Wool-waste valorization: production of protein hydrolysate with high antioxidative potential by fermentation with a new keratinolytic bacterium, Bacillus pumilus A1

Authors


Correspondence

Nahed Fakhfakh, Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax (ENIS) B.P. 1173-3038, Sfax-Tunisia. E-mail: nahedfakh_zouari@yahoo.fr

Abstract

Aims

Wool, a recalcitrant waste mainly composed of keratin, constituted a serious problem for the environment and was not effectively valorized. This study reported the optimization of wool-waste biodegradation by a new keratinolytic bacterium Bacillus pumilus A1. The in vitro digestibility and the antioxidant potential of wool protein hydrolysate (WPH) were also investigated.

Methods and Results

The antioxidant potential of WPH was evaluated using in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power and metal (Fe2+) chelating activity. Cultivation on 50 g l−1 of wool for 2 days, at 45°C and at initial pH of 10, resulted in maximum production of amino acids and peptides (39·7 g l−1). WPH presented a very high in vitro digestibility (97%) as compared with that of the untreated wool (3%).

Conclusions

The keratin present into the wool-waste was completely solubilized. Interestingly, WPH presented an important DPPH radical-scavenging activity with an IC50 value of 0·14 ± 0·01 mg ml−1.

Significance and Impact of Study

WPH would be a very useful source of protein and antioxidants in animals’ diets.

Introduction

Development of meat processing industries and tanneries throughout the world generated annually millions of tons of keratinous wastes such as wool, feathers, hair, nails, hooves, claws, and beaks (Khardenavis et al. 2009; Zheljazkov et al. 2009). Sheep wool has an economic importance and was widely used in textile. However, much wool of insufficient quality was treated as waste. This recalcitrant waste, mainly composed of keratin, constituted a serious problem for the environment and was not effectively valorized (Gousterova et al. 2005). Wool keratin represented a structural protein composed of α-helix, strongly stabilized by several hydrogen and hydrophobic interactions in addition to disulfide linkages. The intensive cross-linkage within keratin residues provided high mechanical stability and resistance to degradation by conventional proteases like trypsin, pepsin, and papain (Papadopoulos et al. 1986).

Despite the high protein content of keratinous wastes, they cannot be utilized efficiently in animal feed under their native state due to their poor digestibility (Moran et al. 1966; Evans et al. 2000). Traditional methods, based on physical and chemical treatments, used for keratin degradation required much energy and resulted in destruction of some amino acids and a loss of nutritional value (Mortiz and Latshaw 2001). Therefore, hydrolysis of keratinous wastes by keratinolytic micro-organisms was an attractive alternative method for its efficient bioconversion and for its ability to improve their nutritional value. In fact, keratinases were the only group of proteases which can completely degrade these highly intricate proteins (Gupta and Rammani 2006). Several keratinolytic micro-organisms were known to degrade keratinous substrates such as fungi (Anbu et al. 2006), actinomycetes (Syed et al. 2009), and some Bacillus species (Fakhfakh et al. 2009; Kumar et al. 2010).

Previous studies have focused on the valorization of chicken feathers waste by biotechnological processes. In our recent study, a keratinolytic Bacillus pumilus A1 was isolated with the aim of effective keratin feather degradation. The production of feathers protein hydrolysate by fermentation of B. pumilus A1, the in vitro digestibility, and the antioxidant activity of this hydrolysate were also investigated (Fakhfakh et al. 2011). However, little attention has been given to another unutilized keratinous waste: sheep wool. In fact, it's very important to optimize the biodegradation of such waste which was present in abundant amounts.

Hydrolyzed proteins from many animal and plant sources have been found to possess antioxidant activity (Blanca et al. 2007; Cumby et al. 2008). However, no antioxidant activity was reported in the wool protein hydrolysate (WPH). To the best of our knowledge, this is the first report highlighting the antioxidant potential of such keratinous waste hydrolysate which could be an important protein and antioxidants source in feed formulations.

This study reports the production of WPH by B. pumilus A1. The in vitro digestibility and the antioxidant potential of WPH were also investigated.

