Enzymatic Degradation of Pretreated Pig Bristles with Crude Keratinase of Bacillus cereus PCM 2849
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The aim of the study was to apply and optimize the process of bioconversion of pig bristle waste using keratinolytic enzymes of Bacillus cereus PCM 2849, and to evaluate the amino acid composition of the resultant hydrolysate.
Hydrolysis with concentrated culture fluid of B. cereus was applied for bioconversion of pig bristles, after thermo-chemical pretreatment with sulfite. The effect of substrate concentration, sulfite concentration during pretreatment and reaction temperature on the release of amino acids was determined using Box-Behnken design. Amino acid composition of the obtained hydrolysate was determined by HPLC. Structural condition and substructural changes of the residual substrate were evaluated with SEM microscopy and FTIR spectroscopy.
The applied enzymatic preparation for bristle biodegradation was verified to contain multiple proteases of a wide molecular weight range. A regression model was developed, in which influential parameters were: linear effect of substrate concentration, followed by quadratic effects of reaction temperature, substrate concentration and pretreatment. Optimum reaction conditions were also determined. The resultant hydrolysate was rich in branched-chain amino acids. Residual substrate was detriorated and sulfitolytic cleavage of disulfides and alteration of protein secondary structures was confirmed.
Application of B.cereus crude keratinase allowed for partial hydrolysis of pig bristles, preceded by sulfitolytic pretreatment. A regression model was built to describe the process of hydrolysis to release free amino acids, at constant enzyme load. Hydrolysis in given conditions allowed to obtain hydrolysate rich in branched chain amino acids. The presented process poses an alternate way of management over pig bristles, a hard-to-degrade keratinous waste.
KeywordsPig bristles Biodegradation Bacillus cereus Keratinase Box-Behnken design Hydrolysis
Keratinous proteins are most recalcitrant constituents of by-products generated by meat and poultry processing industries. The management over this waste remains a challenging issue, due to the unique structure and composition of keratin-based skin appendages, resulting in their exceptional sturdiness. In the face of constantly rising production levels of swine and poultry an increasing inflow of these side-products is expected. As these keratinous materials contain at least 80 % protein, processes aimed at recovery of valuable proteins, peptides or amino acids are highly desirable .
Since application of keratin meals in livestock feeding has been lately subject to severe legal restrictions in most European countries, in response to outbreaks of prion diseases in the past, development of novel conversion methods has become critical. Although utilization of keratinous waste through combustion or co-combustion is widely used and offers simplicity and efficiency, it requires highly specialized facilities. Moreover, the concept of destroying proteinaceous materials instead of their exploitation is questionable.
According to the EU Waste Framework Directive (Directive 2008/98/EC) two types of organic waste management have been defined, comprising aerobic and anaerobic processes. The former include composting and the latter involve biogas production. Composting of keratinous waste appears as a cost-effective and ecologically safe method, especially when closed bioreactors for dynamic composting are used. Nevertheless, to achieve beneficial efficiency of keratin biodegradation it is essential to incorporate keratin-degrading bacteria, fungi or actinomycetes [1, 2, 3]. Biomethanation, anaerobic conversion of organic waste by methanogenic bacteria, is also applicable for feather waste processing. Nevertheless, the acceptable efficiency of the process is achieved in a two-step procedure, where chemical or microbiological degradation of keratin precedes the anaerobic fermentation [4, 5].
Conventional methods used for the bioconversion of keratinous waste, generated during processing of animal raw materials, are based mainly on the thermal and chemical treatment. Steam cooking, a widely used technique for production of feather meal, although energy-consuming, does not employ chemical compounds that become troublesome after use . Application of lime or other alkalies, as well as mineral acids, combined with heat treatment, is a known practice to convert keratinous materials into hydrolysates applicable as feed components, soil fertilizer, etc. and offers high process throughput [7, 8]. Both approaches allow for obtaining by-products of moderately enhanced digestibility, yet with usually deepened imbalance in the essential amino acid content. The implementation of enzymatic or microbial digestion, significantly improves the biological and technological value of keratin meals or hydrolysates by providing more advantageous amino acid balance and high digestibility [9, 10, 11, 12]. Therefore, process designs based on the application of keratinolytic microorganisms or their proteolytic enzymes represent promising alternatives .
