Middle-East Journal of Scientific Research 11 (7): 900-907, 2012

ISSN 1990-9233
© IDOSI Publications, 2012

Production and Optimization of Feather Protein Hydrolysate from

Bacillus Sp. MPTK6 and Its Antioxidant Potential
1
D.J. Mukesh Kumar, 2P. Priya, 3S. Nithya Balasundari, 3G.S.D. Nandhini Devi,
1
A. Immaculate Nancy Rebecca and 1P.T. Kalaichelvan

1
CAS in Botany, University of Madras, Guindy campus, Chennai, TN, India
2
K.S. Rangasamy College of Technology, Tiruchengode, TN, India
3
Kalasalingam University, Virudhunagar, TN, India

Abstract: The production of feather protein hydrolysate (FPH) by Bacillus sp. MPTK6 was investigated for
its in vitro digestibility and antioxidant potential. The effects of feather concentrations (15-60 g/I) and initial
pHs (6-11) on the production of amino acids, peptides and soluble proteins were evaluated for their performance
in FPH production medium. The strain MPTK 6 showed maximum keratinase and caesinase activity of 2.4 U/ml
and 2.1 U/ml in the initial screening process. The strain MPTK6 showed the ability to degrade raw feather to
FPH under optimal conditions (30 g/l chicken feathers, pH 10.0 and 72 h of fermentation). The DPPH free
radical-scavenging activity and reducing power showed the antioxidant potential of FPH of the test isolate.
The FPH showed very high in vitro digestibility compared to that of the raw feathers.

Key words: Feather Protein Hydrolysate In vitro Digestibility Antioxidant Keratinase

INTRODUCTION of keratin protein that has cysteine disulfide bonds [8].

The indigestible structure of raw feather must be
Feathers represent 5-7% (w/w) of the total weight of hydrolyzed to be used as a feed ingredient for non-
mature chickens and are generated in large amounts as a ruminant species. Though keratin can be completely
waste product in commercial poultry-processing plants, dissolved by reducing agents like copper sulphate,
reaching millions of tons per year worldwide [1]. Poultry mercapto acetate, iodoacetic acid, amino, sodium sulphite,
feathers were discharged into the environment after the sodium tetrathionate [9-11] these methods are not suitable
processing which leads to the environmental pollution. for the large scale application. In order to overcome these
Discarded feathers cause various human ailments limitations the use of microbial enzymes which improves
including chlorosis, mycoplasmosis and fowl cholera [2]. the nutritional value of feather wastes have been
Although the feathers are considered as wastages, it implemented in recent years.
contains large amount of protein and this protein can be Alkaline proteases are among the most important
converted into animal feedstuffs which helps to reduce industrial enzymes; they are primarily employed as
the protein shortage and cost effects. Poultry feed detergent additives, accounting for 40-60% of the global
production plays an important role in protein supply and enzyme market. Alkaline proteases are group of
in agricultural economy [3]. proteolytic enzymes that are able to hydrolyze insoluble
The protein shortage for food and feed leads us to hard proteins more effectively than other proteases such
look for a new protein sources from wastage products like as trypsin, pepsin and papain [12-14]. The genus Bacillus
feather wastes [4,5]. Feathers are a significant source of provides most of the alkaline proteases with commercial
protein for livestock because of their high protein content value, offering the advantages of being easy to culture
(>85% CP) [6]. Feathers contain large amounts of and maintain and they are potentially valuable in the
cysteine, glycine, arginine and phenylalanine [7, 46]. Raw bioconversion of keratinous wastes; in the detergent,
feathers, however, are very poorly digested by non- fertilizer, biopolymer, pharmaceutical and animal feed
ruminant animals because they contain a high proportion industries; and in leather processing and hydrolysis of

Corresponding Author: D.J. Mukesh Kumar, CAS in Botany, University of Madras, Guindy campus, Chennai, TN, India.
Tel: +91-9884553310.
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 Middle-East J. Sci. Res., 11 (7): 900-907, 2012

prion proteins as well [15]. Therefore, such amplified in a thermocycler at conditions: 35 cycles of
microorganisms and enzymes have been the focus in 94°C for 1 min, 55°C for 1 min and 72°C for 2 min which
several studies [16-20] and Bacillus alkaline proteases are amplifies the 16S rRNA sequences specifically by using
reported to be among the most efficient keratin degraders the primers [28].
[21, 22].
Against these backdrops, this study aimed to Forward (16F27):5 -AGAGTTTGATCCTGGCTCAG-3
investigate the production of feather protein hydrolysate Reverse (16R1522):5 -AAGGAGGTGATCCAGCCGCA-3
(FPH) by Bacillus sp., isolated from local poultry
industry. The in vitro digestibility and the antioxidant DNA sequencing was performed in a highly
potential of feather protein hydrolysate were also automated gene sequencer. These sequences were read in
investigated. Genbank databases (BLAST) and compared with the other
sequences to analyze the bacterial class and its
MATERIALS AND METHODS phylogeny.