Material and methods

Wool-waste

The wool-waste consisted of unprocessed and unwashed low-grade wool was washed threefold with tap water and finally with distilled water. The washed wool was dried at 90°C for 22 h and then stored at room temperature prior to microbial treatment.

Bacterial strain

A new B. pumilus A1 producing alkaline keratinases was recently isolated from the slaughterhouse polluted water in Sfax city (Tunisia). It was identified on the basis of the 16S rRNA gene sequencing (EU719191) (Fakhfakh-Zouari et al. 2010a).

Culture media and growth conditions

The strain B. pumilus A1 was routinely grown in Luria–Bertani broth medium composed of 10 g l−1 peptone, 5 g l−1 yeast extract, and 5 g l−1 NaCl (Miller 1972). Cells were grown at 37°C under agitation at 200 rev min−1 for 18 h. The initial medium used for WPH production was composed of 30 g l−1 wool-waste, 0·5 g l−1 KH2PO4, 0·5 g l−1 K2HPO4, 2·0 g l−1 NaCl, 0·1 g l−1 KCl, 0·1 g l−1 MgSO4·7H2O, pH 6. The media were autoclaved at 121°C for 20 min. Cultivations were conducted in a 1-l Erlenmeyer flask containing 100 ml culture medium maintained for 5 days at 30°C and 250 rev min−1. To optimize the composition and the condition of the culture, the effect of wool concentration (10–60 g l−1), initial pH, temperature, and inoculum size were individually evaluated for their performance in WPH production. Aliquots of 1 ml were periodically removed, centrifuged for 5 min at 15 000 g, and the supernatant was stored at 4°C for determination of proteolytic activity, amino acids, and peptides concentration. Productivity of amino acids and peptides (g l−1 h−1) was determined after 5 days. Bacterial growth was estimated by a total plate count on nutrient agar. All experiments were performed in duplicate for the mean calculation.

Proteolytic activity

Protease activity of the crude enzyme was measured by the method of Kembhavi et al. (1993) using casein as a substrate (Sigma-Aldrich, St Louis, MO, USA). A 0·5 ml of 100 mmol l−1 Tris-HCl buffer (pH 8·5) containing 1% (w/v) casein was mixed with 0·5 ml of the crude enzyme suitably diluted and incubated for 15 min at 60°C. The reaction was stopped by the addition of 0·5 ml 20% (w/v) trichloroacetic acid, allowed to rest for 15 min at room temperature and then centrifuged for 15 min at 17 000 g. The absorbance of the supernatant was measured at 280 nm (T70, UV/VIS Spectrophotometer; PG. instruments Ltd. Wiftaft, UK) using the appropriate blank. A standard curve was generated using solutions of 0–50 mg l−1 of tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per minute under the experimental conditions used. Protease activity represents the means of at least two determinations carried out in duplicate.

Keratinolytic activity

Keratinase activity was determined by the modified method of Takiuchi et al. (1982). The reaction mixture consisted of 0·5 ml of 100 mmol l−1 glycine–NaOH buffer (pH 9) containing 0·8% (w/v) keratin from hoofs and horns (Sigma-Aldrich) and 0·5 ml of suitably diluted crude enzyme. After 1-h incubation at 55°C, the enzyme reaction was stopped by the addition of 0·4 ml of 10% trichloroacetic acid (TCA). The samples were left at 4°C for 15 min and then centrifuged at 17 000 g for 15 min. The absorbance of the supernatant was measured at 280 nm against a control. The control was treated in the same way, except that TCA was added before incubation. One unit (U) of keratinolytic activity was defined as an increase of corrected absorbance at 280 nm (A280) of 0·1 under the described conditions. The data presented are mean value of two parallel determinations.

Analytical methods

Dry matter was determined by oven-drying at 105°C to constant mass (AOAC 1997). Protein content (dry-weight basis) was analyzed according to the Kjeldahl method (AOAC 1990). Concentration of amino acids and peptides was determined by the ninhydrin method (Moore and Stein 1954). Fat content was determined by Soxhlet extraction with hexane for 8 h at boiling point of the solvent. The ash content was determined by combustion of the sample at 550°C for 8 h.