Hydrothermal pretreatment was proved to be advantageous as a method for preparation of feathers or hooves and horn prior to hydrolysis with bacterial or fungal keratinases [14, 15]. An alternative for keratinous substrate preparation is based on sulfitolytic cleavage of disulfide bonds provided by sulfite. Sulfite enhances keratin feather digestion when present in the reaction environment, but it is especially effective when applied in the thermal treatment of the keratin substrate. Significant improvement of feather and bristle digestion with microbial keratinases, was achieved at relatively low sulfite concentrations of 10 and 100 mM, respectively [16, 17].
Enzymatic digestion of keratins with purified or crude keratinases appears to be one of the preferred approaches to process keratinous waste into valuable hydrolysates. It allows for production of feather meal of improved dietary value, amino acid balance and digestibility [14, 18]. Occasionally, combined chemical and enzymatic hydrolysis also proves to be advantageous [10, 19]. Bacteria-mediated keratin breakdown is known to be a cumulative effect of proteolytic cleavage as well as auxiliary reducing factors, including released reduced thiols or indigenous disulfide reductase enzymes [20, 21]. Nevertheless, enzymatic keratinolysis in vitro, conducted in the absence of red-ox potential of living microbial cells usually requires additional reducing agents to support disulfide bonds cleavage. This could be attained either by introducing chemical reducers into reaction environment or through initial substrate pretreatment [22, 23].
Numerous keratinolytic bacteria, filamentous fungi and actinomycetes have been isolated from various environments and characterized, nevertheless bacteria of the genus Bacillus represent the dominating group. The biotechnological interest in keratinolytic bacilli results mainly from their capability to biosynthesize a diversity of extracellular keratinases, but also from low nutrient requirements that allow growth in simple media where keratin serves as a sole nutrient source, spore forming capabilities that secure culture continuity especially in composting process where disadvantageous conditions may occur, mesophilic growth conditions of most of the species ensuring low energy requirements of cultivation and also occurrence of extremophilic species producing thermostable keratinases. Applicatory potential of keratinolytic proteases or keratin hydrolysates obtained with Bacillus bacteria has been recently explored with special regard to certain branches of industry. Grazziotin et al.  evaluated the nutritional quality of chicken feather hydrolysates obtained in culture of B. licheniformis. Also, purified keratinase from B. licheniformis ER-15 was applied for bioconversion of feathers into feather meal of improved dietary value . Likewise, keratinase from B. subtilis RSE163 proved to be applicable for production of feather hydrolysate for feed supplementation . Keratin hydrolysates may serve as a source of bioactive compounds. Fakhfakh et al.  applied B. pumilus A1 for biodegradation of wool waste, resultant in a product of advantageous amino acid and peptidic composition, exhibiting exceptional antioxidative properties. Similarly, feathers hydrolyzed by fermentation with B. subtilis S1-4 was a source of a specific antioxidative peptide . Applicability of feather hydrolysate as soil fertilizer was confirmed by Bose et al. . Fermentation of feather waste with B. amyloliquefaciens 6B allowed for obtaining a product effective as agricultural nitrogen input. It also exhibited antifungal properties due to the presence of surfactin, that allowed for biocontrol of phytopathogenic fungi. Keratinolytic bacteria found multiple potential applications in leather and textile industry. Proteases from B. subtilis P13 and Bacillus sp. SB12 were effectively used in the process of dehairing, which in contrast to conventional lime-sulfide dehairing, not only allowed for the reduction of effluents, but also positively affected treated leathers [28, 29]. Hoof and horn hydrolysate produced with B. subtilis was also applied as an agent to reduce consumption of chromium salts in the process of tanning, contributing to a significantly lowered discharge of chromium . Finally, production of proteolytic enzymes on keratinous waste materials constantly remains the most frequent application of keratinolytic bacteria. Due to a range of vital technological properties, e.g. thermostability, alkali and solvent stability, detergent compatibility and unique specificity, the enzymes present high predisposition for commercialization .