Feathers and its Processing: Chicken feathers (CF), Feather Protein Hydrolysates (FPH) Production: The
supplied by a local poultry industry were washed strain MPTK 6 was grown in Luria-Bertani broth medium
threefold with tap water and finally with distilled water. which contains 10 g/l peptone, 5 g/l yeast extract and 5 g/l
The washed feathers were dried at 90°C for 22 h and then NaCl. The initial medium used for feather protein
stored at room temperature prior to microbial treatment hydrolysates production composed of 20 g/l chicken
[23]. feather powder (CFP), 0.5 g/l-KH2PO4, 0.5 g/l-K 2HPO4,
2.0 g/l-NaCl, 0.1 g/l-KCl, 0.1 g/l-MgSO4 7H2O at pH
Isolation and Screening of Alkaline Keratinolytic 8.5 autoclaved at 121°C for 20 min. Then the test isolate
Bacteria: The test organisms used in this study were was inoculated in the medium and incubated for 3 d at
isolated from feather composting soil near local poultry 30°C and 250 rpm with a working volume of 100 ml. The
farm in Chennai, India. 1 g of soil sample was added to the cultures were centrifuged at 12,000rpm for 15min at 4°C
keratin enrichment medium which contains (%): NH4Cl- and the cell-free supernatants were used for determination
0.05, NaCl-0.05, K 2HPO4-0.03, KH2PO4-0.04, MgCl2 6H2O- of keratinolytic activity, caseinolytic activity, soluble
0.024, yeast extract-0.01 and 1% feather keratin substrate protein and amino acid concentration. Productivity was
1 at pH 8.5 for 3 d under shaken condition. The enriched determined as soluble protein concentration per time to
sample (1 ml) was suspended in 9 ml of sterilized water reach 3 d concentration (mg/l/h) according to the method
and was spread on the same feather enriched medium by of Fakhfakh et al. [29].
addition of 1.7% agar and incubated at 37°C for 72 h. After
incubation the plates ware stained with Coomassie blue Effect of Physico-Chemical Culture Variables on FPH:
for the observation of clear zones around the margins of The effects of feather concentrations (15-60 g/I) and initial
colonies and were picked up as alkaline keratinase pH (6-11) on the production of amino acids, peptides and
producers. The positive isolates were then screened for soluble proteins were investigated by Bacillus sp.
keratinase [24] and caseinolytic activity [26] in liquid MPTK6 were evaluated for their performance in feather
culture medium using keratin and casein as substrates at protein hydrolysate production.
pH 8.5. The strain which showed maximum activity was
selected for further study. The stock culture of the strain Enzyme Activity: Keratinolytic and Caesinolytic
was maintained on glycerol stocks (50%, v/v) and stored activities were determined using azokeratin or azocasein
at -20°C. (Sigma Co., USA), respectively, as substrates. Azokeratin
was synthesized as described elsewhere [30]. The reaction
Characterization and Molecular Identification of mixture contained 100 ml of enzyme preparation and 100 ml
Bacteria: The preliminary characterization of the isolated of 10 mg ml 1 azokeratin (or azocasein) in 20 mMTris-HCl
strain was done using Bergey’s manual of systemic buffer, pH 8.0. The mixture was incubated for 30 min at
bacteriology [27] and 16S rDNA analysis was used for the 37°C; the reaction was stopped by adding 500 ml of 10%
authentication of the strain. The genomic DNA was (w/v) trichloroacetic acid. After centrifugation (10,000 × g
isolated from the bacteria by the method described by for 5 min) of the reaction mixture, 800 ml of the
Hoseket al. [27]. The highly purified DNA was then supernatant were mixed with 200 ml of 1.8 M NaOH and