Antioxidant activity

DPPH radical-scavenging assay

DPPH radical-scavenging activity of WPH was determined as described by Bersuder et al. (1998). A volume of 500 μl of each sample at different concentrations was mixed with 500 μl of 99·5% ethanol and 125 μl of 0·02% DPPH in 99·5% ethanol. The mixture was then kept at room temperature in the dark for 60 min, and the reduction of DPPH radical was measured at 517 nm using a UV–Visible spectrophotometer. The percentage inhibition of the DPPH radical-scavenging activity was calculated according to the formula:

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where Ac is the absorbance of the control reaction and As is the absorbance of the sample extract. Butylated hydroxyanisole (BHA) was used as a standard. The test was carried out in triplicate.

Reducing power assay

The ability of the hydrolysate to reduce iron (III) was determined according to the method of Yildirim et al. (2001). An aliquot of 1 ml sample of WPH at different concentrations was mixed with 2·5 ml of 0·2 mol l−1 phosphate buffer (pH 6·6) and 2·5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 30 min, followed by addition of 2·5 ml of 10% (w/v) TCA. The mixture was then centrifuged at 15 000 g for 10 min. Finally, 2·5 ml of the supernatant solution was mixed with 2·5 ml of distilled water and 0·5 ml of 0·1% (w/v) ferric chloride. After 10 min reaction, the absorbance of the resulting solution was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The values are presented as the means of triplicate analyses.

Metal (Fe2+) chelating activity

The chelating activity of the WPH for Fe2+ was measured according to the methods described by Dinis et al. (1994). To 0·5 ml of WPH, 1·6 ml of deionized water and 0·05 ml of FeCl2 (2 mmol l−1) were added, followed by the addition of 0·1 ml of (5 mmol l−1) after 15 min. After 10 min at room temperature, the absorbance of the Fe2+-ferrozine complexes with red or violet color was measured at 562 nm. The chelating Fe2+ activity was calculated according to the following formula:

display math

where Ac is the absorbance of the control reaction and As is the absorbance of the sample extract.

In vitro digestibility

In vitro proteolytic digestion of samples was performed with pepsin and pancreatin, as described by Ikeda et al. (1995). One gram of each sample was incubated with 2 mg ml−1 of pepsin (from porcine stomach, Sigma) for 2 h at 37°C in 2 mol l−1 HCl. After incubation, the pH was adjusted to 8 with 2 mol l−1 NaHCO3, and the sample was incubated for additional 16 h with 2 mg ml−1 of pancreatin (from porcine pancreas, Sigma). After digestion, samples were centrifuged and protein concentration was determined in supernatants by the Kjeldahl method (AOAC 1990). Protein digestibility was calculated as: D = content of protein released upon the digestion of 1 g of sample/the content of the total protein of 1 g of sample before digestion.

Results

Production of wool protein hydrolysate

The use of wool as protein source can be of great interest because it is present in abundant amounts and is composed by at least 80·67% of α-keratin (Feughelman 1997). In the present study, the protease produced by B. pumilus A1 allowed the degradation of wool contained in the medium which provoke an increase of amino acids and peptides concentrations. Besides, more the produced proteolytic activity was higher, the degradation was more pushed and the concentrations of amino acids and peptides were more important. For this reason, the caseinolytic activity and end products productions such as free amino acids and peptides were measured during the fermentation process. Thus, the effects of wool concentrations, initial pH, temperature, and inoculum size on the production of amino acids and peptides by B. pumilus A1 were investigated.

Effect of wool-waste concentration

Bacillus pumilus A1 was grown in mineral medium containing different amounts of wool as sole carbon and nitrogen source. The highest levels of proteolytic activities were obtained after 4 days of growth and then activities remained constant. The strain exhibited the highest enzyme production (587 ± 5 U ml−1) in culture medium containing 50 g l−1 of wool-waste (Fig. 1a).

Figure 1.

