used an enzymatic extract produced by a strain of Bacillus subtilis – BLBc 11 – and hydrogen peroxide to conduct enzymatic-oxidative unhairing as an alternative to the conventional process (lime and sodium sulfide). Tests for enzymatic-oxidative unhairing were performed by applying crude enzymatic extract at concentrations of 100Ug−1 and 300Ug−1 of hide and hydrogen peroxide at concentrations of 4% and 8%. Tests were conducted comparing the proposed unhairing method, the conventional unhairing and purely enzymatic unhairing, performed with crud enzymatic extract produced by strain BLBc 11. The results showed that the proposed enzymatic-oxidative unhairing method can be used as an alternative to lime and sodium sulfide.

Environmentally friendly hide unhairing: enzymatic hide processing for the replacement of sodium sulfide and delimig

In this work is described the use of an enzyme preparation produced by a new strain of Bacillus subtilis for hide unhairing aiming at completely replace the chemical process, which uses toxic sulfides and lime. The whole process was designed to be fast and cheap, by applying the crude enzymatic extracts of B. subtilis BLBc 11 cultures on the hides, with good results obtained in just 6 h of process. The treated hides were evaluated for their quality by optical microscopy and analyzes of inter-fibrillary proteins (proteoglycans and glycosaminoglicans). Undesired damage to the hide grain was evaluated by the determination of free hydroxyproline in the process liquor. Furthermore, the environmental impact of chemical and enzymatic treatments were compared, and the processes were evaluated by analyzing the chemical oxygen demand (COD), biological oxygen demand (BOD), total nitrogen, and sulfides in the residual wastewater, as the proprieties of the finished leather. COD reductions were of 42%, 100%, and 82% compared with the chemical process for the unhairing, deliming, and bating steps, respectively. The enzymatic unhairing process showed excellent results, with time reduction for unhairing of more than 50%, without the need for deliming, suggesting that this enzymatic preparation could be used to replace the chemical process.

Kinetics of enzymatic unhairing by protease in leather industry

In leather industry, unhairing is a heavy pollution operation. The conventional lime–sulfide process produces a large amount of sulfide which is toxic to health and difficult to dispose. Moreover, conventional lime–sulfide process leads to the destruction of the hair causing increased COD, BOD and TDS loads in the effluent. As an alternative method, enzymatic unhairing is a promising clean technology. The main utilized enzyme preparations are proteases. There are many reports about enzymatic unhairing, most of which are qualitative. The kinetics of enzymatic unhairing by protease was discussed in this article. It will provide useful information to enzymatic unhairing. In our research, by analysis of the concentrations of released total protein in enzyme bath during protease unhairing, the good linearity between released total protein and square root of time (min 1/2) was obtained at the initial stage. The good linearity suggests that enzymatic unhairing by protease is a diffusion-controlled process at the initial stage. The analysis of kinetics of released saccharides also confirms the same conclusion. On the other hand, the same characteristics between the kinetics of released saccharides and that of released total protein further confirms that it is the hydrolysis of core protein by protease that leads to the degradation of proteoglycans and the release of protein and saccharides. However, in our tests, the kinetics of released collagen indicates that the injury to skin took place in 3–6 h. Therefore, it’s necessary to control the time of protease unhairing within an appropriate limit.

Cleaner Dehairing Technology for Goatskins: Effects of Hydrosulfide and Peroxide on Enzyme Unhairing

Traditional lime and sulfide dehairing process is a major source of the pollution, such as biochemical oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS) and toxic H2S gas, etc. Enzymatic dehairing process is known to be cleaner but its dehairing effects are unsatisfactory. Hence, an attempt has been made to solve those two unhairing methods’ disadvantages through a biochemical approach. Several commercial enzyme formulations were chosen to research effects of hydrosulfide and peroxide on its activities, and then hair-saving enzymatic unhairing experiments with those two reagents were conducted in paste and pile method more effectively and cleanly. Results show that activity of enzyme 2709 is stabilized by the addition of hydrosulfide through activity measurements; Peroxide has some activation effect on protease 3942. Hair removal is found to be complete using scanning electron microscope (SEM) analysis. Those two processes enjoy a significant reduction in BOD, COD, TDS and TSS. The perfromances of the experimental leathers are also comparable to that of conventional ones. Therefore, those are cleaner processing technologies that could be chosen to solve traditional method’s disadvantage.