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the absorbance at 420 nm was measured. One unit of done by preparing different concentrations of
enzyme activity was considered as the amount of enzyme hydrolysates and then adding 2.5 ml of 0.2 M phosphate
that caused a change in absorbance of 0.01 units at the buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide to
above assay conditions. 1 ml sample of each hydrolysate. Then it was kept at 50°C
for 30 min, followed by addition of 2.5 ml of 10% (w/v)
Analytical Assays: Dry matter was determined by oven- trichloroacetic acid. After incubation the sample was
drying at 105°C to constant mass [31]. Protein content centrifuged at 15,000 × g for 10 min. The absorbance of
(dry weight basis) was analyzed according to the the supernatant was done at 700 nm after incubation of
Kjeldahl method [32]. Soluble protein and peptide the mixture containing 2.5 ml of the supernatant solution
concentrations were determined by the method of Biuret with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) ferric
[33] using ovalbumin as the standard. Concentration of chloride for 10 min the sample with high reducing power
amino acids and peptides was determined by the was identified by increased absorbance.
ninhydrin method [34]. Fat content was determined by
Soxhlet extraction with hexane for 8 h at boiling point of In vitro Digestibility: In vitro digestibility of sample was
the solvent. The ash content was determined by done by adding pepsin and pancreatin to the sample [39].
combustion of the sample at 550°C for 8 h. Total protein It is done by adding 2mg of pepsin/ml to 1g of ground
content was measured according to Lowry’s method [35]. sample and incubated for 2 h at 37°C in 2M HCl. After
The amino acid present in the sample were analyzed by incubation, the pH was made to 8 by 2M NaHCO3 and
ion exchange chromatography and its concentration were then adds 2 mg of pancreatin/ml and again incubates it for
estimated by ninhidrin method [34]. Nitrogen content in additional 16 h [40].
the sample was estimated according to the method of
Deivasigamani and Alagappan [36]. D=
Content of protein releasedupon thedigestion of 1g of sample
Content of the total protein 1g of food beforedigestion

Antioxidant Activity: The stable 1,1-diphenyl-2-picryl RESULTS AND DISCUSSION

hydrazyl radical (DPPH) was used for the determination of
the free radical scavenging activity of the extracts (AED Screening of Alkaline Keratinolytic Bacteria: The test
and MED) by the method of Kolevaet al. [37]. For each organisms used in this study was isolated from feather
extract and standard, sample solutions of different composting soil in keratin enrichment medium at pH 8.5.
concentrations (0.5-3.5 mg/ml) were prepared in methanol The isolated organisms were screened in the feather
and added separately to an equal volume of 100 µM DPPH enriched medium. Seven isolates showed a clear zone and
solution in methanol. The reaction mixture was kept at these positive isolates were further screened for
room temperature for 15 min. Then, the absorbance of production of extracellular alkaline keratinase and
the reaction mixture was recorded at 517 nm using a caesinase. The strain MPTK 6 showed maximum
UV-visible spectrophotometer [Shimadzu UVPC-3200 keratinase and caesinase activity of 2.4 U/ml and 2.1 U/ml
(Kyoto, Japan)]. Gallic acid (GA) was used as standard. respectively (Table 1). Hence, this isolate was selected for
Free radical scavenging activity was calculated using the further studies.
following formula:
Characterization and Molecular Identification of
Control − Sample 0D Bacteria: From microscopic appearance and the
% of free redical scavenging activity= × 100
Control 0D
biochemical tests, the isolate was identified as Bacillus
The extract concentration having 50% radical sp. MPTK6 and further confirmation was done by
inhibition activity (IC50) was calculated from the graph of sequencing the 16S rDNA gene. Upon the amplification of
the free radical scavenging activity (%) against the extract 16S rDNA sequence using specific primer, an amplified
concentration. Three replicates were performed for each product of 1099 bp was obtained (Fig. 1) which was then
sample concentration to check the reproducibility of the sequenced and compared with the Genbank databases
experimental result and to get more accurate result. using the BLASTN program. The 16S rDNA sequence of
Results are represented as IC50 ± standard deviation. the isolate revealed a close relatedness to Bacillus sp.
with 97% similarity. Hence the strain was confirmed as
Reducing Power Assay: The iron reducing ability of the Bacillus sp. and the sequence was submitted to Genbank
hydrolysates was analyzed by Yildirim et al. [38]. It was (JQ746528).

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Table 1: Keratinase and caesinase production by bacterial isolates from

poultry farm soil
Isolate Keratinase (U/ml) Caesinase (U/ml)
MPTK 1 1.9±0.17 1.1±0.13
MPTK 2 1.4±0.11 1.7±0.10
MPTK 3 1.4±0.10 1.3±0.03
MPTK 4 1.2±0.61 1.4±0.02
MPTK 5 0.7±0.17 1.3±0.01
MPTK 6 2.4±0.42 2.1±0.03
MPTK 7 1.4±0.04 1.7±0.23

Fig. 2: Effects of feather concentration on caseinolytic

activity

Fig. 1: Photographic representation of PCR amplified Fig. 3: Effects of feather concentration on amino acids
product in Agarose Gel (Lane 1, 15 kbp ladder; and peptides
Lane 2, PCR amplified sample)

Effect of Physico-Chemical Culture Variables on FPH:

Considering that feathers are composed by at least 90%
of keratin, the use of this protein source can be of great
interest. Production of feather protein hydrolysates using
keratinolytic bacteria has been considered as an
interesting alternative [41]. Thus, the effects of feather
concentrations, initial pH on the production of amino
acids, peptides and soluble proteins were investigated on
Bacillus sp. MPTK6.
Fig. 4: Effects of feather concentration on soluble
Effect of Feather Concentrations: The microorganism proteins and peptides
Bacillus sp. MPTK6 was grown in mineral medium
containing different amounts of raw feathers as the sole feathers (Figs. 2 and 3). However, at 60 g/l of feathers,
carbon and nitrogen source. The highest levels of proteases, amino acids, peptides and soluble proteins
proteolytic activities were obtained after cultivation for production were reduced. In fact, it was demonstrated that
72 h and then activities remained constant. The strain high feather concentration may cause substrate inhibition
exhibited the highest enzyme production (620 U/ml) in or repression of keratinase production, resulting in a low
culture medium containing 30 g/l of chicken feathers percentage of feather degradation. The highest ratio of
(Fig. 2). The results are in line with those reported by soluble proteins and peptides per gram of feathers was
Fakhfakhet al. [29] using the strain Bacillus pumilus A1. reached with 10 g/l of feathers (Fig. 4). Maximum
The highest levels of amino acids and peptides productivity of soluble proteins and peptides were
(34±2.0 g/l) and soluble proteins and peptides (5.9±2 g/l) obtained in culture medium containing 30 g/l of feathers
were also obtained in the medium containing 30 g/l of (Fig. 4).

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Fig. 5: Effects of medium pH on caseinolytic activity Fig. 8b: Reducing power of feather protein hydrolysate
(FPH)

Effect of pH: The effect of the initial medium pH values

from 6.5 to 11 on the production of proteolytic enzymes
and on feather protein hydrolysate was studied by
cultivating the strain MPTK6 in the medium containing
30 g/l of intact feathers. As shown in Fig. 5, the highest
protease production level was obtained at pH 6.
Nevertheless, at this pH value no complete feather
degradation was achieved (evaluated by physical
appearance). The optimum initial pH of the strain MPTK6
Fig. 6: Effects of medium pH on amino acids and peptides to produce FPH was observed to be 9-10. The highest
amino acids and peptides (38±1.2 g/l) and soluble proteins
and peptides (19.5±0.2 g/l) productions were observed at
pH 10.0 (Figs. 6 and 7).
Hydrogen ion concentration is one of the most
important factors affecting the growth of the bacteria,
keratinase production and percentage of feather
degradation. Since cultures were conducted in media
containing only chicken feather as nitrogen and
carbon source, complete degradation of chicken
feather to produce a hydrolysate rich in amino acids
and peptides could only be achieved when high
Fig. 7: Effects of medium pH on soluble proteins and keratinolytic activity was attained. Thus, different pH
peptides values were tested for proteolytic enzyme and FPH
production. Complete feather degradation by the MPTK6
strain was observed at initial pHs of 9.0-11.0. This result
could be explained by the fact that the proteolytic
enzymes produced by MPTK6 strain are highly active at
alkaline conditions. This finding is similar to that
published by [42] which shows that complete feather
degradation by the strain Bacillus sp. FK 46 was reached
with initial pH of 9.0. However, Grazziotin et al. [43]
reported that the strain Vibrio sp. Kr2 produced the
maximum level of soluble proteins with initial pH
comprised between 6.0 and 8.0. The MPTK6 strain was
Fig. 8a: Antioxidant activity of feather protein able to complete feather degradation, indicating its
hydrolysate (FPH) strong keratinolytic activity. Maximum amino acids,

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Table 2: Composition and In vitro digestibility of raw feathers and FPH in line with those reported by Fakhfakh et al.,[29] using
Composition Raw feathers FPH the strain Bacillus pumilus A1 who recorded 98% of
Protein 83.1±0.2 84.2±0.1 in vitro digestibility in his study.
Fat 0.041±0.03 1.7±0.5
Moisture 2.92±0.4 5.8±0.6 CONCLUSION
Ash 9.8±0.1 0.52±0.02
In vitro digestibility (%) 96±0.5 1.8±0.5 These results, from the present study, permit us to
conclude that the strain MPTK6 had the ability to
peptides and proteins production as well as more efficient degrade raw feather to FPH under optimal conditions
feather degradation were observed under the following (30 g/l chicken feathers, pH 10.0 and 72 h of fermentation).
conditions (30 g/l of raw feathers; pH 10 and 48 h). The study also showed the DPPH free radical-scavenging
activity and reducing power showing the antioxidant
Antioxidant Activity: DPPH is a stable free radical that potential of FPH obtained from the test isolate. In
shows maximum absorbance at 517 nm. When DPPH addition, it presents a very high in vitro digestibility
radicals encounter a proton-donating substrate such as compared to that of the untreated feathers. Future studies
an antioxidant, the radicals would be scavenged and the should be conducted to evaluate the use of these
absorbance would be reduced [44]. The decrease in hydrolysates as feed additive in vivo.
absorbance is taken as a measure for radical-scavenging
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