Effects of wool-waste concentration on proteolytic activity (a), amino acids and peptides, (b) productions, and on wool protein hydrolysate productivity (c). Cultivations were performed at inoculum size 8% in media with initial pH of 6·0 at 30°C. (♦) 10 g l−1; (■) 20 g l−1; (▲) 30 g l−1; (♢) 40 g l−1; (□) 50 g l−1; (△) 60 g l−1; (image) g of amino acids per g of wool and (■) productivity of amino acids (gh l−1 ×10)

Amino acids and peptides productions increased with increasing wool-waste concentration and reached the highest level (15·3 ± 0·5 g l−1) at 50 g l−1 (Fig. 1b). However, at 60 g l−1 of wool, protease; amino acids; and peptides production were reduced. This could be explained by the fact that high wool concentration may cause low growth of A1 strain. In fact, at 60 g l−1 of wool, the growth of B. pumilus A1 was measured and it was found to be lower than that obtained with 50 g l−1 of wool (data not shown). The highest ratio of amino acids and peptides per gram of wool was reached at 10 g l−1 of wool (Fig. 1c). However, maximum productivity of amino acids and peptides (0·125 g l−1 h−1) was obtained in culture medium containing 50 g l−1 of wool-waste (Fig. 1c).

Effect of initial medium pH

The effect of the initial pH value of the medium within 6–11 on the production of proteolytic enzymes and WPH was studied by cultivating the strain A1 in the medium containing 50 g l−1 of wool-waste. As shown in Fig. 2a, the highest protease production level was obtained at pH 6. Nevertheless, at this pH value, no complete wool degradation was achieved (evaluated by physical appearance). The highest level of amino acids and peptides production was obtained at initial pH medium of 9–11, and complete degradation of wool was obtained after 4 days of fermentation (data not shown). In fact, the highest amino acids and peptides (22·5 ± 0·78 g l−1) production was observed at pH 10 (Fig. 2b).

Figure 2.

Effects of medium pH on proteolytic activity (a) and amino acids and peptides, (b) productions. Cultivations were performed at inoculum size 8% in media containing 50 g l−1 of wool-waste at 30°C. (□) pH 6; (■) pH 7; (△) pH 8; (×) pH 9; (▲) pH 10 and (○) pH 11.

Effect of cultivation temperature

Several works showed that the fermentation temperature has a significant effect on the proteins production (Suntornsuk and Suntornsuk 2003; Zaghloul et al. 2004). In order to optimize WPH production, different temperatures values (between 30 and 50°C) were tested by cultivating the strain A1 in the medium containing 50 g l−1 of wool-waste at initial pH of 10.

As shown in Fig. 3a, at 40, 45, and 50°C, protease production reached a maximum after 24 h of cultivation and then decreased. However, protease production curves were completely different at lower temperatures (30 and 35°C). In fact, at these temperatures, the production of proteolytic enzymes by A1 strain reached highest level after 2 days of fermentation (Fig. 3a). The kinetic production of amino acids and peptides was shown in Fig. 3b. The maximum level of amino acids and peptides (33·9 ± 0·9 g l−1) was obtained at 45°C after 2 days of fermentation (Fig. 3b).

Figure 3.

Effects of cultivation temperature on proteolytic activity. (a) and amino acids and peptides, (b) productions. Cultivations were performed at inoculum size 8% in media containing 50 g l−1 of wool-waste with initial pH of 10·0. (♦) 30°C; (♢) 35°C; (■) 40°C; (□) 45°C and (△) 50°C.

Effect of inoculum size

Media containing 50 g l−1 of wool-waste with initial pH of 10 were inoculated separately with five inocula sizes (between 2 and 10%) and incubated at 45°C. Our results showed that the protease productions were comparable in the different cultures (Fig. 4a). However, a remarkable difference was noticed at the levels of released amino acids and peptides (Fig. 4b). In fact, the production of amino acids and peptides was not proportional with inoculum size. The maximum production of amino acids and peptides (39·7 ± 0·5 g l−1) was attained at inoculum size of 6% which was lower than that used (8%) during the preliminary optimization steps (Fig. 4b).

Figure 4.