Studies on the application of ultrasound in leather enzymatic unhairing

Enzymatic unhairing is a clean technology for leather industry. However, one problem hinders the utilization of enzymes in leather unhairing is the slow diffusion of enzymes through skins. As another clean technology, ultrasound can be applied to accelerate the diffusion of enzymes through skins. In this work, effects of ultrasound on the enzymatic unhairing were investigated. Effect of frequency of ultrasound on the enzymatic unhairing was compared, and 20 kHz was chosen in this work. Under our conditions, ultrasound has little effect on enzyme activities, and also has little influence on the physical and mechanical properties of leather. Ultrasound can enhance the diffusion of enzyme through skins, thus improving the unhairing capacities of enzymes. It is noted that amylase can interact with proteoglycans by attacking saccharide side chains, thus having some unhairing capacity. Ultrasound can improve the unhairing capacity of amylase more effectively. Furthermore, amylase does no harm on the collagen fibrous structure and can be used safely without the need for a high degree of control. So it is possible to use amylase in enzymatic unhairing in combination with protease. In short, using ultrasound in amylase and protease unhairing is a promising clean technology.

Green solution for tannery pollution: effect of enzyme based lime-free unhairing and fibre opening in combination with pickle-free chrome tanning

Growing global concern on environmental health is forcing all the processing industries to adopt greener and cleaner manufacturing practices. Thus, the leather industry is being pressurized to look for cleaner leather processing. The conventional method of leather making involves do–undo logic. This results in the emission of a huge amount of pollution load such as biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total solids (TS). Currently tanners are looking for design and utilization of cleaner and safer technologies. In this approach, a process has been designed to limit the pH range in leather processing from 13.5–2.8 to 8.0–3.8. An experimental process has been developed by combining enzymatic unhairing, enzyme based fibre opening and pickle and basification free chrome tanning. The experimental tanning process provides comparable leather quality with reduced environmental impact and effluent treatment costs. The process explored also appears to be economically viable.

Zero discharge tanning: a shift from chemical to biocatalytic leather processing.

Beam house processes (Beam house processes generally mean liming-reliming processes, which employ beam.) contribute more than 60% of the total pollution from leather processing. The use of lime and sodium sulfide is of environmental concern (1, 2). Recently, the authors have developed an enzyme-based dehairing assisted with a very low amount of sodium sulfide, which completely avoids the use of lime. However, the dehaired pelt requires opening up of fiber bundles for further processing, where lime is employed to achieve this through osmotic swelling. Huge amounts of lime sludge and total solids are the main drawbacks of lime. An alternative bioprocess, based on alpha-amylase for fiber opening, has been attempted after enzymatic unhairing. This totally eliminates the use of lime in leather processing. This method enables subsequent processes and operations in leather making feasible without a deliming process. A control experiment was run in parallel using conventional liming-reliming processes. It has been found that the extent of opening up of fiber bundles using alpha-amylase is comparable to that of the control. This has been substantiated through scanning electron microscopic, stratigraphic chrome distribution analysis, and softness measurements. Performance of the leathers is shown to be on a par with leathers produced by the conventional process through physical and hand evaluation. Importantly, softness of the leathers is numerically proven to be comparable with that of control. The process also demonstrates reduction in chemical oxygen demand load by 45% and total solids load by 20% compared to the conventional process. The total dry sludge from the beam house processes is brought down from 152 to 8 kg for processing 1 ton of raw hides.