Enzymatic capabilities of Bacillus cereus species involving its immense proteolytic potential, frequently becomes a subject of applicatory research. Recently, the ability of keratin biodegradation related to biosynthesis of specific keratinases frequently undergoes detailed investigation. Several keratinolytic B. cereus strains have been described as exceptionally efficient in decomposition of chicken feather keratin, within culture conditions. The strains produced mainly serine proteinases, to utilize keratin as a nutrient source, enabling cell growth. Fermentation process allowed to obtain hydrolysates of improved lysine, methionine and threonine balance, as compared to the crude substrate [32, 33, 34]. In addition, B. cereus strains exhibiting specificity towards biodegradation of other hard keratins, like wool or ram horn, were also described. Their applicatory potential was confirmed through the analysis of obtained fermentation products [35, 36]. Highly proteolytic B. cereus strains were also proved to be applicable for the recovery of proteinaceous fractions form other agro-industrial by-products, e.g. brewer’s spent grain .
Bacillus cereus PCM 2849 was described previously to effectively decompose keratins, mainly feathers, in liquid culture conditions. Likewise, its crude keratinase was readily capable of efficient digestion of chicken feathers and the yield of the process could be further enhanced by substrate pretreatment . The objective of the study was to optimize enzymatic digestion of pretreated pig bristle with concentrated keratinase of B. cereus PCM 2849. A Box-Behnken experimental design was applied to estimate the effect of three influential parameters: substrate content, sulfite concentration during substrate pretreatment and reaction temperature, on the release of amino acids.
Materials and Methods
Bacterial Strain and Culture Conditions
Keratinolytic bacteria B. cereus PCM 2849 applied in the study, formerly referred to as B. cereus B5esz, have been described elsewhere . The strain was deposited in the Polish Collection of Microorganisms at the Institute of Immunology and Experimental Therapy in Wroclaw, Poland.
Keratinase production was carried out in 500 cm3 flasks, at 30 °C, 180 rpm, in 100 cm3 of medium consisting of (%): MgSO4 0.1, KH2PO4 0.01, FeSO4·7H2O 0.001, CaCl2 0.01, yeast extract 0.05 and degreased chicken feathers 1.0. Medium was set to pH 7.1 prior to autoclaving (121 °C, 20 min). Nutrient broth culture (glucose 1.0 % and nutrient broth 0.8 %) of approximately 1.2 × 108 cfu cm−3 served as inoculum (1 cm3 per flask).
Crude Enzyme Preparation
The bacterial culture was terminated on the 3rd day, feather debris was removed on the Whatman no. 2 filter paper and biomass was separated by centrifugation (5000 g, 20 min, 4 °C). The culture fluid was concentrated on the Labscale TFF System (Millipore) with a Pellicon XL 50 casette, Ultracel-10 PLCGC membrane (10 kDa cutoff). The obtained preparation was stored in portions at −24 °C.
Zymographic analysis of the concentrated culture fluid was performed. The sample was mixed at the ratio 6:4 with the sample buffer (Tris–HCl 0.32 M; pH 6.8; glycerol 48 %; SDS 8 %; bromophenol blue 0.06 %). Samples were loaded onto 8 % polyacrylamide gel (5 % staking gel) containing copolymerized casein 0.1 %. Electrophoresis was performed at constant 20 mA, at 2 °C. Subsequently, the gel was washed twice with Triton-X 2.5 %, once with the incubation buffer (Tris–HCl 0.05 M, pH 7.5, containing CaCl2 2 mM and sodium azide 0.02 %) and incubated for 24 h at 30 °C in the same buffer. Staining with Coomassie Blue and decolorization with methanol: acetic acid: water (50:10:40) was applied to visualize the proteolytic activity bands.