Effects of inoculum size on proteolytic activity (a), on amino acids and peptides productions (b), (♦) 2%; (♢) 4%; (■) 6%; (□) 8% and (△) 10%, and kinetics of growth and keratinase production by Bacillus pumilus A1 at inoculum size 6% (c), (▲) Biomass UFC 107 ml−1 and (♢) Keratinolytic activity (U ml−1). Cultivations were performed in media containing 50 g l−1 of wool-waste with initial pH of 10·0 and at 45°C.

In order to explain the kinetic production of protease activity, the time course of keratinase production and growth of B. pumilus A1 in optimized conditions (50 g l−1 wool-waste, 45°C, pH 10, and initial inoculum size 6%) were investigated. The results reported in Fig. 4c showed that keratinase synthesis was associated with the cell growth and reached a maximum activity of 62 U ml−1 at 24 h. However, complete wool degradation was achieved after 48 h of fermentation. Besides, the caseinolytic activity was proportional to keratinolytic activity (Fig. 4c) which confirms that the protease produced by B. pumilus A1 is a keratinase involved in wool biodegradation.

Biochemical and physical characterization of WPH

The A1 strain was able to degrade effectively the wool-waste, indicating its important keratinolytic activity. Maximum amino acids and peptides were obtained under the following conditions: 50 g l−1 of wool-waste; 45°C; pH 10; inoculum size 6%; and 48 h. In order to check that wool was completely degraded, the culture was filtrated through Whatman No. 3 filter paper. Interestingly, after washing and drying, the filter paper at 105°C to constant mass, no residual wool was obtained which demonstrate their complete degradation. Finally, the filtrate was autoclaved, centrifuged to eliminate bacteria biomass, and dried overnight at 90°C to obtain the wool protein hydrolysat.

The physico-chemical characterization of the produced WPH was determined (Table 1). Proteins represent the major component (81·6%) of the WPH followed by ash (12%). The low humidity of this hydrolysate (5·6%) may contribute to the microbiological stability of the product during its storage. The in vitro digestibility of the WPH directed by B. pumilus A1 in the optimized conditions was measured using pepsine and pancreatin. As shown in Table 1, the WPH presented a very high digestibility (97%) as compared to the raw wool (3%).

Table 1. Physico-chemical composition of WPH and raw wool
 WPHRaw wool
Composition (%)
Protein81·6 ± 0·480·67 ± 0·3
Fat0·6 ± 0·051·6 ± 0·4
Moisture5·6 ± 0·46·6 ± 0·7
Ash12 ± 0·45·3 ± 0·08
In vitro digestibility (%)97 ± 0·63 ± 0·3

Antioxidant activity of wool protein hydrolysate

The resulting WPH was subjected to screening for its possible antioxidant activity using three complementary methods: DPPH radical-scavenging activity, reducing power, and metal (Fe2+) chelating activity.

DPPH free radical-scavenging activity

The effect of antioxidant on DPPH radical-scavenging was thought to be due to their hydrogen-donating ability (Shimada et al. 1992). When a solution of DPPH is mixed with that of a substance, it can generate a hydrogen atom. This results in the reduced form of DPPH (nonradical) with the loss of the violet color. The decrease in absorbance is taken as a measure for radical-scavenging activity. The DPPH radical-scavenging activity was investigated at different concentrations (0·1–0·6 mg ml−1) of the WPH. DPPH-scavenging activity is usually presented by IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. Therefore, extract concentrations providing 50% inhibition (IC50) were calculated using the data plotted in Fig. 5a. Lower IC50 value reflects better DPPH radical-scavenging activity. Our results showed that WPH exhibited an interesting radical-scavenging activity with an IC50 value of 0·14 ± 0·01 mg ml−1.

Figure 5.

Antioxidant activity of wool protein hydrolysate (WPH). DPPH-scavenging activity (a), reducing power (b), (♦) WPH and (♢) BHA, and metal (Fe2+) chelating activity (c), (♦) WPH and (♢) EDTA. WPH was obtained after autoclaving and drying the supernatant of culture realized under the following conditions: 50 g l−1 of wool; 45°C; pH 10; inoculum size 6%; and 48 h. Butylated hydroxyanisole (BHA) was used as a standard.

Reducing power

In the reducing power assay, the presence of antioxidants in the sample would result in the reducing of Fe3+–Fe2+ by donating an electron or hydrogen (Yildirim et al. 2001). An amount of Fe2+ complex can then be monitored by measuring the formation of Perl's Prussian blue (Fe4[Fe(CN)6]3) at 700 nm. Increasing absorbance at 700 nm indicates an increase in reductive ability. Figure 5(b) shows the reducing power activities of the WPH at different concentrations (0·1–1·2 mg ml−1) compared with those of BHA as positive standard. The results showed that the reducing power increased linearly with the WPH concentration to reach the same activity obtained with BHA at 1 mg ml−1, while at concentrations up to 0·5 g l−1 the activities were lower than those of BHA.

Metal (Fe2+) chelating activity

The metal chelating activity is also widely used in evaluating the antioxidant activity of different natural products. Transition metal ions can stimulate lipid peroxidation by two mechanisms, namely by participating in the generation of initiating species and by accelerating peroxidation and decomposing lipid hydroperoxides into other components which are able to abstract hydrogen, and thus perpetuating the chain of reaction of lipid peroxidation (Deshpande et al. 1995). The dark color of complex formed by the interaction of ferrozin with Fe2+ ions is decreased by the action of metal chelator compounds that exist in the reaction mixtures. Figure 5(c) presented the chelating activity of WPH at different concentrations (0·5–8 mg ml−1) compared to that of EDTA as positive standard. The results showed that the percentages of inhibition of the ferrozine–Fe2+ complex formation increased linearly with the WPH concentration. Although the chemical EDTA exhibited high antioxidant activity, natural antioxidants are of growing interest.

Discussion

In the present work, B. pumilus A1 was used to degrade wool keratin which represented a structural protein composed of α-helix (Feughelman 1997). Therefore, different wool concentrations were tested for the production of WPH. Our results showed that complete wool degradation was achieved after 5 days of cultivation in the presence of 50 g l−1 wool. However, in the presence of 60 g l−1, residual wool remained after incubation (data not shown). In comparison with chicken feather, which represented a structural protein composed of β-sheet, complete degradation of 50 g l−1 of feathers was also obtained after 5 days of fermentation using the same strain (Fakhfakh et al. 2011). Despite the structural difference between feather and wool, their degradation seems to be similar in terms of concentration (50 g l−1) and fermentation time (5 days) with B. pumilus A1.

pH was one of the most important factors affecting the growth of the bacteria, keratinase production, and wool degradation rate. Because cultures were conducted in media containing only wool-waste as nitrogen and carbon source, complete degradation of wool-waste could only be achieved when high keratinolytic activity was produced. Thus, different pH values were tested for proteolytic enzyme and WPH production. Complete wool degradation by the A1 strain was observed at initial pH from 9 to 11. This result could be explained by the fact that the proteolytic enzymes produced by A1 strain are highly active at alkaline conditions (pH 8·5 on casein and pH 9 on keratin) (Fakhfakh-Zouari et al. 2010b). These findings were in contrast with other studies which stated that measurable keratin hydrolysis directed by Chryseobacterium sp. kr6, Bacillus pumilus FH9, and Vibrio sp. Kr2 was observed between pH values of 6·0–9·0 (El-Refai et al. 2005; Grazziotin et al. 2007; Riffel et al. 2007).

The fermentation temperature was also an interesting parameter that can influence the production of the keratinolytic protease enzyme. Data revealed that the maximum level of amino acids and peptides was obtained after 48 h of fermentation at 45°C. This is in line with previous findings which reported that optimal temperatures for keratin solubilization by B. subtilis mutant cells and B. licheniformis cells were 42°C and 45°C, respectively (Balint et al. 2005; Cai et al. 2008). Conversely, present data were in disagreement with previous reports stating that optimal temperature for keratin solubilization by Vibrio sp. Kr2 (Grazziotin et al. 2007) and B. subtilis recombinant cells (Zaghloul et al. 2011) was 30°C.