Pretreatment of Pig Bristles
Pig bristle was subject to a two-step pretreatment, prior to enzymatic digestion. The first pretreatment step was developed in order to remove tissue remnants, non-keratinous proteins and lipids from the crude pig bristle bulk. It was performed by conducting a short-term culture of B. cereus PCM 2849 in a mixture consisting of pig bristle 10 g, distilled water 250 cm3 and bacterial inoculum in nutrient broth 50 cm3. Afterwards, the solids were separated, washed with tap water and dried. The secondary, thermo-chemical pretreatment was performed by autoclaving the substrate (121 °C, 20 min) in a solution of sodium sulfite (1 g bristle per 100 cm3 of solution), according to Łaba and Szczekała . The pretreated substrate was subsequently washed with tap and distilled water to remove the pretreatment agent and finally dried at room temperature.
Enzymatic Digestion of Pig Bristle
The enzymatic digestion experiment of uncut, pretreated bristle was conducted using the concentrated culture fluid of B. cereus, in a 24-hour reaction in a mixture containing: bristle 0.2–1.0 g, enzyme solution 25 PU, buffer (Tris–HCl 0.05 M, pH 7.5) to 10 cm3, CaCl2 2 mM . After digestion the reaction mixture was subsequently cooled, filtered through glass-fibre filter Marcherey-Nagel GF-1 and centrifuged (12,000 g, 10 min). Concentration of free amino groups assayed according to Snyder and Sobociński  served as a measure of the digestion level.
Optimization of Enzymatic Hydrolysis
Amino Acid Composition of Bristle Hydrolysate
Amino acid composition of bristle hydrolysate, obtained at optimized conditions, was determined using HPLC, according to Henderson et al. . Initial derivatization with O-phthalaldehyde was performed. The analysis made on a HPLC 1100 Series system (Agilent Technologies) equipped with the ZORBAX Eclipse-AAA column, 4.6 × 150 mm, 3.5 µm (Agilent Technologies).
Microscopic Examination of Bristle Degradation
Visual analysis of bristles following enzymatic hydrolysis was performed using scanning electron microscopy (SEM) on a Hitachi S3400 microscope.
The structural changes in bristles after pretreatment and enzymatic hydrolysis were analyzed by Fourier transform infrared spectroscopy (FTIR). Measurements were performed on the VERTEX 70 spectrometer with dedicated program for Fourier transformation. Specimens were prepared as KBr pellets. Measurements were recorded within the 4000–400 cm−1 range with 2 cm−1 resolution.
Results and Discussion
Experimental design layout and obtained results of Box-Behnken design with the independent variables: X1-pig bristle content [%], X2-sulfite concentration during substrate pretreatment [mM], X3-reaction temperature [°C]
Dependent variable [ln (a.a. mg cm−3)]
Box-Cox transformation statistics
Amino acid-ln transformed
Regression results of the Box-Behnken design for the release of amino acids
Analysis of variance (ANOVA) for the obtained regression model
Sum of squares (SS)
Degrees of freedom (DF)
Mean square (MS)
Lack of fit
The plot of bristle load versus temperature confirmed the dominating effect of the former and the convex optimum area of the latter (Fig. 2). Excessive load of proteinaceous substrate could result in low hydrolysis yield due to enzyme inhibition by reaction products. Also keratin substrate reactivity declination might occur during prolonged incubation period .
Amino acid composition of the obtained bristle hydrolysate
Concentration (μg cm−3)
24.2 ± 6.8
61.8 ± 3.2
14.7 ± 5.8
14.5 ± 5.0
37.3 ± 8.0
17.1 ± 9.1
36.7 ± 4.3
325.8 ± 55.4
54.7 ± 5.5
119.5 ± 30.4
269.3 ± 49.6
360.6 ± 134.3
136.0 ± 42.0
25.0 ± 2.7
14.2 ± 6.5
342.3 ± 71.9
953.0 ± 318.7
199.4 ± 29.4
Crude keratinase of B. cereus PCM 2849 was applied in biodegradation of pig bristles, an exceptionally hard to degrade keratinous waste material. As a result, partial hydrolysis of pig bristles was obtained, preceded by sulfitolytic pretreatment of the substrate. A regression model was built to optimize the process of hydrolysis to release free amino acids, at constant enzyme load. The produced amino acid cocktail was especially rich in branched chain amino acids, while residual substrate was characterized by substantial structural deterioration. The presented process of bristles bioconversion into hydrolysate could serve as a way of management of this hardly degradable biomaterial, especially for non-feed applications.
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