Determination of the optimum inoculum size required to direct the biodegradation process was an important aspect to consider when optimizing bioprocesses. In fact, the increase of the inoculum size was accompanied by the increase of amino acids and peptides production. The maximum level of this product was reached at 6%. Further addition of the initial number of bacterial cells decreased the level of amino acids and peptides. The present finding was in disagreement with a previous report stating that a decrease in the proportion of keratin hydrolysis directed by a B. subtilis mutant strain was noticed upon increasing the initial inoculum size from 2–10% (v/v) (Cai et al. 2008). The keratin present into the 50 g of wool-waste (40·33 ± 0·3 g proteins) was completely solubilized to amino acids and peptides, detected by the ninhydrin method (39·7 ± 0·5 g amino acids and peptides). This result was confirmed by the protein concentration in the culture filtrate (39·8 ± 0·4 g proteins) determined by the Kjeldahl method. Because of the total wool degradation, the concentration of amino acids (39·7 g l−1) obtained with the strain A1 was very high as compared to that obtained with the strain Vibrio sp. Kr2 which released only 0·2 g l−1 of amino acids and peptides after 5 days of fermentation in the medium containing 60 g l−1 of keratin (Grazziotin et al. 2007).

The in vitro digestibility of the WPH produced by A1 strain was found to be very close to that obtained with the strain Vibrio sp. Kr2 (Grazziotin et al. 2006). However, the digestibility of keratin hydrolysate obtained by fermentation of the strain Kocuria rosea was found to be 88% (Bertsch and Coello 2005).

The WPH, produced in this study, exhibited high DPPH free radical-scavenging activity (IC50 = 0·14 mg ml−1) which was lower than that of feather protein hydrolysate (IC50 = 0·3 mg ml−1) (Fakhfakh et al. 2011) and that of smooth hound protein hydrolysate (IC50 = 0·6 mg ml−1) (Bougatef et al. 2009). The reducing power of the WPH was investigated at different concentrations and was found to be concentration dependent. Its value increased with the higher WPH concentrations as was reported by several works (Zhu et al. 2006; Bougatef et al. 2009; Manni et al. 2010). The obtained results also revealed that WPH, with high amino acids and peptides contents, could react with free radicals to form stable products. This interesting antioxidant potential of WPH was reported for the first time. In fact, the WPH could be used as a natural source of protein and antioxidants for animal nutrition (fish or domestic animals). Furthermore, when WPH was used in animal feed formulations, it can protect feed against oxidation. The effects of different natural antioxidants such as rosemary extract, ascorbyl palmitate, or ascorbic acid in fish feed made from marine raw materials were studied (Hamre et al. 2010). Moreover, natural antioxidants were found to be important to animal health by enhancing its immunity (Chew, 1996). Indeed, they function to remove harmful free radicals and to strengthen the immune system. Consequently, lower mortality and increased efficiency of livestock production could be reached.

Conclusions

Our results demonstrated that microbial conversion of wool-waste constituted a potential approach for their biodegradation and valorization with respect to cost-effectiveness and environment protection. In the present study, wool-waste degradation was successfully carried out by fermentation of the strain B. pumilus A1. The WPH obtained under optimum conditions (50 g l−1 wool-waste, 45°C, pH 10, initial inoculum size 6%, and 2 days of fermentation) was assessed for antioxidant activities. Interestingly, WPH was found to possess an interesting antioxidant potential. The characterization of the WPH showed that it contained high amounts of peptides and amino acids. Furthermore, it presented a very high in vitro digestibility as compared to that of the untreated wool. The use of WPH in fish feed formulations as a source of protein and natural antioxidants would be an advantage both for the aquaculture industry and for the consumer's health and well-being. Further works should be done to study the in vivo lipid peroxidation of WPH. The oxidative stability during the storage of an animal diet formulated with WPH should also be investigated.

Acknowledgements

The authors would like to thank Dr. Khaled Jebahi (Higher Institute of Applied Studies in Humanities, Medenine, Tunisia) for his kind help with English.

Conflict of Interests

The authors declare that they have no competing interests.

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